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Adaptive patterning during skin formation: assembly of vasculature and adipose tissue

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Adaptive patterning during skin formation: assembly of vasculature and adipose tissue

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ADAPTIVE PATTERNING DURING SKIN FORMATION:
ASSEMBLY OF VASCULATURE AND ADIPOSE TISSUE

by

Kuang-Ling Ou

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CRANIOFACIAL BIOLOGY)

May 2020

ABSTRACT

Making a fully functional skin is a challenge to tissue engineering because the skin has
complex architecture composed of epidermal and dermal layers, as well as appendages such as
hair/ feather follicles, sweat glands and touch domes. Assembly of this architecture requires cell
fate specification and environmental cues at proper time and space. Our long-term objective is to
understand how these components are laid out in development and to apply these principles to
help us reconstitute tissues. The arrangement of dermal condensations in skin, i.e. de novo
patterning, is the first patterning event in skin development. Continued organogenesis may lay
their patterns using patterned structures as reference points via adaptive pattern formation.
Adaptive patterning has been implicated in the formation of intradermal muscles, adipose tissues,
blood vessels and nerves.
In this dissertation, we studied periodic patterning from dissociated human skin progenitor
cells and in wound induced follicle neogenesis in the spiny mouse from the perspective of tissue
engineering. In an effort to generate human hair primordia more effectively, we developed an in
vitro platform to study the self-organization behavior of hair progenitor cells and to identify the
factors that impact feather primordia formation. The hair peg-like structures emerging from this
3D culture system in 96 hours were molecularly validated, resembling hair pegs found in normal
human follicular development. After transplantation to nude mice, mature hair follicles of human
origin were produced. We studied spiny mice (Acomys cahirinus) because of their unique scar-
free wound healing capabilities, producing complete regeneration of epidermal appendages and
dermis after injury. Spiny mouse skin contains a large portion of adipose tissue and has a larger
hair bulge with high stem cell marker expression (K15 and CD34). Also, the spiny mouse hair
cycle is longer and cycles less frequently than that of the laboratory mouse (C57Bl6). The features
we identify by comparing wound healing in spiny and laboratory mice may contribute to improve
the regenerative ability of the spiny mouse which could have broad reaching applications to
regenerative medicine.
Next, we studied adaptive patterning of vasculature and adipose tissue. For the assembly
of vasculature in skin development. Tg(tie1:H2B-eYFP) quail expressing YFP fluorescence
exclusively in the nuclei of endothelial cells (ECs) provides a good model to study the
neovascularization process because it enables us to evaluate the topology of vascular progenitor
cells in living tissues. H2B-eYFP
+
cells were found to aggregate immediately to the newly formed
feather primordia. Angiogenic factors, e.g. FGF2 and VEGF, were found highly expressed in the
primordia epithelium compared to the dermis. These results suggest that the vasculature network
i
is initially patterned in a feather epithelium-dependent manner. On the other hand, by locally
overexpressing sprouty2 and noggin, known to inhibit angiogenesis, feathers showed abnormal
phenotypes at later developmental stages after feather follicles form. Then, we asked whether
the feather vasculatures are newly formed or derived from major vessels. Transcriptome analysis
showed that the profile of H2B-eYFP
+
cells in skin (as the 2
nd
wave of neovascularization)
resembles H2B-eYFP
-
cells in skin more than H2B-eYFP
+
cells in aorta (as the 1
st
wave of
neovascularization), implying new vasculature in the skin is locally induced. Surprisingly, Tie1+
cells in skin, compared to those in aorta, express much higher level of genes for morphogenesis
other than neovascularization, such as smooth muscle formation and adipogenesis, implying an
“interconversion” status exists for the mesenchymal cells during early development.
For the assembly of adipose tissue in skin development, we investigated the distribution
pattern, molecular markers and progression of adipocyte differentiation in embryonic chicken skin.
Interestingly, we identified a close anatomical association between feather tracts and adipocytes
and that the distribution pattern of subcutaneous fat resembled vasculature organization. Within
the feather tract, dermal fat is distributed in parallel with the feather muscle network. Next, we
identified C/EBPa as a reliable preadipocyte marker located in the nucleus. Lineage tracing shows
that some SMA+ cells in developing avian skin give rise to preadipocytes by co-staining with an
anti-C/EBPa antibody. Finally, we found adipogenesis is highly dependent on the presence of a
blood supply because it only occurred in the chorioallantoic membrane (CAM) model but not in
the explant culture model. Taken together, these findings suggest that in gross anatomy, avian
species have a similar arrangement of adipocytes to that in the skin of mammalian species but
the avian dermal adipose layer is different. Avian species have a complex muscle network and
the distribution of dermal fat tissue is associated with feather muscle pattern. A SMA+ cell lineage
tracing study connects the patterning among fat, muscle and vasculature. Some SMA+ cells in
the vasculature and feather muscle were found to become adipose progenitors (C/EBPa+), but
the contribution of this pathway remains to be quantified. The CAM model can support embryonic
skin adipogenesis and we have taken advantage of this model to test the role of “adaptive
patterning” during development.
In summary, we explored the principles of tissue patterning in skin development step-by-
step using different models. It is straight-forward to use human hair follicle precursors to engineer
human hair follicles, transplant them to host skin and study periodic patterning for translational
medicine purposes. Furthermore, we set out to discover the unique spiny mouse skin features
that are responsible for this enhanced regenerative ability. To explore how to improve the hair
peg-like structures that we engineered, we asked how nature builds new structures next to mini-
ii
organs to vitalize and organize them into functional units. Therefore, we studied the adaptive
patterning of vasculature and adipose tissue in avian skin to know how they are assembled into
this network. The phenomena and knowledge acquired in this study will be applied to the
assembly and regeneration of complex tissues to facilitate functional wound healing in
regenerative medicine.
iii
TABLE OF CONTENTS
ABSTRACT i
ACKNOWLEDGEMENTS v
LIST OF FIGURES vi
CHAPTER 1: General introduction 1
Deciphering novel rules of pattern formation in building the skin 1
Follicle neogenesis: tissue engineering and wound regeneration 2
Vasculature: neovascularization, development, VEGF and skin 3
vasculature
Adipose tissue: component, development, difference between 6
species and skin adipose tissue
Findings and significance 9

CHAPTER 2: Follicle neogenesis: tissue engineering and wound regeneration 11
A. Self-organizing hair peg-like structures from dissociated skin 11
progenitor cells: New insights for human hair follicle organoid
engineering and Turing patterning in an asymmetric morphogenetic
field
Introduction 12
Methods 12
Results 13
Discussion 18

B. Comparative regenerative biology of spiny (Acomys cahirinus) and 23
laboratory (Mus musculus) mouse skin
Introduction 23
Material and Methods 24
Results 25
Discussion 27

CHAPTER 3: Avian skin neovascularization: patterning, induction and 31
mesenchymal cell fate plasticity
Introduction 31
Results 34
Discussion 51
Material and Methods 57

CHAPTER 4: Dissection of developmental avian skin adipose tissue: 63
patterning, markers and lineage
Introduction 63
Results 66
Discussion 81
Material and Methods 84

CHAPTER 5: Conclusions and perspectives 87

References 93
iv
ACKNOWLEDGEMENTS

First, I would like to thank my mentor, Dr. Cheng-Ming Chuong, for his guidance during
my graduate study. I appreciate the friendly and collaborative atmosphere he has created
providing strong support from lab members. This research and learning environment allowed me
to be independent and creative. Specifically, the diversity of different professions nourished a
resourceful Chuong lab. None of this dissertation would have been possible without his constant
encouragement and vision, and he helped me become the scientist that I am today. I also want
to thank Dr. Rusty Lansford’s lab for providing transgenic quail eggs that enabled me to visualize
the vasculature. I am very grateful to my committee members, Dr. Michael L. Paine, Dr. Robert
Maxson, Dr. Rusty Lansford, and Dr. Ting-Xin Jiang.
I would like to express my gratitude toward all current and former members of the Chuong
lab for their support and advice. I want to thank Dr. Erin L. Weber and Dr. Ting-Xin Jiang for
making me part of their research in the beginning of my graduate career. I appreciate their
patience to guide me and to teach me experimental logic and techniques required for research.
Also, I want to acknowledge Dr. Stephanie Di-Shan Tsai, Dr. Chi-Kuan Chen, Dr. William Wei-
Jen Chang, Dr. Ya-Chen Liang, Dr. Masafumi Inaba, Dr. Chao-Yuan Yeh, and Dr. Hans I-Chen
Harn. I am very grateful to have an opportunity to work with wonderful colleagues that are also
great scientists. Specifically, I want to thank Dr. Randall Widelitz for proofreading and English
editing this dissertation. And I thank my collaborator, Junxiang Jason Huang, for trying to
understand and solve problems I encountered along the way. He worked tirelessly for these
projects.
I would also like to thank my friends for their love and support. Companionship helped me
keep going at difficult times. In particular, I thank my family, Dr. Po-Wei Chu, Dr. Yu-Chiung Hsu,
Dr. Szi-Heng Hsu, Ms. Yu-Ying Hsu, Dr. Kuang-Wen Ou, Dr. Kuang-I Ou, and grandma, Ms.
Ching-Hsiang Hsu Liu, and members of the Plastic and Reconstructive Division of the Department
of Surgery of Tri-Service General Hospital in Taiwan, Dr. Hsian-Jenn Wang, Dr. Tim-Mo Chen,
Dr. Shyi-Gen Chen, Dr. Nienn-Tzyy Dai, Dr. Yuan-Sheng Tzeng, Dr. Chih-Hsin Wang, Dr. Mao-
Liang Chiang, Dr. Wen-Kuan Chiu, Dr. Chun-Chang Li, Dr. Chin-Ta Lin, Dr. Chi-Yu Chen, Dr.
Chun-Kai Chang, Dr. Chien-Ju Wu, Dr. Hung-Hui Liu and Dr. Ted Huang, although no longer with
us, for supporting me unconditionally for every decision I made in pursuing my Ph.D in the U.S.
and I dedicate this dissertation to them.

v
LIST OF FIGURES

CHAPTER 2: Follicle neogenesis: tissue engineering and wound regeneration
A. Self-organizing hair peg-like structures from dissociated skin progenitor
cells: New insights for human hair follicle organoid engineering and
Turing patterning in an asymmetric morphogenetic field
Figure 1. 13
Figure 2. 15
Figure 3. 16
Figure 4. 17

B. Comparative regenerative biology of spiny (Acomys cahirinus) and
laboratory (Mus musculus) mouse skin
Figure 1. 25
Figure 2. 26
Figure 3. 27
Figure 4. 27
Table 1. 29

CHAPTER 3: Avian skin neovascularization: patterning, induction and
mesenchymal cell fate plasticity
Figure 1. 41
Figure 2. 43
Figure 3. 45
Figure 4. 46
Figure 5. 47
Figure 6. 48
Figure 7. 49

CHPATER 4: Dissection of developmental avian skin adipose tissue:
patterning, markers and lineage
Figure 1. 71
Figure 2. 73
Figure 3. 75
Figure 4. 77
Figure 5. 78
Figure 6. 79
vi
CHAPTER 1: General Introduction

Deciphering novel rules of pattern formation in building the skin
The skin functions as the largest organ in the human body, serves as a barrier to the
outside world, and protects us from environmental harm such as UV light and microbes. It also
regulates temperature and water loss, and senses pain, touch and heat. The skin is actually a
complex system, composed of several distinct mini-organs, including hair follicles, sweat glands
and other sensory units. Understanding the principles underlying pattern formation during the
morphogenesis and regeneration of complex skin architecture would give us insights into ways to
tissue engineer human skin for transplantation. Patients would not only benefit from the rejection-
free transplantation, but also would not need to use their own body part for autologous
transplantation, risking donor site complications, such as painful scars and brittle skin. During the
past decade, three-dimensional (3D) bioprinting technology shed a light on graft substitutes, but
clinical applications of this approach are mostly limited to orthopaedics and craniofacial
reconstruction. Failures of transplanted 3D bioprinted soft tissue products have been reported
and patients suffer from severe complications of graft failure, even death (Day, 2019). We
recognize there is a huge gap of clinical use of 3D printed soft tissue grafts and learning how the
complex pattern is formed from multiple elements may help us identify principles that can be used
in human designed network formation.
The initial patterning events of feather morphogenesis are composed of macro-patterning,
and micro-pattering (Sengel and Mauger, 1976, Sengel, 2003, Dhouailly et al., 2003, Lin et al.,
2006). There are different feather tracts, apteria and scale regions in avian species, and such
regional specificity refers to macro-patterning. The subsequent organization of feather primordia
within specific feather tracts is called micro-patterning, which is mainly periodic/de novo
patterning. Periodic/de novo patterning describes order which emerges from homogeneity
appearing in a spatial or temporal sequence with single or repetitive elements which may be
identical or demonstrate variations (Chuong et al., 2013). Ectodermal organs on the body surface
display diverse and delicate patterns that help the organism cope with external environmental
changes. Our lab previously showed that feather primordia are periodically patterned following a
reaction-diffusion mechanism (Turing, 1952) which occurs through the interactions between a
short-range activator (FGF2) and a long-range inhibitor (BMP4) (Jiang et al., 1999). In the mouse
model, it was demonstrated that hair follicle patterning follows the same mechanism using Wnt
as an activator and DKK as an inhibitor (Sick et al., 2006). After the initial patterning events, other
tissues (i.e., blood vessels, muscles and nerve fibers) are required to be assembled to the system
1
to build the skin as an integral entity. In avian skin, feather muscle development is highly
dependent on feather buds’ locations and our lab named this process “adaptive patterning” (Wu
et al., 2019). In this dissertation, we explored different aspects of skin patterning in the hope that
the future practice of tissue engineering might use the knowledge and patterning principles
derived here.

Follicle neogenesis: tissue engineering and wound regeneration
Hair follicles, constituted by epithelial cells and dermal cells, serve as an ideal model to
study the interplay and communications between ectoderm and mesoderm which are key to hair
follicle development (Headon, 2009). During embryogenesis, the stages of hair follicle
development are divided into: induction, organogenesis, and cytodifferentiation (Schneider et al.,
2009). The stages are characterized by structures as: placode, germ, peg, and the bulbous follicle
(Schmidt-Ullrich and Paus, 2005, Schneider et al., 2009). When the ectoderm covers the embryo,
underlying mesenchymal cells start to interact with it which leads to ectodermal stratification. The
ectodermis then becomes either the epidermis or is specified as part of the appendages. In early
hair/ feather follicle development, Wnt mediated signal transduction arises in mesenchymal cells.
Then the epithelial cells above them form a “placode,” which will then signal to the underlying
mesenchymal cells to form a “dermal condensate”. The dermal condensates will in turn direct
overlying epidermal cells to proliferate and invaginate into the dermal layer. In mammals, the
“dermal papilla” is then distinctly formed and guides the epidermal cells to shape the entire hair
follicle.
Summarized from the developmental process of hair follicles, the ability to form
aggregates, placodes and condensates, is essential to initiate neo-folliculogenesis. Our lab has
developed a simple in vitro skin reconstitution assay to culture cells on transwell membranes that
mimic the support of the basement membrane (Lee et al., 2011). In the mouse model, the
embryonic epidermal cells can first self-assemble into aggregates and then form cyst-like
structures in the presence of dermal cells, followed by branching of the cysts and the formation
of a system-wide two-layer lumen structure (Lei et al., 2017). As for human cells, researchers
have used dermal papilla cells with collagen and matrigel to make aggregates (Havlickova et al.,
2009) (Thangapazham et al., 2014). A hanging drop, 3D culture model was also used to make
dermal papilla spheroids(Higgins et al., 2013). Neonatal foreskin keratinocytes combined with
dermal cells and cultured on transwell membranes formed reconstituted skin. When these were
transplanted to nude mice, the reconstituted human skin formed intact epidermis and dermis with
mature hair follicles (Wu et al., 2014). However, how the mixture of epidermal and dermal cells
2
grow hair follicles in the human model is still unknown and the human hair follicle forming
efficiency is low. Here, we sought to understand the developmental process and cell interactions
of the tissue engineered human skin to improve its efficiency.
Another way to look at neo-folliculogenesis is from the aspect of wound regeneration. In
human beings, wounds heal by forming scars, which deform the skin, are itchy, sometimes painful
and most importantly, do not form appendages in a pilosebaceous unit. In mice, it was discovered
that if a periodic patterning process can be initiated, new hairs can regenerate from a large wound
(>1cm) (Ito et al., 2007). This phenomenon has never been seen in humans. Spiny mice (Acomys
cahirinus) which escapes a predator by autotomizing body parts has the ability to regenerate skin
and skin appendages in a process known as wound induced hair neogenesis (WIHN) (Seifert et
al., 2012). We observed that while WIHN in laboratory mice emerges from the wound center, it
starts from the wound edge in spiny mice. Therefore, we decided to compare the skin
characteristics (including hair types, cycle, hair follicle stem cells, etc.) and reactions to hair
plucking and large wounds between laboratory and spiny mice.

Vasculature: neovascularization, development, VEGF and skin vasculature
Circulation/perfusion is the cornerstone of organ transplantation and microsurgery.
Without meticulous anastomosis of donor’s and recipient’s vasculature, graft failure with severe
complications is expected in patients. Because formation of the vascular system with an adequate
blood supply to provide sufficient oxygen and nutrition vascularization is necessary to maintain
healthy tissues, this is a major limitation of organoids and other three-dimensional culture
systems, that lack a vascular network. In development, the circulatory system begins soon after
gastrulation, and is the first functional organ system to arise. In physiologic and pathologic
processes, neovascularization, the growth of new blood vessels, is an essential step mediated by
coordination of multiple cell types to form and remodel the vascular system.
Vasculogenesis and angiogenesis are the two most common mechanisms of
neovascularization. Vasculogenesis is the de novo formation of new blood vessels, giving rise to
the first blood vessel (Kubis and Levy, 2003, Johnson and Wilgus, 2014). Two distinct steps during
the onset of vascularization, which define vasculogenesis are: 1) angioblasts differentiate to ECs
that 2) self-assemble to form primitive blood vessels. Vessel development proceeds to form a
complex vascular network required for embryonic development. Roles of vasculogenesis have
also been described in adult tissues, especially capillary formation in response to ischemia. In
addition to de novo vessel formation, angiogenesis is also responsible for the vascularization of
organs derived from ectoderm-mesoderm and is studied more frequently. Angiogenesis is defined
3
as new blood vessels forming from the “sprouting” endothelial cells of preexisting vessels.
“Sprouting” is led by a single endothelial cell known as the tip cell, which responds to angiogenic
stimuli and connects to endothelial stalk cells that function in tube formation (Carmeliet, 2003,
Carmeliet and Jain, 2011).
Early blood vessel formation is widely studied in avian systems because of the ease of
access for analysis. The dorsal aorta develops a vascular appearance at E1.5. At this stage, the
first functional intraembryonic blood vessels connect to the nascent heart and are visible shortly
after the heart starts to beat (Sato, 2013, Poole et al., 2001, Krah et al., 1994). The roof of the
dorsal aorta endothelial cells (ECs) is derived from the somite and the floor of the aorta is derived
from splenic mesoderm (Pardanaud et al., 1996) but the floor component will later be replaced by
somitic ECs (Pouget et al., 2006). Notch signaling was shown to regulate the contribution of
somitic cells to the dorsal aorta (Sato et al., 2008). Also, Notch signaling is involved in the
sprouting angiogenesis process (Roca and Adams, 2007, Herbert and Stainier, 2011), arterial EC
specification (Lawson et al., 2002) and the induction of vascular smooth muscle cell
differentiation(High et al., 2008, Liu et al., 2009, Wang et al., 2012). A Tie1 transgenic quail line
Tg(tie1:H2B-eYFP) was made to dynamically analyze vascular morphogenesis (Sato et al., 2010).
The aforementioned dorsal aorta assembles from somite and splenic mesoderm and the motion
of ECs were demonstrated using time-lapse recording in this Tg(tie1:H2B-eYFP) quail sample.
This model was also applied to study avian cornea development and periocular vasculature
formation (Kwiatkowski et al., 2013).
VEGF signaling has essential roles in both vasculogenesis and angiogenesis (Senger et
al., 1990). VEGF binds to tyrosine kinase receptors (VEGFRs) located on the cell surface and
activates them through transphosphorylation (Koch and Claesson-Welsh, 2012). In early
development, VEGFR-2 is the first molecule expressed in the mesenchymal cell population which
gives rise to angioblasts (Yamaguchi et al., 1993, Choi et al., 1998). Genetic studies demonstrate
embryos lacking VEGFR-2 die early in development because they fail to initiate vasculogenesis
and hematopoiesis (Shalaby et al., 1997). Angiogenesis is controlled by changes in the levels of
proangiogenic and antiangiogenic molecules present within the microenvironment surrounding
the vasculature. VEGF is one of the most important proangiogenic mediators. ECs secrete
angiocrine factors that contribute to organogenesis. In early hepatic bud development, newly
specified hepatic endoderm is surrounded by early endothelial cells, which also delimit the
mesenchymal domain in which the liver bud grows (Matsumoto et al., 2001). In flk-1 (VEGFR-2)
mutant models, though hepatic specification occurs in the embryo, liver morphogenesis fails prior
to mesenchymal invasion. Also, hepatic growth was impaired in the absence of ECs indicating
4
ECs are essential for the earliest stages of liver organogenesis, prior to blood vessel function.
Pancreatic growth is initiated where the foregut endoderm epithelium contacts major premature
blood vessel endothelium, before vascular smooth muscle and other perivascular cells developed
(Lammert et al., 2001). Blood vessel endothelium then induced insulin expression in isolated
endoderm. When the dorsal aorta was removed the endoderm failed to express insulin. Also,
ectopic vascularization led to islet hyperplasia and ectopic insulin expression, suggesting the
inductive role of ECs in pancreatic development. Recently, ECs were found to regulate salivary
gland epithelial patterning through VEGFR2-dependant signaling and IGFBP2/3 (Kwon et al.,
2017).
In order to tissue engineer skin, one essential step to make grafts functional is to vitalize
them with blood flow. In chapter 2, we were trying to tissue-engineer human hair follicles and
determine the interactions that took place between vascular cells and hair follicles. It has been
shown that hair follicle growth and cycling is mediated by VEGF-dependent angiogenesis and
Improved follicle vascularization promotes hair growth and increases hair follicle number (Yano
et al., 2001). By comparing characteristics during anagen/telogen, Yano et al. found an
association of hair cycling with cyclic perifollicular angiogenesis, and temporal association of
perifollicular angiogenesis with cyclic changes of follicle size and cutaneous thickness. This
suggests that the angiogenic stimulus was derived from the HF/dermis/subcutis but not from the
interfollicular epidermis. An important role of follicle-derived VEGF in the control of hair
vascularization was identified and a functional perifollicular vascular system is necessary for the
mediation of VEGF effects on follicle growth. Intriguingly, it was also found that rat hair follicle
stem cells (rHFSCs) could induce differentiation into ECs by treating rHFSCs with VEGF165
(Quan et al., 2017). First, although it is not clear how skin vasculature develops during embryonic
stages, summarized from above, it’s possible that the skin vasculature develops within its own
mesenchyme. Second, studies have shown that in some cases, the developing organ produces
paracrine factors that induce blood vessel to form in its own mesenchyme (Auerbach et al., 1985,
LeCouter et al., 2001). Third, even in adult rats, HFSC can be induced to differentiate into ECs.
Avian skin can be an ideal model to uncover new principles because of their accessibility
for experimentation and visibility of phenotypic changes. Like hair follicle development, feathers
are derived from a series of interactions between the epithelium and mesenchyme. Chicken
dorsal dermis is generated from somite dermomyotome and the ectoderm transforms into
epidermis by E5.5(Lei et al., 2016). The dermomyotome develops into three distinct domains:
medial (dorsomedial lip, DML), central and lateral parts and each part contributes to different
regions of the developing skin. However, the origin of the dorsomedial dermis remains
5
controversial: while one study suggested it is derived from the DML(Olivera-Martinez et al., 2002),
another suggested it is derived from the sclerotome(Ben-Yair et al., 2003). Later, during the
initiation of feather buds from embryonic skin starts from a thickening of epidermal cells, followed
by dermal condensation formation. The arrangement of dermal condensates determines the
location of induced “feather primordia.” Feather primordia will differentiate further to form feather
buds, germs, and finally the complex feather follicles (fully formed epidermal collar surrounding
the dermal papilla).
For avian species, the skin vasculature is very different from mammals. Inside the feather,
the pulp is composed of fibroblasts and rich in blood vessels. Also, nerves invade into the pulp
cavity. Outside the feather in the dermis, feathers are connected with blood vessels, nerves, and
muscles. The axial artery of the pulp grows into the feather follicle and the feather during anagen;
at a certain stage, the pulp reabsorbs and makes the feather hollow (Lucas and Stettenheim,
1972). In mice, blood vessels don’t grow into the dermal papilla, but a horizontal plexus under the
hair germ can be found transiently in the neighboring HFSC activation zone (Li et al., 2019). The
vasculature is remodeled during the hair cycle and the cross-talk between HFSCs and ECs is
associated with stem cell activation and tissue homeostasis. In avian species, between early
development of the circulation system and feather vasculature, there is a huge gap of our
understanding about neovascularization. In the 2
nd
part of this dissertation, we aim to characterize
the process of skin neovascularization using a transgenic quail line Tg(tie1:H2B-eYFP), to
elucidate the interactions between feather formation and neovascularization, to identify factors
involved in skin neovascularization, and to try to figure out how neovascularization plays a hinge
in the avian skin development between the formation of dermal condensations, myogenesis and
adipogenesis patterning.

Adipose tissue: component, development, difference between species and skin adipose
tissue
The adipose tissue is a complex organ (Kershaw and Flier, 2004), playing an important
role in physiology, including thermoregulation, metabolism, energy balance and nutritional
homeostasis. Also, it has important mechanical properties, cushioning body parts and protecting
delicate organs. There are two kinds of adipocytes: white adipose tissue (WAT), brown adipose
tissue (BAT) and beige/brite adipose tissue (Rosen and Spiegelman, 2014). WAT makes the bulk
of fat tissue in most animals and usually contains a single large lipid droplet. The primary role of
WAT is energy storage through the control of glucose and lipid homeostasis. BAT has more
abundant mitochondria and many small lipid droplets, which is different form the energy storage
6
abilities of WAT. BAT is specialized for dissipating stored chemical energy in the form of heat
through uncoupling protein 1 (UCP-1). Beige/Brite adipocytes are UCP-1+ cells with a brown fat-
like morphology in WAT caused by cold exposure or adrenergic signaling.
In addition to different types of adipose tissue, regional differences exist in discrete depots
(Hepler et al., 2017). The adipocyte behavior and physiology, such as adipokine secretion and
rates of lipolysis and triglyceride synthesis, are different in distinct depots. Also, preadipocytes
express gene signatures that are specific for their depot of origin, indicating there are intrinsic
differences between depots. Tracing back to the early developmental stage, adipocytes are
thought to be of mesodermal origin. However, mesenchyme derives from the neuroectoderm in
the cephalic region, which means the adipocytes are of ectodermal origin in this part of the body.
Therefore, the precise origin of adipose tissue may vary from depot to depot. In the 1940s, de
novo adipocytes forming along the vasculature was observed in live rabbit ear (Clark and Clark,
1940). On the cellular level, the growth of isolated stromal vascular fractions (SVFs) showed that
fibroblast-like cells from the SVFs are capable of adipogenesis (Cawthorn et al., 2012). This
indicates the presence of adipose precursor cells in the SVF.
PPARγ is the master transcriptional regulator of adipogenesis and a PPARγ reporter
system has been a useful tool to trace adipose cell lineages. Early adipose progenitors within the
fat pads of young mice express PPARγ and are physically associated with the walls of vasculature
within adipose tissues. Tang et al.(Tang et al., 2008) used PPARγ reporter mice to demonstrate
that early adipose progenitors within the fat pads of young mice express PPARγ and localized in
vivo in the vasculature resembling the mural cell compartment. They also showed only some
mural cells express PPARγ and are adipogenic, but almost all adipocytes are derived from mural
cells. Later, the AdipoTrak system (PPARγ-GFP) was developed, with mural cell fate-mapping
(SMA-RFP) and conditional gene knockouts, adult adipocyte fate-mapping was shown from a
mural cell lineage, but not developmental-stage adipocytes(Jiang et al., 2014). Earlier than
PPARγ, ZFP423(zinc-finger protein 423) (Gupta et al., 2010, Gupta et al., 2012) and C/EBPb
(CCAAT enhancer binding protein b) (Lee et al., 1998, Farmer, 2006) can be used to identify
preadipocytes in early lineage commitment. At differentiation stages, committed adipose
progenitors then mature into C/EBPα positive preadipocytes (Farmer, 2006, Wojciechowicz et al.,
2008).
Earlier studies have focused on visceral WAT and subcutaneous (SWAT) for obesity
related metabolic and cardiovascular disorders; dermal WAT(DWAT) was later found as a key
component of the skin defense system, modifying systemic metabolism, immune function and
physiology (Alexander et al., 2015). In humans, verified differences between SWAT and DWAT
7
not only at the anatomical and morphological levels but also in cellular function have been
identified (Kosaka et al., 2016). The dermis, comprised of multiple fibroblast subtypes, has two
distinct layers: superficial papillary and reticular dermis underneath. Dermal fibroblast
heterogeneity increases with development (Lynch and Watt, 2018). PDGFRα was used as a pan-
fibroblast marker to perform lineage tracing of dermal fibroblast populations in the mouse model
(Driskell et al., 2013). Multipotent mesenchymal progenitors are present at E12.5 and then commit
to become dermal papilla (PDGFRα+CD26-SOX2+), papillary (PDGFRα+DLK1-
BLIMP1+LRIG1+) or reticular/hypodermal (PDGFRα+DLK1+BLIMP1-) progenitors by E16.5. At
postnatal day 2, reticular fibroblasts (PDGFRα+DLK1+SCA1-) and adipocyte precursors
(PDGFRα+DLK1+/-SCA1+CD24+) can be differentiated. In other words, preadipocytes in dermis
are fibroblasts with the capacity to differentiate into adipocytes.
For avian species, adipose tissue was studied mainly in poultry and agriculture science
because of concerns in the broiler industry about the quality of domestic chicken meat, a major
protein source. Therefore, there are relatively few avian studies on the morphological,
biochemical, and molecular changes involved in the biological process of adipocyte differentiation.
Chicken preadipocytes prepared from adipose tissue (Ramsay and Rosebrough, 2003) can be
induced to differentiate in culture with exogenous fatty acid (Matsubara et al., 2005). Using this in
vitro system, it was shown that compared to C/EBPa/b/d, PPARγ is the key regulator in the early
stages of chicken preadipocyte differentiation and aP2(adipocyte fatty acid-binding protein)
mRNA and protein levels increased following the increase in PPARγ gene expression. Also,
oleate was demonstrated to induce transdifferentiation of fibroblasts from E9 chicks into
adipocyte-like cells but had no effect on chicken myoblasts from E11 pectoral muscle (Liu et al.,
2010). Other adipogenesis related factors were found playing different roles in chickens than in
mammals (Matsubara et al., 2013). While Zfp423 was identified as a transcriptional regulator of
preadipocyte determination in mammals, chicken ZFP423 appeared to play a role in the late stage
of adipocyte differentiation. However, in Adelie penguins which develop subcutaneous and
retroperitoneal adipose tissues rapidly (weight of subcutaneous and retroperitoneal fat multiplied
by 40) within one month after hatching. C/EBPa was expressed earlier than C/EBPb and PPARγ
during the maturation of adipose precursor cells in vivo (Raccurt et al., 2008). Later, different
adipose depots in the post hatching chicken abdomen, subcutis and supra clavicle space were
shown to have site-specific differences by comparing the developing weight, cellularity and
expression of C/EBPa/b, PPARγ, FABP4(fatty acid binding protein 4)(Bai et al., 2015).
Avian species don’t have brown adipose tissue. Because avian species can adapt to harsh
environments, researchers have looked for brown fat that might generate heat through UCP-1.
8
However, UCP-1 was not found in fat in winter-acclimated Ring-necked Pheasants (Phasianus
colchicus), Japanese quail (Coturnix japonica), Rock doves (Columbalivia), nor in the wild House
sparrows (Passer domesticus), Great tits (Parus major) in winter and Common poorwills
(Phalaenoptilus nuttallii) (Brigham and Trayhurn, 1994). The creatine futile cycle was revealed as
the major component for adipocyte thermogenesis in recent years (Kazak et al., 2015, Kazak et
al., 2017, Kazak et al., 2019). In addition to UCP1 in avian muscle, adipocyte thermogenesis may
also count on creatinine metabolism which needs to be further studied.
Avian skin adipose tissue was described in both the loose layer of the dermis and
subcutis, which are anatomically divided by a layer of elastic lamina (Lucas and Stettenheim,
1972). Subcutaneous adipose tissue was studied in post hatch chickens (as mentioned in the
previous paragraph) but little is known about avian dermal adipose tissue. Our lab specializes in
studying pattern formation using the avian integument system. Actually, adipose accumulation
around the feather follicle base was observed during our study for feather follicle development.
Although previous work has described a close relation between hair follicles and adipogenesis in
the mouse model(Zhang et al., 2016{Festa, 2011 #78, Festa et al., 2011)}, little is known about
avian skin adipose tissue. In the 3
rd
part of this dissertation, we aimed to characterize the process
of skin adipogenesis, to identify distinct markers for skin adipose tissue, to elucidate the spatial-
temporal relationship of neovascularization, myogenesis and adipogenesis and to figure out the
composition and source of avian skin adipose tissue.

Findings and significance
In this dissertation, we experimentally address the skin organization from different levels
of patterning. In Chapter 2, follicle neogenesis was studied from two aspects: human hair follicle
organoid engineering and spiny mouse regenerative wound healing. In “Self-organizing hair
peg-like structures from dissociated skin progenitor cells: New insights for human hair
follicle organoid engineering and Turing patterning in an asymmetric morphogenetic
field”, we specifically focused on the issue of generating human hair primordia more effectively
and identified the principle and factors of periodic patterning in this three-dimensional culture
system. In “Comparative regenerative biology of spiny (Acomys cahirinus) and laboratory
(Mus musculus) mouse skin”, we characterized the skin features and response to small and
large wounds in spiny mice to understand more about the control of skin repair vs regeneration.
After making hair peg-like structures, we were interested in the next step of the assembly
to build the skin organ as an integral entity. Chapter 3 is dedicated to study the adaptive patterning
of neovascularization in developing avian skin. We used the Tie1:H2B-eYFP transgenic quail
9
model to visualize the behavior of vascular progenitor cells. We showed how they are organized
into a vasculature network in a feather epithelium-dependent manner. We compared the
transcriptome of Tie1+ cells in developing avian skin and aorta and found they are very different,
implying endothelial cells of different origins may have been integrated together to form the
vasculature network. Also, we found Tie1+ cells as a new wave of neovascularization in the skin,
may also be involved in other biological processes, such as smooth muscle formation.
In Chapter 4, we studied the adaptive patterning of neovascularization in developing avian
skin. We stained lipid drops with Oil Red O, Bodipy, and LipidTOX to visualize the adipogenesis
pattern in avian skin. We found two waves of adipogenesis in developing avian skin: the first wave
is in the subcutaneous layer and the pattern matches that of the vasculature; while the second
wave is within the dermal layer and the pattern matches that of the feather muscle network. We
used the Cre-loxP lentiviral system to lineage trace SMA+ cells in the avian skin and found some
SMA+ cells may become the precursors of adipocytes.
The lack of genetic functional approaches in avians is still limited and thus lineage tracing
is restricted. Unlike mouse or zebrafish models, either traditional knock out/down or
CRISPR/Cas9 chicken are not technically mature, which limits the genetic functional study mostly
in the RCAS system (with size limitation, and low efficiency in RNAi knockdown), electroporation
(which is efficient only in earlier developmental stages and has a limited half-life) or small
molecules that are commercially available. In this dissertation, we were capable of constructing
the Cre-loxP lentiviral system to lineage trace SMA+ cells in the avian skin, which are novel
techniques in the avian system. Given that these lentiviral tools have been modified to best fit our
needs, tracing the lineage of Tie1+ cells in avian skin is one of our future goals to validate our
findings from RNA-seq results.
Our tentative conclusion summarized from the adaptive patterning studies for
neovascularization and adipogenesis is that morphogenesis is competitive, and the cells that “fail”
in one context can be used for other purposes without being wasted, thus guaranteeing the
robustness of adaptive patterning in the developing dermis. Understanding how the “inter-
conversion” of these mesenchymal cell types is achieved would enhance the success of future
tissue engineering and cell therapy, specifically by manufacturing a certain type of cell that a
patient needs. Yet, detailed mechanisms such as mechanobiology or epigenetics are possible
fields to explore for the inter-conversion of mesenchymal cell types. This would require further
study.
10
Experimental Dermatology. 2019;28:355–366. wileyonlinelibrary.com/journal/exd
|
355 © 2019 John Wiley & Sons A/S.
Published by John Wiley & Sons Ltd

ORIG INAL ARTICLE
Self- organizing hair peg- like structures from dissociated
skin progenitor cells: New insights for human hair follicle
organoid engineering and Turing patterning in an asymmetric
morphogenetic field
Erin L. Weber
1,2
| Thomas E. Woolley
3
| Chao-Yuan Yeh
1
| Kuang-Ling Ou
1,4,5
|
Philip K. Maini
6
| Cheng-Ming Chuong
1,7
1
D e p a r t m e n t o f P a t h o l o g y , K e c k S c h o o l
o f M e d i c i n e o f t h e U n i v e r s i t y o f S o u t h e r n
C a l i f o r n i a , L o s A n g e l e s , C a l i f o r n i a
2
D i v i s i o n o f P l a s t i c a n d R e c o n s t r u c t i v e
S u r g e r y , K e c k S c h o o l o f M e d i c i n e o f t h e
U n i v e r s i t y o f S o u t h e r n C a l i f o r n i a , L o s
A n g e l e s , C a l i f o r n i a
3
C a r d i f f S c h o o l o f M a t h e m a t i c s , C a r d i f f
U n i v e r s i t y , C a r d i f f , U K
4
O s t r o w S c h o o l o f D e n t i s t r y o f t h e
U n i v e r s i t y o f S o u t h e r n C a l i f o r n i a , L o s
A n g e l e s , C a l i f o r n i a
5
D i v i s i o n o f P l a s t i c a n d R e c o n s t r u c t i v e
S u r g e r y , D e p a r t m e n t o f S u r g e r y , T r i - S e r v i c e
G e n e r a l H o s p i t a l , N a t i o n a l D e f e n s e M e d i c a l
C e n t e r , T a i p e i , T a i w a n
6
W o l f s o n C e n t r e f o r M a t h e m a t i c a l
B i o l o g y , M a t h e m a t i c a l I n s t i t u t e , O x f o r d , U K
7
I n t e g r a t i v e S t e m C e l l C e n t e r , C h i n a M e d i c a l
U n i ve r s i t y , T a ich u n g , T a i w a n
Correspondence
C h e n g - M i n g C h u o n g , K e c k S c h o o l o f
M e d i c i n e o f t h e U n i v e r s i t y o f S o u t h e r n
C a l i f o r n i a , L o s A n g e l e s , C A .
E m a i l : cm ch u o n g@ us c .e d u
Funding information
N a t i o n a l I n s t i t u t e o f A r t h r i t i s a n d
M u s c u l o s k e l e t a l a n d S k i n D i s e a s e s , G r a n t /
A w a r d N u m b e r : A R 4 7 3 6 4 a n d A R 6 0 3 0 6 ;
N a t i o n a l S c i e n c e F o u n d a t i o n , G r a n t /
A w a r d N u m b e r : D M S 1 4 4 0 3 8 6 ; C a l i f o r n i a
I n s t i t u t e f o r R e g e n e r a t i v e M e d i c i n e ;
A m e r i c a n C o l l e g e o f S u r g e o n s ; L . K . W h i t t i e r
F o u n d a t i o n ; A . P . G i a n n i n i F o u n d a t i o n
Abstract
H um an s k i n p r o ge n i tor ce l l s wi l l for m n ew h a i r fol l i c le s , a l t h o u g h at a l ow ef f i c i e n c y ,
w h e n i n j e c t e d i n t o n u d e m o u s e s k i n . T o b e t t e r s t u d y a n d i m p r o v e u p o n t h i s r e g e n -
e r a t i v e p r o c e s s , w e d e v e l o p e d a n i n v i t r o s y s t e m t o a n a l y s e t h e m o r p h o g e n e t i c c e l l
b e h a v i o u r i n d e t a i l a n d m o d u l a t e p h y s i c a l - c h e m i c a l p a r a m e t e r s t o m o r e e f f e c t i v e l y
ge n e r ate ha i r p r i m o r d ia . I n t h is t h r e e- d i m e n si o na l c ult u r e , d iss o c iate d h u m a n n e o na -
t a l f o r e s k i n k e r a t i n o c y t e s s e l f - a s s e m b l e d i n t o a p l a n a r e p i d e r m a l l a y e r w h i l e f e t a l
s c a l p d e r m a l c e l l s co a l e s c e d i n t o s t r i p e s , t h e n l a r g e c l u s t e r s , a n d f i n a l l y s m a l l c l u s t e r s
r e s em bli n g der m a l co n dens at io ns . At si te s of der m a l c l u s ter i n g , su b jacent ep i der m a l
c e l l s p r o t r u d e d t o f o r m h a i r p e g - l i k e s t r u c t u r e s , m o l e c u l a r l y r e s e m b l i n g h a i r p e g s
wi t h i n t h e s e qu e n ce of fol l i c u l ar d eve l op m e nt . T h e h a i r p e g - l i k e s t r u c t ur e s e m e r ge d
i n a co o r d i n a t e d , f o r m a t i v e w a v e , m o v i n g f r o m p e r i p h e r y t o c e n t r e , s u g g e s t i n g t h a t
t h e d r o p l e t c u l t u r e co n s t i t u t e s a m i c r o co s m w i t h a n a s y m m e t r i c m o r p h o g e n e t i c f i e l d .
In vivo, hair follicle p opulations also for m in a progressive wave, implying t he summa -
t i o n o f l o c a l p e r i o d i c p a t t e r n i n g e v e n t s w i t h a n a s y m m e t r i c g l o b a l i n f l u e n c e . T o f u r -
t h e r u n d e r s t a n d t h i s g l o b a l p a t t e r n i n g p r o c e s s , w e d e v e l o p e d a m a t h e m a t i c a l
si m ulat i o n usi n g T u r i n g a c t i v ato r - i n h i bi to r p r i n c i p l e s i n a n a s y m m e t r ic m o r p h o ge -
n e t i c f i e l d . T o g e t h e r , o u r c u l t u r e s y s t e m p r o v i d e s a s u i t a b l e p l a t f o r m t o ( a ) a n a l y s e
t h e s e l f - a s s e m b l y b e h a v i o u r o f h a i r p r o g e n i t o r c e l l s i n t o p e r i o d i c a l l y a r r a n g e d h a i r
p r i m o r d i a a n d ( b ) i d e n t i f y p a r a m e t e r s t h a t i m p a c t t h e f o r m a t i o n o f h a i r p r i m o r d i a i n
a n as y m m et r ic m o r p h oge n et ic f ie l d . T his u n d e r s t a n ding w ill e n ha n ce o u r f u t u re a bil -
i t y t o s u c c e s s f u l l y e n g i n e e r h u m a n h a i r f o l l i c l e o r g a n o i d s .
K E Y WO R D S
h a i r f o l l i c l e , o r g a n o g e n e s i s , p e r i o d i c p a t t e r n f o r m a t i o n , s k i n r e c o n s t i t u t i o n , t i s s u e e n g i n e e r i n g

Chapter 2: Follicle neogenesis: tissue engineering and wound regeneration
11
356
|
WEBER Et al .
1 | INTRODUC TION
T h e b a s i c t e n e t o f p l a s t i c s u r g e r y i s t h e r e s t o r a t i o n o f f o r m a n d
f u n c t i o n . H o w e v e r , r e p l a c i n g s k i n a n d f u n c t i o n a l a p p e n d a g e s r e -
m ain s ch a l l e n g in g . T h e h air f o l l ic l e i s a mini- o r g a n , w hich , in a s s o -
c i a t i o n w i t h t h e a t t a c h e d s e b a c e o u s g l a n d , p l a y s a c r u c i a l r o l e i n
s k i n m o i s tu r e , t her m a l r e gu l a t i o n , p r o t e c t iv e s en s a t i o n a n d ae s -
t h e t i c a p p e a r a n c e . F o r b u r n p a t i e n t s , t h e l o s s o f p i l o s e b a c e o u s
u n i t s l e a d s t o d r y , b r i t t l e s k i n w h i c h i s m o r e s u s c e p t i b l e t o i n j u r y .
W h i l e t r a n s p l a n t a t i o n i s c u r r e n t l y t h e b e s t o p t i o n f o r h a i r f o l l i c l e
r e p l a c e m e n t , t h e p r o c e s s r e q u i r e s a l a r g e n u m b e r o f d o n o r f o l l i -
c l e s , w h i c h b u r n p a t i e n t s t y p i c a l l y l a c k , a n d t a r g e t s o n l y t h e s c a l p .
T h e a b i l i t y t o t i s s u e e n g i n e e r a n u n l i m i t e d s o u r c e o f p i l o s e b a c e o u s
u n i t s f or t r an s p l an t a t i on , e i t h e r s i n g l y or ap p r op r i a t e l y p a t t e r n e d
w i t h i n b i o en g i ne er e d s k i n , w ou ld p r o vi de a m u c h - ne e de d so l u t i o n
f o r m a n y p a t i e n t s .
M u l t i p l e d i f f e r e n t a p p r o a c h e s h av e a t t e m p t e d t o p r o d u c e r e -
c o n s t i t u t e d s k i n w i t h h a i r i n m o u s e a n d h u m a n s .
[1]
I n t h e m o u s e ,
w e d e m o n s t r a t e d t h a t d i s s o c i a t e d e p i d e r m a l a n d d e r m a l c e l l s f r o m
newborn mouse skin self- assemble in vitro into multi-layered skin or -
g a n o i d s c o n t a i n i n g p l a c o d e s a n d d e r m a l c o n d e n s a t e s , t h e t w o s t e m
ce l l p op u l at i on s n e ce s s ar y f or h a i r f o l l i c l e d eve lop m e nt .
[2 , 3]
W hen
g r a f t e d o n t o a f u l l t h i c k n e s s d e r m a l w o u n d o n a n u d e m o u s e , t h e
cu lt u re d o r g a n o ids f o r m e d m a t u re , c yc l in g h air f o l l ic l e s w i t hin a p la -
n a r s k i n co nf ig u r at i o n . T r a n s c r i p to m i c a n a l y sis of t h e m u r i n e s k i n o r -
g a n o i d s h a s i d e n t i f i e d f a c t o r s t h a t c a n r e s c u e t h e h a i r f o r m i n g a b i l i t y
o f a d u l t m o u s e c e l l s .
[4]
H o w e v e r , s i m i l a r s u c c e s s w i t h h u m a n c e l l s
h a s b e e n m o r e d i f f i c u l t . A d u l t h u m a n s c a l p c e l l s w i l l p r o d u c e n e w
f o l l i c l e s i n i n v i v o m o u s e m o d e l s , a l b e i t a t l o w r a t e s .
[5 , 6]
T h e u s e o f
f e t a l , r at her t h a n a d u l t , sc a l p en h a n ce s t he ef f i c i en c y of h u m a n h a i r
f o l l i c l e r e g e n e r a t i o n b u t a p e r s i s t e n t l a g t i m e o f 3 m o n t h s t o f o l l i c l e
f o r m a t i o n i n d i c a t e s t h a t m o r e m u s t b e u n d e r s t o o d a b o u t f o l l i c u l a r
mo r pho ge n e sis .
[7 , 8]
D e s p i te s ever a l d i f f er ent a p p r oa c he s , ef f i c i ent ,
l a r ge - sc a l e , t her a p e u t i c t i s s u e en g i ne er i n g a n d t r a n s p l a nt at i o n of
re co n s t i t u t e d h u m a n s k in w i t h p i l o s e b a ce o u s u ni t s re m ain s a ch a l -
l e n g e t o t h e f i e l d .
T h e r e a r e t w o d i f f e r e n t s t r a t e g i e s t o p r o d u c e h a i r f o l l i c l e s
f r o m d i s s o c i a t e d c e l l s . O n e i s t o u s e 3 D p r i n t e d t i s s u e s c a f f o l d s
a n d p l a c e c e l l s a t k e y p o s i t i o n s f o r f u r t h e r m o r p h o g e n e s i s ;
[9]
t h e
o t h e r i s t o r e l y o n t h e s e l f - o r g a n i z i n g a b i l i t y o f s k i n p r o g e n i t o r
c e lls .
[4]
D i f f e r e n t p r o g e n i t o r c e l l s t a t e s c a n b e u t i l i z e d f o r t h e s e l f -
o r g a ni z in g s t r a t eg y , s u ch a s in d u c e d p l u r ip o t e n t c e l l s (iP S ) .
[10]
On
s o m e o c c a s i o n s , c e l l s n e e d “ h e l p ” t o i n t e r a c t w i t h o t h e r c e l l s o r
re q u ire p a r t icu la r m o l e cu la r s i g n a l s t o m o v e f o r w a r d t o t h e n e x t
s t a g e . C u r r en t l y , i n t he emer g i n g f i eld o f s y n t he t i c b i o l o g y , me t h -
o d s a r e u n der de v el o p men t t o p r o vi de c el l s w i t h “ hel p” i n t o p o -
lo g i c a l ar r an g e m e n t
[11 , 1 2]
or molecular signalling at the right time
a n d p l a c e .
[13]
B u t , t o e f f e c t i v e l y a d o p t t h e s y n t h e t i c b i o l o g y a p p r o a c h , w e
m u s t l e a r n m o r e a b o u t o r g a n o i d c u l t u r e s m a d e o f c e l l s f r o m d i f -
f e r e n t a g e s , l o c a t i o n s o r s p e c i e s , s o w e c a n a p p l y k e y m o l e c u l e s t o
re s to re h air f o r min g a bi l i t y .
[4]
T o t h i s e n d , w e s o u g h t t o d e v e l o p a
t h re e- dim e n s i o n a l , cu lt u re s y s t e m in w hich di f f e re n t t y p e s of s k in
p r o gen i to r s , s u c h a s ep i der m a l- o r der m a l- l i ke so m at i c cel l s , emb r y -
o n i c s t e m c e l l s o r i P S c e l l s , c a n b e g u i d e d t o f o r m e c t o d e r m a l o r g a n s
i n a p l a n a r c o n f i g u r a t i o n ( F i g u r e S 1 ) .
[14]
W e h o p e t h a t t h i s c u l t u r e
m o d e l m a y s e r v e a s a p l a t f o r m t o i d e n t i f y t h e c r i t i c a l f a c t o r s n e e d e d ,
s tep by s tep, f o r t he devel o p ment of i n d ivi d u a l e c to der m a l o r g a n s .
H e r e , w e p r e s e n t o u r p r o g r e s s t o w a r d s t h e f o r m a t i o n o f h u m a n h a i r
f o l l i c l e o r g a n o i d s . W i t h i n t h i s i n v i t r o m o d e l , w e o b s e r v e d t w o d i s -
t i n c t a n d n ovel p hen o men a . F i r s t , h a i r p e g - l i ke s t r u c tu r e s emer ge d
a f t e r o n l y 4 d a y s i n c u l t u r e a n d p o s s e s s e d m o l e c u l a r a n d c e l l u l a r
c h a r a c t e r i s t i c s s i m i l a r t o a u t h e n t i c h u m a n h a i r p e g s . S e c o n d , t h e
f o r m a t i v e p r o c e s s o f p e r i o d i c p a t t e r n i n g w a s q u i t e a p p a r e n t : d i s -
s o c i a t e d d e r m a l c e l l s a s s e m b l e d i n t o s t r i p e s , c l u s t e r s , t h e n d i s t i n c t
der m a l co n den s at i o n s , f o l l owe d by ep i der m a l “ s t a l k s” w i t h der m a l
p a p i l l a - l i k e “ c a p s . ” T h e p r o c e s s r e p r o d u c i b l y b e g a n a t t h e d r o p l e t
b o u n d a r y a n d e m a n a t e d a s a c i r c u m f e r e n t i a l w av e t o w a r d s t h e c e n -
t r e o f t h e c u l t u r e .
I n v i v o , p e r i o d i c h a i r a n d f e a t h e r p l a c o d e s f o r m i n a p r o g r e s s i v e
w av e , p r o p a g a t i n g i n d i f f e r e n t d i r e c t i o n s d e p e n d i n g o n b o d y s i t e ( e . g
s c a l p a n d t r u n k ) . T h i s i m p l i e s t h a t t h e p r o c e s s i s a c o m b i n a t i o n o f
l o c a l p e r i o d i c p a t t e r n i n g e v e n t s a n d a n a s y m m e t r i c g l o b a l i n f l u e n c e
t h a t m a k e t h e m o r p h o g e n e t i c f i e l d a s y m m e t r i c . T h e l o c a l p e r i o d i c
p a t t e r nin g e ve n t m ay invo l ve ch e mic a l a n d m e ch a nic a l f e e d b a ck b e -
t we en cel l s a n d t he i r envi r o n ment .
[ 1 5 ,16]
S e v e r a l m o d e l s h av e b e e n
p r o p o s e d , r a n g in g f r o m ch e mic a l- b a s e d re a c t i o n - di f f u s i o n m o d e l s
to o ne s w her e t he “r e a c t a nt s” a r e cel l s t hem sel ve s to me c h a n o -
c hem i c a l m o del s w h i c h cou p l e cel l i nter a c t i o n s w i t h c hem i c a l si g -
n a ls .
[17–19]
T he s el f - o r g a n izi n g pat ter n s o b s er ve d e x p er i ment a l l y i n
o u r c u l t u r e s y s te m r es e m b l e p at te r n s m o s t si m p l y i l l u s tr ate d by th e
T u r in g a c t i v a to r - in hibi to r m o d e l .
[ 2 0 , 21]
T h e g l o b a l b e h av i o u r o f t h e
s y s t e m c a n b e d e s c r i b e d b y t h e o c c u r r e n c e o f a T u r i n g i n s t a b i l i t y o n
a n a s y m m et r ic m o r p h o ge n et ic f i e l d . S u ch a s y m m et r y i s s p e cu la t e d
to b e c a u s e d by m e ch a nic a l o r ch e mic a l f o r ce s o r u n e ve n ce l l p r o l i f -
e r a t i o n o r d e a t h .
[2 2 , 2 3]
T h e d r o p l et cu lt u re s y s t e m d e s cr ib e d h e re p r ov id e s a u niq u e
o p p o r t u n i t y t o s t u d y b o t h p e r i o d i c p a t t e r n i n g a n d g l o b a l e v e n t s i n
h u m a n h a i r f o l l i c l e f o r m a t i o n . T h e f o r m a t i o n o f h a i r p e g - l i k e s t r u c -
t u r e s o c c u r s m o r e r a p i d l y t h a n o t h e r c u r r e n t m e t h o d s a n d , y e t , i s
s l o w e n o u g h t o p e r m i t t h e a n a l y s i s a n d o p t i m i z a t i o n o f t h e s e q u e n c e
of ce l l u l a r eve nt s. M ath e m at i c a l m od e l l i n g of th e fo r m at i o n w ave i n
t h e h a i r p e g p o p u l a t i o n a l l o w s u s t o a n a l y s e t h e s e l f - a s s e m b l y p r o -
c e s s a n d p r e d i c t c o n d i t i o n s t h a t m a y e n h a n c e o r g a n o i d f o r m a t i o n .
T r a n s l a t i o n a l l y , t h i s c u l t u r e s y s t e m p r ov i d e s p r o o f o f c o n c e p t t h a t
s t r u c t u r e s r e s e m b l i n g h u m a n h a i r f o l l i c l e p r e c u r s o r s c a n b e e n g i -
n e e r e d i n v i t r o i n a t i m e - e f f i c i e n t m a n n e r a n d s e r v e s a s a p l a t f o r m
t o i d e n t if y t h e o pt i m a l c o n d i t io ns w i t h w h i c h t o e f f i ci e n t l y e n g i n e e r
h u m a n h a i r f o l l i c l e s f o r t r a n s p l a n t a t i o n .
2 | METHODS
2.1 | In vitro hair follicle reconstitution assay
E p i d e r m a l a n d d e r m a l c e l l s w e r e e n z y m a t i c a l l y a n d m e c h a n i c a l l y
s e p a r a t e d f r o m n e o n a t a l f o r e s k i n a n d s e c o n d - t r i m e s t e r f e t a l s c a l p
12

|
357 WEBER Et al .
( e s t i m a t e d g e s t a t i o n a l a g e ( E G A ) 1 7 - 1 9 w e e k s ) , r e s p e c t i v e l y . 2 × 1 0
6

c u l t u r e d n e o n a t a l f o r e s k i n k e r a t i n o c y t e s a n d 3 × 1 0
6
f r e s h f e t a l
s c a l p d e r m a l c e l l s w e r e r e s u s p e n d e d i n 1 4 0 u l o f F 1 2 : D M E M ( 1: 1 )
m e d i u m w i t h 5 % F B S a n d P / S / A a n d p l a t e d a s a d r o p l e t o n a s i x - w e l l
c e l l c u l t u r e i n s e r t . T h e d r o p l e t s w e r e i n c u b a t e d a t 3 7 ° C a n d 5 % CO
2

f o r 4 - 7 d a y s . G r o w t h f a c t o r s w e r e a d d e d d a i l y . S e e s u p p l e m e n t a l
m e t h o d s f o r d e t a i l s .
2.2 | Patch assay
2 × 1 0
6
n e o n a t a l f o r e s k i n k e r a t i n o c y t e s a n d 3 × 1 0
6
f e t a l sc a l p der -
m a l c e l l s w e r e i n j e c t e d s u b c u t a n e o u s l y i n t o t h e d e e p d e r m i s o f 6 - t o
1 2 - w e e k - o l d h a i r l e s s n u d e m i c e . S u b c u t a n e o u s n o d u l e s w i t h f o r m e d
h a i r f o l l i c l e s w e r e h a r v e s t e d 8 w e e k s l a t e r .
2.3 | Immunostaining, lentiviral vectors and live
cell imaging
S e e s u p p l e m e n t a l m e t h o d , T a b l e s S 1 a n d S 2 .
2.4 | Mathematical modelling
A r e a c t i o n - d i f f u s i o n m o d e l w a s d e v e l o p e d t o s i m u l a t e t h e i n t e r a c -
t i o n o f t w o , a s o f y e t , e x p e r i m e n t a l l y u n i d e n t i f i e d , d i f f e r e n t m o r -
p h o ge n p op u l at i on s . D e t a i l s , e q uat i on s an d p ar am e te r d e f i n i t i on s
a r e i n c l u d e d i n s u p p l e m e n t a l m e t h o d s .
3 | RESULTS
3.1 | Human fetal scalp dermal cells induce the
self- organization of hair peg- like structures in droplet
culture
D i s s o c i a t e d n e o n a t a l h u m a n f o r e s k i n k e r a t i n o c y t e s a n d 1 7 - t o
1 9- w e e k EG A h u m a n f e t a l s c a l p d e r m a l c e l l s w e r e m i xe d a n d
c o- cu lt u re d in t h re e- dim e n s i o n a l d r o p l et s ( F i g u re 1 A ) . W i t hin
2 4 h o u r s , t h e e p i d e r m a l a n d d e r m a l c e l l s s e g r e g a t e d i n t o t w o
l ay er s , w i t h ep i der m a l c el l s a d her i n g t o t he c el l c u l tu r e i n s er t
memb r a ne a t t he b a s e o f t he d r o p l e t a n d der m a l c el l s ov er l y i n g
t h e k e r a t i n o c y t e s i n a m o r e s u p e r f i c i a l l a y e r ( F i g u r e 1B ) . A r o u n d
4 8 h o u r s , d e r m a l c e l l s b e g a n t o o r g a n i z e , f o r m i n g a t r a b e c u l a r
m e s h p a t t e r n , w h i c h t h e n e v o l v e d i n t o p u n c t a t e c e l l c l u s t e r s . B y
7 2 h o u r s , k e r a t i n o c y t e s a b u t t i n g t h e d e r m a l c l u s t e r s r e a r r a n g e d
i n t o a c o n c e n t r i c p a t t e r n a n d , w i t h i n 96 h o u r s , k e r a t i n o c y t e
“s t a l k s ” p r o t r u d e d , a g a i n s t g r av i t y , i n t o t h e d r o p l e t s p a c e , i n a s s o -
c i a t i o n w i t h a d e r m a l c e l l “ c a p ” ( F i g u r e 1B , M ov i e S 1 A - C ) . I n c o m -
p a r i s o n w i t h 1 7 - w e e k EG A f e t a l s c a l p s e c t i o n s , t h e n e w l y f o r m e d
s t r u c t u r e s r e s e m b l e e a r l y h a i r p e g s , a s t a g e i n f o l l i c l e d e v e l o p -
m e n t i n w h i c h th e i nv a g i n a t i n g ke r a t i n o c y t es p r o tr u d e d o w nw a r d
i n t o t h e d e r m a l p l a n e , g u i d e d b y t h e d e r m a l p a p i l l a ( F i g u r e 1 C ) . O f
n o t e , w h i l e t h e r e w a s a c l e a r a n d e a r l y s e g r e g a t i o n o f e p i d e r m a l
a n d der m a l c el l s , w e f r e q u en t l y en c ou n t er e d sc a t t er e d , l a r g e , i n -
t en s el y ker a t i n - p o s i t iv e c el l s i n t er s p er s e d w i t h i n t he der m a l l ay er ,
w hich e x hibi t e d ch a r a c t e r i s t ic s c o n s i s t e n t w i t h t e r min a l l y di f f e r -
e n t ia t e d , a n u c l e a t e d ke r a t i n o c y t e s . T h e s e “c e l l s ” d o n o t a p p e a r t o
p a r t i c i p a t e i n t h e m o r p h o l o g i c a l e v e n t s .
D e r m a l f i b r o b l a s t s a r e k n o w n t o s e l f - a g g r e g a t e i n n o n - a d h e r e n t
c u l t u r e . T o d e m o n s t r a t e t h a t t h e h a i r p e g - l i k e s t r u c t u r e s w e r e n o t
a n a r t i f a c t o f t h e c u l t u r e s y s t e m o r s i m p l y a r e s u l t o f d e r m a l f i b r o -
b l a s t s e l f - a g g r e g a t i o n , h u m a n f e t a l s c a l p d e r m a l c e l l s , i n t h e a b s e n c e
FIGURE 1 H u m a n n e o n a t a l f o r e s k i n k e r a t i n o c y t e s a n d f e t a l
s c a l p d e r m a l c e l l s s e l f - o r g a n i z e d t o f o r m h a i r p e g - l i k e s t r u c t u r e s i n
v i t r o . A , S c h e m a t i c o f t h e i n v i t r o h a i r f o l l i c l e r e c o n s t i t u t i o n a s s a y .
F o l l i c u l a r o r g a n o i d s , c o m p o s e d o f e p i d e r m a l ( g r e e n ) a n d d e r m a l
c e l l s ( r e d ) , p r o t r u d e f r o m a m u l t i - l a y e r e d k e r a t i n o c y t e s h e e t ( g r e e n ) .
h N F K s = h u m a n n e o n a t a l f o r e s k i n k e r a t i n o c y t e s , h F S D s = h u m a n
f e t a l s c a l p d e r m a l c e l l s . B , S e r i a l b r i g h t f i e l d a n d c o n f o c a l i m a g e s o f
t h e c u l t u r e d r o p l e t t a k e n e v e r y 2 4 h d e m o n s t r a t e d t h e f o r m a t i o n
o f p e r i o d i c a l l y a r r a n g e d t h r e e - d i m e n s i o n a l c o n f i g u r a t i o n s b y 9 6 h ,
c o r r e s p o n d i n g t o h a i r p e g - l i k e s t r u c t u r e s c o m p o s e d o f a n e p i d e r m a l
s t a l k a n d d e r m a l c a p . W h o l e m o u n t c o n f o c a l i m a g e s i n t h e s e c o n d
a n d t h i r d r o w s , w i t h p a n c y t o k e r a t i n d e n o t i n g e p i d e r m a l c e l l s i n
g r e e n a n d n u c l e i i n r e d , w e r e t a k e n f r o m t h e p e r i p h e r y o f t h e
d r o p l e t , a s r e p r e s e n t e d b y t h e w h i t e d o t t e d b ox i n t h e b r i g h t f i e l d
i m a g e i n t h e f i r s t r o w . I n t h e s e c o n d r o w , t h e w h i t e d o t t e d l i n e
d e m a r c a t e s t h e p e r i p h e r y o f t h e d r o p l e t . T h e f o u r t h r o w o f
i m a g e s i s t r i p l e - s t a i n e d s e c t i o n s , w i t h p a n c y t o k e r a t i n ( p a n C K )
m a r k i n g e p i d e r m a l c e l l s ( g r e e n ) , v i m e n t i n m a r k i n g d e r m a l c e l l s
( r e d ) a n d n u c l e i ( b l u e ) s t a i n e d w i t h T O - P R O - 3 i o d i d e . T h e s c a l e
b a r i s t h e s a m e f o r a l l i m a g e s p e r r o w . T h e l a r g e g r e e n l o b u l e s i n
t h e 4 8 h s a m p l e a r e d e a d c e l l a r t i f a c t s w h i c h h av e t r a p p e d t h e
f l u o r e s c e n t a n t i b o d y ( n = 2 5 ) . C , I n v i t r o s t r u c t u r e s a t 9 6 h ( l e f t
p a n e l ) r e s e m b l e d h a i r p e g s f o u n d i n 1 9 - w k h u m a n f e t a l s c a l p ( r i g h t
p a n e l ) . p 6 3 i s a m a r k e r o f e p i d e r m a l p r o g e n i t o r c e l l s ( n = 2 5 ) . D ,
H u m a n f e t a l s c a l p d e r m a l c e l l s a l o n e a n d a d u l t s c a l p d e r m a l c e l l s
m i xe d w i t h n e o n a t a l f o r e s k i n k e r a t i n o c y t e s d i d n o t p r o d u c e a n y
h a i r p e g - l i k e s t r u c t u r e s a f t e r 9 6 h i n c u l t u r e . T h e i m a g e s a r e t a k e n
f r o m t h e p e r i p h e r y o f t h e c u l t u r e d r o p l e t , a s e xe m p l i f i e d b y t h e
b l a c k d o t t e d b ox i n B . h A S D s = h u m a n a d u l t s c a l p d e r m a l c e l l s . E ,
W h e n i n j e c t e d s u b c u t a n e o u s l y i n t o a n u d e m o u s e , h u m a n n e o n a t a l
f o r e s k i n k e r a t i n o c y t e s a n d f e t a l s c a l p d e r m a l c e l l s p r o d u c e d m a t u r e
h a i r f o l l i c l e s c o m p o s e d o f c e l l s o f h u m a n o r i g i n . P a n c y t o k e r a t i n
( g r e e n ) a n d v i m e n t i n ( c y a n ) a n t i b o d i e s a r e h u m a n - s p e c i f i c . S e c t i o n s
o f m o u s e s k i n w e r e i n c l u d e d t o c o n f i r m s p e c i e s s p e c i f i c i t y o f t h e
a n t i b o d i e s ( b o t t o m p a n e l s ) ( n = 3 )
Cultured human
foreskin
Kera
nocytes
(hNFKs)
Fresh fetal scalp
dermal cells
17-19 wk EGA
(hFSDs)
3D culture
droplet
Hair peg-like structures
96 h
24
(A) (C)
(D)
(E)
(B)
h 48 h 72 h 96 h
brighield pancytokeran nuclei
panCK vimenn
nuclei
50 μm
50 μm
300 μm
3 mm
Formaon of
keranocyte sheet
Increased dermal
coalescence
Keranocyte
downgrowth
Focal epidermal
reorganizaon
panCK nuclei
p63 nuclei
50 μm 50 μm
Cultured
hNKFs
+
fresh
hFSDs
SQ 8 wks
pancytokeran nuclei vimenn nuclei
pancyotkeran nuclei vimenn nuclei
Mouse Mouse Human Human
100 μm 100 μm 100 μm
100 μm 100 μm 100 μm 100 μm
In vitro whole mount Nave 19 week fetal scalp
hNFKs+ hFSDs hFSDs only hNFKs+ hASDs
1 mm
13
358
|
WEBER Et al .
of f o re s k in k e r a t in o c y t e s , we re cu lt u re d u n d e r id e n t ic a l co n di t i o n s .
F e t a l s c a l p d e r m a l c e l l s a l s o f o r m e d a t r a b e c u l a r p a t t e r n b u t d i d
n ot f o r m a ny t h re e- dim e n s i o n a l s t r u c t u re s (F i g u re 1 D). S imi la r l y ,
a d u lt d e r m a l ce l l s , f r o m h air - b e a r in g a d u lt s c a lp, we re cu lt u re d w i t h
n e o n a t a l f o r e s k i n k e r a t i n o c y t e s . A d u l t s c a l p d e r m a l c e l l s f o r m e d
t hick , d e n s e s h e et s . N ei t h e r co m bin a t i o n p r o d u ce d h air p eg - l ik e
s tr u c t u r es.
3.2 | Hair peg- like structures in vitro displayed
cytoarchitecture and molecular markers similar to
those observed in vivo
U n der def i ne d co n d i t i o n s , ep i der m a l a n d der m a l cel l s r a p i d l y s el f -
a s s e m b l e d a n d t r a n s i t i o n e d t h r o u g h s t a g e s r e m i n i s c e n t o f f o l l i c u l a r
d e v e l o p m e n t t o f o r m h a i r p e g - l i k e s t r u c t u r e s b u t f a i l e d t o p r o g r e s s
f u r t h e r i n v it ro. T o ve r if y t h at t h e h u m a n n e o n at a l fo r e s k i n ke r at i n o -
c y t e s a n d h u m a n f e t a l s c a l p d e r m a l c e l l s p o s s e s s e d f u l l r e g e n e r a t i v e
p o t e n t i a l , t h e s a m e r a t i o o f e p i d e r m a l a n d d e r m a l c e l l s w a s i n j e c t e d
s u b c u t a n e o u s l y i n t o n u d e m i c e i n a t r a d i t i o n a l p a t c h a s s a y .
[2]
E i g h t
we e k s la t e r , co m p l et e h air f o l l ic l e s , in c l u din g h air s h af t s , we re c l e a r l y
v i s i b l e i n t h e s u b c u t a n e o u s t i s s u e e n c i r c l i n g a c e n t r a l k e r a t i n i z e d
m a s s ( F i g u r e 1 E ) . I m m u n o s t a i n i n g w i t h h u m a n - s p e c i f i c a n t i b o d i e s
co nf ir m e d t h a t ce l l s of t h e e p id e r m a l o u t e r r o ot s h e a t h s a n d d e r m a l
p a p i l l a e w e r e o f h u m a n o r i g i n ( F i g u r e 1 E ) .
A k in to h air p eg s in d e ve l o p in g f et a l s k in , t h e re co n s t i t u t e d h air
p e g - l i k e s t r u c t u r e s we r e k e r a t i n - 1 4 p os it i ve a n d k e r a t i n - 1 0 n e g a t i ve
( F i g u r e 2 A ) . K e r a t i n - 1 0 a n d i nv o l u c r i n , m a r k e r s o f s u p r a b a s a l c e l l s ,
w e r e e x p r e s s e d i n a l l c e l l s o f t h e e p i d e r m a l s h e e t e xc e p t t h e b a s a l
l a y e r , c o n s i s t e n t w i t h n o r m a l p a t t e r n s o f e p i d e r m a l s t r a t i f i c a t i o n
( F i g u r e 2 A ) . W h i l e e p i d e r m a l c e l l s o r i g i n a l l y s t r a t i f i e d w i t h b a s e m e n t
m e m b r a n e f a c i n g t h e i n s e r t , t h e p o l a r i t y o f s t r a t i f i c a t i o n w a s a l t e r e d
o n c e e p i d e r m a l d o w n g r o w t h b e g a n , w i t h e p i d e r m a l “ s t a l k s ” a n d a s -
s o c i a t e d d e r m a l “ c a p s ” p r o j e c t i n g u p w a r d s i n t o t h e c u l t u r e t h r o u g h
m o r e d i f f e r e n t i a t e d l a y e r s o f e p i d e r m i s . W e s u s p e c t t h i s i s d u e t o
p h y s i c a l l i m i t a t i o n s o f t h e d r o p l e t c u l t u r e s y s t e m . K e r a t i n o c y t e s o f
t h e e p i d e r m a l s t a l k e x p r e s s e d K 1 7 , K 1 8 a n d E - c a d h e r i n , a l l k n o w n
t o b e e x p r e s s e d i n t h e i n n e r o r o u t e r r o o t s h e a t h l a y e r s o f m a t u r e
f o l l i c l e s , t h o u g h a t t h e h a i r p e g s t a g e , d i s t i n c t e p i d e r m a l s h e a t h l a y -
e r s h av e n o t y e t f o r m e d a n d l e s s i s k n o w n a b o u t t h e e x p e c t e d l o c a -
t i o n s f o r e x p r e s s i o n o f t h e s e p r o t e i n s ( F i g u r e 2 A ) . S o m e o f t h e l a r g e r
h air p eg - l ik e s t r u c t u re s di s p laye d l o n ge r , cu r v in g e p id e r m a l s t a lk s ,
w h i c h w h e n v i e w e d a t t h e r i g h t a n g l e , a p p e a r e d t o p o s s e s s a c e n t r a l
ker at i n- p o si t ive co r e s u r r ou n de d by co n cent r i c a l l y o r i ente d ep i der -
m a l c e l l s , p o s s i b l y i n d i c a t i n g p r o g r e s s i o n i n d e v e l o p m e n t t o w a r d s
t h e b u l b o u s p e g s t a g e ( F i g u r e 2 A ) . T h e s e a d v a n c e d h a i r p e g - l i k e
s t r u c t u re s o c cu r re d inf re q u e n t l y , h owe ve r , m a k in g f u r t h e r ch a r a c -
t e r i z a t i o n di f f icu lt .
p 6 3 , a m a r k e r o f e p i d e r m a l s t e m c e l l s , w a s i n i t i a l l y p r e s e n t i n a l l
ke r ati noc y te s at 2 4 ho u r s . A s is s e e n i n no r m a l h a i r f o l l i c l e d eve l o p -
m e n t , p 6 3 e x p r e s s i o n b e c a m e l i m i t e d t o t h e b a s a l l a y e r f o l l o w i n g
ep i der m a l s t r at i f i c at i o n a n d p 6 3 - p o si t ive cel l s wer e r ep r o d u c i b l y
n ote d at t h e l e a d i n g e d ge of t h e e p i d e r m a l s t a l k , a d ja ce nt to t h e d e r -
m a l c a p ( F i g u r e 2 B ) . P C N A i m m u n o s t a i n i n g d e m o n s t r a t e d a c t i v e c e l l
d i v i s i o n i n b o t h t h e e p i d e r m a l b a s a l l a y e r a n d t h e l e a d i n g e d g e o f
t h e s t a lk , w hi l e t h e re m ainin g e p id e r m a l ce l l s w i t hin t h e s t a lk we re
q u i e scent (F i gu r e 2B). T he p r e s en ce of f o c a l , r ep l i c at i n g ep i der m a l
p r o g e n i t o r c e l l s a t t h e l e a d i n g e d g e o f t h e s t a l k s u g g e s t s t h a t l o c a l -
i z e d p r o l i f e r a t i o n m a y c o n t r i b u t e t o d o w n g r o w t h a n d w e h y p o t h e -
s i z e t h a t t h e s e p r o l i f e r a t i n g c e l l s m a y b e p u t a t i v e h a i r m a t r i x c e l l s .
H o w e v e r , w e c a n n o t r u l e o u t t h e p o s s i b l e c o n t r i b u t i o n o f c e l l m i -
g r a t i o n f r o m t h e a d j a c e n t s t r a t i f i e d e p i d e r m i s i n h a i r p e g f o r m a t i o n
a n d t h e m e c h a n i s m b y w h i c h e p i d e r m a l d o w n g r o w t h o c c u r s i s n o t
y e t k n o w n .
C o n s i s t e n t w i t h a d e r m a l l i n e a ge , d e r m a l c a p ce l l s s y n t h e s i z e d
c o l l a ge n s I a n d I I I ( F i g u r e 2 C ). B a s e m e n t m e m b r a n e p r ot e i n s , c o l l a -
ge n I V a n d l a m i n i n , t y p i c a l l y l o c a t e d a t t h e i n t e r f a ce b e t we e n e p i -
der m a l a n d me s en c hy m a l cel l s w i t h i n t he h a i r fo l l i c l e , wer e p r e s ent
a t t h e j u n c t i o n o f e p i d e r m a l s t a l k a n d t h e d e r m a l c a p ( F i g u r e 2 C ,
Movi e S 2 A ). F u r t her m o r e , t he der m a l c a p cel l s a s so c iate d w i t h t he
h a i r p e g - l i k e s t r u c tu r e s d is p l aye d m a r k er s a l so p r e s ent i n t he der -
m a l c o n d e n s a t e a n d d e r m a l p a p i l l a . T h e d e r m a l c a p w a s c o m p o s e d
of a he ter o gene ou s m ix tu r e of der m a l cel l s w i t h a cent r a l co mpa r t -
m e n t a l i z e d a r e a p o s i t i v e f o r a l p h a - s m o ot h m u s c l e a c t i n (α- SMA,
F i g u r e 2 C , M ov i e S 2B). W h i l e a l k a l i n e p h o s p h a t a s e i s a c l a s s i c m a r k e r
o f t h e m u r i n e d e r m a l p a p i l l a a n d i s e x p r e s s e d i n t h e d e r m a l p a p i l l a e
o f 1 7 - we e k h u m a n f e t a l s c a l p, t h e r e i s l i m i t e d a n d c o nf l i c t i n g d a t a
reg a r din g t h e e x p re s s i o n of a lk a l in e p h o s p h at a s e vs α- S M A in human
d e r m a l p a p i l l a ce l l s i n c u l t u r e . S o m e p u b l i c a t i o n s s h ow p e r s i s t e n t a l -
k a l in e p h o s p h at a s e e x p re s s i o n in cu lt u re d h u m a n d e r m a l p a p i l la ce l l s
but others demonstrate rapid loss of alkaline phosphatase expression
a n d u p reg ulat i o n of α- S M A ex p r e ssi o n .
[24–29]
In reality, the expres -
s i o n o f d e r m a l p a p i l l a m a r k e r ge n e s i s e a s i l y i nf l u e n ce d by c u l t u r e
c o n d i t i o n s . I n o u r s y s t e m , α- S M A e x p r e s s i o n w a s p r e s e n t w h i l e a l -
k a l i n e pho s ph at a s e e x pr e s si o n w a s not . V e r si c a n , a not h e r co m mo n ly
u s e d m a r k e r f o r t h e d e r m a l co n d e n s ate a n d p a p i l l a , w a s s t ro n g l y e x -
p r e s s e d i n t h e d e r m a l c a p ( F i g u r e 2B , C ). W h i l e t h e s e d e r m a l c a p s
r ep r e s ent t he devel o p ment a l p r o g r e s si o n of der m a l s t r i p e s to c l u s -
te r s to co n d e n s at i o n s a n d d e r m a l p a p i l la e- l ik e a g g reg ate s , w hich
c a n f u n c t i o n a l l y i n d u ce h a i r p e g - l i k e s t r u c t u r e s , we b e l i e v e t h e y a r e
i n co mp l e te o r i mm atu r e der m a l pa p i l l ae b e c a u s e t hey e x p r e s s so me ,
b u t n ot a l l , d e r m a l p a p i l l a m o l e c u l a r m a r k e r s a n d i n d u ce t h e f o r m a -
t i o n o f h a i r p e g - l i k e s t r u c t u r e s i n s t e a d o f c o m p l e t e h a i r f o l l i c l e s .
3.3 | The formation of hair peg- like structures
in vitro mimics the sequential stages of development
in vivo
F o r e s k i n k e r a t i n o c y t e s a n d f e t a l s c a l p d e r m a l c e l l s p r o g r e s s e d
t h r o u g h s t a g e s s i m i l a r t o n a t i v e h a i r f o l l i c l e d e v e l o p m e n t .
[30]

B e t w e e n 4 8 a n d 7 2 h o u r s i n c u l t u r e , e p i d e r m a l c e l l s u n d e r l y i n g
f o c a l d e r m a l c e l l c o l l e c t i o n s f o r m e d a c o n c e n t r i c p a t t e r n , d i s t i n c t
f r o m t h e c o b b l e s t o n e p a t t e r n o f t h e s u r r o u n d i n g e p i d e r m a l s h e e t
( F i g u r e 2 D , M ov i e S 3 A , B ) .
[31]
β - c a t e n i n , k n o w n t o b e e x p r e s s e d i n
t h e e p id e r m a l p la co d e a n d re q u ire d f o r h air f o l l ic l e m o r p h o ge n -
e s i s , w a s f o c a l l y e n r i c h e d i n e p i d e r m a l c e l l s a b u t t i n g t h e c l u s t e r e d
d e r m a l c e l l s , b u t a b s e n t f r o m t h e a d j a c e n t e p i d e r m i s a t t h e t i m e o f
14

|
359 WEBER Et al .
ep i der m a l dow n g r ow t h (F i gu r e 2E ).
[32–34]
C e l l s w i t h i n t h e d e r m a l
c l u s t e r s e x p r e s s e d C D 3 4 , a m a r k e r o f t h e h u m a n d e r m a l c o n d e n s a t e
a n d e a r l y d e r m a l p a p i l l a ( F i g u r e 2 E ) .
[35]
T h e f o r m a t i o n o f h a i r g e r m -
l i k e s t r u c t u r e s a n d , t h e n , h a i r p e g - l i k e s t r u c t u r e s e n s u e d b e t w e e n 7 2
a n d 9 6 h o u r s i n c u l t u r e .
L i v e c e l l c o n f o c a l i m a g i n g o f t h e d r o p l e t c u l t u r e w a s d e v e l o p e d
t o v i s u a l i z e t h e c e l l - c e l l i n t e r a c t i o n s a n d c o l l e c t i v e c e l l m ov e -
m e n t s d u r in g h air p eg f o r m a t i o n . V i s u a l di s cr imin a t i o n b et w e e n
e p id e r m a l a n d d e r m a l c e l l s w a s a chi e v e d u s in g e p id e r m a l - s p e c i f ic
p r o m o t e r s. L e n t i v i r a l tr a n s d u c t i o n t o e x p r es s l i n e a g e - s p ec i f i c f l u -
o r e sc en t m a r ker s d i d n o t p er tu r b h a i r p e g de v el o p men t i n vi t r o
( F i g u r e S 2 ) . A v i e w f r o m t h e t o p o f a t w o - c o l o u r , l i v e c e l l c u l t u r e
d r o p l e t d e m o n s t r a t e d d i s t i n c t s p h e r i c a l d e rm a l c a p s a r e s h ow n i n
t he S u p p l emen t M a t er i a l ( F i gu r e S2 , Mo vi e S 4) . N u c l e i o f c el l s a t
t h e p e r i p h e r y o f t h e d e r m a l c a p e x h i b i t e d a c u r v e d m o r p h o l o g y ,
a n d c e l l s n e a r t h e c e n t r e o f t h e d e r m a l c a p d i s p l a y e d i n c r e a s e d
l o c a l c e l l m o t i o n w h i l e c e l l s a t t h e p e r i p h e r y w e r e m o r e s t a t i o n a r y ,
s u g g e s t i n g a h e t e r o g e n e i t y o f d e r m a l c e l l f u n c t i o n . I n c o n t r a s t ,
c e l l s w i t hin t h e e p id e r m a l s h e et re m ain e d s t a t ic . T h re e- c o l o u r l i v e
i m a g i n g d i s t i n gu i s he d K 1 4 + ep i der m a l c el l s (y el l o w) , p 6 3 + ep i der -
m a l p r e c u r s o r c e l l s ( m a g e n t a ) a n d d e r m a l c e l l s (c y a n ) w i t h i n t h e
h a i r p e g a n d a d j a c e n t e p i d e r m a l s h e e t ( F i g u r e S 2 , M ov i e S 5 A , B ) .
A s s e e n i n s t a t i c c o n f o c a l i m a g e s , p 6 3 - p o s i t i v e c e l l s w e r e n o t e d
w i t h i n t h e e p i d e r m a l s h e e t a s w e l l a s t h e e p i d e r m a l s t a l k o f t h e
h a i r p e g . S e v e r a l s t r o n g l y p o s i t i v e p 6 3 c e l l s w e r e p r e s e n t a t t h e
l e a d i n g e d g e o f t h e e p i d e r m a l s t a l k , a b u t t i n g t h e d e r m a l c l u s t e r ,
a n d 1 - 2 c e l l s w e r e c o n s i s t e n t l y n o t e d a t t h e o p p o s i t e p o l e o f t h e
d e r m a l c l u s t e r , a u n i q u e p o s i t i o n w h i c h c o u l d s u g g e s t a n i n s t r u c -
t i v e r o l e i n d i r e c t i o n a l e p i d e r m a l d o w n g r o w t h .
3.4 | Hair peg- like structure formation
in the organoid droplet culture displays
spatiotemporal patterning
L a r g e - sc a l e der m a l c el l pa t t er n s w i t h i n t he d r o p l e t c u l tu r e dem -
o n s t r a t e d a s p a t i o t e m p o r a l p r o g r e s s i o n , w h i c h i n i t i a t e d a t t h e
d r o p l e t p e r i p h e r y a n d a d v a n c e d t o w a r d s t h e c e n t r e ( F i g u r e 3 A ) .
A t 2 4 h o u r s p o s t p l a t i n g , d i s s o c i a t e d d e r m a l c e l l s r e m a i n e d d i s -
t r ib u t e d in a h o m o g e n e o u s la y e r w i t h o u t a di s t in c t m a cr o s c o p ic
p a t t e r n . O v e r t h e n e x t 1 2 h o u r s , d e r m a l c e l l s c o a l e s c e d i n t o l o n g
u n d u l a t i n g s t r i p e s o f h i g h e r d e r m a l c e l l d e n s i t y . B y 4 8 h o u r s i n
c u l t u r e , l o n g s t r i p e s h a d s u b d i v i d e d i n t o s h o r t e r s t r i p e s a n d , ov e r
FIGURE 2 H a i r p e g - l i k e s t r u c t u r e s f o r m e d i n v i t r o e x p r e s s e d
a p p r o p r i a t e e p i d e r m a l a n d d e r m a l m a r k e r s a n d p r o g r e s s e d
t h r o u g h r e p r o d u c i b l e s t a g e s r e m i n i s c e n t o f e a r l y h a i r f o l l i c l e
d e v e l o p m e n t . A , S t a i n i n g o f s e c t i o n s o f h a i r p e g - l i k e s t r u c t u r e s
w i t h k e r a t i n - 1 4 ( g r e e n ) d e m o n s t r a t e d c l e a r s e p a r a t i o n b e t w e e n
e p i d e r m a l a n d d e r m a l c e l l s ( t o p l e f t ) . C o n s i s t e n t w i t h p a t t e r n s o f
k e r a t i n e x p r e s s i o n i n h u m a n f e t a l s c a l p , e p i d e r m a l c e l l s w i t h i n t h e
i n v i t r o h a i r p e g - l i k e s t r u c t u r e s d i d n o t e x p r e s s k e r a t i n - 1 0 ( r e d ) .
I nv o l u c r i n ( g r e e n ) , a m a r k e r o f k e r a t i n o c y t e t e r m i n a l d i f f e r e n t i a t i o n ,
w a s h i g h l y e x p r e s s e d i n o n l y a p o r t i o n o f t h e e p i d e r m a l s h e e t ,
w h i c h , a l o n g w i t h k e r a t i n - 1 0 e x p r e s s i o n , s u g g e s t s s t r a t i f i c a t i o n
( b o t t o m l e f t ) . I nv o l u c r i n a l s o s t r o n g l y m a r k e d c e l l s b e l i e v e d t o
b e t e r m i n a l l y d i f f e r e n t i a t e d , a n u c l e a r c o r n e o c y t e s t h a t b e c a m e
i n a p p r o p r i a t e l y t r a p p e d w i t h i n t h e d e r m a l c e l l c a p . T h e e p i d e r m a l
s t a l k s w e r e k e r a t i n - 1 7 ( g r e e n ) , k e r a t i n - 1 8 ( m a g e n t a ) a n d E - c a d h e r i n
( g r e e n ) p o s i t i v e ( w h o l e m o u n t , t o p r i g h t ) . S e v e r a l o f t h e l a r g e r
h a i r p e g - l i k e s t r u c t u r e s a p p e a r e d t o h av e e p i d e r m a l s t a l k s w i t h
c e n t r a l l u m e n s a n d c o n c e n t r i c a l l y o r g a n i z e d k e r a t i n o c y t e s , m a r k e d
b y k e r a t i n 1 4 ( g r e e n ) ( w h o l e m o u n t , b o t t o m r i g h t ) . B , A t 2 4 h ,
a l l k e r a t i n o c y t e s e x p r e s s e d p 6 3 ( g r e e n ) , a m a r k e r o f e p i d e r m a l
s t e m n e s s ( s e c t i o n s , t o p p a n e l ) . B y 7 2 a n d 9 6 h , p 6 3 - p o s i t i v e c e l l s
w e r e l o c a l i z e d t o t h e b a s a l l a y e r o f t h e e p i d e r m a l s h e e t a n d t h e
l e a d i n g e d g e o f t h e e p i d e r m a l s t a l k a b u t t i n g t h e d e r m a l c a p ( m i d d l e
p a n e l ) . 9 6 - h i m a g e s a r e w h o l e m o u n t s p e c i m e n s . P C N A - p o s i t i v e
( g r e e n ) , a c t i v e l y p r o l i f e r a t i n g c e l l s w e r e p r e s e n t w i t h i n t h e b a s a l
l a y e r o f t h e e p i d e r m a l s h e e t , t h e e p i d e r m a l s t a l k a t t h e i n t e r f a c e
w i t h t h e d e r m a l c a p a n d t h e p e r i p h e r y o f t h e d e r m a l c a p , s i m i l a r
t o 1 7 - w k s e c o n d - t r i m e s t e r h u m a n f e t a l s c a l p ( s e c t i o n s , b o t t o m
p a n e l ) . C , T h e c e l l s o f t h e d e r m a l c a p e x p r e s s e d c o l l a g e n I ( g r e e n ,
s e c t i o n a n d w h o l e m o u n t ) a n d c o l l a g e n I I I ( g r e e n , w h o l e m o u n t )
( t o p l e f t ) . K 1 0 = k e r a t i n - 1 0 . C o l l a g e n I V ( r e d ) a n d l a m i n i n - 3 32
( g r e e n ) , m a r k e r s o f t h e d e r m a l p a p i l l a b a s e m e n t m e m b r a n e , w e r e
e x p r e s s e d a t t h e i n t e r f a c e b e t w e e n e p i d e r m a l a n d d e r m a l c e l l s
w i t h i n t h e h a i r p e g - l i k e s t r u c t u r e s i n v i t r o ( w h o l e m o u n t , b o t t o m
l e f t ) . P a n C K = p a n c y t o k e r a t i n , n u c = n u c l e i , v i m = v i m e n t i n . α -
S M A ( g r e e n ) , a m a r k e r o f h u m a n d e r m a l p a p i l l a c e l l s i n c u l t u r e ,
w a s e x p r e s s e d w i t h i n t h e c e n t r e o f t h e d e r m a l c a p ( w h o l e m o u n t ,
t o p r i g h t ) . H u m a n d e r m a l p a p i l l a c e l l s i n v i v o e x p r e s s a l k a l i n e
p h o s p h a t a s e ( a l k p h o s ( g r e e n ) , l e f t i m a g e ) , a m a r k e r w h i c h i s
t y p i c a l l y l o s t d u r i n g i n v i t r o c u l t u r e . T h e d e r m a l c a p w a s a l s o
p o s i t i v e o r v e r s i c a n , a c o m m o n l y u s e d d e r m a l c o n d e n s a t e o r
d e r m a l p a p i l l a m a r k e r ( w h o l e m o u n t , b o t t o m r i g h t ) . D , S e r i a l o p t i c a l
s e c t i o n s o f a d e r m a l a g g r e g a t e a t 4 8 h i m a g e d a t i n c r e a s i n g d r o p l e t
d e p t h s d e m o n s t r a t e d a r o u n d e d , d e n s e d e r m a l c l u s t e r a t o p a n
e p i d e r m a l s h e e t w i t h c o n c e n t r i c a l l y a r r a n g e d n u c l e i , r e m i n i s c e n t
o f t h e e p i d e r m a l p l a c o d e . E , β - c a t e n i n w a s e x p r e s s e d t h r o u g h o u t
t h e e p i d e r m a l s h e e t a t 4 8 h b u t w a s r e s t r i c t e d t o t h o s e e p i d e r m a l
c e l l s a s s o c i a t e d w i t h t h e d e r m a l c a p b y 7 2 h ( s e c t i o n s , t o p p a n e l s ) .
D e r m a l c e l l s w i t h i n t h e d e r m a l c a p w e r e p o s i t i v e f o r C D 3 4 , a
m a r k e r o f t h e e a r l y d e r m a l p a p i l l a s t e m c e l l ( b o t t o m p a n e l s ) . T h e
4 8 - h i m a g e i s a w h o l e m o u n t s p e c i m e n . A l l o t h e r i m a g e s a r e
s e c t i o n s . A l l s t a i n i n g w a s p e r f o r m e d o n m u l t i p l e h a i r p e g - l i k e
s t r u c t u r e s f r o m a t l e a s t t h r e e b i o l o g i c a l r e p l i c a t e s ( n = 3 )
Keran-14 nuclei
17 wk fetal scalp 96 h
50 μm
Collagen I Keran-10/ nuclei
17 wk fetal scalp 96 h
50 μm 50 μm
96 h
Involucrin
nuclei
50 μm
50 μm
E-cadherin nuclei
96 h
Keran-17 nuclei
50 μm
50 μm
K18 versican
96 h
Keran-14 nuclei
96 h
50 μm
50 μm
96 h
50 μm
96 h 96 h 17 wkfetal scalp
50 μm 50 μm 50 μm
p63 CD34/ nuclei
p63 CD34/ nuclei
24 h 72 h
50 μm 50 μm
17 wkfetal scalp 72 h 96 h
PCNA nuclei
50 μm 50 μm 50 μm
K10 coll I nuclei collagen I nuclei collagen III nuclei
50 μm 50 μm 50 μm
96 h 96 h 96 h
panCK coll IV nuc CD34 coll IV nuc Lam-332 vim nuclei
50 μm 50 μm 50 μm
96 h 96 h 96 h
50 μm 50 μm 50 μm
17 wk fetal scalp 96 h 96 h
α-SMA nuclei alkphos nuclei
versican nuclei
16 wk fetal scalp
50 μm
96 h 96 h
versican p63
50 μm 50 μm
versican collagen IV
pancytokeran / nuclei vimenn / nuclei
50 μm
50 μm
48 h 72 h
β-catenin vimenn/ nuclei
50 μm 50 μm
72 h 48 h
96 h
CD34/ nuclei
17 wkfetal scalp
50 μm
50 μm 50 μm
50 μm
(A)
(C)
(D)
(B)
(E)
15
360
|
WEBER Et al .
t i m e , s h o r t s t r i p e s b e c a m e r o u n d e d , C D 3 4 - p o s i t i v e d e r m a l c l u s -
t e r s ( F i g u r e 2 F ) . B e t w e e n 7 2 a n d 96 h o u r s , h a i r p e g- l i k e s t r u c t u r e s
f o r m e d ( F i g u r e 3 B ) , f i r s t a t t h e d r o p l e t p e r i p h e r y . I n a d d i t i o n t o
f o r m i n g e a r l i e s t , e l o n g a t e d s t r u c t u r e s a p p r ox i m a t i n g m o r e m a t u r e
h a i r p e g- l i ke s t r u c tu r e s f o r me d m o r e den s el y a t t he p er i p her y
( F i g u r e 3 C , D ) . C e n t r a l l y , d e r m a l a g g r e g a t e s w e r e 6 0 % l a r g e r i n
di a m et e r , w hich c o r re la t e d w i t h t h e f o r m a t i o n o f l e s s m a t u re h air
p e g- l i k e s t r u c t u r e s a n d , i n m a n y c a s e s , a b n o r m a l a g g r e g a t e s p o s -
s e s s i n g m u l t i p l e e p i d e r m a l s t a l k s ( F i g u r e 3 D ) . T h e f o r m a t i v e w av e
o f “ l o n g s t r i p e — s h o r t s t r i p e —r o u n d e d c l u s t e r —p e g- l i k e s t r u c t u r e”
a d v a n c e d f r o m t h e p e r i p h e r y t o w a r d s t h e c e n t r e o f t h e d r o p l e t ,
w i t h e a c h n e w c h a n g e i n m o r p h o l o g y , a n d t h e s t r i p e a n d s p o t p a t -
t er n s a r e r em i n i sc en t o f t he p er i o d i c pa t t er n s p r e d i c t e d b y T u r i n g
a c t i v a t o r - in hibi t o r p r in c ip l e s .
[3 6 , 3 7 ]
3.5 | Mathematical modelling simulates the
observed spatiotemporal patterns
Re a c t i on- d i f f u s i on s y s te m s ar e c ap ab l e of s p ont an e o u s l y p r o d u c i n g
s u s t ain e d s p a t i a l p a t t e r n s . S p e c i f ic a l l y , s p ot s , s t r ip e s a n d la by r in t hin e
p a t t e r n s a r e a l l p o s s i b l e w i t h i n t h e f r a m e w o r k o f d i f f u s i o n - d r i v e n
i n s t a b i l i t y , k n o w n a s T u r i n g p a t t e r n s . O n c e f o r m e d , g e n e r a l l y o n l y
o n e o f t h e s e p a t t e r n s i s s e l e c t e d a n d r e m a i n s f i xe d .
[38]
I n co nt r as t , in
o u r d r o p l et cu lt u re , m u lt ip l e di s t in c t p a t t e r n s o ccu r s im u lt a n e o u s l y
a n d a r e f o r m e d i n s e r i a l p r o g r e s s i o n a t d i f f e r e n t l o c a t i o n s w i t h i n t h e
d r op l e t . A s m e nt i on e d , s u c h p at te r n i n g com p l e x i t y c an ar i se f r om
s e v e r a l d i f f e r e n t s o u r c e s . W e c h o s e t o u s e a r e a c t i o n - d i f f u s i o n d e -
s c r i p t i o n b e c a u s e t h e t r a n s i t i o n b e t w e e n s p o t s a n d s t r i p e s i s w e l l
u n der s to o d .
[3 8 , 3 9]
C r i t i c a l l y , i n t w o d i m e n s i o n s , T u r i n g p a t t e r n s c a n
p r o d u c e s p o t s a n d / o r s t r i p e s , b u t t y p i c a l l y n o t a t t h e s a m e t i m e .
[21]

I t i s s i m p l y t h e c o m p e t i t i o n b e t w e e n t h e q u a d r a t i c a n d c u b i c t e r m s
o f t h e a c t i v a t o r k i n e t i c s t h a t d e t e r m i n e w h i c h p a t t e r n m o d e i s o b -
tained.
[21]
T h us , if t h e co r re c t p at te r n k in et ic s a re ch os e n to p ro d u ce
i n p h a s e , o r o u t o f p h a s e , c o n c e n t r a t i o n p a t t e r n s , t h e n a n y T u r i n g
s y s t e m c a n b e g u i d e d t o g i v e r i s e t o s p o t s a n d / o r s t r i p e s . F u r t h e r ,
a p a r a m e t e r ' s i n f l u e n c e i s e x t r e m e l y l o c a l i n T u r i n g p a t t e r n s .
[22]

T h u s , a l l w e r e q u i r e t o c o nv e r t a s y s t e m f r o m s p o t s t o s t r i p e s i s t o
u s e a g r a d i e n t t h a t i n f l u e n c e s t h e c o m p e t i t i o n b e t w e e n t h e c u b i c
a n d q u a d r a t i c t e r m . H e n c e , t h i s i s a c o m p l e t e l y g e n e r a l a n d r o b u s t
m e c h a n i s m f o r p r o d u c i n g s u c h d y n a m i c s . B a s e d o n t h i s , w e p r o p o s e
t h a t a n a s y m m e t r i c s p a t i o t e m p o r a l g r a d i e n t i s p r e s e n t t o e x p l a i n t h e
m i xe d s p e c t r u m o f p a t t e r n s w i t h i n t h e s p a c e ( o r g a n o i d d r o p l e t ) a n d
t h e t r a n s i t i o n of p a t t e r n s ove r t i m e . T h e w o r k i m p l i e s t h a t t h e d r o p -
l e t r ep r e s ent s a n a s y m me t r i c m o r p h o gene t i c f i eld . I n de e d , i n emb r y -
o n i c d e v e l o p m e n t , h a i r s a n d f e a t h e r s f o r m i n p r o p a g a t i v e w av e s i n
di f f e re n t b o d y d o m ain s , r a t h e r t h a n s im u lt a n e o u s l y .
[40–42]
T h i s g av e
us t he m otivation to develop a simulation of T ur ing pat ter ning occur -
r i n g i n a n a s y m m e t r i c m o r p h o g e n e t i c f i e l d ( F i g u r e 3 E ) .
T o m a k e t h e s i m u l a t i o n m o d e l m o r e b r o a d l y a p p l i c a b l e , w e p u r -
p o se l y a s s i g n e d t h e m or p h o ge n s ge n e r i c a c t i v ator or i nh i b i tor f u n c -
t i o n s , r a t h e r t h a n f o c u s i n g o n s p e c i f i c s i g n a l l i n g m o l e c u l e s ( p l e a s e
r e f e r t o t h e s u p p l e m e n t a l m e t h o d s f o r a m o r e d e t a i l e d d e s c r i p t i o n ) .
C r i t i c a l l y , t h i s w o r k i s n o t a b o u t s p e c i f y i n g t h e e x a c t u n d e r l y i n g
FIGURE 3 C o l l e c t i v e l y , a s t r i p e - t o - d o t f o r m a t i v e g r a d i e n t f o r m s
f r o m t h e c e n t r e t o p e r i p h e r y o f t h e c u l t u r e d r o p l e t a n d s u g g e s t s
a T u r i n g p e r i o d i c p a t t e r i n g p r o c e s s o n a n a s y m m e t r i c f i e l d . A ,
B r i g h t f i e l d i m a g e s o f a s i n g l e c u l t u r e d r o p l e t t a k e n e v e r y 1 2 h
d e m o n s t r a t e d t h e f o r m a t i o n o f a n i n i t i a l t r a b e c u l a r p a t t e r n , w h i c h
t h e n g av e w a y t o t h e p e r i o d i c a l l y a r r a n g e d s t r i p e s a n d c e l l c l u s t e r s
( t o p r o w ) . L i n e d r a w i n g s c r e a t e d f r o m b r i g h t f i e l d i m a g e s ( m i d d l e
r o w ) a n d a s c h e m a t i c w h e r e r e d r e p r e s e n t s d e r m a l c e l l s a n d g r e e n
r e p r e s e n t s e p i d e r m a l c e l l s ( b o t t o m r o w ) e m p h a s i z e t h e t r a n s i t i o n s
i n d i s t i n c t p e r i o d i c p a t t e r n s a s t h e y r e l a t e t o d e v e l o p m e n t a l
s t a g e s ( n = 4 ) . B , H i g h - p o w e r m a g n i f i c a t i o n d e m o n s t r a t e s t h e h a i r
p e g - l i k e a r c h i t e c t u r e u n d e r b r i g h t f i e l d i m a g i n g ( n = 4 ) . C , T h e f i e l d
w a s d i v i d e d i n t o f i v e c o n c e n t r i c z o n e s . A n a t o m i c h a i r p e g - l i k e
s t r u c t u r e s d e v e l o p e d a t a h i g h e r d e n s i t y i n z o n e s 4 - 5 , t o w a r d s
t h e p e r i p h e r y o f t h e d r o p l e t . I n p a i r e d t t e s t c o m p a r i s o n s , t h e
av e r a g e d e n s i t y o f h a i r p e g - l i k e s t r u c t u r e s a t t h e p e r i p h e r y o f t h e
c u l t u r e d r o p l e t w a s s t a t i s t i c a l l y d i f f e r e n t f r o m m o r e c e n t r a l z o n e s
(*P < 0 . 0 5 ) . E r r o r b a r s r e p r e s e n t s t a n d a r d e r r o r o f t h e m e a n ( n = 1 1 ) .
D , D e r m a l c l u s t e r s o f a s m a l l e r d i a m e t e r w e r e m o r e l i k e l y t o b e
a s s o c i a t e d w i t h s i n g l e s t a l k e d h a i r p e g - l i k e s t r u c t u r e s t h a n w e r e
l a r g e r d e r m a l c l u s t e r s f o u n d a t t h e c e n t r e o f t h e d r o p l e t . A v e r a g e
c l u s t e r d i a m e t e r i s p l o t t e d . P a i r e d t t e s t c o m p a r i s o n s w e r e u s e d t o
e x a m i n e s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n g r o u p s . E r r o r
b a r s r e p r e s e n t s t a n d a r d e r r o r o f t h e m e a n . *P < 0 . 0 5 , * *P < 0.01
( n = 3 ) . E , N u m e r i c a l s i m u l a t i o n s o f r e a c t i o n - d i f f u s i o n e q u a t i o n s
a r e p r e s e n t e d i n t h e m e t h o d s s e c t i o n . T h e t o p r o w i l l u s t r a t e s h o w
t h e d e n s i t y o f a c t i v a t o r ( u ) a l t e r s ov e r t i m e , w i t h d a r k e r c o l o u r s
p r e s e n t i n g h i g h - d e n s i t y r e g i o n s a n d l i g h t e r c o l o u r s r e p r e s e n t i n g
l o w - d e n s i t y r e g i o n s . T h e b o t t o m r o w i l l u s t r a t e s t h e r a d i a l l y
s y m m e t r i c s p a t i o t e m p o r a l g r a d i e n t f i e l d t h a t a l t e r s t h e p r o p e r t i e s
o f t h e r e a c t i o n - d i f f u s i o n e q u a t i o n s h e t e r o g e n e o u s l y a c r o s s t h e
d o m a i n . A s t i m e i n c r e a s e s , t h e v a l u e o f t h e f i e l d a t t h e b o u n d a r y
i n c r e a s e s t o a m a x i m u m v a l u e a n d t h e g r a d i e n t g e t s s t e e p e r . W e
s e e t h a t a s t h e g r a d i e n t s t e e p e n s , t h e a c t i v a t o r p a t t e r n t r a n s i t i o n s
f r o m s p o t s a t t h e p e r i p h e r y t o l a b y r i n t h i n e p a t t e r n s i n t h e c e n t r e ,
w h i c h r e c a p i t u l a t e s t h e i n v i t r o p e r i o d i c p a t t e r n s . A d d i t i o n a l
p a r a m e t e r v a l u e s a r e g i v e n i n T a b l e S 3
*
Hair peg-like
structures
100 μm
96 h
(B)
1
mm
1 mm 1 mm
Hair peg-like
structures
1
2
3
4
5
(C)
6
5
4
3
2
1
0
Density of hair pegs (per mm 2 )
1 2 3 4 5
zones
160
120
80
40
0
Diameter of dermal condensate (μm)
Peg
Mul-stalk
No peg
24 h36 h48 h 60 h72 h 84 h
1 mm
(A)
1 mm
**
** *
*
(D)
(E)
16

|
361 WEBER Et al .
k i n e t i c s . I n d e e d , w e d o n o t h av e s u f f i c i e n t l y d e t a i l e d i n f o r m a t i o n t o
d e t e r m i n e t h e s y s t e m t o t h i s a c c u r a c y . T h e s p e c i f i c u s e o f o u r m o d e l
i s t o h i g h l i g h t t h a t t h e t r a n s i t i o n s e e n i n t h e e x p e r i m e n t s c a n b e
c a p t u r e d , q u i t e g e n e r a l l y , u s i n g a s i m p l e r a d i a l l y s y m m e t r i c , l i n e a r ,
t i me - dep en dent g r a d i ent . T he o b s er ve d r e s u l t cou ld b e o b t a i ne d i n
a n i n f i n i t e n u m b e r o f m o r e c o m p l i c a t e d w a y s . H o w e v e r , o u r r e s u l t s
h av e p u t a l o w e r b o u n d l i m i t o n t h e c o m p l e x i t y r e q u i r e d t o m a k e a
m o d e l c o n s i s t e n t w i t h t h e o b s e r v e d r e s u l t s .
S i n c e w e o b s e r v e t h a t h a i r p e g - l i k e s t r u c t u r e s f i r s t f o r m i n t h e
p e r i p h e r y , th e a s y m m e tr y s u g ges t s th at th e a c t i v ato r beco m es i n -
cre a s in gl y s e n s i t i ve to t h e in hibi to r m o r p h o ge n ν , a t t h e p e r i p h e r y ,
o r , a l ter n at ivel y , t he a c t iv ato r b e co me s de c r e a si n g l y s en si t ive to
t h e a c t i v a t o r ' s s e l f - a c t i v a t i o n r e s p o n s e . S u c h a g r a d i e n t c a n e a s i l y
a r i s e a s t h e ex p e r i m e nt a l dr o p l e t i s a n i s ot r o pi c , a n d co ul d b e d u e to
c h e m i c a l s i g n a l l i n g ( e . g g r o w t h f a c t o r s ) o r p h y s i c a l f o r c e s i n n a t u r e ,
o r b o t h . T h u s , t h e p r i n c i p l e s o f T u r i n g a c t i v a t o r a n d i n h i b i t o r r e m a i n
t h e s a m e , b u t i n d i f f e r e nt r e g i o n s , we a nt i c i p ate t h e f i e l d c a n b e h e t -
e r o g e n e o u s l y p r e d i s p o s e d w i t h p a r a m e t e r s t h a t f av o u r o r s u p p r e s s
p e r i o dic p a t t e r nin g . A s t im e p r o g re s s e s , t h e g r a di e n t in cre a s e s to -
w a r d s t h e p e r i p h e r y ( b ot t o m s i m u l a t io ns of f i g u r e “ s i m u l a t io n ” ), a n d
p a t t e r n s t r a n s i t f r o m l a b y r i n t h i n e s t r i p e s t o s p o t s ( t o p s i m u l a t i o n s
o f f i g u r e “ s i m u l a t i o n ) ( F i g u r e 3 E ) . T h e s p a t i o t e m p o r a l h e t e r o g e n e i t y
i s m o d e l l e d a s a l i n e a r s p a t i a l g r a d i e n t t h a t i n c r e a s e s a t t h e d r o p l e t
b o u n d a r y a n d f i xe s ov e r t i m e ( F i g u r e 3 E , M ov i e S 6 ) . T h e v i s u a l i z a -
t i o n o f t h e g r a d i e n t e x h i b i t s i t s e l f a s a h a i r p e g f o r m a t i v e w av e t r av -
e l l i n g f r o m t h e p e r i p h e r y t o w a r d s t h e c e n t r e o f t h e f i e l d , m a t c h i n g
e x p e r im e n t a l re s u lt s o bs e r ve d in t h e d r o p l et cu lt u re s . C r i t ic a l l y , t h e
p r o p o s e d a s y m m e t r y c o u l d b e w r a p p e d u p i n s i d e t h e e q u a t i o n s , b u t
t h i s wou ld o b sc u r e t he e s s ent i a l r e q u i r ement of a s pat i otemp o r a l
g r a d i e n t a p p e a r i n g . T h u s , w e c h o o s e t o b e e x p l i c i t w i t h t h e a d d i t i o n
o f s u c h c o m p l e x i t y .
3.6 | A platform to modulate hair peg
morphogenesis in vitro
T o i n c r e a s e t h e n u m b e r o f h a i r p e g - l i k e s t r u c t u r e s a n d t o s t i m u l a t e
d e v e l o p m e n t b e y o n d t h e h a i r p e g s t a g e , w e m o d u l a t e d m u l t i p l e p a -
r a m e t e r s w i t h i n t h e d r o p l e t c u l t u r e s y s t e m . T h e g r e a t e s t n u m b e r
o f h a i r p e g - l i k e s t r u c t u r e s p e r d r o p l e t c u l t u r e w a s g e n e r a t e d w i t h
a 150 μ L v o l u m e d r o p l e t , 5 % F B S c o n c e n t r a t i o n a n d e p i d e r m a l t o
d e r m a l c e l l r a t i o o f 2 : 3 ( F i g u r e 4 A ) . A n av e r a g e o f 28 6 h a i r p e g - l i k e
s t r u c t u r e s ( ± 1 3 8 ) p e r c m
2
w i t h a n i n t e r f o l l i c u l a r d i s t a n c e o f 3 5 0 μm
w a s p r o d u c e d u n d e r o p t i m a l c o n d i t i o n s ( F i g u r e 4 B ) . F o r c o m p a r i s o n ,
e n d o g e n o u s h a i r p e g s f r o m 1 7 - w e e k f e t a l s c a l p a r e s p a c e d , o n av e r -
a g e , 23 5 μ m a p a r t . T h e i n v i t r o h a i r p e g - l i k e s t r u c t u r e s w e r e s i m i l a r
i n ov e r a l l s h a p e t o h a i r p e g s o f 1 7 - w e e k f e t a l s c a l p b u t e x h i b i t e d
s i g ni f ic a n t l y di f f e re n t s t r u c t u r a l p r o p o r t i o n s . T h e re co n s t i t u t e d h air
p e g - l i k e s t r u c t u r e s p o s s e s s e d s h o r t e r , n a r r o w e r k e r a t i n o c y t e s t a l k s
a n d w id e r d e r mal ca ps w hil e t h e h eight of t h e d e r mal ca p re main e d
c o n s i s t e n t w i t h e n d o g e n o u s f e t a l h a i r p e g s ( F i g u r e 4 B ) . N a t i v e f e t a l
s c a lp e x hibi t e d h air p eg s of v a r i o u s e p id e r m a l s t a lk h ei g h t s . T h e re -
c o n s t i t u t e d h a i r p e g - l i k e s t r u c t u r e s w e r e , o n av e r a g e , s h o r t e r t h a n
t he en do gen ou s h a i r p e g s b u t m o r e c l o s el y r e s emb l e d t he s h o r ter ,
e a r l y h a i r p e g s i n n a t i v e s k i n , s u g g e s t i n g t h a t , a c c o r d i n g t o n o r m a l
devel o p ment a l pat ter n s , t he r e co n s t i tu te d h a i r p e g - l i ke s t r u c tu r e s
c o u l d b e e x p e c t e d t o e l o n g a t e f u r t h e r b e f o r e t r a n s i t i o n i n g t o t h e
b u lb o u s p eg s t a ge . H owe ve r , o u r re co n s t i t u t e d h air p eg - l ik e s t r u c -
t u r e s f a i l e d t o p r o g r e s s f u r t h e r w h e n t h e y w e r e m a i n t a i n e d f o r t h r e e
a d d i t i o n a l d a y s i n c u l t u r e . C l e a r l y , o t h e r f a c t o r s a r e r e q u i r e d .
A l t h o u g h w e h av e n o t b e e n a b l e t o a c h i e v e m o r e m a t u r e h a i r
f o l l i c l e f or m at i on , t h e se l f - or g an iz at i on of p e r i o d i c a l l y ar r an ge d h a i r
p e g - l i k e s t r u c t u r e s f r o m d i s s o c i a t e d c e l l s i s a r e m a r k a b l e p r o c e s s .
D e t a i l e d a n a l y s i s o f t h e p r o c e s s e n a b l e s t h i s d r o p l e t c u l t u r e t o s e r v e
a s a p l a t f o r m f o r l a r g e - s c a l e s c r e e n i n g o f e x p e r i m e n t a l c o n d i t i o n s
FIGURE 4 M o d u l a t i o n o f h a i r p e g m o r p h o g e n e s i s i n v i t r o . A ,
H e r e , w e e x a m i n e t h e c o n d i t i o n s t h a t c a n i n f l u e n c e t h e n u m b e r ,
s i z e a n d p r o g r e s s i o n o f t h e h a i r p e g - l i k e s t r u c t u r e s . H a i r p e g - l i k e
s t r u c t u r e s f o r m e d m o r e f r e q u e n t l y w h e n c u l t u r e m e d i u m c o n t a i n e d
5 % F B S a n d w h e n a n e p i d e r m a l t o d e r m a l c e l l r a t i o o f 2 : 3 w a s
u s e d . T h e av e r a g e n u m b e r o f h a i r p e g - l i k e s t r u c t u r e s f o r m e d p e r
c o n d i t i o n i s p l o t t e d , w i t h e r r o r b a r s r e p r e s e n t i n g s t a n d a r d e r r o r o f
t h e m e a n a n d s t a t i s t i c a l s i g n i f i c a n c e a s s e s s e d w i t h p a i r e d t t e s t s .
*P < 0 . 0 5 ( n = 6 ) . B , T h e i n v i t r o h a i r p e g - l i k e s t r u c t u r e s ( b l a c k b a r s )
s h o w s i m i l a r a r c h i t e c t u r e t o h a i r p e g s i n 1 7 - w k h u m a n f e t a l s c a l p
( g r e y b a r s ) . H o w e v e r , t h e av e r a g e s t a l k h e i g h t , s t a l k w i d t h a n d
c a p w i d t h o f t h e i n v i t r o h a i r p e g - l i k e s t r u c t u r e s w e r e s i g n i f i c a n t l y
s m a l l e r . W h i l e t h e i n v i t r o h a i r p e g - l i k e s t r u c t u r e s f o r m e d a t
r e g u l a r i n t e r v a l s , t h e r e w a s a l a r g e r av e r a g e i n t e r f o l l i c u l a r d i s t a n c e
t h a n i s f o u n d i n f e t a l s c a l p t i s s u e . A n a s t e r i s k d e n o t e s a P- valu e
o f < 0 . 0 5 , w h e n c o m p a r e d t o n a t i v e h u m a n f e t a l s c a l p , v i a p a i r e d
t t e s t s . E r r o r b a r s r e p r e s e n t s t a n d a r d e r r o r o f t h e m e a n ( n = 5 ) .
C , T h e a d d i t i o n o f g r o w t h f a c t o r s t o t h e d r o p l e t c u l t u r e s c a u s e d
s i g n i f i c a n t c h a n g e s i n c e r t a i n a s p e c t s o f t h e e p i d e r m a l s t a l k a n d
d e r m a l c a p d i m e n s i o n s , b u t d i d n o t i n d u c e f u r t h e r d e v e l o p m e n t
i n t o a b u l b o u s p e g s t r u c t u r e . D i m e n s i o n s m e a s u r e d t h e f o l l o w i n g :
1 ) e p i d e r m a l s t a l k w i d t h , 2 ) e p i d e r m a l s t a l k l e n g t h , 3 ) e p i d e r m a l
s t a l k a r e a a t t h e s t r u c t u r e m i d p o i n t , 4 ) d e r m a l c a p w i d t h , 5 ) d e r m a l
c a p h e i g h t , 6 ) e p i d e r m a l s t a l k - d e r m a l c a p o v e r l a p a n d 7 ) d e r m a l
c a p a r e a a t t h e s t r u c t u r e m i d p o i n t . G r o w t h f a c t o r s : A ) n e g a t i v e
c o n t r o l , B ) S h h 1 μ g / m L , C ) T g fβ2 0.5 μ g / m L , D ) R A R a n t a g o n i s t
ER50891 1 μ m o l /L , E ) F G F 7 + F G F 1 0 1 μ g / m L , F ) F G F 2 1 μ g / m L , G )
F G F 2 + S h h 1 μ g / m L , H ) F G F 2 + W n t 7 a 1 μ g / m L , I ) P K C i 6 6 0 n m o l /L
c h e l e r y t h r i n e c h l o r i d e a n d 1 0 n m o l /L b i s i n d o l y l m a l e i m i d e I a n d
J ) N o g g i n 1 μ g / m L . A l l m e a s u r e m e n t s w e r e n o r m a l i z e d t o t h e
n e g a t i v e c o n t r o l , a n d av e r a g e d i m e n s i o n s a r e s h o w n . E r r o r b a r s
r e p r e s e n t s t a n d a r d e r r o r o f t h e m e a n . S t a t i s t i c a l s i g n i f i c a n c e
b e t w e e n t w o g r o u p s w a s c a l c u l a t e d u s i n g p a i r e d t t e s t s . A n a s t e r i s k
d e n o t e s a P - v a l u e < 0 . 0 5 , c o m p a r e d t o t h e n e g a t i v e c o n t r o l w i t h o u t
a d d e d f a c t o r s ( n ≥ 3 )
Cap width (4)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
Normalized length
or area
Cap height (5) Cap-stalk overlap (6) Cap area (7)
Stalk width (1)
A B C D E F G H I J
Stalk area (3) Stalk height (2)
2.0
1.5
1.0
0.5
0
Normalized length
or area
1
4
2
5
6
3
7
*
*
*
*
*
*
*
*
A B C D E F G H I J A B C D E F G H I J
A B C D E F G H I J A B C D E F G H I J A B C D E F G H I J A B C D E F G H I J
*
*
*
*
*
*
vimenn nuclei
400
300
200
100
Height or width (μm)
Stalk height
Stalk width
Cap height
Cap width
Inter-peg
distance
*
*
*
*
17 week human fetal scalp
50 µm
0% 1% 5% 10%
FBS concentraon
300
(A) (B) (C)
250
200
150
100
50
0
HF precursors per droplet
2:1 2:3 1:5 1:9 1:12
Epidermal:Dermal rao
HF precursors per droplet
300
250
200
150
100
50
0
*
17
362
|
WEBER Et al .
t o o p t i m i z e i n v i t r o f o l l i c l e f o r m a t i o n . W e i d e n t i f i e d f o u r p o s s i b l e
s i g n a l s t h a t m i g h t s u p p o r t d e v e l o p m e n t a l p r o g r e s s i o n : ( a ) i n c r e a s e d
der m a l si g n a l l i n g f o r ep i der m a l dow n g r ow t h (S h h , T g fβ 2 ) , ( b ) s t r o n -
g e r d e r m a l p a p i l l a i n d u c t i v i t y ( W n t 7 a , F G F 2 ) , ( c ) i n h i b i t i o n o f p r e -
m a t u r e k e r a t i n o c y t e d i f f e r e n t i a t i o n ( p r o t e i n k i n a s e C ( P K C ) , N o g g i n ,
ret in o ic a c id re ce p to r s (R A R )) a n d ( d ) s t im u la t i o n of k e r a t in o c y t e di f -
f e r e n t i a t i o n a n d / o r s t r a t i f i c a t i o n ( F G F 2 , F G F 7 / 1 0 ) .
[43–50]
E xo g e n o u s g r o w t h f a c t o r s w e r e a d d e d t o t h e c u l t u r e m e d i u m
e v e r y 2 4 h o u r s . A r a n g e o f c o n c e n t r a t i o n s w a s t e s t e d f o r e a c h p r o -
t e i n ; r e s u l t s f o r t h e c o n c e n t r a t i o n w h i c h p r o d u c e d t h e g r e a t e s t
e f f e c t a r e s h o w n ( F i g u r e 4 C ) . T h u s f a r , n o n e o f t h e a d d e d f a c t o r s
h av e r e s u l t e d i n p r o g r e s s i o n t o t h e n e x t s t a g e , t h e b u l b o u s h a i r
p e g . H o w e v e r , a d e t a i l e d a n a l y s i s o f d e r m a l c a p a n d e p i d e r m a l s t a l k
w i d t h , h e i g h t a n d a r e a i d e n t i f i e d s i g n i f i c a n t c h a n g e s m e d i a t e d b y t h e
a d d e d g r o w t h f a c t o r s , w h i c h , w i t h m o r e i nv e s t i g a t i o n , m a y h o l d t h e
k e y t o s t i m u l a t i n g t r u e f o l l i c l e f o r m a t i o n i n c u l t u r e . C a p a n d s t a l k
s a g i t t a l ar e a s m a i nt a i n e d a l i n e ar r e l at i on s h i p w i t h t h e tot a l c ap an d
s t a l k v o l u m e s , e m p h a s i z i n g t h e r a d i a l s y m m e t r y o f t h e s e s t r u c t u r e s
a n d a l l o w i n g u s t o s i m p l i f y a n a l y s i s b y m e a s u r i n g t h e a r e a o f e a c h
s t r u c t u re a t t h e mid p o in t co r re s p o n din g to m a x im a l w id t h (F i g u re
S 3 ) . D u r i n g t h e e a r l y p e g t o b u l b o u s p e g t r a n s i t i o n , t h e d e r m a l c a p
b e co m e s m o r e co m p a c t a n d is e n c a p su l ate d by t h e b as e of t h e e l o n -
g at i n g ep i der m a l s he at h .
[30]
T h e a d d i t i o n of 1 μ m o l /L S h h s t i m u l a t e d
e p id e r m a l d ow n g r ow t h , re s u lt in g in l o n ge r e p id e r m a l s t a lk s , a s we l l
a s a c h a n g e i n t h e d e r m a l c a p s h a p e , w i t h a n i n c r e a s e d w i d t h a n d
c a p - s t a l k ov e r l a p , s u g g e s t i n g t h a t S h h m a y s t i m u l a t e d e r m a l c e l l m i -
g r at i o n p r oxi m a l l y a l o n g t he ep i der m a l s t a l k o r , co nver s el y , ep i der -
m a l s t a l k d i s p l a c e m e n t o f d e r m a l c a p c e l l s ( F i g u r e 4 C ) . T h e p r o t e i n
k in a s e C in hibi to r s , ch e l e r y t h r in e ch l o r id e a n d bi s in d o l y lm a l eimid e
I , p r o d u c e d a s i m i l a r e f f e c t , w i t h i n c r e a s e d e p i d e r m a l s t a l k l e n g t h
a n d ov e r a l l s t a l k a r e a , a s w e l l a s i n c r e a s e d d e r m a l c a p a r e a a n d c a p -
s t a lk ove r la p. FG F2 , in co m bin a t i o n w i t h S h h , d e cre a s e d d e r m a l c a p
h e i g h t a n d a r e a a n d t h e r e t i n o i c a c i d r e c e p t o r a n t a g o n i s t E R 5 0 8 9 1
e n h a n c e d t o t a l s t a l k a r e a . T h o u g h s u b t l e c h a n g e s w e r e e v i d e n t
w h e n e xo g e n o u s f a c t o r s w e r e a d d e d , t h e y w e r e i n s u f f i c i e n t t o a l t e r
t he g r o s s m o r p h o l o g y of t he h a i r p e g - l i ke s t r u c tu r e a n d p u s h devel -
o p m e n t i n t o t h e b u l b o u s p e g s t a g e .
4 | DISCUSSION
T h e a b i l i t y t o t i s s u e e n g i n e e r h u m a n h a i r f o l l i c l e s f o r t r a n s p l a n t a t i o n
wou ld el i m i n ate a t r e at ment g a p f o r n u mer ou s pat i ent s . O ver t he
y e a r s , o u r g r o u p ' s r e s e a r c h h a s f o c u s e d o n t h e m o r p h o g e n e s i s o f
s ki n a p p e n d a ges. Rece nt l y , we e x a m i n e d th e s e l f - o rga n izi n g be h av -
i ou r of d is so c i ate d ep i der m a l a n d der m a l ne w b o r n m ou s e cel l s a n d
t h e i r a b ili t y t o r e c o ns t i t u t e f u n c t io n a l f o lli c l e s .
[4]
S imi la r s t u di e s of
h u m a n f o l l icu la r m o r p h o ge n e s i s h ave b e e n di f f icu lt to a chi e ve , d u e
t o t h e l o w e f f i c i e n c y o f f o l l i c l e f o r m a t i o n f r o m r e a d i l y av a i l a b l e a d u l t
cel l s a n d t he l o n g t i me to f o l l i c l e f o r m at i o n . H er e , we dem o n s t r ate d
t h e p r o d u c t i o n o f h u m a n h a i r p e g - l i k e s t r u c t u r e s i n v i t r o f r o m a w e l l -
d e f in e d mi x t u re of p r o ge ni to r ce l l s . I n t hi s t h re e- dim e n s i o n a l o r g a -
n o id d r o p l et cu lt u re , di s s o c i a t e d n e o n a t a l e p id e r m a l a n d f et a l d e r m a l
cel l s p r o g r e s s e d , vi a s el f - o r g a n iz at i o n , t h r ou g h t he f o l l ow i n g r ep r o -
d u c i b l e an d r e co g n iz ab l e s t a ge s ak i n to e ar l y f o l l i c l e d eve lop m e nt
t o r e a c h c e l l u l a r c o n f i g u r a t i o n s s i m i l a r t o h a i r p e g s i n s i t u : ( a ) m i xe d
d i s s o c i a t e d c e l l s , ( b ) c e l l s h e e t s , ( c ) d e r m a l s t r i p e s a n d c l u s t e r s , ( d )
der m a l c l u s ter s w i t h a s so c i ate d ep i der m a l p l a co de - l i ke co l l e c t i o n s
a n d ( e ) di s t in c t h air p eg - l ik e s t r u c t u re s w i t h s p a t i a l p e r i o dic i t y .
[30]

T h e d e v e l o p m e n t a l p r o c e s s p r o c e e d e d r a p i d l y w i t h i n 9 6 h o u r s a n d
w a s dep en dent o n ep i der m a l:der m a l cel l r at i o a n d f a c to r co n cent r a -
t i o n , s u g g e s t i n g t h e n e e d f o r a n a p p r o p r i a t e b a l a n c e o f e p i t h e l i a l -
m e s e n c h y m a l s i g n a l l i n g f a c t o r s o r c e l l - c e l l i n t e r a c t i o n s . T h i s i n v i t r o
c u l t u r e s y s t e m d e m o n s t r a t e s t h e i n i t i a t i o n a n d r a p i d p r o g r e s s i o n o f
e a r l y s t a g e s o f h u m a n f o l l i c l e - l i k e d e v e l o p m e n t . I t a l s o s h o w s t h a t
h u m a n a n d m o u s e c e l l s u t i l i z e d i f f e r e n t m o r p h o g e n e t i c p a t h s i n t h e
m o r p h o s p a ce of e p id e r m a l- d e r m a l m u lt ice l l u la r co nf i g u r a t i o n s a n d
m ay e x p lain w hy i t h a s b e e n di f f icu lt to a chi e ve r o b u s t h u m a n h air
r eco n s t i t u t i o n . W e hy poth esize th at th e d i f f e r e n ces be t wee n h u m a n
a n d m o u s e h a i r f o l l i c l e r e c o n s t i t u t i o n m a y b e d u e t o t h r e e f a c t o r s :
epidermal cell plasticity, the inducing ability of dermal cells and morpho-
genetic field competence.
1. The plasticity of foreskin keratinocytes is known to wane with
p r o l o n ge d cu lt u re , re s u lt in g in re d u ce d h air f o l l ic l e f o r m at i o n .
[51]

W e h y p ot h e s iz e d t h at a l o s s o f e p i d e r m a l p l a s t i c i t y i n h i b i te d
f u r t h e r f o l l i c l e o r g a n o i d d ev e l o p m e nt i n v i t r o b ey o n d t h e p e g
s t a ge . P r ote i n k i n a s e C (PK C ) a n d r e t i n o i c a c i d p at h w a y s p l a y
a r o l e i n e p i d e r m a l d i f f e r e nt i at i o n a n d s t r at i f i c at i o n d u r i n g s k i n
d ev e l o p m e nt . E xce s s i v e r e t i n o i c a c i d c a u s e s ce s s at i o n o f h a i r
f o l l i c l e d ev e l o p m e nt at t h e ge r m s t a ge i n m i ce , w h i l e i n h i b i t i o n
o f PK C p r o m ote s f o l l i c u l o ge n e s i s f r o m a d u l t m o u s e ce l l s .
[4 ,4 7 , 49 ]

T h e a d d i t i o n o f PK C i n h i b i to r s a n d a n R A R a nt a g o n i s t e x h i b i te d
p o s i t i v e e f f e c t s o n t h e l e n g t h a n d d i a m e te r o f t h e e p i d e r m a l
s t a l k b u t w a s i n s u f f i c i e nt to d r i v e f u r t h e r f o l l i c u l o ge n e s i s , s u g -
ge s t i n g t h at ot h e r f a c to r s a r e r e q u i r e d f o r p r o g r e s s i v e d ev e l -
o p m e nt . W e s u s p e c t t h at t h e l e s s p r i m i t i v e e p i ge n e t i c s t ate
o f t h e k e r at i n o c y te s u s e d m a y b e t h e m o l e c u l a r b a s i s f o r
s u b o p t i m a l co m p e te n ce . I n f u t u r e s t u d i e s , we w i l l s e a r c h f o r
f a c to r s t h at c a n “ r e p r o g r a m ” t h e s e k e r at i n o c y te s o r u s e m o r e
res po nsi ve k e r at in o c y tes.
2. The inducing ability of dermal cells is a second critical component
f o r f o l l icu l o ge n e sis . T h e d e r m a l p a p i l la re l e a s e s m u lt ip l e f a c to r s ,
w hich p a r t ic ip ate in e p id e r m a l-m e s e n ch y m a l si g n a l l in g d u r in g f o l -
l i c u l o ge n e s i s . S h h a n d T g fβ a r e ne ce s s a r y fo r ep i der m a l dow n -
g r ow t h a n d mice w hich la ck S h h si g n a l l in g p o s s e s s h air f o l l ic l e s
w h i c h a r e s t a ll e d at t h e ge r m/ p e g s t a ge .
[4 3 , 4 4]
T h e a d d i t io n of S h h
to t h e d r o p l et cu lt u re s s t im u late d a d di t i o n a l e p id e r m a l d ow n -
g r o w t h b u t d i d n ot c a u s e s t r u c t u r a l p r o g r e s s i o n to t h e b u l b o u s
p e g s t a ge . W e a l s o e x a m i n e d W nt 7 a a n d F G F2 , w h i c h h av e b e e n
s h ow n to m aint ain p r o l i f e r at i o n a n d in d u c t i v i t y in cu lt u re d m u r in e
d e r m a l p a p ill a ce lls .
[45 , 4 6 ]
Y et , we did not obser ve signif ic ant pro -
gress in organoid development . The dermal papilla-like cells in our
culture do not appear fully func tional as they c an only suppor t the
in d u c t i o n o f h air p eg -l ik e s t r u c t u re s , n ot m at u re f o l l ic l e s .
H o wev e r , t h i s s y s te m p r ov i d e s a p r o m i s i n g p l at f o r m f o r t h e
18

|
363 WEBER Et al .
cont i n u e d se ar c h for f a c tor s or con d i t i on s w h i c h e nh an ce
inductivity.
3. The morphogenetic field, co mp r is e d of ep i der m a l cells , der m a l cells
a n d e x t r a ce l l u l a r m at r i x to ge t h e r , m u s t e nte r a co m p e te nt s t a ge
f o r p e r i o d i c p at te r n i n g to b e g i n . T h e d ev e l o p i n g e m b r y o i s a h e t -
e r o ge n e o u s m o r p h o ge n et ic f i e l d w i t h a nis ot r o p ic g r ow t h in w hich
c h e m i c a l f a c to r s , ce l l t y p e s a n d m e c h a n i c a l f o r ce s a r e u n ev e n l y
d i s t r i b u te d i n t h r e e s p at i a l d i m e n s i o n s a n d o n e te m p o r a l d i m e n -
sio n . H er e , ou r o r g a n o i d c u l tu r e dem o ns t r ate s o bviou s as y mme -
t r y w i t h i n t h e d r o p l e t , a s p at te r n s b e g a n at t h e p e r i p h e r y a n d
migrated centrally . L abyrinthine stripes of dermal cells were noted
ini t ia l l y , w hich s u bs e q u e nt l y t r a n s f o r m e d into p e r i o dic a l l y a r -
r a n ge d d e r m a l ce l l c l u s te r s . B ot h s t r i p e s a n d d e r m a l c l u s te r s c a n
b e p ro du ce d by a sim p le T ur ing m o d e l an d c an ref le c t an inte r m e -
diate s t age of the f inal periodic pat terns if there is an uneven mor -
p h o gene t i c f i eld .
[38]
What can account for the difference in
p r o g r e s s i o n t h r o u g h t h e p e r i o d i c p at te r n i n g p r o ce s s ? W h i l e a
sim p le ge n e r ic r a dial gr a die nt ef fe c te d by o n e co m p o n e nt may b e
s u f f i c i e nt f o r a T u r i n g a c t i v ato r - i n h i b i to r s y s te m to p r o d u ce t h e
pat ter n here, it may not be suf f icient to produce the complex spa -
t iotemp o r a l pat ter n i n g t r a nsi t io ns we o b s er ve d e x p er i men -
tally.
[3 6 , 37 ]
H er e , we p u r p o s el y de sig ne d a gener i c m o del , to h ave
w id e r co n ce p t u a l a p p l ic at i o n . S im u lat i o n w i t h m at h e m at ic a l m o d -
e l l in g s u g ge s t s simi la r p at te r nin g s e q u e n ce s c a n b e a chi ev e d
t h r ou g h u neven c hem i c a l sig n a lli n g a c t ivi t i e s
[52]
(e.g higher con -
ce nt r at i o n o f a c t i v ato r m o r p h o ge n s at t h e d r o p l e t p e r i p h e r y o r
h ig her s ensi t ivi t y of cells at t he d r o p l e t p er i p her y) o r u neven me -
ch a nic a l f o r ce s
[53]
( e . g ce l l u l a r te n s i o n o r m at r i x r i g i d i t y f av o u r
p er io d i c fo r m at io n at t he d r o p l e t p er i p her y). T he m at hem at i c a l
a n d e x p er i ment a l m o dels p r e s ente d her e w ill hel p u s i dent if y t he
m o l e c u l a r b a s i s o f t h e s e p at te r n i n g p r o ce s s e s i n t h e a n a l y s a b l e
d r o p l e t i n v i t r o a n d i n t h e co m p l e x d ev e l o p i n g e m b r y o i n v i v o i n
t h e f u t u r e .
In viv o , chick en fea ther buds form ex quisit e hexag onal pa t t erns
progressiv ely from the midline t o the flank . Earlier w ork s hav e sug -
g es t ed this result s from a loc al T uring e v en t and a global propaga ting
event.
[54]
However , the nature of the global event was unknown. This is
par t of the motivation for this study , to use the organoid droplet to un -
derstand more about the nature of the sequential appearance of hair or
f e a t her p r im or di a . I t i s t imel y t h a t a pap er rep or t in g a global E da wa v e
s p re a din g f r om t he midl ine t o t he f lank i s j u s t rep or t e d , w hich s ug g e s t s
Eda induces F G F20, f ollo wed b y dermal cell ag grega t e f orma tion , thus
f a c i l i t a t in g T ur in g pa t t er nin g vi a me ch an o- chemic al c oup l in g.
[55]
Based
on this and other studies, we propose a new understanding on the pro -
c ess o f propaga tiv e forma tion o f fea ther arr a ys in viv o : a loc al T uring
periodic pa t t erning oc cur s and w ork s t og ether with a global propaga -
t i on me ch ani s m .
[56]
In this new study , progressive expression of Eda
and ac tiva tion o f E da signalling from the midline t o the flank is sho wn
to mediate the global process by lowering the threshold of dermal con -
dens a tion .
[55]
Y et, what is upstream to Eda remains unclear . Thus, the
global mechanism can be chemical or mechanical in nature, as long as it
c an tilt the T uring ac tiva t or /inhibit or sys t em r a tio .
[56]
T he a s y mm et r ic
m or p h o g enet ic f i eld in t he or g an o id cu lture s tu di e d here p re sen t s a
g ood model t o fur ther inv es tiga t e ho w this global asymmetr y mech -
anism w ork s.
W i t h t w o c e l l u l a r c o m p o n e n t s , t h e i n i t i a l e p i d e r m a l a n d d e r m a l
c e l l r a t i o w i l l i n f l u e n c e t h e f i n a l s t a b l e p o s i t i o n i n t h e m o r p h o - s p a c e
of t wo- co m p o n e n t m u lt i- ce l l u la r a s s e m b l i e s .
[4]
T h e ini t i a l co n di -
t i o n s , d e t e r m i n e d b y t h e p r o b a b i l i t y o f c e l l c o l l i s i o n a n d t h e r e l a t i v e
s t r e n g t h o f c e l l a d h e s i o n , c o n t r o l t h e i n i t i a l m u l t i - c e l l u l a r c o n f i g u -
r at i o n . I n t he h u m a n cel l c u l tu r e d r o p l e t , ep i der m a l- m at r ix a d he -
s i on s ap p e ar to d om i n ate , l e a d i n g to t h e f or m at i on of t h e e p i d e r m a l
l ayer f i r s t . D er m a l- der m a l i nter a c t i o n s a r e s t r o n ger t h a n ep i der m a l-
d e r m a l a d h e s i o n s , l e a d i n g t o t h e f o r m a t i o n o f d e r m a l s t r i p e s a n d
der m a l c l u s ter s . H owever , d u r i n g m o r p h o gene t i c p r o ce s s e s , t her e
c a n b e “ q u a l i t a t i v e c h a n g e s ” o f c e l l u l a r c o l l e c t i v e s . F o l l o w i n g t h e
f o r m at i o n of der m a l co n den s at i o n s , ep i der m a l- der m a l co n den s ate
a d h e s i o n i n c r e a s e s a n d c a n i n d u c e t h e f o r m a t i o n o f h a i r p e g - l i k e
s t r u c tu r e s , u p to t he e x tent t h at ep i der m a l b a s ement memb r a ne p o -
l a r i t y i s r e v e r s e d . T h u s , t h e h i g h - r e s o l u t i o n a n a l y s i s o f t h e p r o c e s s
o f h a i r p e g f o r m a t i o n p r ov i d e s a n e xc e l l e n t o p p o r t u n i t y t o f i n e - t u n e
k e y c e l l u l a r e v e n t s .
I n s u m m a r y , w e h av e d e m o n s t r a t e d t h a t h u m a n f e t a l s c a l p d e r -
m a l c e l l s , i n a s s o c i a t i o n w i t h c o m p e t e n t e p i d e r m a l c e l l s , c a n d i r e c t
t h e r a p id rege n e r a t i o n of h u m a n h air p eg - l ik e o r g a n o ids in v i t r o.
T h e o p p o r t u n i t y t o s t u d y t h e a s y m m e t r i c s p a t i o t e m p o r a l s e q u e n c e
o f p e r i o d i c p a t t e r n i n g w i t h i n t h e d r o p l e t p r ov i d e s i n s i g h t s i n t o t h e
s e l f - o r g a n i z i n g b e h av i o u r o f s k i n p r o g e n i t o r c e l l s . F u r t h e r m o r e , t h i s
i n v i t r o c u l t u r e s y s t e m p r ov i d e s a n o p p o r t u n i t y t o s t u d y w a y s t o r e -
s to re a n d o p t imi ze h air f o l l ic l e rege n e r a t i o n f r o m e a s i l y o b t ain a b l e
a d u l t d e r m a l c e l l s a n d m a y s u p p o r t t h e p r o d u c t i o n o f c o m p l e t e h a i r
f o l l i c l e s f o r t r a n s p l a n t a t i o n i n t h e f u t u r e .
[57]
ACKNOWLEDG EMENTS
T h i s w o r k w a s s u p p o r t e d b y f u n d i n g f r o m t h e A m e r i c a n C o l l e g e o f
S u r g e o n s ( E . L . W . ) , t h e C a l i f o r n i a I n s t i t u t e f o r R e g e n e r a t i v e M e d i c i n e
( E . L . W . ) , t h e A . P . G i a n n i n i F o u n d a t i o n ( E . L . W . ) , a g r a d u a t e f e l l o w -
s h i p f r o m t h e M i n i s t r y o f N a t i o n a l D e f e n s e o f T a i w a n ( K . L . O . ) , t h e L .
K . W h i t t i e r F o u n d a t i o n ( C . M . C . ) , t h e N a t i o n a l I n s t i t u t e o f A r t h r i t i s
a n d M u s c u l o s k e l e t a l a n d S k i n D i s e a s e s o f t h e N a t i o n a l I n s t i t u t e s o f
H e a l t h ( A R 4 7 3 6 4 , A R 6 0 3 0 6 ; a n d G M 1 2 5 32 2 t o C . M . C . ) a n d t h e
N a t i o n a l S c i e n c e F o u n d a t i o n ( D M S 1 4 4 0 3 8 6 t o T . E . W . a n d P . K . M . ) .
W e t h a n k D r . C h i n L i n G u o f o r h e l p f u l d i s c u s s i o n a n d a c k n o w l e d g e
t h e U S C R e s e a r c h C e n t e r f o r L i v e r D i s e a s e C e l l a n d T i s s u e I m a g i n g
C o r e a n d t h e U S C Ste m C e l l M i c r o s co py C o r e F a c i l i t y f o r t h e i r a s s i s -
t a n c e a n d p a r t i c i p a t i o n . W e t h a n k D r . R . B . W i d e l i t z f o r h e l p f u l i n p u t .
T . E . W . a n d P . K . M . t h a n k t h e M a t h e m a t i c a l B i o s c i e n c e s I n s t i t u t e
( M B I ) a t O h i o S t a t e U n i v e r s i t y f o r h e l p i n g t o i n i t i a t e t h i s r e s e a r c h .
M B I r e c e i v e s i t s f u n d i n g t h r o u g h t h e N a t i o n a l S c i e n c e F o u n d a t i o n
g r a n t D M S 1 4 4 0 3 8 6 .
CONFLIC T OF INTERE S T
T h e a u t h o r s h av e d e c l a r e d n o c o n f l i c t o f i n t e r e s t .
19
364
|
WEBER Et al .
AUTHOR CONTRIBUTIONS
E . L . W . a n d C . M . C . c o n c e i v e d t h e i d e a a n d e x p e r i m e n t a l d e s i g n .
E . L . W . a n d K . L . O . c o n d u c t e d t h e e x p e r i m e n t s . C . Y . Y . p r ov i d e d i m a g -
i n g e x p e r t i s e . T . E . W . a n d P . K . M . p r o d u c e d m a t h e m a t i c a l m o d e l l i n g .
E . L . W . , T . E . W . a n d C . M . C . p r e p a r e d t h e m a n u s c r i p t .
ORCID
Kuang-Ling Ou ht tps:/ / orcid.org /00 00 - 0001 -9536- 26 77
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365 WEBER Et al .
SUPPORTING INFORMATION
A d d i t i o n a l s u p p o r t i n g i n f o r m a t i o n m a y b e f o u n d o n l i n e i n t h e
S u p p o r t i n g I n f o r m a t i o n s e c t i o n a t t h e e n d o f t h e a r t i c l e .
Figure S1 . A g e n e r ic w o r k ing m o d e l fo r p la na r sk in re co ns t it u t io n
w it h hair fo r m at io n . Epid e r m a l a n d d e r m a l c e lls a re mi xe d a n d p late d
o n t issu e cult u re ins e r t in hig h c e ll d e nsit y as a dro p l et . D if fe re nt
ep ider m a l a n d der m a l c ells c a n b e u s e d , t h os e der iv e f ro m new -
b o r n sk in , a d ult sk in , a d ult hair fo llicl e , a n d E S o r iP S d e r i v e d c e lls
Figure S2 . L i v e c e ll im aging o f hair p eg - lik e s t r u c t u re s . A , O v e rex p re ssio n
o f f l u o re s c e nt p ro teins did n o t a f fe c t t h e a bilit y to fo r m hair p eg - lik e
structures. In this image, epidermal cell nuclei were marked with green
f l u o re s c e n t p r o t ein a n d d e r m a l c e ll n u c l ei w e re p refe re n t ia ll y m a r k e d
w it h o r a n g e f l u o re sc ent p ro tein . T he do t te d line ou t line s t he ep ider -
m a l s t a lk . Epi = e pid e r m a l , D = d e r m a l , E = e pid e r m a l ( n = 7 ) . B , A s t ill
im ag e f ro m a t w o- co l o r li v e im aging v id e o (F ig u re S 5 A ) , l o o k ing d o w n
f ro m t h e to p o f t h e cult u re dro p l et , d e m o ns t r ate s a d e r m a l c a p. p 6 3-
p ositive k er atinocy te nuclei are magent a , der mal cell nuclei are cyan .
Epi S C = e pid e r m a l s te m c e ll ( n = 5) . C , A singl e late r a l im ag e t a k e n
from a three- color live imaging video (Figure S5B) demonstrates a hair
p eg - lik e s t r u c t u re. K e r at in o c y te n u cl ei a re o r a ng e , p 6 3- p osit i v e k e -
r at in o c y te n u cl ei a re m ag e nt a , a n d d e r m a l c e ll n u cl ei a re c y a n ( n = 5)
Figure S3 . R e c o ns t i t u t e d h air p eg - lik e s t r u c t u re s dis p la y e d r a dia l s y m -
m et r y . C a p a n d s t a lk s a g i t t a l a re as m ain t ain e d a lin e a r re la t io ns hip
with the total cap and stalk volumes, emphasizing the radial symmetr y
o f t h e s e s t r u c t u re s a n d a ll o w ing us to sim p lif y a na l ysis b y m e asu r ing
t h e a re a o f e a ch s t r u c t u re at t h e midp oint co r re sp o n ding to m a x im a l
wi dt h
Movie S1 T h re e- dim e n s i o n a l z- s t a c k r e c o n s t r u c t i o n s o f h u m a n
h air p eg- l ike s t r u c t u re s f o r m e d in cu lt u re . A , W h o l e m o u n t c o n -
f o c a l z- s t a ck im a g e s o f m u lt ip l e h air p eg- l ike s t r u c t u re s im m u -
n o s t ain e d w i t h ke r a t in - 1 4 ( g re e n) , v im e n t in (re d ) , a n d TO - P R O - 3
i o d i d e ( n u c l e i , b l u e) d e m o n s t r a t e t h e p e r i o d i c p a t t e r n i n g a n d f o r -
m a t i o n o f d i s t i n c t s t r u c t u r e s w i t h i n a 42 5 μm
2
a re a . C e l l s w i t hin
t h e ke r a t ini ze d s h e et w e re di f f icu lt t o s t ain w i t h p a n c y t o ke r a t in
d u e t o p o o r a n t i b o d y p e n e t r a t i o n . B , W h o l e m o u n t c o n f o c a l z -
s t a ck im a g e s o f h air p eg- l ike s t r u c t u re s im m u n o s t ain e d w i t h p a n -
c y t o ke r a t in ( g re e n) a n d p r o p idi u m i o did e (n u c l ei , re d ) d e m o n s t r a t e
t h e s p h e r i c a l c o n f i g u r a t i o n o f t h e d e r m a l c a p a n d t h e t u b u l a r
s t r u c tu r e o f t he ep i der m a l s t a l k . S a n dw i c h i n g o f t he w h o l e m ou n t
cu lt u re b e n e a t h a c o v e r s l ip f o r c o n f o c a l im a g in g c a u s e d t h e h air
p e g s t o a p p e a r b e n t o r f l a t t e n e d a g a i n s t t h e k e r a t i n o c y t e s h e e t .
C , H i g her m a g n i f i c a t i o n vi e w o f a s i n g l e r e c o n s t i tu t e d h u m a n h a i r
p eg- l ike s t r u c t u re im m u n o s t ain e d w i t h p a n c y t o ke r a t in ( g re e n) a n d
p r o p i d i u m i o d i d e ( n u c l e i , r e d ) . N o t e e p i d e r m a l c e l l s s t a r t t o w r a p
a r o u n d t h e d e r m a l c a p
Movie S2 T h re e- dim e n s i o n a l z - s t a c k r e c o ns t r u c t io ns of r e c o ns t i -
t u t e d h u m a n h a i r p e g - l i k e s t r u c t u r e s d e m o n s t r a t e m a r k e r s o f d e r m a l
p a p i l l a g e n e e x p r e s s i o n . A , W h o l e m o u n t c o n f o c a l z - s t a ck im a g in g
d e m o n s t r ate s α - S M A s t a i n i n g i n a c e n t r a l l o c a t i o n w i t h i n t h e d e r m a l
c a p o f a h a i r p e g - l i k e s t r u c t u r e . α- S M A ( g re e n ), p r o p idi u m i o did e
( r e d ) . B , W h o l e m o u n t c o n f o c a l z - s t a c k i m a g i n g d e m o n s t r a t e s t h e
p r e s en ce of e x t r a cel l u l a r co l l a gen I V at t he ep i der m a l- der m a l i nter -
f a c e . C D 3 4 - a n e a r l y d e r m a l p a p i l l a m a r k e r ( g r e e n ) , c o l l a g e n I V ( r e d ) ,
T O - P R O - 3 i o d i d e ( b l u e ) . T h e l a r g e g r e e n l o b u l e s a r e a r t i f a c t s r e p r e -
s e n t i n g d e a d c e l l s w h i c h h av e t r a p p e d t h e d y e
Movie S3 T h re e- dim e n s i o n a l z - s t a c k r e c o n s t r u c t i o n s o f e p i d e r m a l
p l a c o d e - l i k e s t r u c t u r e s a n d d e r m a l c l u s t e r s a t 4 8 h o u r s . A , W h o l e
m o u n t c o n f o c a l z - s t a ck im a g in g d e m o n s t r a t e s m u lt ip l e d e r m a l c l u s -
t e r s a t o p a k e r a t i n o c y t e s h e e t a n d a l t e r e d k e r a t i n o c y t e a r r a n g e m e n t
pat ter n a r ou n d t he der m a l c l u s ter s . P a n c y to ker at i n ( g r e en), p r o p i d -
i u m i o d i d e ( r e d ) . T h e l a r g e g r e e n l o b u l e s a r e a r t i f a c t s r e p r e s e n t i n g
d e a d c e l l s w h i c h h av e t r a p p e d t h e d y e . B , W h o l e m o u n t c o n f o c a l
z - s t a ck im a g in g d e m o n s t r a t in g v im e n t in - p o s i t i ve im m u n o s t ainin g
o f t h e d e r m a l c l u s t e r s a t 4 8 h o u r s p o s t - p l a t i n g . V i m e n t i n ( r e d ) , T O -
P R O - 3 i o d i d e ( b l u e )
Movie S4 L i v e c e l l i m a g i n g o f a r e c o n s t i t u t e d h u m a n h a i r p e g - l i k e
s t r u c t u r e . T i m e - l a p s e m ov i e h i g h l i g h t i n g d e r m a l c e l l s h a p e a n d
m ov e m e n t w i t h i n t h e d e r m a l c a p o f a r e c o n s t i t u t e d h u m a n h a i r p e g -
l i k e s t r u c t u r e , a s v i e w e d f r o m t h e t o p o f a c u l t u r e d r o p l e t . T h e e p i -
d e r m a l s t a l k i s n o t v i s i b l e i n t h i s v i e w . A z - s t a c k i m a g e w a s r e c o r d e d
e v e r y 1 0 m i n u t e s f r o m 1 01 - 1 0 3 h o u r s p o s t - p l a t i n g a n d i s r e p l a y e d a t
a r a t e o f 5 f r a m e s p e r s e c o n d . T h e e n t i r e c u l t u r e d e s c e n d e d a l o n g t h e
z - a x i s d u r i n g i m a g i n g , r e s u l t i n g i n p a r t i a l m ov e m e n t o u t o f t h e f o c a l
p l a n e ove r t i m e . p 6 3 - p os it i ve e p i d e r m a l ce l l s we r e l a b e l l e d w it h n u -
c l e a r e G F P f l u o r e s c e n t p r o t e i n ( m a g e n t a ) . D e r m a l c e l l s w e r e l a b e l l e d
w i t h n u c l e a r m C e r u l e a n 3 f l u o r e s c e n t p r o t e i n ( c y a n ) . N o t e t h e v a r i e d
d e r m a l c e l l m ov e m e n t a n d n u c l e a r s h a p e w i t h i n t h e d e r m a l c a p . F e w
p 6 3 - p o s i t i v e e p i d e r m a l c e l l s a r e v i s i b l e i n t h i s t o p - d o w n v i e w , a s t h e
e p i d e r m a l s t a l k i s o b s c u r e d b y t h e c e l l s o f t h e d e r m a l c a p . H o w e v e r ,
r e p r o d u c i b l y , 1 - 3 p 6 3 - p o s i t i v e e p i d e r m a l c e l l s w e r e n o t e d w i t h i n t h e
d e r m a l c a p , f r e q u e n t l y a t t h e a p e x , a s s e e n h e r e
Movie S5 L i v e c e l l i m a g i n g o f a r e c o n s t i t u t e d h u m a n h a i r p e g - l i k e
s t r u c t u re . T im e- la ps e m ov i e of a re co n s t i t u t e d h u m a n h air p eg - l ik e
s t r u c t u r e , v i e w e d f r o m t o p - d o w n ( A ) a n d l a t e r a l ( B ) o r i e n t a t i o n s .
A z - s t a c k i m a g e w a s r e c o r d e d e v e r y 1 0 m i n u t e s f r o m 8 3 - 8 5 h o u r s
p o s t - p l a t i n g a n d i s r e p l a y e d a t a r a t e o f 1 0 f r a m e s p e r s e c o n d . A l l
ep i der m a l n u c l e i wer e p r e - l a b el l e d w i t h m O r a n ge 2 f l u o r e scent p r o -
t e i n ( y e l l o w ) . p 6 3 - p o s i t i v e e p i d e r m a l n u c l e i w e r e l a b e l l e d w i t h e G F P
(m a gent a). D er m a l n u c l e i wer e l a b el l e d w i t h m Cer u l e a n 3 f l u o r e scent
p r o t e i n ( c y a n ) . N o t e t h a t t h e e n t i r e s p e c i m e n d r i f t s d u r i n g i m a g i n g .
H owever , t he ep i der m a l cel l s w i t h i n t he ep i der m a l s he e t r em a i n
s t a t i c , a s e v i d e n c e d b y n o c h a n g e i n p o s i t i o n a l r e l a t i o n s h i p w i t h a d -
j a c e n t e p i d e r m a l c e l l s . T h e p o s i t i o n o f t h e d e r m a l c a p d o e s m ov e i n
s pa ce , r el at ive to t he ep i der m a l s he e t , b e c a u s e t he ep i der m a l s t a l k is
f l e x i b l e a n d s w a y s w i t h i n t h e d r o p l e t c u l t u r e m e d i u m
Movie S6 M a t h e m a t ic a l s im u la t i o n of h u m a n h air f o l l ic l e p e r i o dic
p a t t e r n f o r m a t i o n i n v i t r o . T h e c h a n g e s i n p e r i o d i c p a t t e r n i n g f r o m
l o n g s t r i p e s t o s h o r t s t r i p e s t o p u n c t a t e c l u s t e r s , c o r r e s p o n d i n g t o
21
366
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WEBER Et al .
der m a l c l u s ter s a n d t hen h a i r p e g - l i ke s t r u c tu r e s , i s r ep r e s ente d her e
b y a T u r i n g - b a s e d m a t h e m a t i c a l s i m u l a t i o n . T h e p e r i o d i c p a t t e r n s
fo r m s e q u e nt iall y o n t h e l ef t , as t h e r a diall y s y m m et r ic sp at iote m p o -
r a l g r a d i e n t i n c r e a s e s i n t h e m i d d l e a n d r i g h t - s i d e d d i a g r a m s .
Appendix S1 S u p p l ement a l M e t h o d s
Table S1 A nt i bod i es
Table S2 P r i m e r s f o r l e nt i v i r a l ve c to r co n s t r u c t i o n . g D N A = ge n o mic
DN A
Table S3 P a r a m e t e r v a l u e s f o r e q u a t i o n s ( 1 ) - ( 4 )
How to cite this article: W eber E L , W oolle y TE , Y eh C - Y , O u
K -L , Maini P K , Chuong C -M . Self - organi z ing hair peg - lik e
s truc tures from dissocia t ed skin prog enit or c ells: New insigh t s
for human hair follicle organoid engineering and T uring
pa t t erning in an asymmetric morphog enetic f ield. Exp
Dermatol . 201 9;28: 3 5 5- 366. https:/ / doi.org/ 1 0. 1111/
exd.13891
22
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wileyonlinelibrary.com/journal/exd Experimental Dermatology. 2019;28:442–449.
© 2019 John Wiley & Sons A/S.
Published by John Wiley & Sons Ltd
1 | INTRODUC TION
Successful wound repair usually ends with a scar that restores
the continuity of injured tissue but which does not function like
the original, uninjured tissue. Human adults are unique in our
ability to “overheal” ourselves with hypertrophic or patholog-
ical scars.
[1]
Patients who underwent surgical procedures or had
major trauma, especially burn injuries, suffered from disfiguring,
sometimes painful, scarring which leads to permanent functional
loss.
[2]
In contrast, regeneration, which means nearly total reca-
pitulation of the original tissue architecture after injury, occurs in
the African spiny mouse (Acomys cahirinus)
[3]
and is the ultimate
goal of the clinical treatment of wounds. The repair of most injuries
in human adults results in a patch of fibroblasts and disorganized

Received: 7 October 2018
|
Revised: 2 January 2019
|
Accepted: 22 January 2019
DOI: 10.1111/exd.13899
ORIG INAL ARTICLE
Comparative regenerative biology of spiny (Acomys cahirinus)
and laboratory (Mus musculus) mouse skin
Ting-Xin Jiang
1
| Hans I-Chen Harn
1,2
| Kuang-Ling Ou
1,3,4
| Mingxing Lei
5,6
|
Cheng-Ming Chuong
1,2,5
1
Department of Pathology, School of
Medicine, University of Southern California,
Los Angles, California
2
International Research Center of Wound
Repair and Regeneration (iWRR), National
Cheng Kung University, Tainan, Taiwan
3
Ostrow School of Dentistry, University of
Southern California, Los Angles, California
4
Divison of Plastic and Reconstructive
Surgery, Department of Surgery, Tri-Service
General Hospital, National Defense Medical
Center, Taipei, Taiwan
5
Integrative Stem Cell Center, China
Medical University Hospital, China Medical
University, Taichung, Taiwan
6
Institute of New Drug
Development, College of Pharmaceutical
and Food Sciences, China Medical
University, Taichung, Taiwan
Correspondence
Cheng-Ming Chuong, Keck School of
Medicine, University of Southern California,
Los Angeles, CA.
Email: cmchuong@usc.edu
Funding information
National Institute of Arthritis and
Musculoskeletal and Skin Diseases, Grant/
Award Number: AR060306; National
Institute of General Medical Sciences,
Grant/Award Number: GM125322; Ministry
of Education, Taiwan; Ministry of Science
and Technology, Taiwan, Grant/Award
Number: 107-3017-F-006-002
Abstract
Wound- induced hair follicle neogenesis (WIHN) has been demonstrated in labora-
tory mice (Mus musculus) after large (>1.5 × 1.5 cm
2
) full- thickness wounds. WIHN
occurs more robustly in African spiny mice (Acomys cahirinus), which undergo autot-
omy to escape predation. Yet, the non- WIHN regenerative ability of the spiny mouse
skin has not been explored. To understand the regenerative ability of the spiny
mouse, we characterized skin features such as hair types, hair cycling, and the re-
sponse to small and large wounds. We found that spiny mouse skin contains a large
portion of adipose tissue. The spiny mouse hair bulge is larger and shows high expres-
sion of stem cell markers, K15 and CD34. All hair types cycle synchronously. To our
surprise, the hair cycle is longer and less frequent than in laboratory mice. Newborn
hair follicles in anagen are more mature than C57Bl/6 and demonstrate molecular
features similar to C57Bl/6 adult hairs. The second hair cycling wave begins at week
4 and lasts for 5 weeks, then telogen lasts for 30 weeks. The third wave has a 6- week
anagen, and even longer telogen. After plucking, spiny mouse hairs regenerate in
about 5 days, similar to that of C57Bl/6. After large full- thickness excisional wound-
ing, there is more de novo hair formation than C57Bl/6. Also, all hair types are pre-
sent and pigmented, in contrast to the unpigmented zigzag hairs in C57Bl/6 WIHN.
These findings shed new light on the regenerative biology of WIHN and may help us
understand the control of skin repair vs regeneration.
K E Y WO R D S
hair cycle, regeneration, scar, spiny mouse, wound healing
23

|
443 JIANG et Al .
extracellular matrix. Acomys can heal full- thickness skin wounds in
a scar- free manner with the complete regeneration of hair follicles,
sebaceous glands and dermis after skin autotomy induced by self-
defense to escape predation.
[3]
Thus, Acomys provides a versatile
model to study regeneration of mini- organs in skin. We can learn
more about regeneration vs repair by comparing wound healing in
Acomys with Mus.
As a mini- organ in skin, the hair follicle is composed of different
layers and each of them encompasses numerous cell types charac-
terized by specific programs of differentiation.
[4]
In addition to the
delicate and complex structure, hair follicles also provide a unique
stem cell niche so that they undergo regeneration and involution
cycles.
[5]
There are four major stages of each hair cycle: anagen
(growth), catagen (degeneration), telogen (rest) and exogen (shed-
ding). The quiescence and activation of hair follicle stem cells drive
the hair cycles.
[6]
In full- thickness skin wounds, stem cells also play
an important role in restoration and repair of lost tissue. Moreover,
while de novo hair follicle generation has never been observed in
humans after injuries, wound- induced hair neogenesis (WIHN)
[7]

was found in the central area of the healing skin of adult mice (Mus).
In the WIHN model, endogenous reprogramming occurs to generate
not only new hair follicles but also sebaceous glands and dermal ad-
ipose tissue. Additionally, Plikus et al
[8]
showed the ability of wound
myofibroblasts to convert to fat cells indicating the opportunity to
influence regeneration over scarring after wounding. Therefore, we
also want to compare the WHIN model between Acomys and Mus.
To examine the similarities and differences in the morphology of
Acomys and Mus, we compared their gross view, skin sections and
hair types first.
[9]
Like Mus, Acomys has zigzag, awl and guard spiny
hair. Second, we studied the hair cycle, regeneration cycle after shav-
ing and plucking and skin section staining with epithelial and dermal
stem cell markers (K15, CD34 and Sox2). Unlike Mus, the hair cycle of
Acomys begins before birth and is as long as 7- 8 months after shaving.
We observed that Acomys already has a fur coat, and the hair folli-
cles are in anagen status at birth. After hair plucking, Acomys regains
hair follicles with full recovery in 20 days. Compared to Mus, Acomys
has more K15- , CD34- and Sox2- expressing cells in the hair follicle.
Full- thickness skin excisional wounds (1.5 × 1.5 cm
2
) in Acomys heal
quickly (20 days for full closure) with all three types of de novo hair
regeneration (occurring at 30 days in the periphery; 40 days in the
wound centre) and pigmentation. By comparing the response of
Acomys and Mus to the WIHN model, we set to decipher how regen-
eration vs repair can be achieved. These data serve as the first step to
study further fundamental principles to regeneration in and beyond
the skin system.
2 | MATERIAL AND METHODS
2.1 | Spiny mouse
African spiny mice, A cahirinus, are from Dr. Malcolm Maden at
the University of Florida and Dr. Ashley W Seifert of University of
Kentucky. The mice are transferred to our facility through a MTA. A
colony of captive- bred A cahirinus was established at the University
of Southern California (USC), and all experiments were performed
with protocols approved by the USC IACUC. The spiny mice are
fed on a mixed diet containing rabbit diet, sunflower seed and wild
bird seed mix. C57Bl/6 mice are from the Jackson laboratory and
fed a regular rodent diet. We use 3- to 6- month- old adult female
spiny mice for wound experiments unless otherwise specified. All
mice used in this project were determined to be healthy by a USC
veterinarian.
2.2 | Histology and immunostaining
Mouse dorsal skin samples were dissected from different ages and
fixed with 4% paraformaldehyde in PBS at 4°C for overnight followed
by procedures described by Jiang and Chuong
[10]
for histology and im-
munohistochemistry. Paraffin section immunostainings were performed
using following primary antibodies: mouse anti- K14 (1:200, Thermo
Fisher, Waltham, MA, Cat# MA1- 34677, RRID:AB_2134821), mouse
anti- K10 (1:200, Thermo Fisher, Cat# 39- 5300, RRID:AB_2533418),
mouse anti- AE13 (1:200, Abcam, Cambridge, UK, Cat# ab16113,
RRID:AB_302268), mouse anti- AE15 (1:200, Santa Cruz, Dallas, TX, Cat#
sc- 80607, RRID:AB_2303435), rabbit anti- TRP63 (p63, 1:200, Santa
Cruz, Cat# sc- 8343, RRID:AB_653763), goat anti- K15 (1:500, Thermo
Fisher, Cat# MA1- 90929, RRID:AB_2132754), goat anti- CD200 (1:200,
R&D systems, Minneapolis, MN, Cat# AF3355, RRID:AB_2073945),
goat anti- Sox 2 (1:200, R&D systems, Cat# AF2018, RRID:AB_355110),
rat anti- CD34(1:200, eBioscience, San Diego, CA, Cat# 14- 0341- 85,
RRID:AB_467211), PCNA (1:200, Millipore, Burlington, MA, Cat#
CBL407, RRID:AB_93501) and mouse anti- MitfD5 (1:200, Abcam, Cat#
ab3201, RRID:AB_303601). Whole- mount immunostaining of mouse
epidermis was performed with rabbit anti- K17 (1:200, Cell signaling tech-
nology, Danvers, MA, Cat# D32D9 , RRID:AB_10830066). Colour was de-
veloped with the peroxidase substrate AEC kit (Vector Labs, Burlingame,
CA) or visualized with fluorescent secondary antibodies.
2.3 | Spiny mouse hair plucking
Spiny mouse hairs were plucked with wax stripping or forceps on
anaesthetized animals. The proportion of hair types was evaluated
by counting the total numbers of all de novo hairs in the wound and
comparing them to the hairs in a 1 × 1 cm
2
square area of normal
skin. The percentage is calculated based on the sum of all hair types
in either the wound or the skin. Percentages of different hair types
were collected from three different mouse wounds and normal dor-
sal skins. SD was calculated from three independent biological sam-
ples (N = 3). More than 400 hairs were calculated in each sample.
2.4 | Spiny mouse wounding
Mice were anesthetized, and a 1.5 × 1.5 cm
2
section of dorsal skin
was excised. Ito et al
[7]
used a 1 × 1 cm
2
area as large wound model.
Here, we optimized it from 1 × 1 cm
2
to 1.5 × 1.5 cm
2
because of the
size of Acomys is larger than Mus.
24
444
|
JIANG et Al .
2.5 | Whole- mount staining of spiny mouse wound
epidermis and dermis
Wound epidermis was separated from dermis as reported by Ito
et al.
[7]
Briefly, the specimens of full- thickness wounds were incu-
bated in 20 mmol/L EDTA in PBS at 37°C overnight. Epidermis was
gently peeled off from dermis with fine watchmaker's forceps and
fixed in 1:4 DMSO/methanol for one hour and incubated with 3%
H
2
O
2
for 20 minutes. The specimen was washed with PBS, blocked
with FBS for 1 hour and then incubated with rabbit anti- K17 an-
tibody at 4°C overnight. After washing with PBS, the secondary
antibody was added and incubated for another one hour at room
temperature. The specimen then was washed with PBS again and
colour developed using AEC kit (Vector Labs). The dermis was fixed
in acetone at 4°C for overnight, rinsed with PBS and then incubated
in NBT/BCIP to develop colour.
3 | RESULTS
3.1 | Spiny mice show distinct hair types, with
different ratios in different body regions
The adult spiny mouse shows different hair types and colour on its
dorsal (Figure 1A1), lateral (Figure 1A2) and ventral (Figure 1A3)
skin. The dorsal hairs are brown, the lateral hairs are lighter and more
yellowish than those in the dorsum, and the ventral hairs are white.
H&E sections show that spiny mouse skin contains a very high pro-
portion of adipose tissue (~85% of full thickness) regardless of its
location on the body (Figure 1B1- B3). The spiny hairs (awl hairs) are
thick and have a large hair bulge (Figure 1B1’- B3’). Dimension wise,
dorsal hairs are the longest and thickest, followed by lateral and lastly
ventral. All hair types can be found in dorsal, lateral and ventral parts
of the spiny mouse skin (Figure 1C1- C3); however, they differ in per-
centage. The hair type percentages in the order of guard, awl and
zigzag hairs are 18%, 33% and 49% (total hair = 473 hairs/cm
2
) in the
dorsum, 6%, 41% and 53% (total hair = 854 hairs/cm
2
) in the flank,
and 2%, 43%, and 55% in the ventral skin (total hair = 1060 hairs/
cm
2
), respectively (Figure 1D). Using Student's t test, we found it sig-
nificantly different (P < 0.01) for each hair type between each sam-
ple (N = 3) in the dorsal, flank and ventral regions.
3.2 | Hair follicle stem cell markers CD34 and
K15 are highly expressed in the bulge region of adult
spiny mouse hairs
We perform immunohistochemistry to characterize the expression
pattern of skin and stem cell markers in adult spiny mouse skins.
K10, a marker for differentiated keratinocytes, is only expressed in
the suprabasal layer of spiny mouse skin. AE15, a marker for inner
root sheath cells, and AE13, a marker for upper hair cuticle cells,
are not expressed in spiny mouse telogen hair follicles. CD200, a
hair germ marker, is expressed in the hair follicle upper bulge. The
basal layers of skin and hair follicles stain positively for K14 and p63.
Furthermore, hair stem cell markers CD34, K15 and Sox2 are highly
expressed in the bulge of all three hair types. Sox2, in particular, is
indeed expressed in the bulge region (red arrow) and dermal papil-
lae (red arrowhead) of telogen hairs, but in anagen phase is only ex-
pressed in the dermal papillae (Figure 1F, F’).
3.3 | The spiny mouse shows slower hair
cycling, and all hair types cycle synchronously
The spiny mouse pup is born with a fur coat that exhibits all three
hair types in anagen (Figure 2A, A’, A’’), indicating the spiny mouse
hairs start to grow in the embryo. We shaved the dorsal hair of the
2- week- old mouse to observe hair growth and found the first hair
wave persists past week 2 as it enters early catagen (Figure 2B,
FIGURE 1 Spiny mouse hair types. A1, Dorsal view of adult
spiny mouse with dark brown spiny hairs. A2, Lateral view of adult
spiny mouse with yellow spiny coat. A3, Ventral view of adult spiny
mouse with white spiny coat. B1, 2 and 3. H&E staining of dorsal,
lateral, ventral spiny mouse skin. B1’, B2’, B3’, the magnified view of
B1, B2 and B3, respectively. C1, C2 and C3, all hair types (Z: zigzag
hair; G: guard hair; A: awl hair) from dorsal, lateral and ventral side
of the skin, respectively. D, The percentage of hair types from
1 cm
2
area of each respective body part. SD is calculated from 3
independent samples (N = 3); more than 400 hairs are calculated
in each sample. E, Immunostaining of K10, K14, p63, AE13, AE15,
CD200, CD34, K15 and Sox2. Control: secondary antibody
negative control. Arrowhead: zigzag hair; arrow: guard hair;
asterisk: awl hair. F, Sox2 immunostaining of telogen and anagen
hair follicles. White arrow: hair bulge, arrowhead: dermal papilla.
All photographs are representative of the data acquired from all
specimens
(A1) (B1) (B1′) (C1)
(A2) (B2) (B2′) (C2)
(A3) (B3) (B3′) (C3)
(D)
(E)
(F)
(F′)
25

|
445 JIANG et Al .
B’, B”), but the posterior part of the skin is still visibly darker than
the anterior side (Figure 2B, blue bar). This initial hair wave ceases
3 weeks after birth (Figure 2C, C’, C”). All hair types, guard, awl and
zigzag, cycle synchronously. The second hair cycle wave begins
on week 4, starting from the dorsal mid- body region (Figure 2D).
While most of the dorsal skin is still in early anagen, the anterior
part of the skin (Figure 2D arrowhead, 2D’) is at an earlier hair cycle
stage than the posterior side (Figure 2D double arrowhead, 2D’’).
On week 6, the hair fibres are visibly thicker, and the skin also con-
tains a thick adipose layer (Figure 2E, E’, E’’). On week 8, most of
the dorsal skin of the mouse is in anagen except for the neck re-
gion (Figure 2F. orange bar). Zigzag (Figure 2F’ arrowhead), guard
(Figure 2F’’ arrow) and awl (Figure 2F’’ asterisk) hairs from the mid-
body region are all found in anagen. By week 10, most of the hairs
have entered telogen (Figure 2G), and only the anterior dorsal skin
near the neck is in catagen (Figure 2G arrowhead, Figure 2G’). The
adipose tissue occupies the majority of the thickness during telogen
(Figure 2G’’). In addition, this second hair wave coincides with the
change in hair colour, as the appearance of the newly grown hairs
is brown (Figure 2H). However, after shaving and revealing the hair
close to the skin, the proximal end of the hairs appears to be grey
(Figure 2I, 9- 13 weeks), reflecting the differential pigmentation of
the hair fibre in different body regions and locations along the hair
fibre. These differences give rise to distinct colour patterns on the
dorsal, lateral and ventral sides. The third hair wave begins much
later on week 42 through week 47, and telogen is even longer. This
is evident from new hair growth on a shaved 3- month- old mouse
(Figure 2J, 40- 45 weeks, green bar). Generally speaking, the spiny
mouse hair wave lasts around 6 weeks, with a long ~27- week gap
between the second and third wave. The timeline of the spiny hair
cycle is illustrated in Figure 2K. While in C57Bl/6 mice, anagen oc-
curs in complex domain patterns throughout different parts of the
body after the first two synchronous cycles, and in spiny mice, each
hair wave does not fragment into domains but appears to be a co-
ordinated, slow cycling wave, with a longer anagen, and a very long
telogen phase.
3.4 | Hair plucking initiates hair regeneration in
spiny mice
Here, we plucked all the hairs within a 2 × 4 cm
2
area on the dorsal
skin, and hence created micro- injuries to track hair regeneration
after plucking (Figure 3A). Gross observation shows that mature
hairs are visible 14 days after plucking (Figure 3A, D14), and new
hairs repopulated the plucked area after 20 days (Figure 3A, D20).
H&E sectioning of the skin shows new hair follicles began to re-
generate as early as 5 days after hair plucking (Figure 3B, D5),
and these hairs mature to full size after 12 days (Figure 3B, D12).
Moreover, all hair types regenerated in sync and were observed
on days 5, 7 and 12 after plucking. PCNA staining, a marker for
cell proliferation, shows that cells were positive, though sparingly
on day 5 after plucking. On days 7 and 12, most of the hair cells,
including those in the infundibulum and the bulge region, were
positive for PCNA (Figure 3B. arrow: guard; arrowhead: zigzag;
asterisk: awl.)
3.5 | All hair types regenerate while awl and guard
hairs are pigmented in WIHN of spiny mice
Ito et al
[7]
demonstrated that C57Bl/6 mice have the ability to regen-
erate hairs in a large full- thickness wound, yet only zigzag hairs were
observed. Seifert et al have shown spiny mice shed and regenerate
their skin after wounding. To characterize the regenerative responses
FIGURE 2 Spiny mouse hair cycle. A, The newborn spiny mouse
has a fur coat when it is born. (Red arrow indicates where the
section was taken from. Arrowhead: zigzag hair; arrow: guard hair;
asterisk: awl hair) A’, A’’, H&E staining of skin section shows that all
three hair types have developed in this stage. The hairs across the
whole dorsal body are already in anagen at birth (red arrowhead,
blue bar). B, This hair wave continues 2 wk after birth (blue bar),
but parts of the body (red arrowhead) begins to enter catagen
(B’, B’’). C, Hairs were shaved at 3 wk after birth. H&E staining
showed that hairs have entered to telogen stage (arrowhead, C’,
C’’). D, The spiny mouse hairs were shaved at 4 wk after birth. D’,
H&E from red arrowhead, D’’, H&E from double red arrowhead,
where the second hair waves start to regenerate, and hair stages
are in exogen (D’) and early anagen stages (D’’). E, shaved 6- wk- old
mouse, in which hair waves begin to propagate from the mid- body
region. E’ and E’’, H&E from mid- body region (red arrowhead). F,
shaved 8- wk- old mouse. F’ and F’’, the zigzag (green arrowhead),
guard (green arrow) and awl (green asterisk) hairs are all in anagen
stage. G, shaved 10- wk- old mouse, end of hair wave. H&E section
showing hairs in catagen (red arrowhead, G’) and telogen (double
red arrowhead, G’’). Scale bar A’, B’, C’, D’, D’’, E’, G’, G’’, 200 μm. A’’,
B’’, C’’, E’’, F’, F’’, 100 μm. H, Hair wave propagation from 4 to 9 wk.
I, Hair wave sustaining from week 8 to wk 13. J. The third wave did
not start until 40 wk after birth and lasted until 45th wk (green bar).
K, Comparison of hair cycle waves in spiny and C57Bl/6 mice. In
C57Bl/6 mice, first two cycles are in general synchronous, and then
they break into multiple complex cycling domains. In spiny mice,
waves do not break into subdomains and the waves traverse the
skin more slowly, with longer anagen and very long telogen phases.
All photographs are representative of the data acquired from all
specimens
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(A′)
(B′)
(C′)
(D′)
(E′)
(F′)
(G′)
(A′′)
(B′′)
(C′′)
(D′′)
(E′′)
(F′′)
(G′′)
(H) (I) (J)
(K)
26
446
|
JIANG et Al .
of the spiny mouse to a large wound, we created a 1.5 × 1.5 cm
2

wound on the dorsal skin of the spiny mouse and allowed it to heal by
secondary intention. After approximately 35 days, we found that all
three hair types were regenerated (Figure 4A1- 3). Comparatively, the
original hair fibres (Figure 4B, mid- upper panel) are still thicker than
newly regenerated ones (Figure 4B, mid- lower panel). Furthermore,
the respective percentage of hair types (guard, awl and zigzag) re-
mained very similar to unwounded skin (22%, 27%, 51% in regener-
ated hair vs 20%, 35%, 45% in normal skin, Figure 4B graph).
In response to wounding, the regenerated awl and guard hairs
can be pigmented (Figure 4C), while the zigzag hairs remain un-
pigmented. The hair placodes are positive for MitfD5, a marker
for melanocytes, as early as 15 days after wounding (Figure 4C1).
MitfD5 localizes to the hair follicles 30 days after wounding, reflect-
ing the role of melanocytes in regenerating pigmented hair fibres
(Figure 4C2). All three types of hairs are completely repopulated
throughout the whole wound (Figure 4D, D1 and D2). Six months
after wounding, we plucked the regenerated new hairs. All three
types of hairs grow back (Figure 4E), which means de novo hair can
recycle after being plucked.
4 | DISCUSSION
In this study, we characterized the spiny mice's skin tissue, hair fibre
types, molecular expression, hair cycle pattern and its response after
hair plucking or wounding. Compared to laboratory mice (Mus), the
spiny mouse skin contains a very large portion of adipose tissue
in the hypodermis (Figure 2). The spiny hair fibres are thicker and
FIGURE 3 Spiny mouse hair regeneration after hair plucking.
A, Spiny mouse hair was plucked and observed on day 0 (D0), day
10 (D10), day 14 (D14) and day 20 (D20). B, H&E staining of spiny
mouse dorsal skin at 5, 7 and 12 d after hair plucking. Noted all
hair types regenerated after plucking. Immunofluorescent PCNA
staining shows proliferation during the hair cycle. Arrowhead:
zigzag hair; arrow: guard hair; asterisk: awl hair. All photographs are
representative of the data acquired from all specimens
(A)
(B)
FIGURE 4 Regeneration of hair type, pigmentation and
recycle. A, All three hair types, zigzag (A1, arrowhead), guard (A2,
arrow) and awl (A3, asterisk), are regenerated 35 days after large
1.5 × 1.5 cm
2
wounding. B, The morphology of regenerated hairs.
(A, awl; G, guard; Z, zigzag). The normal hairs (mid- upper panel) are
thicker than regenerated hairs (mid- lower panel). The percentage of
the de novo hair types is similar to the normal skin hair (B, graph).
SD is calculated from 3 independent wounds and normal mouse
dorsal skin samples (N = 3). C, The pigmented hair shaft can be
observed 40 d after wounding. Dotted line: original wound margin.
C1, MitfD5 immuno- reactivity was expressed 15 d after wounding
in hair placodes (arrows). C2, MitfD5 immuno- reactivity was
expressed in new hair follicles after 30 d (arrows). D, Whole- mount
staining of the wound epidermis and dermis (D, D1, D2) shows
regeneration of all hair types across the wound bed. Arrowhead:
zigzag; arrow: guard; asterisk: awl. E, Six months after wounding,
the regenerated hairs are plucked. All three hair types, zigzag
(arrowhead), guard (arrow) and awl (asterisk) grow back
(A)
(A1) (A2) (A3)
(B)
(C) (C1) (C2)
(D) (D1) (D2)
(E)
(E1)
(E2)
(E3)
27

|
447 JIANG et Al .
longer, and the percentage of hair type is different from Mus (18%
guard, 33% awl, 49% zigzag in Acomys, vs 2% guard, 6% auchene, 7%
awl and 85% zigzag in Mus) (Figure 1).
[11]
The hair bulges are larger
and show high expression of stem cell markers K15, CD34 and Sox2
(Figure 1). The Acomys skin undergoes 2 rounds of rapid hair cycles
starting from embryonic day 24,
[12]
in contrast to postnatal day 3 in
Mus. This can be attributed to the precocial nature of spiny mice;
though the time points seem different (hair grows in Acomys be-
fore birth and in Mus after birth), they are actually in the same hair
cycle. The hair cycle period is longer in Acomys than Mus. The ana-
gen in Acomys each lasts around 5- 6 weeks, and telogen of the first
three hair cycles is 2 weeks, 30 weeks and even longer, respectively
(Figure 2). Interestingly, when observing the spatiotemporal pattern
of hair cycling in the spiny mice, we noted that there were no com-
plex hair cycle domain patterns as previously observed in C57Bl/6
mice,
[13,14]
which suggests a higher threshold to activate telogen to
anagen transition for spiny mouse hairs. We have learned that this
domain pattern is regulated by BMP4 from intradermal adipose tissue
that inhibits Wnt/β- catenin signalling in the skin. We speculate that
this “less restricted” hair wave observed in the spiny mice may imply
less local dermal control and allows a more robust response to occur
across the skin during homeostasis and regenerative wound healing.
We will need to characterize intradermal adipose tissue in the spiny
mouse to see if they also express cyclic BMP4 expression.
[12]
It is intriguing that, compared to Mus dermal white adipose tis-
sue (dWAT) in telogen, which reduces in size by ~50%,
[15]
there is no
obvious thinning of the thick adipose layer in Acomys skin. In this re-
view article, Guerrero- Juarez and Plikus
[15]
report evidence that der-
mal adipocytes are heterogeneous in development, in sensitivity to
catagen and spatial arrangement. We speculate that: (a) only a rela-
tively small portion of the hypodermis adipose tissue responds to HF
cycling in Acomys and (b) the adipose layer maintains its thickness at
all times which may aid in wound healing. Due to the fragile nature
of the Acomys skin, it becomes imperative for the skin to maintain
its niche for fast regeneration, and hence, this adipose tissue layer
is uncoupled with HF cycling. These interesting possibilities will re-
quire further investigation. Furthermore, a better microenvironment
provided by the thickened adipose tissue may be an important rea-
son underlying spiny mouse's regenerative ability. Previous studies
have identified reciprocal signalling that takes place between dWAT
and hair follicles to regulate their progression through cycle stages.
Anagen hair follicles secrete multiple BMP ligands and activate tran-
scription regulator Zfp423, which is necessary for adipogenesis in
skin. In telogen, dWAT expresses BMP2, which maintains hair fol-
licle stem cells in a quiescent state, to prevent hair over production
on the skin. Considering the long telogen in Acomys, it is plausible
that BMP2 may play a role and further characterization is required
in future study.
Several previous reports have tried to unravel the mechanism
behind the spiny mice's superior ability to regenerate after wound-
ing.
[3,12,16–18]
Brant et al have analysed and compared the gene ex-
pression profiles during skin regeneration in Mus and Acomys, and
identified the differences in the extracellular matrix profiles. Mus
wounds express high levels of TIMP1 and low levels of Fn1, MMP9
and MMP13 and a high collagen I to III ratio. In contrast, Acomys
wounds express high Fn1, MMP9 and MMP13 and a high collagen
III to I ratio.
[3,19,20]
On a side note, these results are based on gene
expression data generated from microarrays and RT- PCR data, and
from cytokine arrays all designed for mouse. However, Simkin et al
pointed out the shortcomings of comparing across species by mi-
croarray data analysis.
[21]
To further explore the differences in
molecular expression between Mus and Acomys, Gawriluk et al
[20]

acquired RNA- seq data. Many of the findings of Brant et al were also
observed in the transcriptome analysis with the exceptions of Fn1
which was high in Mus and low in Acomys and collagen III which is
expressed to higher levels in Mus than in Acomys. The spiny mouse
genome should be publicly available in early 2019.
The spiny mouse wounds are described to have a profile more
similar to foetal wounds.
[17]
The immune responses of the two spe-
cies are also rather different. Mus expresses high interleukins (IL),
CXCLs, MCPs and CSFs, while the cytokine responses in Acomys
is generally low, IL- 1 and MIP- 1 being the only exception.
[17,19]
Hair
plucking in Mus also activates Ccl2, Cxcl2 and IL- 1, and initiates
hair wave and hair regeneration.
[22]
In Acomys, our study shows all
three hair types begin to regenerate as early as 5 days after plucking
(Figure 3). Despite the seemingly similar ability to heal a small wound
(<6 mm), Seifert et al
[3]
have shown that the spiny mice can close a
6 mm wound rapidly, and the wound bed is characterized by low ex-
pression of myofibroblasts, a stark contrast to the normal response
of a Mus. Lastly, although Ito et al
[7]
successfully demonstrated that
Mus can regenerate hair by WIHN, and other investigations identi-
fied additional regulatory factors,
[8,23,24]
WIHN in Mus has not yet
shown: (a) regeneration of hair types other than zigzag, (b) pigmen-
tation of regenerated hairs and (c) de novo hair follicles that cover
the entire wound bed, as observed in this study. The comparison is
summarized in Table 1.
Why does the spiny mouse skin contain a thick layer of adipose
tissue? Seifert et al
[3]
show that the spiny mouse skin is weak, very
deformable and tears easily. We postulate the high adipose con-
tent in skin partially contributes to this mechanical characteristic,
as the ultimate tensile strength of obese mouse skin is much lower
to that of lean mice (45.9 vs 107 N/cm
2
).
[25]
Although adipose tissue
seems to constitute only a small portion of the regenerated wound
(Figure 4C), it has been identified as an important regulator of hair
follicle growth.
[26]
Adiponectin- deficient mice showed severely de-
layed re- epithelialization, and cultured keratinocytes treated with
adiponectin showed increased proliferation and migration,
[27–29]

suggesting adipokines are essential for normal wound healing.
Moreover, regenerative healing has been shown to be heavily age-
dependent,
[30–32]
and signals from the adipose tissue also modulate
hair stem cell behaviour and ageing.
[33]
Another remarkable observation is the regeneration of pig-
mented de novo hairs. In the standard Mus WIHN model, 3- to
4- week- old mice were used, in which little to no regenerated hairs
were pigmented as reported.
[7,23]
However, Yuriguchi et al used
5- week- old mice and created wounds on the anagen skin and
28
448
|
JIANG et Al .
observed pigmented regenerated hairs. They attribute this pigmen-
tation to the elevated level of Wnt7a in keratinocytes during anagen,
which may direct melanocyte stem cells to produce pigmented hairs
in the regenerated follicles. This notion was supported by the finding
that Kitl (melanocyte stimulatory factor) expressing transgenic mice
regenerated pigmented hairs regardless of age or hair cycle stage.
[34]

While we still do not have a clear mechanism, the high proportion
of adipose tissue, enlarged bulge and high expression of hair stem
cell markers CD34, K15 and Sox2 may provide important clues. The
development of the thick and spiny awl hair in the spiny mouse is
attributed to hair cells undergoing an additional step of prolifera-
tion and differentiation.
[12]
We postulate the presence of stem cell
marker- positive cells around the hair bulge probably facilitated cell
proliferation (via p63) and differentiation (via Sox2, CD34 and K15)
to allow pigmented hairs of all types to regenerate, which include the
essential interaction and activation of MitfD5 positive cells.
In summary, by characterizing the African spiny mouse, this
study shows its unique skin composition, molecular expression,
hair cycle and response to wounding. Surprisingly, the normal
hair cycle is not faster and more frequent; rather it is longer and
slower. All three hair types cycle synchronously suggest the cy-
cling is under a local dermal control, not autonomously in each hair
follicle or each type of hair follicle. The slow progression of sim-
ple waves, in contrast to the complex hair domains in C57Bl/6,
[13]

suggest a different local dermal control. The facts that all three
hair types form and they are pigmented suggest there may be dif-
ferent molecular mechanisms, different paths for cells to achieve
de novo hair formation. Though further studies focusing on the
molecular and even mechanical signalling would be necessary to
reveal its mechanism, this study provides fundamental character-
istics and clues to understand how to elicit regenerative wound
healing.
ACKNOWLEDG EMENTS
This project is supported by NIH grant NIH GM125322 and
AR 060306, the International Center for Wound Repair and
Regeneration at National Cheng Kung University from The Featured
Areas Research Center Program within the framework of the Higher
Education Sprout Project by the Ministry of Education (MOE) in
Taiwan, and by the grant of the Ministry of Science and Technology
(MOST 107- 3017- F- 006- 002) in Taiwan. The authors would like to
thank Dr. Seifert and Dr. Maden for providing the spiny mice. We
thank Dr. R. B. Widelitz for editing the manuscript.
CONFLIC T OF INTERE S T
The authors have declared no conflicting interests.
TABLE 1 Comparison of skin characterization and regenerative response to wounding in Mus and Acomys mice
Mus Acomys References
Skin tissue Epidermis, dermis and hypodermis with a thin
layer of adipose tissue
Epidermis, dermis and a very thick portion of adipose
layer in the hypodermis
Figure 1
Hair types 2% guard, 6% auchene, 7% awl, 85% zigzag 18% guard, 33% awl, 49% zigzag in dorsum skin. All
three hair types are thicker and longer. Awl hairs
undergo a second round of anisotropic proliferation
during growth
[11,12],
Figure 1
HFSC markers Lower K15, CD34, Sox2 expression in adult hair
follicles
High expression of K15, CD34 and Sox2 in the large hair
bulges
Figure 1
Hair cycle 1st hair cycle ends synchronously at about 3 wk
of age. 2nd hair cycle ends about 7 wk of age.
Complex hair cycle domains form after 2 mo
All hair types cycle synchronously. 1st hair cycle ends at
about 3 wk of age. Anagen of the 2nd hair cycle occurs
between 4- 9 wk followed by a long telogen (until
38 wk). Anagen of the 3rd hair lasts from 39- 45 wk
followed by a very long telogen. No complex hair cycle
domains are observed
[5,12],
Figure 2
In response to wounding
ECM High collagen I to collagen III ratio*, high TIMP1.
Lower Fn1*, MMP9, MMP13.
(*controversial)
High collagen III to collagen I ratio*, High Fn1*, MMP9,
MMP13.
(*controversial)
[3,17,20]
Immune High ILs, CXCLs, MCPs, CSFs, etc Low cytokines, macrophages, high IL- 1, high MIP- 1.
Lower CD86+
[19,20]
Hair plucking Ccl2, Cxcl2, IL- 1 activation, initiation of hair
wave, and hair regeneration on day 5
Regeneration of all three hair types beginning on day 5 [22],
Figure 3
Small wound More myofibroblasts, scarring. Direct contrac-
tion to close the wound
Rapid wound closure, fewer myofibroblasts.
Regeneration of all hair types
[3]
Large full-
thickness wound
(WIHN)
Regeneration of zigzag hair only. Incomplete
coverage of wound bed. Pigmented regener-
ated hairs only from wounds created on
5- wk- old anagen skin regenerated hairs
Faster regeneration of all three hair types and they can
recycle after plucking. Complete repopulation of
wound. Normal pigmentation in awl and guard
regenerated hairs
[7,34],
Figure 4
29

|
449 JIANG et Al .
AUTHOR CONTRIBUTION
T.X.J. and C.M.C. conceived the idea and experimental design. T.X.J.,
H.I.H. and M.L. conducted the experiments. T.X.J., H.I.H., K.L.O. and
C.M.C. prepared the manuscript.
ORCID
Hans I-Chen Harn https://orcid.org/0000-0001-8222-5614
Kuang-Ling Ou https://orcid.org/0000-0001-9536-2677
Mingxing Lei https://orcid.org/0000-0002-4271-2714
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How to cite this article: Jiang T-X, Harn HI-C, Ou K-L, Lei M,
Chuong C-M. Comparative regenerative biology of spiny
(Acomys cahirinus) and laboratory (Mus musculus) mouse skin.
Exp Dermatol. 2019;28:442-449. https://doi.org/10.1111/
exd.13899
30
CHAPTER 3: Avian skin neovascularization: patterning, induction and
mesenchymal cell fate plasticity

INTRODUCTION

Neovascularization is a critical step in development needed to establish an adequate
blood supply for oxygen, nutrients, circulation, immune response, and connections with other
body systems and to remove CO2 and metabolic wastes. Avian skin development is achieved
through a series of events: 1) First, there is the initial induction, followed by 2) de novo patterning
which gives rise to feather primordia, 3) feather primordia serve as landmarks upon which
adaptive patterning, such as the formation of blood vessels, nerves, muscles and pigmentation
patterns, occurs. These sequential steps are required to form a fully functional tissue. Primary
angiogenesis is a prominent feature of developing skin during embryogenesis (Detmar, 1996).
On the other hand, studies have indicated that the developing organ produces paracrine factors
that induces blood vessels to form in its own mesenchyme in some cases (Auerbach et al., 1985,
LeCouter et al., 2001). It is unknown how early in skin formation, de novo patterning producing
feather primordia affects neovascularization. This is the next level of avian skin development.
Evidence has shown that endothelial cells (ECs) emerging as important signaling centers regulate
tissue morphogenesis (Ramasamy et al., 2015). In our present study, we sought to reveal which
endothelial progenitors are capable of affecting the next level of avian skin development.
Understanding how vasculature is formed and the identity of endothelial progenitor cues that
influence the next patterning event in developing skin may shed light on vascularizing tissue-
engineered products or organoids for translational medical purpose.
There are two common mechanisms of neovascularization: vasculogenesis and
angiogenesis. Vasculogenesis, defined as the de novo formation of new blood vessels from the
differentiation of embryonic stem cells, endothelial progenitor cells (EPC) or angioblasts, gives
rise to the first blood vessels (Kubis and Levy, 2003, Johnson and Wilgus, 2014). Angiogenesis,
defined as the growth of vasculature from the extension of an existing blood vessel, characterizes
embryonic development (Carmeliet and Jain, 2011, Patel-Hett and D'Amore, 2011). It is not clear
whether skin neovascularization starts from vasculogenesis or angiogenesis. To better
understand avian skin vascular patterning, investigators have relied on known molecular markers.
The expression of vascular endothelial growth factor-2 (VEGFR-2) (also known as quail
endothelial kinase (Quek1) or fetal liver kinase (Flk-1)), an early marker for endothelial and
31
hematopoietic precursor cells(Choi et al., 1998, Yamaguchi et al., 1993), is seen from day 7
onwards after feather primordia form in quail (Nimmagadda et al., 2004). At earlier times, quail-
chick-transplantations revealed that the epithelial somite contains angioblasts (Scaal and Christ,
2004). Later, angioblasts in the dorsomedial quadrant dermamyotome (DM) migrate
predominantly into the dorsal dermis(Wilting et al., 1995). However, little is known about how
neovascularization and vasculature patterning occur between the DM stage and day 7 feather
bud. It is known that fibroblast growth factor 2 (FGF2) is implicated in both mesodermal induction
and the induction of angioblasts from the mesoderm(Cox and Poole, 2000). Several studies have
previously shown that VEGF signaling is essential for both vasculogenesis and angiogenesis
(Patel-Hett and D'Amore, 2011). Currently in developing avian skin, it is not clear how FGF2 and
VEGF may be involved in vascular patterning. In addition, it is also not clear how perturbing
neovascularization will feedback to affect avian skin development.
After neovascularization occurs, myogenesis is the next event that takes place in
developing avian skin. Our previous work characterized the development of the avian skin muscle
network into 7 stages between E7-12 using the chicken model (Wu et al., 2019). Unlike trunk
muscles which originate from four locations (dorsomedial, ventrolateral, rostral and caudal)
located opposite to the neural tube and lateral plate mesoderm of the DM, feather muscle
develops within the avian dermal layer, indicating that they probably originate from the central DM
with the dermal layer. The fate of the central DM was analyzed and a single DM progenitor which
gave rise to both dermogenic and myogenic lineages was revealed by in-ovo clonal analysis (Ben-
Yair and Kalcheim, 2005). In contrast to the central DM, the segregation of endothelial and mural
cells is already underway in lateral epithelial somites (Ben-Yair and Kalcheim, 2008). However, it
is still unclear what the compositions or status of skin mesenchymal cells are after skin starts to
form. Transgenic mice and quail expressing specific fluorescent colors under the control of the
Tie1 promoter have been generated to monitor and analyze embryonic vascular morphogenesis
(Iljin et al., 2002, Sato et al., 2010). Intriguingly, Tie1: LacZ has promiscuous expression both in
vascular smooth muscle cells (vSMC) and ECs, indicating a common subpopulation of vascular
progenitor cells predisposed to vSMC fate are present in the embryo (Chang et al., 2012). In
developing avian skin, we proposed performing transcriptome analysis of H2B-eYFP
+
(enhanced
yellow fluorescent protein) flow cytometry sorted cells to study their differentially expressed genes
(DEGs). This would enable us to identify genes associated specifically with this cell population
and to examine skin mesenchyme diversity and plasticity specifically after de novo patterning.
Previous studies led us to propose a multiscale model for tissue patterning of the skin.
First, from the non-patterned morphogenic field, feather primordia formed in a “de novo periodic
32
patterning” process. Next, skin mesenchymal cells (e.g. adipocytes, endothelium, and smooth
muscle cells) are assembled using the existing feather patterns as the reference points in an
“adaptive patterning” process. In our present study, initially, we used Tg(tie1:H2B-eYFP) quail to
visualize the behavior of vascular progenitor cells. We reasoned that angiogenic stimulus is
derived from the epidermis, not the dermis (Malhotra et al., 1989, Detmar, 1996) and showed how
H2B-eYFP
+
cells are organized into a vasculature network pattern in a feather epithelium
dependent manner. We constructed retroviral vectors expressing sprouty2 (spry2) and noggin,
known to inhibit neovascularization. For this approach we chose to use the RCAS (replication-
competent avian sarcoma-leukosis virus (ASLV) long-terminal repeat (LTR) with splice acceptor)
system. High titer virus was injected into early chicken embryos (E2-3). After feather follicle
forming stage (E12), both RCAS-noggin and RCAS-spry2 induced abnormal embryonic feather
development. Later, we sought to investigate the distinct transcriptional programs active within
H2B-eYFP
+
cells, as well as the surrounding tissues, to better understand the genetic programs
and signaling interactions that govern the ongoing developmental events. Also, we compared the
transcriptome of H2B-eYFP
+
cells in developing avian skin with that of the aorta and found they
are very different, implying ECs of different origins may have integrated together to form the
vasculature network. Intriguingly, we found H2B-eYFP
+
cells which function as a new wave of
neovascularization in the skin, may also be involved in other biological processes, such as smooth
muscle formation.

33
RESULTS

Vasculature develops in parallel with feather buds
Skin formation is an integral event and neovascularization is an indispensable process
during feather development. There are five successive phases of feather morphogenesis: macro-
patterning, micro-patterning, intra-bud morphogenesis, follicle morphogenesis, and regenerative
cycling (Lin et al., 2006). We therefore mapped the vascular growth to the initial feather cycle. We
chose to use the chicken model, so we could grossly observe the vascular network and blood
flow in the intra-bud and follicle. At E10, major vessels running through the interbud area connect
every feather bud with branches invaginating into each feather bud (Fig. 1A&B). When feather
buds grow from short to long at E11, the blood vessels branch further and develop inside the
feather bud to support feather development. Feather follicles are formed after E12; feathers start
to keratinize at E14 and the space of vasculature inside the feather becomes narrower. The
vasculature becomes shaped to accommodate the narrower space inside the feather. Intriguingly,
as the distal feather becomes progressively keratinized, the vasculature begins to be absorbed
at E16. At E18, no vasculature is seen inside the feather.
The feather undergoes regenerative cycling and so does the vasculature. The feather pulp
contains the vasculature, which includes a central axial artery, veins (Fig. 1C) and capillaries. This
network supplies nutrients to the developing feather epidermal cells. Also, it exerts outward
pressure which supports the feather structure (Lucas and Stettenheim, 1972). During feather
maturation, the pulp develops first as seen during growth phase and then degenerates so that
when an adult feather is plucked, it is usually hollow in the center. The septa above the pulp are
pulp caps (Fig. 1C). While the pulp is actively being absorbed at the apex, the cap composed of
pulp epithelium forms at periodic intervals.

Early neovascularization takes place before feather buds form
Tie1 is a receptor tyrosine kinase that is specifically expressed in the endothelial lineage
(Sato et al., 1995). In the mouse model, Tie1 is first expressed on E8-8.5 in differentiating
angioblasts of the head mesenchyme, in migrating ECs of the aorta and developing heart and in
the blood islands of the yolk sac (Sato et al., 1993, Korhonen et al., 1994). The expression persists
in ECs throughout embryogenesis. Tg(tie1:H2B-eYFP) quail line that expresses H2B-eYFP
specifically under the Tie1 promoter was generated for dynamic analysis of vascular
morphogenesis (Sato et al., 2010). This model was validated by overlapping expression of
endogenous TIE1, VE-CADHERIN mRNA, the QH1 antibody (a reference standard for the
34
vascular endothelial linage in quail) and the H2B-eYFP signal. The expression of H2B-eYFP is
first observed at late HH stage 6 within extraembryonic blood islands. It was shown at HH18, the
H2B-eYFP expression pattern (Fig. 1E) matches that of endogenous VEGFR-2 mRNA showing
expression in the lateral portion of somites and in regions surrounding them (Nimmagadda et al.,
2004). On the E6 embryo limb, stronger H2B-eYFP expression is seen at the boundary of the
digits (Fig. 1D) similar to that of endogenous VEGFR-2 mRNA. The pulp of feathers plucked after
hatching also shows an H2B-eYFP pattern resembling the vasculature (Fig. 1C). In E8 quail skin,
similar patterns showing vascular loop formations developing inside the feather buds were
observed in the Tg(tie1:H2B-eYFP) model and by immunostaining with an anti-quail endothelial
antigen (Fig. 1F).
To visualize vascular patterning in the feather primordia of developing avian skin prior to
blood flow, we peeled off the back skin of Tg(tie1:H2B-eYFP) from E5.5 to E8 (Fig. 2A&B). At
E5.5, there is no obvious feather primordium formation, but a dense midline across the whole
piece of skin is grossly observed with different skin densities in the cranial (back skin cranial to
the wing bud), middle (back skin between the wing bud and limb bud) and caudal portion (back
skin caudal to the limb bud) (Fig. 2B,C &3C). In the cranial part of the back skin, more tissue is
seen close to midline; H2B-eYFP
+
cells distribute with this layer of tissue and a primitive
vasculature is formed. In the middle part of the back skin, no extra tissue or H2B-eYFP
+
cells are
seen. In the caudal part of the skin, more tissue is seen in the lateral side of the trunk; H2B-eYFP
+

cells distribute accordingly in the lateral trunk but do not form vasculature as mature as that seen
in the cranial part. At E6, the first column of feather primordia forms symmetrically but the caudal
part has an extra feather primordia along the midline (Fig. 2B&C). H2B-eYFP
+
cells aggregate
immediately to the newly formed feather primordia. H2B-eYFP
+
cells in the cranial part of the skin
are connected to the extra layer of tissue, in the middle part skin are connected to the midline
vascular network, and in the caudal part of the skin are connected to the lateral trunk vasculature.
At E6.5, feather primordia elevate to become feather buds; the number of feather buds in a row
is 1-2 (cranial)/5 (middle)/2-3 (caudal) plus one feather bud along the midline (Fig. 2B,C&3C).
H2B-eYFP
+
cells develop inside the feather buds accordingly and inter-bud connections between
each feather bud are observed. At E7, feather buds grow taller, and the shape becomes narrower;
1-2 newly formed feather buds are added to the lateral side at each feather row (Fig. 2B&C). H2B-
eYFP
+
cells keep developing inside the feather bud. At E7.5, the innermost two columns have
black and yellow pigmentation on feather buds and a newly formed column of feather primordia
are observed in the middle part, along the midline (Fig. 2B,C&3C). H2B-eYFP
+
cells are more
organized; linear patterns with inter-connections are observed from the base to inside of the
35
feather bud. At E8, the feather buds become sharp at the distal tip and grow taller (Fig. 2B&C).
H2B-eYFP
+
cells seem to enter the feather buds in a narrow tunnel and then spread out to several
linear tracks, growing together with the feather buds.

Dynamic imaging of the avian skin vasculature
We asked what model we could use to study the dynamic changes of H2B-eYFP
+
cells
between the aforementioned stages of skin development. In chicken, the skin explant culture
model (Fig. 3A) has been used to analyze cellular and molecular events during feather
morphogenesis (Jiang et al., 2011). HH 28-34 (E6-8) chicken embryo dorsal skins are usually
used to observe feather development (Chuong, 2000). It was shown that explant culture
conditions are capable of supporting E8 feather bud development for 10 days and invagination to
form feather follicles can be observed under these conditions, although there is a clear delay in
the developmental timing (Jiang et al., 2011). The skin explant culture method was also used to
study quail pigment formation (Inaba et al., 2019). Here, we verified that the skin explant model
could be used to observe neovascularization by comparing samples grown in culture for one day
with skins grown in ovo (Fig. 3B&C ). For E5.5 Tg(tie1:H2B-eYFP) quail skin grown in culture for
1 day (+1D) (Fig. 3C), feather primordia form and the H2B-eYFP
+
cells aggregate to the newly
formed feather primordia just like they do in vivo (Fig. 2B&3C). However, H2B-eYFP
+
cells didn’t
seem as bright if the skins were cultured for more than 1 day (Fig. 3B). We also characterized the
distribution of H2B-eYFP+ cells in E6 and E7 cultures grown for one day (E6+1 and E7+1) and
compared them to E7 and E8 skins grown in vivo (Fig. 3C).
After characterizing the explant model, dynamic live imaging was performed to capture
vascular pattern formation focusing on the middle to caudal parts of the back skin. In the E5.5+1D
movie, disseminated H2B-eYFP
+
cells in the lateral trunk migrate both inwards and outwards (Fig.
4A). Meanwhile, they pattern and connect themselves forming vasculature and so do cells at the
midline. Cells in specific regions of the lateral trunk migrate further towards the midline
(speculated to be the direction towards future feather primordia location). In the E6+1D movie,
the first column of feather primordia form; a few H2B-eYFP
+
cells have already aggregated to
feather primordia locations, proliferated, interconnected, and patterned themselves into
vasculature (Fig. 4B); cells in the midline also migrate and build connections with those located
within feather. In the E6.5+1D movie, additional columns of feather primordia form; H2B-eYFP
+

cells in feather primordia are organized into a polygonal pattern; vasculature in earlier developed
primordia are more compact than those in newly formed ones (Fig. 4C ); cells in each feather
primordia build connections with other feather buds. In the E7+2D movie, feather buds elevate
36
from the skin surface; H2B-eYFP
+
cells in feather buds develop in accordance to feather buds in
a balloon shape; melanocytes are seen at the feather epithelium (Fig. 4D). Intriguingly, the
vascular network in interbud skin seems to disperse after a day in culture, but the H2B-eYFP
+

cells in feather buds continue to grow for another two days. In the E8+1 movie, large caliber
vasculature is established between feather buds, suggesting blood flow is supplied into the skin
tissue. However, the eYFP signal is obscured by melanin and it is difficult to study the pattern
formation.

Expression of pro-angiogenic factors in developing avian skin
Between E5.5 to E6, the pattern of H2B-eYFP
+
cells changed rapidly once feather
primordia formed. We wondered what molecules might mediate this seemingly interactive change.
Because both VEGF and FGF2 are known strong pro-angiogenic factors in development, we first
performed section in situ hybridization staining to see if VEGF and FGF2 are expressed within
feather primordia. These were performed in E6-8 chickens (Fig. 5A&B) to eliminate the concern
that quail pigmentation might obscure our ability to detect expression. We observed stronger
expression of both VEGF and FGF2 in feather primordia. Specifically, the expression is stronger
in the epithelium than in the dermis. To functionally test whether VEGF and FGF2 promote local
H2B-eYFP
+
cell aggregation, beads coated with VEGF and FGF2 were placed on top of the
explant skin which then was cultured for 1 day. The data show that both VEGF and FGF2 coated
beads caused H2B-eYFP
+
cells to aggregate in skin explants under the beads (Fig. 5C).

Antagonistic regulation of neovascularization resulted in an abnormal feather phenotype
Noggin, known as a BMP inhibitor, can control embryonic blood vessel formation via
inhibition of VEGFR-2 (Nimmagadda et al., 2005). Noggin was expressed to higher levels in
feather primordia and buds than in interfollicular regions starting from E6 (Fig. 5E). To examine
how perturbing neovascularization will feedback to affect avian skin development, we made an
RCAS-noggin retrovirus and injected it into early stage chicken embryos. When the chicken skin
developed to E12-E14, formation of follicles and the beginning of feather keratinization were
observed in non-viral affected areas; in the RCAS-noggin infected area, an abnormal phenotype
was observed, forming an enlarged balloon shape and increased vascularity inside the feather
(Fig. 5F).
Spry2 attenuates both VEGF and FGF signaling (Patel-Hett and D'Amore, 2011).
Overexpression of spry2 in ECs inhibits VEGF- and FGF-induced ERK activation (Yang et al.,
2015). In contrast, spry2 was shown to express higher levels in the interfollicular region than in
37
feather buds starting from E8, which was later compared to noggin (Fig. 5D). We also made
RCAS-spry2 and injected it into early stage chicken embryos. The abnormal feather phenotype
produced by RCAS-spry2 showed an ampulla shape at the base of the feather and increased
vascularity inside the feather, too (Fig. 5F). In addition to the abnormal feathers on the back and
head region, an ectopic induction of abnormal bubble shaped feathers was noted on the scales
of the feet. A delay of feather follicle invagination was also observed in the RCAS-spry2 infected
area.

Vascular network pattern in a feather epithelium dependent manner
Epidermal-mesenchymal interaction is known to play an important role in organogenesis.
In human epidermis, VEGF most likely represents the major angiogenic activity to enhance
dermal blood supply to the avascular epidermis for high metabolic demands (Detmar, 1996).
Together, with the stronger expression of VEGF and FGF2 we observed in feather primordia
epidermis led us to the question: is the neovascularization process in developing avian skin
guided by epithelium? To test this, we first separated the epithelium and the mesenchyme of the
skin and observed how it affected the vascular pattern (Fig. 6A). The polygonal pattern of H2B-
eYFP
+
cells in feather primordia at E6.5 gradually dispersed without the epithelium (Fig. 6B).
Later, after separation of skin epithelium and the mesenchyme, the epithelium was rotated 90°
and placed black on the mesenchyme (Fig. 6C). Surprisingly, the developing axis of H2B-eYFP
+

cells were redirected to the orientation of the epithelium and not the mesenchyme (Fig. 6D). This
result revealed a guidance role of epithelium in skin neovascularization.

Vasculogenesis or angiogenesis in developing avian skin: mesenchymal cell fate plasticity
Next, we were interested in discerning where the H2B-eYFP
+
cells in feather primordia
originate and what roles they play in the developing avian skin. Specifically, we wanted to know
if the H2B-eYFP
+
cells come from vasculogenesis, differentiation of stem cells in skin due to local
signal inductions or angiogenesis, the extension of already existed blood vessels. Theoretically,
if the H2B-eYFP
+
cells come from skin stem cells, their transcriptome profile should resemble
those of other skin cells ; if they come from the extension of existing blood vessels, their
transcriptome profile should resemble H2B-eYFP
+
cells in the existing vasculature (ie.,
vasculature found in the aorta that forms very early) (Fig. 7A ). Also, to identify the regulators of
skin neovascularization (vasculogenesis and angiogenesis), a different time point is needed to
explore the process. Therefore, we dissected the skin and aorta of the Tg(tie1:H2B-eYFP) quail
38
embryos at E6 and E7, and obtained eight groups of cells (E6, E7 dermis H2B-eYFP
+ and –
and E6,
E7 aorta H2B-eYFP
+ and -
) by fluorescent-activated cell sorting (FACS) (Fig. 7B). The sequencing
reads from the eight transcriptomes were mapped to the Coturnix Japonica genome (the statistics
of sequencing reads are listed in Materials and Methods).
Principal component analysis (PCA) showed that each cell type clustered together: E6
and E7 dermis H2B-eYFP
+
cells, E6 and E7 dermis H2B-eYFP
-
cells, E6 and E7 aorta H2B-eYFP
+

cells and E6 and E7 aorta H2B-eYFP
-
cells (Fig. 7C). Intriguingly, dermal H2B-eYFP
+
cells are
closer to dermal H2B-eYFP
-
cells than aorta H2B-eYFP
+
cells, implying a higher similarity
between dermal H2B-eYFP
+
cells and dermal H2B-eYFP
-
cells rather than aorta H2B-eYFP
+
cells.
On the other hand, aorta H2B-eYFP
+
cells and aorta H2B-eYFP
-
cells separated well, suggesting
more mature cell types in aorta. Based on the PCA plot, our data suggest that vasculogenesis
rather than angiogenesis is involved in skin neovascularization process.
To understand the differential expression profile of aorta H2B-eYFP
+
cells, as the 1
st
wave
of neovascularization and dermal H2B-eYFP
+
cells, as the newly formed 2nd wave of
neovascularization, the differentially expressed genes (DEGs) with a padj value (adjusted p-
value) <0.05 among both groups (n=4243) were analyzed (Fig. 7D). Using H2B-eYFP
-
cells in
both groups as a control, there were 2518 annotated DEGs among dermal and aorta H2B-eYFP
+

groups. We functionally profiled these DEGs using g:Profiler (Raudvere et al., 2019) and found
many biological process involved, specifically those related to early tissue development and
biological process. We then mapped DEGs of Coturnix Japonica to Gallus Gallus using the
orthology search function on g:Profiler. There are 185 transcription factors (TFs) (padj<0.05)
involved in these DEGs. E2F and E2F-1,4 are important for cell cycle control and tumor
suppressor protein binding. Also, by repressing CEBPa binding to its target gene promoters, E2F
can block adipocyte differentiation (Zaragoza et al., 2010). Other TFs related to adipogenesis
such as CEBPa,β are also present in this DEGs. Pax3, myogenin and myoD are involved in
muscle development. The ETS family, known to be important in embryonic vascular development
(Craig and Sumanas, 2016), is shown in the DEG list, too.
Furthermore, we added the time factor to assess the differential expression profile of the
neovascularization process of dermis and aorta. Within dermis, there are 474 DEGs (padj<0.05)
in the E6 to E7 neovascularization (H2B-eYFP
+
cells) process (Fig. 7E); within aorta, it’s 29 DEGs
only (Fig. 5D). Using H2B-eYFP
-
cells in both groups as a control, there were 284 annotated
DEGs(padj<0.05) among dermal E6 and E7 H2B-eYFP
+
groups and 27 for the aorta group. We
also functionally profiled these DEGs using g:Profiler and mapped DEGs of Coturnix Japonica to
Gallus Gallus. Intriguingly, while TFs (n=69, padj<0.05) related to myogenesis (Pax3 and
39
myogenin), adipogenesis (CEBPa,β and E2F) and vascular development (ETS family) were found
in the dermal group, there’s only one TF, Cdx1, present in the aorta group. The H2B-eYFP
+
cells
in dermal neovascularization seem to have fate plasticity in biological processes compared with
those in the aorta.

40

Figure 1. Development of avian skin vasculature and the Tg(tie1:H2B-eYFP) quail model
A. B.
D.
E10
E11
E14
E18
C.
Tie1:H2B-EYFP VEGFR2
E6
VEGFR2 Tie1:H2B-EYFP
F.
E.
Tie1:H2B-EYFP Anti-quail
endothelial
antigen
VEGFR2
Tie1:H2B-EYFP
HH16
E10
E11
E14
E18
Section
Whole mount
41
(A). The patterns of vessel growth in chicken feather from E10 to E18. The vessels are indicated by red
lines in the schematics.
(B). H&E stain of chicken skins from E10 to E18. The vessels in the skin and the feather are in red. The
left column shows whole-mount samples; the right column shows skin sections where there are
developing feather follicles. Left column, scale bar: 200 μm; right column, scale bar: 100 μm.
(C). Plucked chicken or quail feathers. Starting from the left, are whole mount, sheath-off whole mount
and sectioned chicken feathers stained with VEGFR2 (red). VEGFR2 is used to identify endothelial cells of
the vessels. The most right is a whole mount feather from the Tie1:H2B-EYFP transgenic quail. The EYFP
is shown in green. Scale bar: 500 μm.
(D). Paws of an E6 Tie1:H2B-EYFP transgenic quail (left) and an E5 quail (right) with ISH (in situ
hybridization) stain. The EYFP is shown in green; the ISH signal is in dark blue (Nimmagadda et al., 2004).
Scale bar: 500 μm.
(E). Embryos of an E2 Tie1:H2B-EYFP transgenic quail (top) and a HH16 quail (bottom) with ISH stain. The
EYFP is shown in green; the ISH signal is in dark blue (Nimmagadda et al., 2004). Scale bar: 500 μm.
(F). Whole mount skins of E8 quails. On the left, it’s a wildtype quail stained with quail endothelial
antigen (red); on the right it’s Tie1:H2B-EYFP transgenic quail and the EYFP is shown in green. Scale bar:
100 μm.

42

Figure 2. Neovascularization in avian skin before circulation established
A.
E5.5
B.
C.
E5.5
E6
E6.5
E7
E7.5
E8
ㄏㄏ
E5.5
E6 E6.5 E7 E7.5 E8
43
(A). Tie1:H2B-EYFP transgenic quail embryo at the age of E5.5. The left shows the brightfield image of
the embryo; the right shows the EYFP (green) signals from the same embryo. Scale bar: 1 mm.
(B). Whole-mount skins of Tie1:H2B-EYFP quails from E5.5 to E8. The left column shows the brightfield
skin images (with midline in the middle); the right column shows the EYFP (green) channel of the same
region. Scale bar: 300 μm.
(C). Patterns of the Tie1+ cells and feather follicles (condensations, buds) in developing quail skin by age.
The Tie1+ cells are drew as green dots, thin or thick lines depending on its appearing continuity and
thickness. Feather follicles (condensations, buds) are drew as circles or ovals depending on its shape.
The yellow circles indicate newly-formed feather condensation; the orange ones are condensations
appeared in the previous stage already. Feather buds with keratinization are drew with black caps.

44

Figure 3. Explant culture system can be used for dynamic imaging
(A). Schematics of the explant culture system for dynamic imaging. In brief, whole-mount skin is
dissected, flatten and cultured on a porous insert, immersed in culture media.
(B). E5 skin explant from Tie1:H2B-EYFP quails cultured over time. The white dash lines indicate the
midline of the skin. Scale bar: 500 μm.
(C). Skin explant from Tie1:H2B-EYFP quails before and after 24 hours culture. Both brightfield and EYFP
images are shown. Skin are sampled from E5.5, 6.5 or 7.5 quail embryos. Top row, scale bar: 2000 μm;
second row, scale bar: 200 μm.

A.
B.
0hr 24hr
C.
Explant culture system for dynamic imaging
E7.5
0hr 72hr 24hr
E6.5 E5.5
45

Figure 4. Time-lapse images of H2B-eYFP+ cells in developing avian skin
(A) (B) (C) (D). Frames from the dynamic imaging of the explant cultures. Skin specimen sampled from
Tie1:H2B-EYFP quails with different ages are shown in panel A, E5.5; B, E6; C, E6.5 and D, E7. In panel A-
C, skin explants are cultured for about 15 hours and focused on the epithelium level. Single frames from
every 3 hours are shown. In panel D, the skin explant is cultured for almost 3 days and focused on the
feather bud level. The timing of the shown frames is listed to the left of each image. In each panel, a
schematic shows the approximate position of the field of view (FOV) on an embryonic skin. Scale bar:
200 μm.

0hr
3hr
6hr
9hr
12hr
15hr
A.
C.
B.
E5.5
D.
E6 E6.5
E7
D0+
0hr
D1+
0hr
D2+
0hr
D0+
15hr
D1+
15hr
D2+
15hr
46

Figure 5. VEGF, FGF2, noggin and spry2 expression and functional studies
(A) (B) (D) (E). ISH stains of embryonic quail sections. The genes stained against are indicated at the left
in each panel (A. FGF2; B. VEGF; D. Sprouty2; E. Noggin). Samples from E6-E8 are listed from the left to
the right in each panel. The lower row in each panel shows a zoom-in view of the top row. Scale bar: 500
μm.
(C). Skin explants with agarose beads after 24-hr culture. The skins are sampled from E7 Tie1:H2B-EYFP
(green) quails. The agarose beads appear as dark green circles in the EYFP channel. The lower row in
each column shows a zoom-in view of the top row. Scale bar: 300 μm.
(F). Whole-mount and sectioned skins with the overexpression of Sprouty2 or Noggin. The RCAS virus
expressing Sprouty2 or Noggin are injected to E2 embryos and the skins are sampled at E14. Scale bar:
200 μm.

A.
B.
C.
D.
E.
F.
FGF2
E6 E7 E8
Sprouty2
E6 E7 E8
E6 E7 E8
VEGF
Noggin
E6 E7 E8
BSA FGF2 VEGF Control Sprouty2 Noggin
47

Figure 6. H2B-eYFP+ cells pattern is epithelial oriented.
(A) (C). Schematics demonstrating the manipulations done with the skin explant. The epithelium in the
skin are indicated in white, while the dermis is in grey. For the “no epithelium” condition in panel A, the
epithelium is removed from the dissected skin and the remaining dermis is cultured on the insert. For
the “recombination” condition, the epithelium is first separated from dermis but then rotated 90 degree
and put back onto the dermis. The recombinant skin is cultured on the insert together.
(B). Skin explants before and after 24-hr culture. The control sample is an intact piece of skin. The “no
epithelium” sample is a dermis-only skin. The first rows are brightfield images and the second rows are
EYFP images from the same FOV. Scale bar: 300 μm.
(D). The recombinant skin explant cultured for 24 hours. EYFP is shown in green. A zoom-in view of the
explant is shown on the right. The original orientation of the epithelium is 90 degree counterclockwise.
Scale bar: 300 μm.

A. C.
Recombination
B.
A P
No epithelium
A P
A P
90
∘
Recombination
D.
Control
0hr 24hr
No epithelium
0hr 24hr
48

Figure 7. RNA-seq analysis of skin and aorta neovascularization at E6 and E7.
(A). The workflow of the RNA-Sequencing experiment. First, skin (dermis) and aorta are separately
dissected from E6 or E7 Tie1:H2B-EYFP quails. Then, cells are dissociated from the tissues and sorted
into EYFP-positive and -negative groups by FACS. mRNA from EYFP+ and EYFP- cells are extracted and
A.
B.
C.
PC1(47.5)
-50 0 50 100
0
80
40
-40
PC2(22.3)
-80
Tie1 +
Tie1 -
Aorta
Dermis
D.
E.
2518
DEGs between dermal
and aorta cells
Tie1+
Tie1−
Identify related TFs in g:Profiler
Myogenesis Adipogenesis
Vascular
development
Cell cycle
others
DEGs between
E6 and E7
dermal cells
29
284
DEGs between
E6 and E7
aorta cells
Identify
related
TFs in
g:Profiler
Myogenesis Adipogenesis
Vascular
development
Cell cycle
others
Tie1+ Tie1−
49
sequenced separately. Thus, there are 8 types of samples generated different by their Tie1 expression,
tissue types and embryonic ages.
(B). An example FACS sorting result of the cells from a E6 quail. The rightest graph shows the expression
level of Tie1 gene in those EYFP+ and EYFP- samples.
(C). PCA analysis result indicating the similarity of overall RNA expression profile between samples.
Samples from dermis are shown as triangles; samples from aorta are shown as circle dots. Tie1+ samples
are in red; Tie1- samples are in blue. The dash lines in the graph indicates the possible grouping scheme
of the data points.
(D) (E). Workflows of related transcription factor (TF) ontology mapping. The number in the red shapes
are the total DEG number input to g;Profiler. The biological processes that the identified TF involves are
listed in colored rectangles. The hexagons in panel D represent the DEGs between dermal and aorta cells
that are either Tie1+ (with red outline) or Tie1- (with red outline). The triangles in panel E represent the
DEGs between E6 and E7 dermal cells that are either Tie1+ (with red outline) or Tie1- (with red outline),
and the circles represent DEGs of aorta cells. DEGs, differentially expressed genes.

50
DISCUSSION

Avian skin is unique in studying pattern formation. Our lab proposed that the integument
pattern is built at multi-levels: first, the rising of feather primordia from the non-patterned
morphogenic field is achieved by de novo patterning following rules such as Turing principles
(Turing, 1952, Turing, 1990, Maini et al., 2006); second, we demonstrated that the feather muscle
network is patterned using feather primordia as reference points to pattern themselves, in a
process known as “adaptive patterning” (Wu et al., 2019). In this developing process, several
pieces are still missing. Among which, neovascularization is the most vital step for tissue
assembly. The Tg(tie1:H2B-eYFP) quail model is a powerful approach in studying the early
vascular formation process. The early expression of Tie1 in ECs makes it possible to observe
dynamic neovascularization before there are tubular structures and blood flow. This study finds
that skin vasculature is assembled in a stepwise fashion, linked tightly to feather primordia
formation and epithelial-dependence. On the molecular level, we found VEGF and FGF2 are
involved in this process, just like in other tissues, and perturbation of the neovascularization
process eventually leads to abnormal feather morphology. These conclusions guided us back to
question whether neovascularization is induced locally during skin development (vasculogenesis)
or formed by the migration of existing ECs (angiogenesis). We took advantage of the Tg(tie1:H2B-
eYFP) quail model and FACS H2B-eYFP
+
and H2B-eYFP
-
cells in developing skin and aorta,
representing local and migrating groups of cells and performing RNA-seq to profile each group of
cells. Transcriptional analysis gave us clues regarding the identity of H2B-eYFP
+
cells in skin.
First, PCA revealed a closer relationship between H2B-eYFP
+
and H2B-eYFP
-
cells in the skin
than to H2B-eYFP
+
cells in the aorta, suggesting a similar trait between the H2B-eYFP
+
and H2B-
eYFP
-
cells in the skin. Second, from the DEGs of skin and aorta H2B-eYFP
+
cells, TFs related
to biological processes other than neovascularization were also found. Specifically, we added a
time factor comparing E6 and E7 H2B-eYFP
+
cells in each group and found that TFs related to
muscle formation, adipogenesis…etc. were in the skin group, indicating a potential pluripotent
status of H2B-eYFP
+
skin cells at this developmental stage. Nevertheless, future studies are
required to uncover the specific identify of the H2B-eYFP
+
skin cells and to determine the
molecules or mechanisms capable of inducing these cell fate lineage decisions.
A major difference of avian integument from mammalian skin is that every feather has its
own blood circulation system developing with the feather which is not seen in human or mouse
hair. In E10 chickens, a circulatory loop visible within feather buds is connected at the feather
base to a major vessel running across the skin. This vascular structure resembles those of
51
mammalian digits, a loop circulation in each digit connected to palmar arches. During E11-12,
vasculature develops further, branching inside the feather. Intriguingly, the pulp, rich in blood
vessels supporting feather development, is derived from the dermal papilla (Yu et al., 2004) but
at this stage, the dermal papilla doesn’t seem to have formed yet. Either the pulp is a 3
rd
wave of
neovascularization inside the feather and the vasculature formed at an earlier stage isn’t involved
in the new blood network, or there is still something missing in this pulp forming process.
Furthermore, pulp degeneration is an important process in the feather cycle. It starts around the
8th day after hatching. At the pulp tip, the arteries grow so many branches that the apical blood
vessels begin to disintegrate, releasing blood and the pulp tip recedes (Lucas and Stettenheim,
1972). The cavity of pulp cap then forms by coalescence of spaces and the pulp cap itself starts
to keratinize. How the pulp is self-programmed to reabsorb themselves at certain stages and
trigger the subsequent events to make the hollow quill is still unknown.
Our study focused on the blood vessel initiation within feathers and how this is achieved
from an early developmental stage because in tissue engineering, one essential step to make
grafts functional is to vitalize them with blood flow. At E5.5 quail skin, differences in skin tissue
density can be seen in different regions, and specifically, a coexistence of this layer of tissue and
H2B-eYFP
+
cells were observed. First, what’s this layer of tissue? In humans, it was recently
found by confocal laser endomicroscopy that an unrecognized interstitium with a reticular pattern
exists in the submucosae of the gastrointestinal tract, urinary bladder, the dermis, the peri-
bronchial and peri-arterial soft tissues, and fascia (Benias et al., 2018). To our understanding,
skin is formed from DM and by epidermal-mesenchymal interactions. But this unevenly distributed
layer of tissue has never been mentioned during skin development. Second, it was shown that
this layer developed earlier in the cranial and caudal parts then in middle part of the back skin.
Does this layer migrate to the middle of the back skin and fuse to a whole piece or does it develop
later onsite and fuse with the cranial and caudal part? Third, the coexistence of H2B-eYFP
+
skin
cells: are H2B-eYFP
+
skin cells induced within this layer of tissue or is this layer of tissue
necessary for H2B-eYFP
+
cells to form a vascular network, perhaps by providing an appropriate
environment for H2B-eYFP
+
cells to migrate? In an E4 Tg(tie1:H2B-eYFP)

quail lateral view image,
H2B-eYFP
+
cells were shown to have a linear pattern between vertebrae and no skin H2B-eYFP
+
cells were observed at this stage. Although spatially closely related, it is not clear if the
intervertebral H2B-eYFP
+
cells underlying forming skin directly contribute to the skin H2B-eYFP
+

cells. Later, when feather primordia start to develop, H2B-eYFP
+
cells showed a rapid pattern
change according to the feather primordia position. Likewise, our previous feather muscle
research demonstrated that during E7-E12, the muscle fibers emit outward from feather buds in
52
every direction but only those muscle fibers connecting two feather buds become stabilized (Wu
et al., 2019). In both cases, feather primordia/buds guide other structures to assemble themselves.
It was also shown that in zebrafish, osteoblasts create paths in scales guiding nerves and blood
vessels during both development and regeneration (Rasmussen et al., 2018).
The Tg(tie1:H2B-eYFP) quail model makes studying vascular pattern formation possible
in the avian system by providing a means to perform dynamic imaging to observe the
morphogenesis and cell-cell interactions. However, in the avian skin forming stage, the embryo
sinks to the egg center and in-ovo imaging is difficult to set up because of the long working
distance and movement of the embryo. The Egg-in-Cube model improves the observability and
accessibility of the embryo (Huang et al., 2015, Huang et al., 2017), but further optimization of the
system is required to reduce the working distance. For these reasons, we chose to study the well-
established skin explant model for the dynamic imaging presented in our study. We tested if the
explant model represents the in vivo condition and though it did, generally speaking, the process
is slower than in vivo and florescence of H2B-eYFP
+
cells became dim after one day in culture.
Therefore, we weren’t able to record a complete process of feather primordia formation and H2B-
eYFP
+
cell movement beginning at E5.5. We had to break the process up into smaller steps. In
the E6+1D movie, H2B-eYFP
+
cells aggregate according to feather primordia positions and
proliferate; in the E6.5+1D movie, polygonal patterns form at the feather bud base and vascular
structure in each feather bud seems to try to make connections with each other. Here, we
observed the transformation of a previous capillary network to a branching structure. The
formation of the branching pattern was computer simulated using the blood vessels in the wall of
the avian yolk sac (Honda and Yoshizato, 1997). It was shown that the polygonal capillary network
enlarged leading to a subdivision of finer polygons adapting to the embryo body size. In the E7+1D
movie, multiple connections between each feather bud have become established to form a
network, yet no double walled lumen structure can be seen at this stage. Inside the feather bud,
it is still unclear how H2B-eYFP
+
cells adapt to the elongating feather and how the initial H2B-
eYFP
+
cells participate in pulp formation. In the E8+1D movie, large caliber vasculature formed.
Our study demonstrated a transition status of skin vasculature from the irregularly disseminated
H2B-eYFP
+
cells to an organized tubular structure from E5.5 to E8 Coturnix Japonica.
While angioblasts (EC precursors) are induced by FGF2, VEGF is critical for growth and
morphogenesis of angioblasts into the initial vascular pattern (Poole et al., 2001). During
gastrulation, the endoderm plays an important role in angioblasts specification from the overlying
mesoderm. FGF2 was detected at high levels in the endoderm (Riese et al., 1995) and beads
coated with FGF2 could induce ectopic vessel formation just like the endoderm (Cox and Poole,
53
2000). As a chemoattractant for ECs, VEGF is expressed to slightly higher levels in the endoderm.
In our data, both FGF2 and VEGF are initially expressed higher in the epithelium than in the
dermis. When feather primordia formed, both FGF2 and VEGF are expressed higher in feather
placodes than in inter-primordia regions. Later, it appeared that both FGF2 and VEGF are
expressed higher in feather buds than in inter-bud regions. And similarly, we ectopically induced
H2B-eYFP
+
cells or promote the migration of H2B-eYFP
+
cells by FGF2 and VEGF coated beads.
Since FGF2 and VEGF are both expressed higher in epithelium than in dermis, separation of the
epithelium and dermis diminished the vascular pattern. Also, FGF2 and VEGF are expressed
higher in feather placodes than in inter-primordia regions so that the recombination of rotated
epithelium by 90° and dermis resulted in changing the H2B-eYFP
+
cell growth axis. On the other
hand, vascular cells were found to play an important role in pattern formation during
organogenesis. Prior to blood vessel function, hepatic growth was found to fail in the absence of
ECs (Matsumoto et al., 2001). In pancreatic development, ECs play an inductive role: ectopic
vascularization leading to islet hyperplasia and ectopic insulin expression (Lammert et al., 2001).
Here, we used RCAS-noggin and RCAS-spry2 to interfere with the neovascularization process
via the VEGF and FGF signaling pathways and feather morphogenesis was impaired and the
vascularity in infected regions was shown to be higher than in normal conditions. It was shown in
quail embryo VEGF loss of function studies that ECs aggregate into clusters rather than forming
normal cord-like structures and there is a lack of polygonal structures of the primary vascular
plexus (Drake et al., 2000). We speculate that the resemblance of our results from overexpressing
RCAS-noggin and RCAS-spry2 to those resulting from VEGF loss of function is because noggin
and spry2 are acting via VEGF and FGF signaling.
From avascular to vascular, vasculogenesis and angiogenesis mechanisms together build
complex vascular networks to support the embryo to meet its high metabolic requirements. Our
observation of H2B-eYFP
+
cell transitioning from disseminated cells to a polygonal pattern with
lumen and loops makes us wonder if these skin H2B-eYFP
+
cells form from vasculogenesis or
angiogenesis. During organogenesis, capillary endothelial cells express on their cell surface an
array of antigens that manifest organ selectivity in brain, ovary and lung(Auerbach et al., 1985).
Also, an endocrine-gland-derived vascular endothelial growth factor (EG-VEGF) exclusively for
either steroidogenic glands, ovary, testis, adrenal and placenta is identified, suggesting a highly
specific mitogen regulating the proliferation and differentiation of the vascular endothelium in a
tissue specific manner (LeCouter et al., 2001). However, in addition to skin derived from DM, little
is known about the details of origin of the skin vasculature. Intriguingly, researchers were
specifically interested in studying the origin of skin lymphatic ECs (LECs). Studies have shown
54
that although most LECs derive from the cardinal veins and intersomitic veins (Srinivasan et al.,
2007, Yang et al., 2012), a blood capillary plexus-derived population of progenitor cells also
contributes to the dermal lymphatic vasculature during embryonic development (Pichol-Thievend
et al., 2018). Our PCA plot showing a higher similarity between skin H2B-eYFP
+
cells and skin
H2B-eYFP
-
cells than aorta H2B-eYFP
+
cells is in favor of vasculogenesis process for skin
neovascularization. Indeed, in pre-circulation stage, when there’s no blood flow, vasculogenesis
is the dominant mechanism of vascular morphogenesis (Poole et al., 2001). The Tie1 gene
expression level (Fig. 7B) in H2B-eYFP
+
and H2B-eYFP
-
cells confirmed that our FACS process
was sound. The expression of a series of marker genes determining EC lineage were found all
significantly higher in aorta than in skin H2B-eYFP
+
cells, meaning a more committed cell fate in
aorta than in skin H2B-eYFP
+
cells, and were higher in skin H2B-eYFP
+
than in skin H2B-eYFP
-
cells, implying not only a sound FACS strategy but also Tg(tie1:H2B-eYFP) quail as a reliable
model in which to study neovascularization at this developmental stage. The TF list exploited from
skin neovascularization during E6-7 includes multiple biological processes while there’s only one
TF in the aorta group. For neovascularization, in addition to the ETS family in the list, Nkx2.5 was
identified to contribute in hemoangiogenic lineage specification and diversification (Zamir et al.,
2017). For myogenesis, the feather muscle network develops between E7-12 and overlaps with
neovascularization. It is not clear if H2B-eYFP
+
cells participate in the feather muscle forming
process, but it was shown that the Tie1 promoter is active in vSMC precursors and Notch signaling
is essential for the differentiation from Tie1
+
precursors to vSMC (Chang et al., 2012). For
adipogenesis, although no mature adipose tissue is observed at this stage in avian skin,
C/EBPa,β and E2F were both found in the TF list. However, future work, such as building a
lineage tracing system using the Tie1 promoter, is required to elucidate the role of the Tie1
promoter in mesenchymal cell fate determination.
In conclusion, we have demonstrated the spatiotemporal vascular pattern formation in
avian skin development both after and before circulation using the Tg(tie1:H2B-eYFP) quail model
and a transition status of skin vasculature from irregularly disseminated H2B-eYFP
+
cells to an
organized tubular structure. Proangiogenic factors, FGF2 and VEGF, were found highly
expressed in epithelium and dermis of the developing feather primordia. We separated the
epithelial and dermal layer and observed the organization process of H2B-eYFP
+
cells related to
feather primordia is epithelial-dependent. On the other hand, we constructed RCAS-noggin and
RCAS-spry2 and showed the feather development was impaired by this perturbation. We FACS
E6/7 skin/aorta H2B-eYFP
+/-
cells to perform transcriptome analysis. The profile of H2B-eYFP
+
cells in dermis and aorta are very different and H2B-eYFP
+
cells are more similar to H2B-eYFP
-
55
cells in dermis. Furthermore, we discovered the fate plasticity of H2B-eYFP
+
cells in E6-7 dermis
in skin development implying the inter-conversion status of Tie1
+
precursors play an important
role in mesenchymal cell differentiation. These findings highlight the importance of further
characterizing the mechanism for mesenchymal cell fate determination in the light of a cell therapy
era nowadays.
56
MATERIALS AND METHODS

Experimental model
Fertilized pathogen-free (SPAFAS) chicken eggs were from and staged according to the
method described by Hamburger and Hamilton(Hamburger and Hamilton, 1951). Fertilized wild-
type Japanese quail eggs were from (Westminster, CA); fertilized Tg(tie1:H2B-eYFP) and
Tg(tie1:H2B-eYFP) X Tg(PGK:H2B-mcherry) quail eggs were from USC translational imaging
center and are staged according to Ainsworth et al.(Ainsworth et al., 2010). Eggs were incubated
at a temperature of 38°C with 60-65% humidity with turning every 2 hours.

Specimen harvesting and processing
Chicken embryos staging from HH36~44 and quail embryos staging from HH26~35, for
section purpose, we removed head, four limbs, internal organs and feathers, and only back area,
from skin deep to spine and dorsal ribs, is preserved. The samples are fixed with 4%
formaldehyde (Sigma) overnight at 4ºC. For cryosection, we changed samples to phosphate
buffered saline (PBS) and then 15% sucrose in PBS the next day. After samples sunk to the
bottom of the vial, samples were changed from 15% sucrose in PBS to 30% sucrose in PBS. And
then the samples were embedded in mounting media (Tissue-Tek
R
O.C.T™ Sakura) and snap
frozen on dry ice, and stored at -80ºC. For wholemount purpose, quail embryos staging from
HH13~25 and skin samples were peeled off from the back area of HH26~35 quail embryos
including dorsal-pelvic, apteric, and femoral tracts. And then fixed with 4% formaldehyde (Sigma)
overnight.

Imaging
Imaging of wholemount embryos and skin samples was performed using a Nikon
SMZ1500 or Olympus MVX10 or Leica TCS SP8 confocal microscopy. Sectioned samples were
imaged with a Leica TCS SP8 confocal microscope or Keyence BZ-X700.

Explant Imaging Condition
Skin samples from quail embryos staging from HH28~35 were peeled off for dynamic
analysis. We placed the embryonic skin on a culture insert in 6-well culture plate (Falcon)
containing Dulbecco’ modified Eagle’s medium (DMEM) supplemented with 2% fetal bovine
serum (FBS, GEMINI#100-106), 10% chicken serum (CS, Sigma C5405) and Penicillin-
Streptomycin (Gibco) (1:1000). The explants were incubated at 38 ºC at an atmosphere of 5%
57
carbon dioxide and 95% air. Dynamic imaging was performed using an Olympus MVX10 or Leica
TCS SP8 confocal microscopy or Keyence BZ-X700.

In situ hybridization
Gene-specific fragments were amplified (SuperScript III First-Strand Synthesis system,
Invitrogen) from RNA extracted from the dorsal skin of quail and chicken embryos by using Trizol
reagent (Invitrogen), and subsequently cloned into pDrive cloning vector system (Qiagen). PCR
primers for the cDNA amplifications are listed below. Both sense and antisense RNA probes were
made by in vitro transcription according to manufacturer’s instructions (Roche). Wholemount in
situ hybridization was done according to the procedure described in (Chuong et al., 1996). Briefly,
after fixation in 4% formaldehyde (Sigma) overnight at 4ºC, samples were sequentially
dehydrated with methanol. For section in situ hybridization, after dehydration with methanol,
samples were embedded in paraffin wax (McCormick) and sections were prepared as 7um
thickness. Hybridizations with probes were carried out overnight at 65 ºC in the hybridization
buffer, containing a cocktail of the digoxigenin-labeled RNA probes. After wash and
blocking, color reaction was done with NBT/BCIP substrate (Promega).
Primers Forward Reverse Product
VEGF AGCGGAAGCCCAATGAAGTT TCTTTGGTCTGCAGTCACATT 300bp
FGF2 AGCATCACCACGCTGC GATTCCAAGCGCTCAAAAA 314bp
*Other probes: noggin (Tzahor et al., 2003), spry2 (Minowada et al., 1999, Yue et al., 2012)

Retrovirus production and mis-expression (This part of work is done by Dr. Ting-Xin Jiang)
For RCAS production, retroviruses were cultured and harvested as described (Morgan
and Fekete, 1996). RCAS-noggin was a gift from Dr. R. Johnson (Johnson and Tabin, 1997, Jiang
et al., 1999) and RCAS- spry2 was a gift from Dr. G. Martin (Minowada et al., 1999, Yue et al.,
2012). Both viruses were injected into E3 chicken embryos. The presence of virus was detected
by in situ hybridization using a probe to the viral polymerase gene (Crowe et al., 1998).

Epidermis removal and epidermis-dermis recombination
The separation of epidermis and dermis was performed according to the procedure
described in (Chuong, 1998). Briefly, dissected quail skin samples were incubated in 2X calcium-
58
magnesium free saline with 0.25% EDTA on ice for 10-15 minutes. Epidermis and dermis were
separated by forceps carefully. For recombination, epidermis was rotated 90º and recombined on
top of dermis. The recombinants were incubated in the culture insert.

Isolation of H2B-eYFP
+/-
cells, immunofluorescence staining and FACS
1. Isolation of H2B-eYFP
+/-
cells from Tg(tie1:H2B-eYFP) quail dermis and aorta
After removal of epidermis as described in previous paragraph, dermis was broken
up into small pieces with forceps. After dissected out from the circulation system, aorta was
broken up into small pieces, too. Samples were incubated in 0.1% trypsin and 0.1% collagenase
in calcium-magnesium free Hanks’ Balanced Salt Solution in 37ºC water bath for 5 minutes.
The cell suspension was agitated and inactivated with 100% FBS. The cell suspension was then
diluted with DMEM and filtered through a 0.7um cell strainer. The filter cells were spun down at
low-speed centrifugation (300g, 1221rpm) at 4ºC for 15 minutes. After removal of the
supernatant, cells were resuspended and filtered through a 0.4um cell strainer into a FACS
tube.
2. Immunofluorescence staining with DAPI and Draq5
The filtered cells were resuspended with DAPI (1:100,000) and Draq5 (1:2500) in
staining buffer (2% FBS in PBS).
3. FACS
FACS was done using ARIA I and program. The excitation was available from
405nm (for measurement of DAPI), 510nm (for measurement of eYFP) and 633nm (for
measurement of Draq5). FSC (Forward-Scattered) versus SSC (Side-Scattered) plot was run
first to adjust the voltage on each detector. And then DAPI and Draq5 was run to ensure the
viability of each cell sample. The compensation was adjusted between channels to eliminate
overlap in the florescence signal. Finally, eYFP gate was defined and H2B-eYFP
+ and –
cells were
collected into microfuge tubes containing lysis buffer RA1 and reducing agent TCEP
(NucleoSpin RNA XS, Takara Clontech #740902.50) for RNA extraction. The samples were
kept at -80 ºC preparing for RNA extraction.

Total RNA isolation
After thawing, the samples were homogenized and total RNA extracted using NucleoSpin
RNA XS kit (Takara Clontech #740902.50). The 15min rDNase treatment was done at room
temperature as described in the manual to remove the DNA thoroughly.
Sample Sequencing ID
(biological repeats)
E7 dermis Tie+ 695, 699
59
E7 dermis Tie- 696, 700
E6 dermis Tie+ 675, 737
E6 dermis Tie- 676, 738
E7 aorta Tie+ 697, 701
E7 aorta Tie- 698, 702
E6 aorta Tie+ 677, 739
E6 aorta Tie- 678, 740

Stranded RNA sequencing
Total RNA concentrations from sixteen samples (eight samples with two biological
repeats) were measured by NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific), and
quality was assessed by BioAnalyzer 2100 RNA Pico kit (Agilent). The samples had RNA integrity
number (RIN) values ranging from 8.5 to 10. Ten nanogram of total RNA from each sample was
used for library construction using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Kit
(Takara Bio), following the manufacturer’s instructions. Deep sequencing of single-end 75 nt was
carried out on NextSeq500 (Illumina). The Illumina sequencing was conducted by USC Molecular
Genomics Core.

Libraries Raw reads Processed
reads
Mapping rate
Chuong675 29,681,072 28,220,335 73.83%
Chuong676 29,477,793 27,983,967 73.49%
Chuong677 27,607,414 26,215,226 72.31%
Chuong678 27,570,230 26,163,957 72.62%
Chuong695 30,754,020 30,072,354 78.98%
Chuong696 24,591,999 24,079,882 78.15%
60
Chuong697 27,504,374 26,935,623 79.73%
Chuong698 30,666,419 29,985,709 78.50%
Chuong699 26,800,723 26,234,859 78.63%
Chuong700 27,317,784 26,736,640 78.36%
Chuong701 31,765,782 31,071,406 76.86%
Chuong702 30,559,056 29,870,444 74.86%
Chuong737 29,087,674 28,190,085 74.77%
Chuong738 27,767,609 26,911,764 75.95%
Chuong739 22,158,973 21,462,867 73.36%
Chuong740 36,018,724 34,812,579 73.68%

61
RNA-Seq data analysis (This part of work is done by Dr. Chi-Kuan Chen)
Low-quality bases and reads were removed by using Trimomatic (Bolger et al., 2014)
according to the following procedure: 1) Remove adaptors; 2) remove leading low quality bases
below Q score 15; 3) remove trailing low quality bases below Q score 15; 4) scan the read with
a 4-base wide sliding window, cutting when the average quality per base drops below 15; and 5)
drop trimmed reads below 36 bases long. The latest Japanese quail genome assembly (version
Coturnix_japonica_2.0) and its annotation file were downloaded from Ensembl FTP. The
processed reads were mapped to the genome using HISAT2 (Kim et al., 2015) with the
parameter: --score-min L,0,-0.7. The statistics of processed and mapped reads were listed in
the table below. The read counts for each gene were obtained from the mapping files using
StringTie (Pertea et al., 2016, Pertea et al., 2015) with the default parameter and the genome
annotation file.

Identification of differentially expressed genes (DEGs)
We identified the DEGs through several sets of comparisons. Set 1: Gene expressions
between dermis and aorta were compared. Set 2: Gene expressions between dermis and aorta
in Tie1+ samples were compared. Set 3: Gene expressions between Tie1+ and Tie1- in dermis
samples were compared. Set 4: Gene expressions in E6 Tie1+ versus those in E6 and E7 Tie1-
dermis samples were compared. Set 5: Gene expressions between E6 and E7 in Tie1+ dermis
samples were compared. Set 6: Gene expressions between Tie1+ and Tie1- in E6 dermis
samples were compared. Set 7: Gene expressions between Tie1+ and Tie1- in E7 dermis
samples were compared. Set 8: Gene expressions between E6 and E7 in Tie1+ dermis samples
were compared. Set 9: Gene expressions between E6 and E7 in Tie1- dermis samples were
compared. Set 10: Gene expressions between E6 and E7 in Tie1+ aorta samples were compared.
Set 11: Gene expressions between E6 and E7 in Tie1- aorta samples were compared. Set 12:
Gene expressions between Tie1+ and aorta dermis samples were compared. Set 13: Gene
expressions between Tie1- and aorta dermis samples were compared. Set 14: Gene expressions
between Tie1+ and Tie1- in aorta samples were compared. These two replicates were compared
with each other. The DEGs from the comparisons were computed by DESeq2 (Love et al., 2014).
Only the genes with q value < 0.05; fold change > 1 were defined as DEGs.

62
CHAPTER 4: Dissection of developmental avian skin adipose tissue:
patterning, markers and lineage

INTRODUCTION

The adipose tissue serves as a multifunctional organ, providing thermoregulation,
metabolism and components of stem cell niches. Adipocytes neighboring other tissues (ie. bone
marrow/hematopoietic cells, muscle, heart/blood vessels) exert a profound influence on the
tissues in several ways (Rosen and Spiegelman, 2014). In the skin, adipocytes play a critical role
in dermal homeostasis and respond to cold exposure, wounding and bacterial infection
(Alexander et al., 2015, Kruglikov and Scherer, 2016). On the other hand, the skin, composed of
several distinct mini-organs, including hair follicles, sweat glands and other sensory units, is one
of the most complicated and the largest organ in the human body. Understanding how the proper
layering and complex skin architecture is made will provide insights into the process of
organogenesis. We are motivated to learn how adipogenesis is incorporated in this process which
may provide us with clues toward tissue engineering skin for clinical practice.
In terms of skin development, avian skin can be an ideal model to uncover new principles
because of their accessibility for experimentation and visibility of phenotypic changes. The pattern
formation in developing avian skin is multiscale on different levels (Chuong et al., 2013): de novo
periodic patterning including epithelial-mesenchymal interactions, reaction-diffusion system and
Turing principles guiding feather primordia formation from the non-patterned morphologic field
(Jiang et al., 1999); the subsequent events (blood vessels, muscle and nerve fibers) use feather
primordia locations as reference points to assemble themselves in the complicated system, called
“adaptive patterning” (Wu et al., 2019). Actually, the concept of adaptive patterning has been
applied to study the relation between hair follicles, nerve innervation and adipose tissue in
mammals. Researchers have shown that during the development of hair follicles, heterogenous
hair follicle stem cells provide a specific niche for nerve innervation (Cheng et al., 2018). Also,
transplanting hair follicles to the skin restores arrector pili muscle and nerve connections (Sato et
al., 2012). Dermal adipose tissues grow concomitantly with hair follicle down growth and the hair
follicle transit-amplifying cells govern adipose tissue and hair follicle expansion through Sonic
Hedgehog (Zhang et al., 2016). Unlike hairs in mammals, avian feathers are interconnected with
each other by integumentary muscles. The topology of feather muscle arrangements differ in the
apteria, femoral tract and dorsal pelvic tracts (Lucas and Stettenheim, 1972). In the integumentary
63
muscle system, we demonstrated muscle fibers extend from feather buds in every direction but
only those able to connect to neighboring buds are eventually stabilized and the network can be
reconfigured, adapting to experimentally altered feather bud arrangements (Wu et al., 2019). After
we explored the developmental principle behind integumentary muscle, we wanted to know if the
adaptive patterning rule also applies to skin adipose tissue.
There are two anatomically distinct skin associated adipose tissues: subcutaneous white
adipose tissue (SWAT) and dermal white adipose tissue (DWAT) in mammals (Driskell et al.,
2014). In rodents, they are separated by the panniculus carnosus, a striated muscle; in humans,
there’s no clear separation of SWAT and DWAT, but several studies have shown that histology,
morphology and metabolism differ between the superficial and deeper skin adipose layer (Kosaka
et al., 2016). Studies have shown murine DWAT has a common precursor with dermal fibroblasts
(Driskell et al., 2013) and develops independently of SWAT (Wojciechowicz et al., 2013). In avian
species, a thin layer of elastic fibers separates the dermis and subcutis. Both SWAT and DWAT
exist in avian species, too. However, the composition of avian and mammalian skin shows many
differences. In addition to the essential differences between feathers and hair follicles, the
epidermis of birds over most of the body is very thin, usually 3-5 cells thick (Pass, 1995) while
mammalian epidermis is usually composed of several different layers with different thickness.
Plus, the whole epidermis of birds is considered to be an oil-producing holocrine gland in which
keratinocytes produce lipids (Spearman and Hardy, 1985), which is important for birds in water
resistance and skin protection. Avian species don’t have brown adipose tissue, but they do have
muscular uncoupling protein-1 (UCP-1) for a non-shivering mode of thermoregulation (Mozo et
al., 2005). The other major difference is the existence of a complicated feather muscle network in
avian dermis as mentioned in the previous paragraph. With all the similarities and differences
between mammals and avian species, we are also interested in the developmental origins of
avian skin adipose tissue which has yet to be explored.
Although previous work has described a close relationship between follicles and
adipogenesis in the mouse model (Zhang et al., 2016{Festa, 2011 #78, Festa et al., 2011)}, little
is known about avian skin adipose tissue. On a macroscale, we examined the patterns of adipose
tissue in embryonic avian skin. We observed that while subcutaneous fat patterns in parallel to
the vasculature, intriguingly, dermal fat macro-patterns match to feather tracts and micro-
patterns to feather muscle. We also sought to find developmental markers for chicken skin
adipose tissue. We found that the CCAAT enhancer-binding protein alpha (C/EBPa) is a reliable
indicator of early adipocyte differentiation, while proliferator-activated receptor-gamma (PPARg)
and fatty acid-binding protein 4 (FABP4) are both expressed in feather muscle and adipose tissue.
64
On a microscale, we use the Cre-loxP lentiviral system to trace the lineage of alpha-smooth
muscle actin-alpha positive (aSMA+) cells in the avian system based on the observed dermal
adipose pattern. By co-staining with CEBPa, we discovered that at least part of the dermal
adipose cell is derived from aSMA+ cells. In the end, we applied an explant and chorioallantoic
membrane (CAM) model to functionally test the “adaptive” rule in adipogenesis. This is the first
research reporting the development of avian skin adipogenesis.

65
RESULTS

Adipose tissues in avian skin display region-specific patterns
To have a general idea of the topological distribution of skin adipogenesis, a deep layer
skin dissection technique is performed under a microscope on E11-E20 chicken embryos. Starting
from E13, we observed some Oil Red O (ORO) mature adipose tissue in the subcutaneous layer
in the dorsal pelvic tract (DPT) (Fig. 1C&D). It is present in a trident pattern originating from the
posterior part of the DPT with its tip at thigh level. As the embryo grows, SWAT doesn’t grow in
proportion to the anterior-posterior axis of the DPT and the SWAT trident sticks to the posterior
part of the DPT. DWAT (Fig. 1C&D) can be separated manually from SWAT in the DPT. DWAT
is not observed until E16, starting from the midline of the DPT and then develops lateral symmetry.
On a macroscale, DWAT is only observed in feather tracts and not in apteria. Initially, DWAT dots
form in the intersection of feather buds and feather muscles at E16 (Fig. 1C&D). Later, the adipose
tissue develops alongside the feather muscle and a grid pattern is formed. This pattern is not
restricted to the DPT, we observed the same DWAT pattern in the sternal, ventral and humeral
tracts. A transverse section of skin showed both SWAT and DWAT develops into depots enriched
with mature adipocytes with lipid drops at E18 (Fig. 1A). Subcutaneous (SQ) and dermal spaces
are anatomically separated by a specific elastic fiber layer (EFL) (Fig. 1A&B) (Pass, 1995). DWAT
was observed above the EFL, under feather muscles and adjacent to feather follicles. To validate
the deep layer skin dissection technique, we cut the skin piece into transverse sections and
immunostained them with an anti-elastin antibody to fluorescently visualize the EFL (Fig. 1B).
Instead of ORO, Bodipy was used to visualize adipose tissue. Intact depots of SWAT and DWAT
suggested that used a sound dissection technique to preserve both adipose layers.

Stepwise assembly of the avian skin adipose tissues
We sought to correlate patterns of different avian skin tissues with their topological
adipose distribution. To achieve higher-level functions, blood vessels and muscles interconnect
feather follicles in avian skin. Their sequential developmental sequence is 1) feather buds, 2)
blood vessels and finally 3) muscles. We therefore used LipidTOX Neutral Lipid Stains available
in three colors, green (lx/em=495/505), red (lx/em=577/609), and deep red (lx/em=637/655), to stain
adipose tissue (Fam et al., 2018) and specific antibodies to detect different tissues in an E16
piece of chicken skin. Tenascin, an adhesion molecule known to be expressed early in feather
buds (Jiang and Chuong, 1992) and follicular mesenchyme (Tucker, 1991), is used to identify
feather tracts (Fig. 2B). Huge adipose patches mainly composed of SWAT are observed in both
66
FTs and DPT. This SWAT patch is located in the middle of the feather tracts and does not cover
the whole feather tracts. LipidTOX positive dots are located in the periphery of feather tracts and
are next to feather follicles. From previous observations, these should be DWAT. Both SWAT and
DWAT patterns are correlated with feather follicles, but in a different way: SWAT appears to be
relegated to feather tracts while DWAT localizes to individual feather follicles.
We asked if the SWAT and DWAT patterns that correlate with feather follicles are also
associated with subsequent vascularization and myogenesis events in developing avian skin.
TIE1:H2B-eYFP transgenic quail (Sato et al., 2010), which express YFP fluorescence exclusively
in the nuclei of endothelial cells, enabled us to determine the relative topology of vasculature and
adipose tissue. Because SWAT develops earlier than DWAT and vascularization develops earlier
than myogenesis, we focus on SWAT and vasculature first. On an E11 TIE1:H2B-eYFP
transgenic quail transverse section, co-localization of eYFP+ and LipidTOX+ areas are observed
in SQ skin (Fig. 3A). (Our study focused before E12-13, when DWAT starts to develop in quail
skin.) To have a general idea of the topological distribution of skin vasculature, a deep layer skin
dissection technique was performed under a microscope on E9-E12 quail embryos. A complex
vascular network is observed in feather skin with higher density in feather tracts then in apteria
(Fig. 3A). Two major vessels in the lateral neck support blood flow to the DPT anterior to humoral
tracts. Posterior to the humoral tracts, the blood network is concentrated in the middle of the DPT.
In a high-power magnification view, LipidTOX+ clusters grow along with vasculature and larger
clusters of fat depots are seen in the neck then those in the back (Fig. 3B). Compared to the
correlation of SWAT and feather follicles, the overlapping pattern of SWAT and vasculature in the
middle of the DPT seems to have a more direct relationship (Fig. 3D).
Next, we focused on the relationship between DWAT and the muscular pattern. Both
alpha-smooth muscle actin-alpha (aSMA or ACTA2) and smooth muscle protein 22-alpha
(SM22a) are differentiation markers of smooth muscle cells. While SM22a is expressed
specifically to smooth muscle cells, aSMA can also be detected in many other cell types such as
pericytes (Skalli et al., 1989). In avian vascular and visceral muscles, aSMA appears 1-2 days
earlier than SM22 (Duband et al., 1993). On E16 chick skin, we found SM22a antibody specifically
stains feather muscles presenting hexagonal and grid patterns in the DPT and the LipidTOX+
dots appear at the intersections of feather follicles and muscle fibers and between muscle fibers
(Fig. 3C). This SM22a staining pattern in E16 chickens correlates to a later DWAT pattern seen
at E20 (Fig. 1C&D).

Expression of adipogenic related markers in avian skin development
67
We asked what differentiation markers can be used to specifically identify preadipocytes
in avian skin. A series of adipose lineage differentiation markers have been discovered in the
mouse model (Fig. 4A). The transition from adipose precursor cells to committed preadipocytes
marks the “determination” of the adipose lineage (Rosen and Spiegelman, 2014). Zfp423, a
critical transcriptional regulator involved early in preadipocyte commitment, amplifies the effect of
BMPs to induce adipose lineage commitment (Gupta et al., 2010). However, Zfp423 is not a
specific marker for preadipocytes in avian skin as it also stains muscle tissues and is not
expressed in nuclei as a transcription factor (Fig. 4B). Other chicken preadipocyte markers
including Delta-like protein 1 (DLK1)(Shin et al., 2008) and Kruppel-like factor 7 (Klf7) modulate
the differentiation of chicken preadipocytes (Zhang et al., 2013). Although, Klf7 is not unique to
preadipocytes as it is expressed in other tissues.
During the transition from preadipocytes to mature adipocytes, there are “early lineage
development” and “terminal differentiation” stages. The early transition from preadipocytes to
adipocytes are orchestrated by transcription factors involving PPARg and members of the C/EBP
family (Farmer, 2006); mature adipocytes express FABP4, leptin and perilipin. Though PPARg is
well known as the master regulator in adipocyte differentiation, in our system, it is also expressed
in muscles (Fig. 4C). Likewise, FABP4 is not specific to fat tissue either. Sequential expression
of C/EBPs is observed during adipocyte differentiation: C/EBPb and C/EBPd are expressed first
with a subsequent increase in the levels of C/EBPa and PPARg (Farmer, 2006). Studies have
shown that among the C/EBPs, C/EBPa might be the most reliable marker of early adipocyte
differentiation in vivo (Lee et al., 1998). In the mouse and rat model, C/EBPa identifies
differentiating preadipocytes in embryonic and neonatal skin (Wojciechowicz et al., 2008). On the
other hand, it was demonstrated that C/EBPa could activate the PPARg gene promoter using a
reporter gene assay in the chicken model (Ding et al., 2011). Therefore, we used a C/EBPa
antibody to study the timing and location of early adipocyte differentiation in chicken skin.
In E13 chick transverse sections, when SWAT is just about to develop, cells with C/EBPa+
nuclei are observed in the subcutaneous layer and do not overlap with SM22 staining in the
feather muscle (Fig. 4D). At E16, SWAT develops into larger adipose depots with both positive
C/EBPa+ and Bodipy staining (Fig. 4E). On the other hand, DWAT is just about to develop with
C/EBPa+ cells in close proximity to feather follicles, but without positive Bodipy staining. Later, at
E18, double positive C/EBPa and Bodipy staining adipose depots can be observed in both the
subcutaneous and dermal layers.

68
SMA+ cells contribute to skin adipose tissue
We wondered about the origin of these two waves of adipogenesis in avian skin. We found
that the SWAT pattern, the first wave of adipogenesis, matches that of the vasculature (Fig. 3D);
while the DWAT pattern, the second wave of adipogenesis, matches that of the feather muscle
network (Fig. 3E). The vasculature is composed of mainly endothelial cells with different kinds of
peri-endothelial mesenchymal cells: pericytes, vascular smooth muscle cells (vSMCs),
fibroblasts, macrophages, and even epithelial cells (Armulik et al., 2011). Pericytes and vSMCs
together, are referred to as mural cells. A combination of PDGFRβ, SMA and NG2 can be used
to identify mural cells (Vishvanath et al., 2017). aSMA+ cells are present in vasculature and
feather muscles. Indeed, using aSMA antibody stains both vasculature and feather muscles.
To track their origin, we used the Cre-loxP lentiviral system to trace the lineage of SMA+
cells in avian skin. The aSMA promoter controls expression of the Cre recombinase with the blue
fluorescent protein (FP)-TagBFP codon (Fig. 5A). This is crossed to a reporter line with a red FP-
DsRed, which is flanked by two loxP sites as the stop cassette and a green FP-EGFP. In this
configuration, when Cre is driven by the aSMA promoter, the stop cassette is excised and then
EGFP is expressed so we can trace all of the daughter cells. Each construct was inserted into a
lentivirus and injected into E2-3 chick somites. Cells infected with SMA-TagBFP-Cre are blue;
those infected with loxP-DsRed-loxP-EGFP are red; SMA+ cells containing both constructs are
green.
By co-staining with a C/EBPa antibody, we can identify the SMA+ cells becoming
preadipocytes. At E15, one day before DWAT, a grid pattern of EGFP expressing feather muscles
is observed (Fig. 5D). After section and quenching the lentiviral colors, several cells with C/EBPa
+ nuclei and GFP cytoplasm were found in close proximity to feather follicles (Fig. 5B). These
were not only present in feather tracts, but we also found such cells in the apteria. Previously in
our feather muscle study, muscle bundles failed to stabilize in the apteria (Wu et al., 2019). We
carried out a TUNEL assay to evaluate if muscle cells undergo apoptosis if they are not stabilized
between feather buds. We found very few TUNEL+ cells. Here, we speculate that cells that “fail”
in one context may be used for other purposes without wasted a valuable cellular resource, thus
guaranteeing the robustness of adaptive patterning in the developing avian dermis.

The skin adipose tissue is adaptive
Lastly, to find a model for adipogenesis to test the idea of “adaptive patterning” we tested the
explant model from E9-12 (to characterize SWAT adipogenesis) to E15 (to characterize DWAT
adipogenesis). However, no adipogenesis was observed in tests using the skin explant culture
69
model (Fig. 6A). We wondered if failure to undergo adipogenesis was because the model lacks
blood flow. We next tried the CAM model, which can provide blood flow to donor tissue (Fig. 6B).
We cultured chick skin, prior to the appearance of adipose tissue, on transgenic quail (PGK:H2B-
mcherry) CAMs and characterized whether adipogenesis occurred. The transgenic quail
ubiquitously express histone 2B-mCherry FP under the control of the human PGK promoter (Huss
et al., 2015), thus allowing us to distinguish donor from recipient cells in the host skin sample.
Adipogenesis can be observed adjacent to feather follicles in the CAM system (Fig. 6C). Here,
recipient cells contribute to muscle cells (Fig. 6D) and adipose cells (Fig. 6C). Thus, we conclude
that while feathers can grow on an explant culture model for more than one week, blood flow is
crucial to adipogenesis. And the principle of adaptive patterning is verified by using the CAM
model in which 1. adipogenesis resembled the in vivo situation in that only occurs adjacent to
feather follicles; 2. while the donor sample is in the process of myogenesis, recipient cells also
participate in myogenesis and a later adipogenesis process.

70

A.
B.
C.
D.
Elastic fiber layer (EFL)
SWAT
DWAT
FF
E14
E19
NO
DWAT
until E16
DWAT SWAT
Bodipy
ELN
DAPI
E14 E15 E16 E17 E18 E19
71
Figure 1. SWAT and DWAT developing patterns in developing avian skin
(A). H&E stain image of an E20 (HH46) chicken dorsal pelvic tract section. Mature adipose tissue is red.
Scale bar: 300 μm.
(B). On the left, a skin section of an E18 chick. Mature adipose tissue is stained with Bodipy (green);
elastic fiber layer (EFL) is visualized by elastin (ELN) stain (red); blue is DAPI. Scale bar: 300 μm. On the
right, the schematic shows the location of DWAT (dermal white adipose tissue) and SWAT
(subcutaneous white adipose tissue), as well as their relative position to feather follicles (FFs) and the
elastic fiber layer (pink).
(C). Mature adipose tissue stained with Oil Red O (ORO, in red) in whole-mount chicken skins from the
deep dissection. The top row shows the specimens with SWAT attached. The middle row shows the
same specimens but with SWAT removed. The bottom row shows the zoom-in view of the dorsal pelvic
tract region from the middle row specimens. Skins from different ages are shown in different columns.
Scale bar: 2 mm (top and middle row), 500 um (bottom row).
(D). The schematics summarize the changing patterns of SWAT and DWAT in the chicken skin from E14
to E19. Mature adipose tissue visualized by ORO are drew in orange. Different grey shades represent
different feather tracts. The grey circles in the lower column represent feather follicles.

72

Figure 2. Patterns of skin adipose tissue vs feather follicle
73
(A). The schematic shows how the “skin stripes” in panel B and figure 3 are obtained. The stripes
generated contain 3 different feather tracts with different densities of feather follicles.
(B). A general view of the tissue stripe containing feather tracts with different follicle densities is shown
on top. Zoom-in views of each tract are listed below. Both SWAT and DWAT are visible on the stripe.
Tenascin (green) labels the feather follicles; LipidTOX (red) labels the mature adipocytes. The tissue is
from a E16 chick. Scale bar: 500 μm.

74

Figure 3. Match SWAT pattern to vasculature; DWAT pattern to feather muscle network
(A). The panel is organized similarly to figure 2B. The stripe here is obtained from a E12 Tie1:H2B-EYFP
transgenic quail so the vessels are labeled in green. LipidTOX (red) labels the mature adipocytes. Scale
bar: 500 μm.
(B). Whole-mount skin with SWAT of a E12 Tie1:H2B-EYFP quail. Vessels are labeled with EYFP (green);
LipidTOX (red) labels the mature adipocytes. Scale bar: 500 μm.
(C). The panel is organized similarly to figure 2B. SM22 (green) specifically labels the feather muscles;
LipidTOX (red) labels the mature adipocytes. The stripe is from a E16 chick. Scale bar: 500 μm.
(D). The schematic shows the relative position between SWAT (blue) and vessels (green), summarized
from panel A and B.
75
(E). The schematic shows the relative position between DWAT (blue) and feather muscles (orange),
summarized from panel C.

76

Figure 4. C/EBPα is expressed in the subcutis and dermis
(A). The major steps of adipocyte development in mammalian systems is summarized here. The
molecular markers of each step are listed underneath.
(B). E13 chicken skin stained with Zfp423 (green) and SMA (red). The SMA or SM22 are used to identify
the myocytes. Zfp423 labels both myocyte-like and adipocyte-like cells. The asterisk indicates myocyte-
like cells. The arrows indicate adipocyte-like cells. Scale bar: 100 μm.
(C). E14 chicken skin stained with PPARγ (green) and SM22 (red). PPARγ labels both myocyte-like and
adipocyte-like cells. The asterisk indicates myocyte-like cells. The arrows indicate adipocyte-like cells.
Scale bar: 100 μm.
(D). E14 chicken skin stained with SM22 (green) and C/EBPα (red). C/EBPα also labels adipocyte-like cells
only in chicken skin. The asterisk indicates myocyte-like cells. The arrow indicates adipocyte-like cells.
Scale bar: 100 μm.
(E). Consecutive E18 chicken skin sections stained with either SM22 (green) and C/EBPα (red) or Bodipy
(green) and elastin (ELN, red). The C/EBPα-positive cells predominantly co-localize with Bodipy-positive
cells, indicating that they would differentiate into mature adipocytes. Scale bar: 300 μm.
77

Figure 5. Lineage tracing SMA+ cells in developing avian skin
(A). The schematics shows the design of the viral lineage tracing system, the experimental workflow, and
outcome interpretation. The tracing system consists of 2 vectors: the first one is a Cre recombinase and
TagBFP expression vector driven by novel identified chicken SMA promoter. The second one is a
reporter vector that expresses DsRed-Express2 in the absence of Cre and expresses EGFP in the
presence of Cre. Therefore, in the cells that are co-transfected with 2 viruses, SMA-lineage cells will
express EGFP and maybe BFP if itself still expresses SMA. On the other hand, non-SMA-lineage cells will
only express DsRed-Express2.
(B). Sections of E15 chicken skin stained with GFP (green) and C/EBPα (red). The fluorescent proteins in
the specimen are all quenched first so that immunofluorescence staining is possible later. C/EBPα is
used to identify adipocyte-lineage cells. The arrows indicate cells that are both GFP and C/EBPα-positive.
Scale bar: 50 μm.

A.
B.
GFP
C/EBPα Merge
Feather
tract
Apteria
78

Figure 6. CAM model for adipogenesis
(A). Oil Red O stain of the chicken skin and cultured skin explant. The second-row images are zoom-in
view of the images in the first row. Scale bar: 2 mm.
(B). Schematic for the hybrid embryo chorioallantoic membrane (CAM) culture system. Wildtype chicken
skin is grafted to the CAM of the PGK:H2B-mCherry transgenic quail. In this system, the cell origin can be
easily identified because the host’s cells are mCherry+.
A. B.
C.
D.
Bodipy mCherry Merge
E11 +
10 days
SMA mCherry Merge
E11 +
10 days
E9
E9 + 8days
79
(C). Section of CAM-cultured chicken skin stained with Bodipy (green). The skin was transplanted from a
E11 chick and cultured for 10 days on CAM. Bodipy is used to identify mature adipocytes. Scale bar: 50
μm.
(D). Section of CAM-cultured chicken skin stained with SMA (green). The skin was transplanted from a
E11 chick and cultured for 10 days on CAM. SMA is used to identify muscle cells. Scale bar: 50 μm.

80
DISCUSSION

Understanding the principles of adaptive patterning during the building of complex skin
architecture would give us insights pertaining to the tissue engineering of human skin for future
transplantation. Over the years, our group’s research has focused on pattern formation in
developing avian skin. Recently, we examined the feather muscle and revealed possible
mechanisms that established muscle patterning (Wu et al., 2019). Yet, we know little about the
patterning of adipose tissue in developing avian skin. Here, we demonstrated that adipose tissue
is assembled in a stepwise fashion just like the feather muscle network. In the very beginning of
embryonic skin development, the vasculature formed after feather condensations earlier than
feather muscle patterning. Later, feather muscles using feather follicles as reference points
develop superficial to the vasculature in skin. Then, in the subcutaneous layer, the first-wave of
adipose tissue grows alongside the vasculature, consistent with the long-held notion that adipose
stem cells are present in a vascular niche. Finally, in late embryonic stage, the second-wave of
adipose tissue grows alongside the feather muscle networks in the dermal layer. And because
both vasculature and feather muscle networks are patterned according to feather follicle positions,
the developmental process of skin adipose tissue also follows feather tracts. This avian skin
development system demonstrates the principle of adaptive patterning: tissue is assembled in a
stepwise fashion, progressing through de novo patterning, neovascularization, myogenesis and
adipogenesis. Tissues that form early serve as signaling centers which promote the development
of later structures. The lineage tracing result demonstrates that mesenchymal cells maintain
plasticity for alternate fate determination during development. The multi-potent SMA+ cells,
poised to become vasculature or feather muscle can have their fate changed under the right
environmental conditions to become adipose tissue. Nevertheless, future studies are required to
uncover what molecules or mechanisms are capable of inducing such cell fate lineage decisions.
Regarding the SWAT, our results are consistent with previous studies. From the
developmental perspective, the earliest fat pad derived from a cluster of blood vessels was
observed in reptiles, chickens, mice and humans (Wasserman F., 1965). The tight apposition of
the vasculature and adipogenesis led to the concept that pericytes and vSMCs together, referred
to as mural cells, are the precursor of adipocytes. Tang et al.(Tang et al., 2008) used PPARγ
reporter mice to demonstrate that early adipose progenitors within the fat pads of young mice
express PPARγ and localized in vivo in the vasculature resembling the mural cell compartment.
They also showed only part of the mural cells express PPARγ and are adipogenic, but almost all
adipocytes are derived from mural cells. Jiang et al. (Jiang et al., 2014) applied the AdipoTrak
81
system (PPARγ-GFP) with mural cell fate-mapping (SMA-RFP) and conditional gene knockouts
to show that adult adipocytes are derived from a mural cell lineage, but not from developmental-
stage adipocytes. However, Guimarães-Camboa et al. (Guimaraes-Camboa et al., 2017) using a
similar lineage strategy with a different marker, Tbx18- CreERT2; Rosa26RtdTomato mice, found
the labeled cells didn’t significantly contribute to other cell lineages. From the avian perspective,
quail-chick chimeras provide a powerful lineage tracing tool, but the drawbacks are that the tissue
contributions are attributed to groups of cells, and thus not precise (Bower et al., 2011). Indeed,
for tissue-specific lineage tracing, the Cre-loxP system is more precise. Though rarely used in
avian systems, Leighton et al. (Leighton et al., 2016) produced a transgenic chicken expressing
Cre recombinase to target excision of a loxP-floxed genome. Here, we constructed the Cre
recombinase under the control of the SMA promoter because we observed the SWAT and DWAT
patterns. In the mouse model, aSMA was expressed in stromal vascular cells, adipose stem cells
and committed preadipocytes (Cawthorn et al., 2012). Our result is somewhat consistent to the
mouse model but identification of each adipose lineage stage in the avian system requires further
studies. In avian skin, SMA+ cells become preadipocytes in the vicinity of feather follicles, but
also within feather follicles adjacent to the major feather blood vessel.
Regarding the DWAT, adipose tissue develops initially around E16 feather follicles and
then grows into a grid pattern matching that of the feather muscle network. The first part of DWAT
development around feather follicles is similar to that found in the mouse model. In mouse skin,
adipose precursors develop independently of early stage hair follicles (Guerrero-Juarez and
Plikus, 2018). Later at the maturation phase, adipose progenitors are coupled with hair follicle
signaling. When hair follicles develop fully in anagen phase, feather follicles appear to be floating
within an adipose cushion. DWAT then is remodeled periodically in coordination with hair follicle
cycle. Though we haven’t dug into the relationship between the feather follicle cycle and DWAT,
the fact that DWAT can develop around the feather follicle in vivo and on the CAM implies a
crosstalk between feather follicles and the DWAT surrounding them. The most distinguishing
feature of avian DWAT is the grid pattern intermingled with feather muscle. One possible
explanation is that feather muscle progenitor cells become preadipocytes. And our lineage tracing
system using SMA promoter serves as a piece of evidence supporting this hypothesis. In the
mouse model, Plikus et al. (Plikus et al., 2017) discovered adipocytes regenerate from
myofibroblasts during wound healing. Long et al. (Long et al., 2014) used Myh11-driven Cre fate
mapping to demonstrate that at least a subset of beige cells arise from a smooth muscle-like
origin and suggested perivascular UCP-1 positive adipocytes are beige cells. Intriguingly, avian
species don’t have brown, beige or UCP-1 positive adipocytes. But UCP-1 is expressed in the
82
muscle system. Further studies are required to reveal whether avian muscle cells share more in
common with preadipocytes or have more plasticity than mice. On the other hand, many cell types
are SMA positive in addition to muscle cells. In a section view, adipose tissue is observed adjacent
to rather than within feather muscles. To our knowledge, muscles are surrounded by muscle
fascia. Su et al. (Su et al., 2016) discovered in rat that fascia serves as a source of adipocytes. In
the future, many questions will have to be answered regarding the origin of DWAT in avian skin.
Here we addressed a question that arose from our previous feather muscle work (Wu et
al., 2019). In the integumentary muscle system, we demonstrated muscle fibers extend from
feather buds in every direction but only those able to connect neighboring buds are eventually
stabilized. In apteria, where the distance between feather buds is long, the muscle fibers are not
stabilized between the feather buds and become long and thin. TUNEL assays were carried out
to see if the muscle progenitor cells in apteria undergo apoptosis if not stabilized. However,
apoptosis was only observed infrequently. We were curious about how those muscle progenitor
cells end up if they don’t become mature muscles or undergo apoptosis. Here, using the SMA+
lineage tracing system, we discovered that SMA+ cells which do not become muscle cells may
choose another path and become adipocytes. The inter-conversion of the mesenchymal cell types
in the dermis, suggest that morphogenesis is competitive, and yet cells that “fail” in one context
can be used for other purposes in another context without being wasted, thus guaranteeing the
robustness of adaptive patterning in the developing dermis.
In summary, we demonstrated the process of adipose pattern formation in avian skin
development both in subcutaneous and dermal layers and discovered a reliable marker, C/EBPa,
for avian skin preadipocytes. We used a lentiviral Cre-lox system, a powerful tissue-specific
lineage tracing tool rarely applied in the avian model and found SMA+ cells in each layer give rise
to preadipocytes and thus SWAT, the first wave of adipogenesis in avian skin development; The
SMA+ pattern matches that of the vasculature; The pattern of DWAT, the second wave of
adipogenesis in avian skin development, matches to that of the vasculature. Furthermore, we
used the CAM system to observe adipogenesis in skin development. The versatile approach of
this study, covering pattern formation, marker identification, and cell lineage differentiation, helps
us reveal the “adaptive patterning” principle and provides us an opportunity for further studies.

83
MATERIALS AND METHODS

Experimental model
Fertilized pathogen-free (SPAFAS) chicken eggs were from and staged according to the
method described by Hamburger and Hamilton(Hamburger and Hamilton, 1951). Fertilized wild-
type Japanese quail eggs were from (Westminster, CA); fertilized transgenic Tie1:H2B-eYFP
and PGK:H2B-mcherry quail eggs were from USC translational imaging center and are staged
according to Ainsworth et al. (Ainsworth et al., 2010). Eggs were incubated at a temperature of
38°C with 60-65% humidity with turning every 2 hours.

Specimen harvesting and processing
Embryos staging from HH39~45, for section purpose, we removed head, four limbs,
internal organs and feathers, only back area, from skin deep to spine and dorsal ribs, is preserved.
The samples are fixed with 4% formaldehyde (Sigma) overnight. For cryosection, we changed
samples to PBS and then 15% sucrose in PBS the next day. After samples sunk to the bottom of
the vial, samples were changed from 15% sucrose in PBS to 30% sucrose in PBS. And then the
samples were embedded in mounting media (Tissue-Tek
R
O.C.T™ Sakura) and snap frozen on
dry ice, and stored at -80ºC. For wholemount purpose, skin samples were taken from the back
area including dorsal-pelvic, apteric, and femoral tracts. After removal of feathers, skin was peeled
and a No.15 scalpel blade was used to assist preservation of deep layer skin fat (deep layer skin
dissection technique). And then fixed with 4% formaldehyde (Sigma) overnight. For DWAT
dissection, after removal of feathers, the subcutaneous layer was removed together with elastic
fiber layer after ORO staining.

Lipid detection: Oil Red O, Bodipy and HCS LipidTOX Deep Red neutral lipid staining
Oil Red O: Sections between 10 and 16um thick were cut on a cryostat (Microm HM505E),
air dried for up to 90 minutes at room temperature, and then were washed in PBS or running
water. Next, sections were rinsed in 60% isopropanol (Sigma) and stained with a filtered Oil Red
O solution (0.5g Oil Red O in 100ml isopropanol and then diluted with ddH2O in a 3:2 ratio) for
15 minutes (wholemount samples: 30-60 minutes). Section slides were then washed in 60%
isopropanol, ddH2O and then counterstained in Mayers Hematoxylin Solution (Thermo) and
mounted in 50% glycerol (Sigma) in PBS (no counterstaining for wholemount samples). ORO
analysis was performed using a Keyence BZ-X710 microscope for section slides and a Nikon
SMZ1500 microscope for wholemount sample.
84
Bodipy and HCS LipidTOX Deep Red neutral lipid staining: We performed this in the
process of immunofluorescence staining after the secondary antibody staining was done or on
transgenic quail samples. Following PBS wash, section slides were incubated with Bodipy (diluted
in PBS or DMSO at a concentration of 1mg/ml, ThermoFisher) for or HCS LipidTOX Deep Red
neutral lipid stain (1:200, Invitrogen) at RT for 45 minutes. Section slides were then stained with
4’, 6-diamidine-2’- phenylindole, dihydrochloride (DAPI) (Sigma) for 15 minutes and mounted in
50% glycerol (Sigma) in PBS (no DAPI staining for wholemount samples).

Immunofluorescence staining
Cryosections slides were air dried for up to 90 minutes at room temperature, and then
were washed 3 times in 0.1% Triton-100 (BIO-RAD) in PBS (wholemount samples were washed
with 0.5% Triton-100 in PBS). Alternatively, skin samples after 4% formaldehyde (Sigma) fixation
overnight were sequentially dehydrated (in 50% to 75% to 85% to 95% to 100% ethanol (Gold
Shield) and then xylene (Thermo)), and embedded in paraffin wax (McCormick). Immunostaining
was performed on paraffin sections with heat-based antigen retrieval as required. Sections slides
were then blocked with 0.1% BSA(Sigma) in PBS for 30 minutes (wholemount samples were
blocked with universal blocking buffer for 2 hours). Both sections and wholemount samples were
incubated with the primary antibodies overnight in 4 ºC. The primary antibodies used were mouse
anti-elastin (1:100; Millipore), rabbit anti-ZFP423 (1:50; Abcam), mouse Klf7 (1:100; Santa Cruz),
mouse anti-PPARγ (1:100; Santa Cruz), mouse anti-C/EBP-a (1:100; Santa Cruz), goat anti-
FABP4 (1:50; R&D), mouse anti-SMA (ready to use), rabbit anti-SM22 (1:100, Abcam), rabbit
anti-GFP (1:200, Abcam). The next day, sections and samples were incubated with the secondary
antibodies after 0.1% Triton-100 in PBS (wholemount samples were washed with 0.5% Triton-
100 in PBS). Alexa Fluor anti–mouse-488 (1:200), anti-rabbit-488 (1:200), anti–mouse-546
(1:200), anti–mouse-594 (1:200), anti-rabbit-594 (1:200) from Invitrogen were used as secondary
antibodies. DAPI was used to visualize the nuclei for section slides. Both stained sections and
wholemount samples were mounted in 50% glycerol in PBS and were imaged with a Leica
confocal microscope.

Lineage tracing (This part of work is done by Dr. Stephanie Di-Shan Tsai and Dr.
Masafumi Inaba)
1) Identify SMA promoter in chicken
For the SMA promoter-reporter in vivo system, the chicken SMA promoter sequence was
amplified from ACTA2 (-1039~+2337, NC_006093.5) and then subcloned between the PacI and
85
BamHI sites of FUGW, which was a gift from David Baltimore (Addgene plasmid # 14883;
http://n2t.net/addgene:14883; RRID:Addgene_14883).
2) Plasmid construction
For the lentiviral loxP reporter construct, the loxP-DsRed-loxP-EGFP sequence was amplified
from pLV-CMV-LoxP-DsRed-LoxP-eGFP, which was a gift from Jacco van Rheenen (Addgene
plasmid # 65726; http://n2t.net/addgene:65726; RRID: Addgene_65). This loxP sequence was
then subcloned between the AgeI and BsrGI sites of FUGW. For the lentiviral reporter construct
containing SMA promoter and Cre: TagBFP was amplified from pHR-SFFV-dCas9-BFP-KRAB,
which was a gift from Stanley Qi & Jonathan Weissman (Addgene plasmid # 46911;
http://n2t.net/addgene:46911; RRID: Addgene_46911). Cre was amplified from Cre ERT
plasmid, a gift from Marianne lab. The SMA promoter, TagBFP, P2A, and Cre sequences were
later subcloned into the PacI and Bam sites of pUltra, which was a gift from Malcolm Moore
(Addgene plasmid # 24129; http://n2t.net/addgene:24129; RRID: Addgene_24129).
3) Lentivirus production
To be short, each lentiviral construct was co-transfected with pCMV-dR8.2 dvpr (a gift from Bob
Weinberg, Addgene plasmid # 8455; http://n2t.net/addgene:8455 ; RRID: Addgene_8455) and
pCMV-VSV-G (a gift from Bob Weinberg, Addgene plasmid # 8454; http://n2t.net/addgene:8454
; RRID: Addgene_8454) into 293T cells culturing with DMEM (containing 1% P/S and 10%
FBS). The suspensions are ultra-centrifuged (Beckman L8-80M Ultracentrifuge), resuspended
with HBSS, and injected at HH 18 chicken embryos.

Chorioallantoic membrane (CAM) culture model
Donor: an area of interest about 5*5mm
2
piece of skin was dissected out on chick back
before there is adipose tissue development around E9-E11. Recipient: E6-7.5 transgenic quail
(PGK:H2B-mcherry), which ubiquitously express histone 2B-mCherry FP under the control of the
human PGK promoter (Huss et al., 2015), eggs were prepared for transplantation. A window and
its underlying membrane were meticulously opened on shell to expose CAM. Donor skin piece
from chicken was carefully placed on CAM. The window was resealed with adhesive tape and
eggs were returned to the incubator until harvested at estimated day of adipogenesis.
86
CHAPTER 5: Conclusions and perspectives

Starting from tissue engineering, we made hair peg-like structures from dissociated
human skin progenitor cells and revealed periodic patterning principles. From the perspective of
wound regeneration, we studied the spiny mouse hair cycle and features of spiny mouse skin.
To understand how the next level of tissue assembled in the developing skin, we used the avian
skin model to decipher the principles. We revealed the spatial temporal distribution of vascular
and adipose tissue and how vascular and adipose cells were incorporated in the developing
avian skin. We connected sequential events after de novo patterning in developing avian skin,
neovascularization, myogenesis and adipogenesis using RNA-seq and a cell lineage tracing
system. The versatile approach of this dissertation, covering tissue engineering, regeneration
and adaptive pattern formation, helps us understand how Nature makes a complex skin system
and provides opportunities for further studies.

Tissue engineering the human hair follicle
In “Self-organizing hair peg-like structures from dissociated skin progenitor cells:
New insights for human hair follicle organoid engineering and Turing patterning in an
asymmetric morphogenetic field”, we have demonstrated that the dissociated human fetal
scalp dermal cells between E17 to E19 and neonatal foreskin keratinocytes together can form
hair peg-like organoids with a specific cell ratio to obtain the best efficiency. After injection to the
subcutis of nude mouse skin, mature hair follicles composed of cells of human origin can be
observed 2-3 months after the injection. Compared to the mouse reconstitution model (Lee et al.,
2011), the developmental process is very different (Lei et al., 2017), and the efficiency of hair
follicle production is low. In mice, the epidermal cells aggregate first. And the epidermal cysts
formed with dermal cells surround them. The cysts coalesce and the epidermal and dermal layers
are polarized. Thus, a planar skin forms, and dermal condensations form later at D11 in an in vitro
culture. However, in the human reconstitution system, the keratinocytes form a sheet at the
bottom first. Then, dermal aggregates form on the sheet. The dermal aggregates develop into
dermal caps with an epidermal stalk after 4 days in culture, looking like a hair peg structure. We
contributed the differences and the low efficiency to epidermal cell plasticity, the inducing ability
of dermal cells and morphogenic field competence.
Another way to analyze the process is from the perspective of biomechanics. The fast
spread of epidermal cells onto the supporting membrane in the human system suggests that
87
human epithelial cells might exhibit a stronger cell-substrate adhesion capacity than that of mouse
embryonic epithelial cells. Collapse of the entire epidermal-dermal mixture in the human system
when the ratio of epidermal to dermal cells is compatible with that of the mouse system suggests
that human dermal cells might exhibit a stronger contractility than that of mouse dermal cells, a
behavior that is consistent with why keloids exist primarily in humans. Thus, if we can a) prevent
human dermal cells from collapsing the entire co-culture system and b) allow human epidermal
cells to develop layer-like structures with a proper polar orientation facing the dermal cells, we
might be able to reproduce mouse reconstitution results in the human system.
We speculated that human dermal cells exhibit a strong contractility to collapse the entire
co-culture. Such force arises from long-range cell-ECM-cell interactions and can be attenuated
by adjusting the density of dermal cells or the density/composition of the ECM. Human epidermal
cells possess a strong tendency to develop a monolayer on supporting membranes, which can
be used to develop two-layer lumen-like structures by overlaying cells with ECM. In the future, we
plan to optimize the 3D culture system with a type I collagen gel (Fratzl, 2008, Guo et al., 2012)
to enable epidermal cells to self-organize into a system-wide two-layer system. Intermediate
application of scattering factors such as hepatic growth factor (HGF) and TGF-beta might be used
to facilitate the remodeling of epidermal structures. A small fraction of ECM that does not interact
with dermal cells (such as laminin and matrigel) might be added into the mixture to attenuate the
long-range contractility of dermal cells, without a significant influence on the motility of epidermal
cells.

Spiny mouse wound regeneration
The ultimate goal of treating defects in patients is regeneration, which is regaining the
original tissue architecture with normal functions after injuries. However, wound healing in
humans are achieved by repair, which causes scar, pain and deformity. In “Comparative
regenerative biology of spiny (Acomys cahirinus) and laboratory (Mus musculus) mouse
skin”, we used the African spiny mouse (Acomys spp.) and laboratory mouse (C57Bl/6, Mus) as
models with exceptional and limited regenerative abilities to reveal the differences in their wound
healing processes. We compared and identified several new findings between Acomys and Mus,
including: the composition of hair types, hair follicle stem cells, hair cycle and responses to
different kinds of wounds. These data highlight the regenerative biology of the spiny mouse skin
and serve to inspire potential new principles to promote human skin regeneration.
One interesting finding of our results is spiny mouse skin contains a high content of
adipose tissue. The thick adipose layer may contribute to the skin regenerative ability in two ways:
88
providing a stem cell niche and the formation of a lower tension wound. First, the soft wound bed
found in adiponectin-deficient mice produced severe delays in wound epithelialization (Shibata et
al., 2012). Adipoctyes increase in number and serve as a niche component and synthesize stem
cell factor to promote hematopoietic regeneration after endothelial cells and leptin receptor+
stromal cells are depleted (Zhou et al., 2017). Second, in mechanobiology, it was suggested
tension, which means a stiff environment, can induce an immune response (Harn et al., 2019).
Clinically, manipulating wound tension with a pressure garment or purse-string suture was
demonstrated to be effective for scar prevention and treatment. The thick adipose layer indeed
provides a softer environment for wound healing in terms of mechanobiology. Since skin autotomy
is a survival instinct enabling the spiny mouse to escape predation, it’s possible this thick layer of
adipose tissue is the key to regeneration. We will focus on this unique spiny mouse feature in
future studies.

Adaptive patterning during skin formation: the assembly of vasculature
In chapter 3, we explored the features, induction and mesenchymal cell fate plasticity of
the developing avian skin vasculature. Before dermal condensation induction, endothelial cells
(ECs) were seen in lateral and medial line skin. After feather buds form, the endothelial cells under
each feather bud, starts to build connections. The patterns of Tie1+ cells are correlated with
feathers buds. Epithelial-mesenchymal interactions were associated with expansion of the
vascular structure in avian skin: the vascular pattern diminished without epidermal cues; after
separating and recombining the epidermis rotated 90
o
to the original skin dermis orientation, we
observed neovascularization follows epidermal cues. We found pro-angiogenic factors of feather
placode and condensations induce neovascularization. Like in other organs, avian skin expressed
VEGF and FGF2 in feather placodes and condensations to induce neovascularization. Ectopically
induced FGF2 and VEGF further induced endothelial cell differentiation or triggered existing
endothelial cell migration. On the other hand, interference with the neovascularization process
using RCAS-noggin and RCAS-spry2 in turn resulted in abnormal feather development. Lastly,
the transcriptome profile of developing skin neovascularization and the aorta demonstrated a cell
fate plasticity in skin neovascular cells.
Morphologically, the avian vasculature developmental pattern described in this
dissertation was analyzed before E8 because after E8 the fluorescent signal would be obscured
by melanin. But really, there is a lot left for us to explore after E8. While short buds grow into long
buds, the invagination begins at HH37. At HH38, feather follicles form and pulp cells are produced
by the papilla. Feather keratinization starts at HH40. At HH42, the pulp begins to be resorbed.
89
We are also interested in how vasculature participates and reorganizes itself in these important
feather development events. We tried to use a very high-power laser for confocal imaging to see
the vascular pattern inside the feather but only obtained a vague signal in the presence of melanin.
In the future, we plan to make a lentiviral Tie1 reporter system and inject it to chick embryos so
that we would be able to see vascular development inside the feather. Also, we want to improve
our time-lapse movie quality to make cell tracing possible.
Mechanistically, we identified several transcription factors involved in skin
neovascularization in the RNA-seq data. Further quantitative RT-PCR for FACS H2B-eYFP
+
cells
is required to validate the RNA-seq results. Also, overexpression and knockout functional studies
or suppressing gene expression using RNAi and small molecules are required to make this
research more complete. Since we identified the cell fate plasticity of the H2B-eYFP
+
cells, we
plan to make a Cre-lox system using the Tie1 promoter to trace the lineage of these cells.
Furthermore, while Notch activity is necessary for smooth muscle development, BMP signaling is
required for endothelial cell differentiation (Ben-Yair and Kalcheim, 2008). Besides, the
ectodysplasin receptor (Edar)-BMP signaling pathway is essential for the primary hair follicle
pattern in the mouse skin explant model(Mou et al., 2006) and BMP/Smad was found to regulate
the vegf gene in the angioblast of zebra fish embryos(He and Chen, 2005). Thus, BMP signaling
may be a connection between de novo pattering and neovascularization, yet we haven’t explored
it yet in the avian skin model. In addition to the vasculogenesis and angiogenesis processes,
arterial, venous and lymphatic specification that happens later in vascular lineage development
hasn’t been explored yet in avian skin. Other than that, we haven’t yet applied tissue engineering
to neovascularization, which is the next step towards our goal to apply the principles we’ve learned
translationally.

Adaptive patterning during skin formation: the assembly of adipose tissue
Adipose tissue was found to contribute to the stem cell niche and has a great potential in
cell-based therapies. Here, we demonstrated that tissue is assembled in a stepwise fashion and
we dissected the anatomy, correlated our findings with other tissues and used lineage tracing of
the developing avian skin adipose tissue. First, we identified that the patterns of white adipose
tissue in the subcutis (SWAT) and dermis (DWAT) are different. Here, the SWAT develops and
matures earlier than dermal fat. Second, we identified regional specificity of the developing SWAT
and DWAT. While the SWAT pattern correlates to blood vessels, DWAT is mainly observed in the
feather growing area (Macro-patterning) and the pattern follows feather muscles (Micro-
patterning). Third, we identified that C/EBPa (CCAAT enhancer-binding protein alpha) can be
90
used as an adipose lineage marker in avian skin because it is expressed within the nucleus of
adipocytes, not in feather muscle. A common factor was identified in both the vascular system
correlating to SWAT and the feather muscle network correlating to DWAT: smooth muscle cells.
Therefore, we traced the lineage of SMA+ cells using a Cre-lox lentiviral system in avian skin and
revealed that SMA+ cells function as part of the vasculature and feather muscle; some of those
will turn into adipose progenitor cells (C/EBPa+). Lastly, we found that adipogenesis didn’t occur
in explant cultures but could happen on when explants were grown on the CAM (chorioallantoic
membrane) model. Skin explants cultured on the CAM before adipose tissue develops, can form
adipose tissue which develops adjacent to feather follicles. Here, the recipient not only provides
blood flow, but recipient cells also participate in musculogenesis and adipogenesis of the donor
skin.
One interesting finding of our results is that SMA+ cells, as part of the vasculature and
feather muscle, turn into adipose progenitor cells. Associated with vasculature, it is known that
under certain conditions, part of the adipose tissue is derived from mural and smooth muscle-
related cells (Jiang et al., 2014). Associated with myogenesis, it was shown that adipocytes
regenerate from myofibroblasts during wound healing (Plikus et al., 2017). However, researchers
haven’t identified adipogenesis from embryonic feather muscle progenitors. This observed
phenomenon gave us an explanation that arose from our previous study of the feather muscle
network (Wu et al., 2019). While muscle progenitors arise from all directions, no matter whether
they initiate in the feather tract or apteria, only those attached between feather buds are stabilized.
We were wondering where the muscle progenitors went or if they underwent apoptosis. However,
experiments blocking neovascularization or musculogenesis processes are needed to further
address the adaptive patterning principle. Also, the mechanism of how muscle progenitors
become adipose progenitors still needs further study. It was demonstrated that dermal progenitor
aggregates broke morphological and molecular symmetries through contractility-driven cellular
pulling (Shyer et al., 2017). Concurrently, dermal cell aggregates trigger the mechanosensitive
activation of b-catenin in adjacent epidermal cells, initiating the follicle gene expression program.
For myogenesis development, mechanobiology specifically determines cellular proliferation and
differentiation (Brosig et al., 2010). Therefore, we plan to use ROCK inhibitors, known to relax
skin or blebbistatin, the myosin II inhibitor, first to manipulate the feather muscle forming process
and then to further explore the role of mechanobiology.
Taken together, this dissertation encompasses tissue engineering, wound regeneration,
and the adaptive patterning of avian skin vasculature and adipose tissue. In Chapter 2, we
explored de novo patterning using a human hair peg-like organoid model and characterized spiny
91
mouse skin with unique regenerative abilities. In Chapters 3 and 4, we revealed the adaptive
patterning principles in developing avian skin and in both vasculature and adipose tissues. We
found a common feature of mesenchymal cell fate plasticity by transcriptome profiling vascular
progenitors and SMA+ cells. We hope that the progress encapsulated within this dissertation can
be applied to guide stem cells and support organoid development to form specific tissues and
organs required for medical treatment.
92
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Abstract (if available)

Abstract Making a fully functional skin is a challenge to tissue engineering because the skin has complex architecture composed of epidermal and dermal layers, as well as appendages such as hair/ feather follicles, sweat glands and touch domes. Assembly of this architecture requires cell fate specification and environmental cues at proper time and space. Our long-term objective is to understand how these components are laid out in development and to apply these principles to help us reconstitute tissues. The arrangement of dermal condensations in skin, i.e. de novo patterning, is the first patterning event in skin development. Continued organogenesis may lay their patterns using patterned structures as reference points via adaptive pattern formation. Adaptive patterning has been implicated in the formation of intradermal muscles, adipose tissues, blood vessels and nerves. ❧ In this dissertation, we studied periodic patterning from dissociated human skin progenitor cells and in wound induced follicle neogenesis in the spiny mouse from the perspective of tissue engineering. In an effort to generate human hair primordia more effectively, we developed an in vitro platform to study the self-organization behavior of hair progenitor cells and to identify the factors that impact feather primordia formation. The hair peg-like structures emerging from this 3D culture system in 96 hours were molecularly validated, resembling hair pegs found in normal human follicular development. After transplantation to nude mice, mature hair follicles of human origin were produced. We studied spiny mice (Acomys cahirinus) because of their unique scar-free wound healing capabilities, producing complete regeneration of epidermal appendages and dermis after injury. Spiny mouse skin contains a large portion of adipose tissue and has a larger hair bulge with high stem cell marker expression (K15 and CD34). Also, the spiny mouse hair cycle is longer and cycles less frequently than that of the laboratory mouse (C57Bl6). The features we identify by comparing wound healing in spiny and laboratory mice may contribute to improve the regenerative ability of the spiny mouse which could have broad reaching applications to regenerative medicine. ❧ Next, we studied adaptive patterning of vasculature and adipose tissue. For the assembly of vasculature in skin development. Tg(tie1:H2B-eYFP) quail expressing YFP fluorescence exclusively in the nuclei of endothelial cells (ECs) provides a good model to study the neovascularization process because it enables us to evaluate the topology of vascular progenitor cells in living tissues. H2B-eYFP⁺ cells were found to aggregate immediately to the newly formed feather primordia. Angiogenic factors, e.g. FGF2 and VEGF, were found highly expressed in the primordia epithelium compared to the dermis. These results suggest that the vasculature network is initially patterned in a feather epithelium-dependent manner. On the other hand, by locally overexpressing sprouty2 and noggin, known to inhibit angiogenesis, feathers showed abnormal phenotypes at later developmental stages after feather follicles form. Then, we asked whether the feather vasculatures are newly formed or derived from major vessels. Transcriptome analysis showed that the profile of H2B-eYFP⁺ cells in skin (as the 2nd wave of neovascularization) resembles H2B-eYFP⁻ cells in skin more than H2B-eYFP⁺ cells in aorta (as the 1st wave of neovascularization), implying new vasculature in the skin is locally induced. Surprisingly, Tie1+ cells in skin, compared to those in aorta, express much higher level of genes for morphogenesis other than neovascularization, such as smooth muscle formation and adipogenesis, implying an “interconversion” status exists for the mesenchymal cells during early development. ❧ For the assembly of adipose tissue in skin development, we investigated the distribution pattern, molecular markers and progression of adipocyte differentiation in embryonic chicken skin. Interestingly, we identified a close anatomical association between feather tracts and adipocytes and that the distribution pattern of subcutaneous fat resembled vasculature organization. Within the feather tract, dermal fat is distributed in parallel with the feather muscle network. Next, we identified C/EBPα as a reliable preadipocyte marker located in the nucleus. Lineage tracing shows that some SMA+ cells in developing avian skin give rise to preadipocytes by co-staining with an anti-C/EBPα antibody. Finally, we found adipogenesis is highly dependent on the presence of a blood supply because it only occurred in the chorioallantoic membrane (CAM) model but not in the explant culture model. Taken together, these findings suggest that in gross anatomy, avian species have a similar arrangement of adipocytes to that in the skin of mammalian species but the avian dermal adipose layer is different. Avian species have a complex muscle network and the distribution of dermal fat tissue is associated with feather muscle pattern. A SMA+ cell lineage tracing study connects the patterning among fat, muscle and vasculature. Some SMA+ cells in the vasculature and feather muscle were found to become adipose progenitors (C/EBPα+), but the contribution of this pathway remains to be quantified. The CAM model can support embryonic skin adipogenesis and we have taken advantage of this model to test the role of “adaptive patterning” during development. ❧ In summary, we explored the principles of tissue patterning in skin development step-by-step using different models. It is straight-forward to use human hair follicle precursors to engineer human hair follicles, transplant them to host skin and study periodic patterning for translational medicine purposes. Furthermore, we set out to discover the unique spiny mouse skin features that are responsible for this enhanced regenerative ability. To explore how to improve the hair peg-like structures that we engineered, we asked how nature builds new structures next to mini-organs to vitalize and organize them into functional units. Therefore, we studied the adaptive patterning of vasculature and adipose tissue in avian skin to know how they are assembled into this network. The phenomena and knowledge acquired in this study will be applied to the assembly and regeneration of complex tissues to facilitate functional wound healing in regenerative medicine.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Ou, Kuang-Ling (author)

Core Title Adaptive patterning during skin formation: assembly of vasculature and adipose tissue

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 08/14/2020

Defense Date 01/15/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag adaptive patterning,adipogenesis,integument,mesenchymal tissue,neovascularization,OAI-PMH Harvest,transgenic quail

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chuong, Cheng-Ming (committee chair), Jiang, Ting-Xin (committee member), Lansford, Rusty (committee member), Maxson, Robert (committee member), Paine, Michael L. (committee member)

Creator Email kuanglio@usc.edu,kuanglio085@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-270787

Unique identifier UC11673608

Identifier etd-OuKuangLin-8178.pdf (filename),usctheses-c89-270787 (legacy record id)

Legacy Identifier etd-OuKuangLin-8178.pdf

Dmrecord 270787

Document Type Dissertation

Rights Ou, Kuang-Ling

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA