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Macroenvironmental regulation of hair follicle stem cells

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Macroenvironmental regulation of hair follicle stem cells

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Content MACROENVIRONMENTAL REGULATION OF HAIR FOLLICLE STEM CELLS
by
Damon Nino de la Cruz
___________________________________________________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)
August 2012
Copyright 2012 Damon Nino de la Cruz
Dedication
This work is dedicated to people without whom this Dissertation would not have
materialized:
My wife Shawna, thank you for everything
My parents, all the work and sacrifice was worth it
To those people whose negative reinforcement propelled me toward my goals
ii
Acknowledgements
Without these people this Dissertation would not be possible:
My mentor Dr. Cheng-Ming Chuong for guiding me through the Amazon
Dr. Randall Widelitz, his companionship and insight were invaluable
Dr. Maksim Plikus for helping me explore Nature and Science.
iii
Table of Contents
Dedication ii
Acknowledgements iii
List of Figures viii
Abstract x
Chapter 1: Introduction 1
The Regenerative Hair Cycle 3
Circadian Rhythms: An external signalling phenomena 8
Neuroendocrine signalling in hair follicle maintenance: a remote signalling
phenomena 13
Subcutaneous Adipose tissue: an adjacent signalling phenomena
21
Macroenvironmental regulation of hair follicle stem cells: understanding extra-niche
signalling phenomena 32
Figures 33
Chapter 2: The Macroenvironment 39
Background 39
Results 42
Cyclic Dermal BMP Signalling Regulates Stem Cell Activation During Hair
Regeneration -Nature Jan 2008 42
Self-Organizing and Stochastic Behaviors During the Regeneration of Hair Follicles-
Science April 2011 44
Summary 45
Figures 46
Chapter 3: Circadian Rhythms as a stem cell modulator 50
Background 50
Results 53
Summary 55
Figures 57
Chapter 4: Pregnancy Telogen Hair Wave Reset 63
Background 63
Results 67
Summary 70
Figures 75
iv
Chapter 5: Subcutaneous Adipose Development 85
Background 85
Results 89
Summary 91
Figures 93
Chapter 6: Discussion and Future Directions
99
Circadian Rhythms as a Stem Cell Modulator 100
Pregnancy Telogen Hair Wave Reset 103
Subcutaneous Adipocyte Development 110
Future Directions 114
Materials and Methods 117
References 125
v
List of Figures
Chapter one Introduction
Figure 1: Hair Cycle 33
Figure 2: Biosynthetic pathways of steroid hormones 34
Figure 3: Steroid hormone signal transduction 35
Figure 4: Prolactin signal transduction 36
Chapter Two: The Macroenvironment
Figure 5: A propagating hair wave 46
Figure 6: Days of Anagen/Telogen 46
Figure 7: Pregnancy induced hair wave reset 46
Figure 8: Bmp signalling in the subcutaneous adipocytes 47
Figure 9: Hair wave propagation in rabbit 47
Chapter Three: Circadian Rhythms as a stem cell modulator
Figure 10: Circadian rhythm core loop 57
Figure 11: K14cre transgene 57
Figure 12: Mouse genotype 58
Figure 13: K14cre, R26R cross 58
vi
Figure 14 :Bmal1 null mouse hair wave 59
Chapter Four: Pregnancy Telogen Hair wave Reset
Figure 15: Hormone signature during pregnancy 75
Figure 16: Hair wave during pregnancy 76
Figure 17: Signalling morphogens in the skin during pregnancy 78
Figure 18: Bead implantation experiment 79
Figure 19: Prolactin receptor expression in skin compartments during
pregnancy/lactation 80
Figure 20: Morphogens downstream from Prolactin signalling 80
Chapter 5: Subcutaneous Adipose Development
Figure 21: Adipocyte maturation relative to Hair follicle development in mouse
skin 93
Figure 22: In Situ Hybridization of adipocyte markers in developing skin 94
Figure 23: Summary of signalling molecule expression 96
vii
Abstract
The burgeoning field of stem cell research is expanding beyond the initial
studies that involved localizing, isolating and culturing of the cells. How stem cell
homeostasis is regulated is now the focus of current research. Even more
nascent is the idea of how homeostasis is regulated from signals outside the
defined stem cell niche or microenvironment. The extra-niche area is defined as
the Macroenvironment. This includes adjacent tissue, distant organ systems and
even the external environment. To investigate the macroenviroment as a stem
cell regulator, hair follicle stem cell interactions with 1)adjacent tissues
(subcutaneous adipose tissue), 2)remote signalling (neuroendocrine signalling),
and 3)the external environment (circadian rhythms) were studied. Capitalizing on
recent work that highlighted inhibitory Bmp2 signalling from the subcutaneous
adipose tissue to be in phase with quiescent telogen and non-propogating
autonomous anagen portions of the hair cycle we investigate the post-natal
development of the dermal fat layer in relation to hair follicle development.
During pregnancy in humans and mice modulation to the hair wave, hair cycle
and ultimately hair follicle stem cells has been recorded in both classical and
current literature yet no studies have been recorded that investigate this
phenomena in detail. Using a murine system it is shown show that the hair wave
viii
is reset to a telogen resting phase as a consequence of prolactin signalling late in
pregnancy and throughout lactation. Additionally, it is observed using in situ
hybridization that the prolactin signalling is mediated via the increased
expression of prolactin receptors associated with the dermal fat layer and
subsequent expression of Bmp2. In the murine model hair grows in a cyclic
fashion. Although much of the cycle has been elucidated the source of cycle
timing still eludes hair biologist. Using current mouse technologies from the field
of circadian rhythms we investigate the possible role of peripheral clocks in the
skin by ablating the core component Bmal1 from the circadian clock system in a
skin specific knock out model, K14creER
tam
, Bmal1 mice. These studies have
impact on not only the field of hair biology but on a larger scale stem cell
research, regenerative medicine and pathologies involved with adipose tissue,
hormonal signalling involved with cancers, and circadian rhythms.
ix
Chapter 1: Introduction
The 1831 voyage of the H.M.S. Beagle is well known. Observations made
on that journey by the ship’s naturalist named Charles Darwin significantly
changed the way humans evaluate the interplay of organisms and the
environment in which they reside (Darwin, C. 1839). Undeniably, for an organism
to succeed it must adapt to the surrounding environment. Evolution occurs when
an organ or an organ system changes to impart a selective advantage. Therefore
the success of an organism is contingent on evolving tissues and by necessity,
its component cells. Years beyond Darwin’s postulations we understand that
evolution begins at the cellular level. The idea that a successful response to the
environment starts at the cellular level is an underlying theme in cell biology.
Stem cells can be loosely defined as a subset of unspecialized cells that
have the potential to self-renew while in an undifferentiated state, the capacity to
give rise to various differentiated cell types and the ability to replenish lost cells
throughout an organism’s life. The idea that a cell possesses the ability to self
renewal has massive implications in human health and disease and regenerative
medicine. Although these cells are inherent to all systems and tissues they are
considered to reside in a specialized niche in which the stem cell associates
closely with other cells that determine its behavior (Schofield, R. 1978). This
idea was been expanded to encompass all of the cellular components of the
microenvironment surrounding stem cells as well as the signals that emanate
1
from the support cells (Li et al., 2005). Because stem cells could offer obvious
utility in medicine as well as broad implications in many disciplines of research
including but not limited to developmental biology, efforts to isolate these cells
from tissues became an important part of research.
Because stem cells can be derived from embryos there are ethical issues
surrounding the use of these cells. One way to circumvent ethical issues is to
reprogram somatic cells, this was first achieved through transferring the nuclear
contents of somatic cells into oocytes, recall “Dolly” the cloned sheep (Wilmut et
al., 1997). More recently differentiated cell reprogramming was shown to be
achieved by introducing four factors, Oct3/4, Sox2, c-Myc and Klf4, to adult
mouse fibroblasts in culture (Takahahi et al. 2006). Later it was described how
these same four factors could reprogram differentiated cells when delivered via
one vector (Carey, B.W. et Al. 2008). Even more recently the efficacy of hair
follicle cells that had been reprogrammed to promote the regeneration of nerve
tissue was reported (Yasuyuki, A et Al. 2009).
2
The Regenerative Hair Cycle
The idea that hair follicles can be important in stem cell research and
regenerative medicine is fairly novel. It is generally accepted that the hair follicle
is a “mini-organ” that arises from embryonic skin progenitor cells, and house a
distinct stem cell population (Millar 2005). The hair follicle “mini-organ” was
thought to form only during development and if lost, i.e. injury etc., the loss was
permanent. However, contrary to this thinking it was observed that de novo hair
follicle regeneration does occur in the healing field of large wounds (Ito et al.
2009).
Hair follicle development is fairly well understood. Briefly, after
gastrulation a layer of multipotent epithelial cells that comprise the skin develops.
Underlying this field is a layer of mesenchymal cells, the dermis. The
mesenchymal cells interact with the overlying epithelium to induce the formation
of follicular placodes. After the formation of the bud there is a downgrowth of the
epidermis and the hair follicle matures (Fuchs 2007). Hair follicle spacing is
determined via WNT and its associated inhibitor DKK (Dickkof related protein)
signals (Sick et al. 2006). In adult mice hair fiber integrity is maintained by cyclic
regeneration of the hair follicle (Stenn and Paus, 2001). Hair fibers are produced
3
during the anagen growth phase as proliferative cells at the base of the hair shaft
differentiate from matrix cells. After a period matrix cell proliferation ceases and
the follicle regresses into the catagen phase. The termination of catagen is
marked by the onset of the telogen phase a resting period. Telogen is followed
by a return to the growing phase, anagen. Therefore, the phases of the hair
cycle can be directly correlated to stem cell activity (Figure one). (Goldstein et al.
2012).
The hair follicle stem cell niche was first described to reside in the matrix
area of the hair bulb. These studies were done by taking advantage of the fact
that stem cells are slow cycling in nature. Tritaited thymidine was used to label
populations of cells, the slow cycling cells would retain the radioactive signal,
conversely cells that were proliferating at a faster rate would lose the signal
(Cotsarelis et al. 1990). Later, the same group identified that cytokeratin 15 was
a unique marker for label retaining cells in the hair follicle bulge (Cotsarelis et al.
1998). More recently a second set of stem cell markers has been delineated. A
small intestine stem cell marker, Lgr5 (leucine-rich G Protein coupled receptor 5),
was observed to be expressed in hair follicles. In the intestine Lgr5 is a marker
for cells that are actively proliferating and multipotent stem cells. In the hair
follicle Lgr5 expression is observed in the bulge region and the lower outer root
sheath (Jaks et al. 2008). Lgr6 (leucine-rich G Protein coupled receptor 6) a
close relative of Lgr5, is expressed in the central isthmus, the area that lies
between the sebaceous gland opening and the bulge (Snippert et al. 2010).
4
Defining the mechanisms that drive the various stages of the hair cycle is
important to understanding how stem cells are regulated. When a hair is in the
anagen phase the stem cells are activated and during telogen stem cells are
quiescent. Several activating molecules have been implicated in anagen, Noggin
(Botchkarev et al. 2001; Plikus et al. 2008), Follistatin (Nakamura et al. 2003)
Beta-catenin (Fuchs et al. 2004), Fgf7 (Fibroblast Growth Factor), Tgf-beta 2
(Transforming Growth Factor) (Plikus 2012). Several members of the
transforming growth factor family, generally considered inhibitory molecules,
have been implicated in telogen including activin and Bmp2/4 (Bone
Morphogenic Protein) (Nakamura et al. 2003, Plikus et al. 2008). In a biological
system fluctuating levels of the above diffusible activators and inhibitors manifest
as the hair cycle.
The study of one hair follicle as a single unit becomes geometrically
interesting when one takes into consideration thousands of hair follicles over the
entire organism. Hair biology research focused on individual hair follicle until
recently. These studies were able to discern regulatory factors residing within the
hair follicle but largely ignored a role for extra-follicular regulation. Work from
the Chuong lab demonstrated that at the population level, in mice and other
organisms, a follicle communicates with its neighboring follicles, the result is a
coordinated phasic timing. This produces a propagating hair wave, where a
follicle that is in anagen stimulates its neighbors to enter anagen, similar to a
slow moving forest fire. This mechanism of this coordination is not yet fully
5
understood but molecular interplay between activators such as Wnt and Noggin
and inhibitors such as BMP have been strongly implicated (Plikus et al. 2008,
2011). Signal transduction between neighboring follicles can be visualized in a
noninvasive manner in the murine model. When a mouse is shaved areas of
anagen can be seen as dark pigmented areas. The dark coloration is an
accumulation of hair fiber associated pigments. On the same mouse there may
be areas that are not dark but rather, pink, these areas are where the hair
follicles are in telogen. By shaving a mouse to reveal its underlying pigmentation
pattern it is possible to see which stem cell populations are active, represented
as follicles in anagen, and which populations are quiescent visualized as follicles
telogen (Figure 5).
The idea that stem cell populations are activated over a field as observed
in the propagating hair wave is a novel concept. This idea moves away from the
concept first suggested that described stem cells as being closely associated
with support cells within the specialized niche in which they reside (Schofield, R.
1978, Li et al., 2005), their microenvironment. Extra-niche signalling has been
termed Macro-envrionmental. The idea of macro-enviromental regulation is
emerging in the literature as it pertains to hair follicle stem cells. Extra-niche
signals such as BMPs, FGFs, PDGFs (platelet derived growth factors), and Wnts
can activate stem cell activity in the hair follicle (Festa et al. 2011). A number of
intrinsic molecular mechanisms gate the transition of follicular stem cells from
quiescence to activation, additionally numerous cell types influence the activity
6
and characteristics of hair follicle stem cells (Goldstein et al. 2012). Observations
have been made that a quiescent state in the follicular system is maintained not
solely by the niche microenvironment but also by the larger dermal
macroenvironment (Plikus 2012).
Pioneering papers in the burgeoning field address some of the pertinent
questions that are relevant to the concept that hair follicle stem cells and
therefore the hair cycle are modulated by the environment outside the stem cell
niche (Festa et al. 2011; Plikus2012). Although the hair cycle is fairly well
defined the major control of the hair cycle remains a mystery, potentially the cycle
is governed to some degree by a well known oscillator, the circadian clock.
Recent papers have also illustrated that physiological states such as pregnancy
can have profound effects on the hair cycle. Additionally, the dermal fat layer, a
tissue compartment distinct from the hair follicle, has been implicated in
maintenance of a quiescent state in hair follicle stem cell (Plikus et al. 2008;
Festa et al. 2011). Although these factors that regulate hair follicle stem cell
activity have been eluded to in the literature they have not been well defined.
These three macroenvironmental regulators will be addressed in greater detail
within this dissertation.
7
Circadian Rhythms: An external signalling phenomena
As the earth revolves around the sun on an annual basis seasonal
changes are experienced. These changes are marked by alterations in the
intensity of sunlight exposure to the surface of the earth resulting in variations in
day length relative to night length as well as fluctuations in temperature. In a
smaller temporal span, the earth’s 24 hour rotation on its axis results in light
oscillations that we understand to be day and night. Adaptation to these
environmental oscillations is necessary for the biological organisms on earth to
survive.
Members of the animal kingdom respond to seasonal changes in a variety
of ways; examples are seen throughout nature. Seasonal migration and mating
in response to food sources and climates in loggerhead turtles and humpback
whales among many other species have been reported (Wibbels et al. 1990;
Stevick, et al. 2010). Hibernation, as observed in bears and squirrels, is a
physiological adaptation that at the molecular level is marked by a myriad of
biochemical alterations including seasonal variations in lipid management
(Platner et al. 1971). The timing of physiological modulations, such as the molting
of antlers and coat modifications in red deer, are in response to changes in
season. These modifications have been observed to, at least in part, be acting
through hormonal pathways (Goss 1984; Thornton 2001). Additionally, avia are
well known for seasonally driven migrations for reproduction as well as molting in
8
response to seasonal cues (Gwinner 2003).
Similarly, organismal responses to daily light fluctuations are observed
throughout nature. One of the most massive migrations on earth occurs on a
diel, or daily, basis. Nightly, an enormous mass of zooplankton migrate upward
in oceanic water columns to warmer water, during daylight hours these
organisms swim downward in the water column to escape predation (Reichwaldt
et al. 2005). Bats are often considered the quintessential nocturnal animal, but
many other animals operate primarily during dark hours including mice and rats.
Conversely, there are many examples of diurnal species in nature including man
and dog (Plytycz et al. 1997; Saper et al. 2005).
Because light cues appear to have a marked impact throughout the five
kingdoms of life there has been a significant amount of research invested in
elucidating how light cues are integrated by organisms. Coordination with the
external environment is maintained by an internal clock that regulates Circadian
rhythms of gene expression (Bell-Pendersen 2005). The entomology of circadian
literally translates from Latin Circa( meaning “around”) dia (“day”), this term is
applied to the endogenous biological clock system that governs rhythmic output.
These endogenous clocks impart survival by enabling organisms to make
behavioral and physiological changes by anticipating daily environmental
changes. Buttressing the idea that circadian clocks are important to the survival
and adaptation is the fact that the genetics of this system are conserved across a
broad range of species (Panda et al. 2002). Unicellular alga have been observed
9
to possess as few as two circadian oscillators (Morse et al. 1994), whereas
higher organisms possess multiple clocks.
In mammals there exists a central clock that governs all other peripheral
clocks. This clock resides in the suprchiasmatic nucleus, a small region of the
brain that sits on top of the optic chiasm in the anteroventral region of the
hypothalamus. (Welsh et al. 1995; Bell-Pendersen 2005). Environmental light
cues are entrained by the retinohypothalamic tract connecting the SCN and the
eyes (Klein et al. 1991). Ablation and rescue experiments with the SCN in rats
determined the importance of the SCN in endogenous oscillator maintenance
(Ralph et al. 2009). The core of the oscillatory process is controlled by a delayed
negative feedback loop (Harmer et al. 2001). The favored model of the
mammalian circadian oscillator is comprised of four clock genes. The negative
aspects of the feedback loop, Period (Per1,2) and Cryptochrome (Cry1,2) are
activated early in circadian day by heterodimers of CLOCK and BMAL1. As
nuclear levels of PER and CRY elevate transcription of Clock and BMAL1 cease.
Eventually levels of PER and CRY in the nucleus decrease and CLOCK and
BMAL1 are again transcribed (Hastings et al.2004) .
In addition to the central clock housed in the SCN there are peripheral
clocks in multiple systems. Using a PER2 luciferase reporter Yamazaki et al.
2000, were able to show that liver, lung and skeletal muscle also expressed
circadian oscillations. Following this study, peripheral circadian clocks have been
shown to be important in a variety of other tissues and organs. Rhythmicity in
10
the liver has been studied extensively. The liver is important in metabolism and
energy homeostasis of the entire organism. This is achieved by rhythmic control
of biochemical reactions maintained by the circadian clock of the liver distinct
from the SCN (Schmutz et al. 2011) . Expanding on the study by Yamazaki that
illustrated a peripheral clock existed in skeletal muscle, it was recently shown
that the muscle clock phase can be shifted temporally by altering exercise times.
This suggests that there are multiple inputs for timing synchronization of
circadian clocks (Wolff and Esser 2012).
Limited Circadian Rhythm studies have been performed on skin and even
fewer studies have occurred in an attempt to correlate the periodicity of follicular
regeneration to a circadian oscillations. In a study involving human subjects
mRNA levels of the circadian genes Clock, Per1, Cry1 and Bmal1, were studied
within biopsies from oral mucosa and skin. It was observed that in skin there is a
circadian gene profile that is consistent with that previously published on the
SCN of mice, peaking in early morning, late afternoon and at night. Additionally,
using a thymidylate synthase marker for the synthesis phase of the cell cycle
illustrated that there was some coordination of cycle based on circadian gene
expression (Bjarnason et al. 1999,2001). In mouse studies a differential
expression of mPer1 was observed in skin when mice were subjected to different
lighting conditions, mPer2 mRNA levels were similar between both groups. This
was interpreted as a differential regulation of mPer1 and mPer2 mRNA in both
the SCN and peripheral tissues (Oishi et al. 2002).
11
The basis of the hair cycle is a perplexing question to hair biologists. A
consequence of molting, a means to control the length of the hair, a function to
cleanse the surface of the body, even to adapt to social conditions and to allow
flexibility to adapt to the surrounding environment (i.e. seasonal changes),
however the forces that perpetuates the cycle are unknown (Stenn and Paus
2011). To date few studies have been done in an attempt to link regulation of the
hair cycle to circadian rhythms. A recent study revealed that in mice with mutant
CLOCK and Bmal1 genes there was a delay in first anagen progression and that
the hair germ compartment of the hair follicle lacked mitotic cells. Molecular
analysis of these mice showed an upregulation of p21, a gene significant in the
block at the G1 phase of the cell cycle suggesting the delay in first anagen to be
based at gating during the cell cycle (Lin et al. 2009). While the proceeding
work certainly expanded the knowledge of how circadian genes may play a role
in follicular maintenance there still remains a question of what governs the
regenerative cycling in adult mice that have moved beyond the synchronized first
anagen to a state in which multiple hair wave domains have begun to cycle
independent of each other. It is my goal to investigate the role that circadian
rhythms might play in the regulation of the hair cycle.
12
Neuroendocrine signalling in hair follicle maintenance: a remote
signalling phenomena
Procreation is inherent to all living things. Inheritance of genetic make up
allows for propagation of successful traits while allowing for modifications to less
successful traits that may impart advantage. In mammals, procreation is
comprised of multiple phases, one of which is pregnancy, the carrying of
developing offspring within an individual. Pregnancy is marked by myriad of
physiological and biochemical changes to the individual such as alterations to
protein metabolism, immune function, neuroendocrine signalling and food
consumption (King 1975, Roberts et al. 1996; Neville et al. 2002; Douglas et al.
2007) to accommodate the reproductive state and modified needs of the
individual(s).
In humans, pregnancy related modification to the hair cycle has been
reported, in one study catagen was described as delayed during pregnancy
(Ebling 1964; Winton and Lewis 1982). In classical literature it has been
described that during the postpartum period there is a diffuse loss of hair. Using
a dissecting microscope to examine depilated hairs and morphological
comparison of anagen and telogen hairs it was observed that at the onset of
pregnancy an average of 85% of hairs were in anagen while in the postpartum
13
months the average number of anagen hairs dropped to 65%( Lynfield 1960).
The author speculates that the alterations to the hair cycle are based on a
changed endocrine constellation experienced during pregnancy. The author also
notes that in animal models hormonal modifications have been observed but
cautions the reader when comparing human models to mouse models because
in the mouse hair growth is coordinated over a region and in humans, each hair
grows autonomously.
Interestingly, modulation to the mouse hair cycle during pregnancy has
also been observed. In classical literature, studies done on sexually dimorphic
hair growth on mice included a brief description of the hair growth of pregnant
mice as “naked” (Fraser and Nay 1953). More recently, the modulation to the
hair wave was described in greater detail, during pregnancy and lactation female
mouse hair follicles transition to telogen and are unable to re-enter anagen until
after lactation has ceased (Plikus et al. 2008). Although this phenotype has been
loosely described further research needs to be done to elucidate the timing of the
transition to telogen and the molecular pathways that might be involved.
The observation that hair follicles, an epithelial organ, are being modulated
during pregnancy is not surprising. There are other examples of epithelial
reorganization during pregnancy. The mammary gland is remodeled during
pregnancy where a pre-pregnant branched duct system develops into a
lobuloalveolar expansion seen through pregnancy and lactation. In the post
lactation period the mammary gland involutes, decreasing to a pre-pregnant
14
branched ductal system (Hennighausen and Robinson 2001). At the cellular
level, in uterine epithelium, alterations are seen in both cytoskeletal elements and
junctional adhesions (Luxford and Murphy 1991, and Murphy 2006). Epithelial
organ modification to accommodate the reproduction needs is also observed in
avia. In birds, around the time of egg laying, a “brood patch” develops. This
phenomena involves feather loss, and vascularization of the ventral apterium
(bare area on birds) and an increase in tactile sensitivity. Brood patch formation
has been postulated to allow for heat transfer during egg incubation and to be
mediated by a combination of prolactin, estrogen and progesterone (Hutchison et
al. 1967). In rabbits, nest building during pregnancy occurs during the last third
of pregnancy and is a systematic process. It is comprised of digging a burrow,
lining the burrow with collected straw and plucking hair with which to line the
straw. These activities have been correlated to changes in plasma
concentrations of estradiol, progesterone and prolactin(respectively). In
experiments administering bromocriptine, a prolactin inhibitor, to rabbits in the
last third of the nest building phase a decrease in straw collecting and hair pulling
was observed relative to controls (Gonzales-Mariscal et al. 1996).
Modifications to hair follicle epithelium as a result of fluctuations in
circulating levels of hormones is not surprising. Several receptors for pregnancy
related hormones are reported to be expressed in various compartments of the
hair follicle compartment. These receptors are Estrogen receptors alpha and
beta, Progesterone and Prolactin. (Ohnemus et al. 2004; Pelletier and Ren,
15
2004; Craven et al. 2006). Estrogen and Progesterone are steroid hormones,
the sex steroid biosynthetic pathway has been previously described. Briefly,
steroids are synthesized from cholesterol, which is converted to pregnenolone by
p450scc in mitochondria by the removal of a side chain. At this point synthesis
pathways can be directed towards progesterone and the corticosteroids or to the
androgen pathway. Estrogens are converted from androgens. Pregnenolone is
17alpha hydroxylated by P450c17, the subsequently converted to
dehydroepiandrosterone by the 17-20-lyase activity of P450c17. 3-beta-
hydroxysteroid activity converts dehydroepiandrosterone to androstenedione, this
reaction is reversible. In another reversible reaction androstenedione is
converted to testosterone by 17-beta hydroxysteroid dehydrogenase.. In a
nonreversible reaction testosterone is converted to 5 alpha dihydrotestosterone
(DHT) in a NADPH dependent reaction by 5alpha-reductase. Androgens,
testosterone, or androstenedione can be converted to estradiol, by the enzyme
17-beta-hydroxysteroid dehydrogenase. This reaction is reversible (Figure two)
(Mayer et al., 2004). Androgens are secreted by the adrenal glands and the
ovaries (Burger 2002). The mediators of hormone actions are their
corresponding receptors. Hormones are lipid soluble and pass through the cell
membrane, bind their cognate receptors in the cytosol of the cell and are
translocated to the nucleus (Figure three) (Mangelsdorf et al. 1995).
16
Estrogens are synthesized primarily in the ovaries and adrenal glands,
there are however, other sites of aromatase activity that contribute to peripheral
estrogen including but not limited to the mesenchymal cells of the skin and
adipose (Simpson 2000, 2003) and the central nervous system (Roselli and
Resko 1987). Estrogen is important in the development of males and females
and is required for maturation of the neuroendocrine-gonadal axis in both sexes
(Alonso and Rosenfield 2002). Estrogen has two cognate receptors, estrogen
receptor alpha and estrogen receptor beta. These two receptors have been
observed to have exhibit dynamic responses to ligand binding (Damdimopoulos
et al. 2007). Estrogen receptor beta is known to have a wider tissue distribution
than Estrogen receptor alpha including the gastrointestinal tract and lung
(Younes and Honma 2011). In the skin, estrogen is known to accelerate wound
healing (Gilliver et al, 2007; 2010). Estrogen related receptor skin expression
has been studied, estrogen receptor alpha is limited to the sebocytes while beta
is expressed in the dermal papilla, inner sheath cells, matrix cells and outer
sheath cells including the bulge region (Thornton et al. 2003; Pelletier and Ren
2004). There is conflicting data on estrogen receptor expression in the hair
follicle, presumably this may be due to studies performed at different times of the
hair cycle that may lead to differential gene expression. In a later study both
subunits of the estrogen receptor were detected at the mRNA and protein level
during the hair cycle in mice. This group showed that estradiol can arrest the
hair wave in telogen and induce catagen. However, an antagonist for the
17
receptors does not induce anagen. Immunostudies by the group report
coexpression of the receptor types throughout the skin and hair follicle. However,
expression fluctuates temporally (Ohnemus et al. 2005).
Progesterone is a steroid receptor that binds one of the two the
progesterone receptors with high specificity. The two receptors are similar in
structure but progesterone receptor b is the longer of the two. As a lipophilic
steroid hormone, progesterone diffuses easily through the lipid bilayer of the cell
membrane. Once in the cytoplasm it binds one of the receptors and translocates
to the nucleus, binds DNA and transcription occurs(Yang et al. 2011). In
progesterone receptor null mice ovulation fails due to a disabled follicular rupture
in response to gonadotropin stimulation suggesting a role in ovulation
maintenance (Conneely 2010). During pregnancy progesterone signalling has
been observed to maintain myometrial relaxation and as parturition ensues a
decrease in progesterone receptors from myometrial cells is observed,
suggesting a dynamic role of progesterone during pregnancy (Mesiano et al.
2011).
Progesterone receptors have also been localized to the skin and some
follicle compartments. Radio-receptor assays were used to detect progesterone
receptor in the skin of pre and postmenopausal women. Additionally
progesterone immunostudies revealed that the nuclei of some keratinocytes
throughout all layers of the epidermis and some sebocytes were positive for
receptor expression. However, hair follicle reactivity was not observed (Pelletier
18
and Ren 2004), and in some reports skin tissue was negative for progesterone
receptor (Li et al. 2008).
Prolactin is secreted by the pituitary gland. Levels of prolactin are
moderated by dopamine (Bernichtein et al. 2010). There are three forms of the
prolactin receptor a long form composed of 610 amino acids, an intermediate
form composed of 412 amino acids, and a short form composed of 310 amino
acids. The three forms have common extracellular domains but differ in the
sequence composition and length of their cytoplasmic domains. Upon ligand
binding, receptors dimerize and a signals are transduced via the Jak/stat
pathway to act upon target genes (Figure 4) (Hu et al. 1998). Prolactin was
originally identified for its role in mammary gland development but is now known
to have more than 300 separate actions such as salt regulation, growth and
development and behavior (Bouilly et al. 2011).
Expression of both prolactin and the prolactin receptor have been reported
in human hair follicles (Foitzik et al. 2006).Immunohistochemistry exhibited
expression of prolactin receptor to the hair follicle, epidermis and sebaceous
glands of mice (Craven et al. 2001). Later, prolactin and its associated receptor
have also been shown to be expressed in different compartments of the hair
follicle based on phases of the hair cycle. Immunostudies performed during
telogen illustrated prolactin positive cells in the outer root sheath of the hair
follicle. During anagen both the inner root sheath and the outer root sheath were
positive for prolactin (Foitzik et al. 2003) . Studies using prolactin receptor
19
knockout mice revealed decreased telogen, conversely prolactin treatment
delayed re-entry into anagen (Craven et al. 2006). Recent studies have shown
that ectopic estrogen treatment upregulated the expression of both prolactin and
its receptor in the skin (2009).
Taken together this information points in a direction that the hair wave
reset observed in mice during pregnancy could be a manifestation of
neuroendocrine signalling experienced during the pregnancy and lactation
period. This phenomena may be similar to the nest building requirements
observed in rabbits, hair plucking driven by hormonal cues. The temporal
parameters of the reset have only been briefly described and require further
analysis. Additionally, the mechanism through which the hair cycle locks in
telogen has yet to be elucidated. It is my goal to elucidate the mechanism
through which the pregnancy associated hair wave reset occurs.
20
Subcutaneous Adipose tissue: an adjacent signalling
phenomena
A tissue or organ that is not necessary for survival is usually either
eliminated or reduced greatly during development. These vestigial organs were
thought by Darwin to be evidence of evolution (Scadding 1981; Naylor 1982).
Although not considered a vestigial organ adipose tissue was thought to exhibit
limited function as a support tissue and to provide protection from the external
environment in the form of impact resistance and thermoregulation. However the
past fifteen years have seen an accumulated interest in adipose. The increased
interest can be attributed discoveries in regulation of adipocytes, adipocyte
specific pathologies and utility in stem cell research.(Rosen et al, 2006; Rasheed
et al. 2008; Sugii et al. 2010). Currently, according to the center for disease
more than one third of Americans are obese
( www.cdc.gov/obesity/data/trends.html). Additionally, obesity is associated with
other disease states such as diabetes and cardiovascular disease. These facts
poise adipose research at a significant position in health care research.
Adipocytes are emerging in the field of stem cell research due to the abundant
store of stem cells contained within the adipocyte depot milieu (Ferris and
Crowther 2011). Adipocytes are mesenchymal cells and compared to other
mesenchymal sources of stem cells such as umbilical cord, or bone marrow,
21
these adipocyte stem cells are relatively easy to harvest and are a practical
source of material for experimental and possibly clinical applications (Ding et al.
2011). Additionally, it was recently reported that adipose tissue house a variety
of multipotent cells including endothelial progenitor cells, white fat progenitor
cells and immune progenitor cells (Tallone et al. 2011). Importantly, adipose
derived stem cells fit the criteria of a mesenchymal stem cell. This is based on
several characteristics, the marker expression of CD73, CD90, CD105. The
ability to differentiate into a variety of cells including adipogenic, chondrogenic
and osteogenic cell lines and they lack expression of hematopoietic lineage
markers (Witkowska-Zimny et al. 2011). When adipose derived stem cells are
collected typically mature adipocytes are discarded. Adding to the utility of
adipose tissue, recent work has shown that these mature adipocytes can be
collected and dedifferentiated to a lipid free fibroblast state.
Adipose stem cells are multipotent and have the ability to differentiate into
not only adipocytes, but also chondrocytes, myocytes, epithelial, neuronal and
osteoblast lineages. In addition to the differentiation potentials, these cells have
they can also serve in other capacities, for example as support cells and aid in
gene transfer (Gimble and Guilak 2003a, Baer 2011). Differentiation of adipose
derived stem cells into a several tissues has been shown. Chondrogenic tissue
was generated using transforming growth factor beta and muscle tissue was
generated using myoD and myogen. In the presence of ascorbate, beta
glycerophosphate and dexamethasone adipose stem cells differentiated into
22
osteoblasts (Gimble and Guilak 2003b). Hepatocyte-like cells were the result of
adipocyte stem cells cultured with Vascular endothelial growth factor(VEGF),
Interlukin 6( Il-6), neural growth factor( NGF) and hepatocyte growth factor(HGF)
(Battah et al. 2011).
Because adipose derived stem cells are now recognized as a versatile
source of material for regenerative medicine cell culture systems are being
improved. Using subcutaneous adipocytes cultured in a 3D spheroid manner
resulted in cells that maintained both proliferative and multipotential properties
(Kapur et al. 2012). Regenerative medicine is capitalizing on the advantages of
adipose derived stem cells. In nerve regeneration adipose stem cells are
important in protection of the distal end of the nerve where schwann cell
migration occurs (Dadon-Nachum et al. 2011). Recent autologous
transplantation studies have shown the effectiveness of adipocytes in the
treatment of systemic sclerosis, a cutaneous disease (Scuderi et al. 2011). In
skin, adipose derived stem cell have been shown to be beneficial to regeneration
of cutaneous wounds (Cherubino et al. 2010, Collawn et al. 2011, Reichenberger
et al. 2012).
Undoubtedly one of the primary functions of adipose tissue is storage of
lipids for times of feast and lipid mobilization in times of famine. In an organism
these functions are regulated at the cellular level and systemically by circulating
hormones and signals from the nervous system. The mature adipocyte is
comprised of a single lipid droplet in a thin layer of cytoplasm, the cellular
23
membrane hosts a variety of receptors specific to adipogenic function.
Adipocytes are derived from pluripotent mesenchymal stem cells. Many of the
details of adipocyte differentiation have been elucidated from in vitro studies
due to the inherent difficulties experienced when studying lipids in vivo (Tang and
Lane 2012). Generally adipogenesis is thought of as occurring in two phases.
The first involves commitment of pluripotent stem cells to the adipocyte lineage.
In this stage there is a conversion of mesenchymal a stem cell to a pre-
adipocyte, this adipocyte precursor is morphologically similar to to the stem cell
except it has lost the potential to differentiate into other cell types, C/ebp alpha is
a marker for committed but immature adipocytes (Wojciechowicz et al. 2008).
The second phase of adipogenesis is known as terminal differentiation. In this
phase pre-adipocytes take on the characteristics of mature adipocytes. Mature
adipocytes are defined as possessing the machinery for lipid transport and
synthesis, insulin sensitivity and secretion of adipocyte specific proteins (Rosen
et al. 2006). Pre-adipocyte factor-1 is known to inhibit adipocyte differentiation
(Shen et al. 2009). Conversely, several transcription factors are important in
adipogenesis including but not limited to, CCAAT/ enhancer binding protein alpha
(C/EBPa), Peroxisome proliferator-activated receptor gamma (PPARg), and
adipocyte fatty acid binding protein, (ap2 or FABP4) (Nam and Lobie 2000;
Poulos et al. 2010).
24
Pre-adipocyte factor-1 (Pref-1 also referred to in the literature as Delta-like
protein 1 DLK1) is an epidermal growth factor transmembrane protein. Pref-1 is
synthesized as transmembrane protein but is cleaved to create a soluble form,
the structure of this molecule is conserved over several species including human,
mouse and cow (Deiuliis et al. 2006; Kim et al. 2006; Andersen et al. 2009).
Although associated with several tissues, in adipocytes pref-1 is specific to pre-
adipocytes. Pref-1 inhibits adipogenesis via sex determining region-Y box 9
(Sox-9). Expression of Sox-9 results in the suppression of C/ebps. Pref-1
increases MEK/extracellular signal regulated kinase phosphorylation in a time
and dose dependent manner. Activation of MAPK kinase/ERK prevents down
regulation of Sox-9( Kim et al. 2006; Sul, 2009). Therefore, for adipogenesis to
occur Sox-9 must be down regulated via pref-1 modulation. A down regulation of
pref-1 is essential for adipogenesis to occur (Sumuano et al. 2008). By inhibiting
adipogenesis mesenchymal stem cells are directed to other lineages such as
skeletal muscle (Waddell et al. 2010). Misregulation of adipogenesis as a result
of pref-1 misexpression can result in human pathologies related to obesity
(O’Connell et al. 2011). Recently it was shown that fibronectin and pref-1 interact
to mediate the inhibition of adipogenesis (Wang et. al. 2010). Luciferase reporter
assays suggest that the transcription factor E2F1 regulates the activity of pref-1,
this evidence was further substantiated in knockdown experiments using shRNA
against E2F1 to decrease mRNA of pref-1 (Shen et al. 2009). Foxa-2, a
transcription factor, has also been implicated in the activation of pref-1.
25
C/EBPs are members of the basic leucine zipper family of transcription
factors (House et al. 2010). C/EBPa is activated early in adipogenesis. Culture
system treatment of adipocytes with differentiation inducers (DMEM with 10%
fetal bovine serum., 1uM dexamethasone and 0.5 mM 3-isobutyl-1-
methylzanthine) triggers an adipogenic cascade which involves the early
expression of C/EBPa (Tang, et al. 2004). The expression of C/EBPa in
adipogenesis is conserved across several species including human, mouse and
porcine (Ding et al. 1999; Schmidt t al. 2011) suggesting an important role in the
evolution of adipocyte differentiation. During development C/EBPa expression
increased in prenatal periods based on western blot analysis and
immunohistochemistry (Lee et al. 1998). In addition to a role in adipogenesis
C/EBPa has a role in the development of lung (Martis et al. 2006), sebaceous
gland (House et al.), and tumor suppression, via cell cycle regulation, at the G1
checkpoint. (Loomis et al. 2007; Thompson et al. 2011).
At the molecular level C/EBPa maintains adipose tissue by activating ppar
gamma expression (Sumuano et al. 2008). During adipogenesis SIRT1 mRNA
expression is upregulated, overexpression experiments suggested that C/EBPa
was instrumental in the regulation of SIRT1 during adipogenesis. SIRT1 is
known to play an important role in insulin sensitivity (Jin et al. 2010). Adiponectin
is also known for its role in insulin maintenance, luciferase assays have
suggested that C/EBPa has a role in adiponectin activity (Qiao et al. 2005).
Additionally, it has been suggested that glucose uptake is regulated by C/EBPa
26
via GLUT4 expression (Cha et al. 2008). Regulation of C/EBPa is not well
understood but one study suggests that transforming growth factor beta (TGF-
beta) may be acting through the Smad 3,4 pathway to repress C/EBP function
(Choy and Derynck 2002). Interestingly, immunohistochemistry analysis of
developing mouse skin showed that there is a close association of C/EBPa
expressing adipocytes and developing hair follicles in mouse skin
(Wojciechowicz et al. 2008).
Fatty acid binding proteins are a family of proteins that are essential to
intracellular fatty acid transport and comprised of nine tissue specific members
(thus far) (Chmurzynska 2006). There is homology among FABPs across tissues
and species suggesting that they are important from an evolutionary aspect
(Fischer et al, 2006; Mercade, et al. 2006; Akiduki and Imanishi 2007). These
proteins reversibly bind fatty acids thereby maintaining cell membrane integrity
by protecting the cell from accumulating free fatty acids by transporting these
free fatty acids to various compartments in the cell (Gorbenko et al. 2006,
Ordovas 2007). Fatty acid binding protein 4 (FABP4) is believed to be important
in maintaining the balance between lipogenesis and lipolysis in differentiating
adipocytes (Samulin et al. 2008). In human pathologies FABP4 has been
reported to be linked to obesity and diabetes (Maeda 2007). FABP4 becomes
transcriptionally active when its promoter is bound by Ppar gamma(Rival et al.
2004). Interestingly, A study using null mice elucidated that BMP expression in
27
adipocytes is, to some degree, under the regulation of FABP4 (Witthuhn and
Bernlohr 2001).
Members of the Wnt family are known to exert different effects on
adipogenic precursors. Wnt10b completely abolishes adipogenesis (Bennett et
al. 2004; Longo et al. 2004; Bowers and Lane 2008; Isakson et al. 2009), and
Wnt3a can inhibit adipocyte differentiation in vitro (Li et al. 2008). Conversely,
Wnt-5a has been observed to upregulate adipocyte differentiation transcription
factors (Bilkovski et al 2010). According to the literature canonical WNT
signalling has been shown to inhibit adipogenesis. But there is evidence that the
noncanonical path may impart survival on adipocyte precursors. Wnt10b and
Wnt3a are canonical , act through beta catenin signalling, and Wnt -5a is
noncanonical (Crisancho and Lazar 2011). In fact, blocking the canonical Wnt
signalling pathway is sufficient for initiating adipogenesis (MacDougald 2001,
Laudes et. al 2011). Specifically, when Il-6 was used to block Wnt10b adipocyte
differentiation occurred (Gustafson and Smith 2006) It has been suggested that
beta catenin, acting through the canonical Wnt pathway may be repressing
C/EBPs and ppar gammas because activation of these transcription factors
would lead to a substantial reduction in beta catenin levels (Leftrova and Lazar
2009). Conceivably, because both bone tissue and adipocyte are derived from
the same mesenchymal precursor cells and Wnt signalling is crucial for
osteogenesis there is a shunt that directs mesenchymal stem cells to the
osteocyte lineage that disengages the adipose tract (Gunther and Schule 2007).
28
Not surprisingly, ectopic expression of Dkk mediated a significant uptake in lipids
by inhibiting Wnts (Straovsky and Pan 2011).
Multiple depots of adipose tissue exist throughout the body. These depots
are now known to be both morphologically and functionally unique. For example
visceral adipocytes are smaller than subcutaneous adipocytes and differences in
insulin action in the two depots have been reported (Giorgino et al. 2005;
Spalding et al. 2008). The idea that the subcutaneous fat layer may function
independent of other fat depots is exciting in the field of hair research. Classical
literature reported alterations of the dermal fat layer that suggested a relationship
with the overlying hair follicles. It was noted that during activation of hair growth,
the expansion of the intra-dermal adipocyte layer in the skin doubles the skin’s
thickness(Butcher, 1934; Chase et al., 1953; Hansen et al., 1984)
Recently the role of subcutaneous adipose tissue as a modulator of stem cell
activity of adjacent hair follicles has emerged. One set of experiments utilized a
double staining technique of in situ hybridization for mBmp2 mRNA transcripts
along with a lipid specific stain to show that inhibitory Bmp signals were in phase
with refractory telogen and autonomous anagen and that the transcripts were
restricted to the subcutaneous fat layer (Plikus et al. 2008). This experiment
elegantly illustrated a role for subcutaneous adipocytes beyond the classically
thought function of thermoregulation and compression protection. Data from this
study has not gone unnoticed. Prompted by the results, another group more
closely investigated the role that the dermal fat layer may play in the propagation
29
of the hair wave. Using transgenic mouse systems with aberrations in adipose
development as well as using a pharmacological agent to block adipocyte
differentiation the group investigated how adipose development affected the first
synchronized anagen. Evidence from the study suggests that in mammalian skin
there is an adipocyte niche that regulates epithelial stem cells. Additional,
studies suggest that follicular regulation may be occurring through PDGF ligands
being expressed by adipocytes (Festa et al. 2011). Information from this study
has yet to be applied to the regenerative hair cycle in the adult mouse. In
another study a group capitalized on the information regarding PDGF signalling
and using a reporter mouse showed that there existed two distinct populations of
subcutaneous adipocytes. Of these two populations the lower subcutaneous
adipocytes harbor adipocyte precursors which are sensitive to Wnt signalling.
Using a Wnt/beta catenin knockout model the group was able to show that in
dermal development Wnt inhibits adipocyte maturation (Collins and Driskell et al.
unpublished data). The reports from this study certainly move the field closer to
an understanding of the development of the dermal fat layer as well as
interactions between hair follicle development and the underlying fat yet more
work needs to be done to understand how the interactions between the two
dermal compartments interact in the regenerative hair wave.
Certainly what appears to be cyclic interactions between two adjacent
tissues where at least one, possibly two, population of stem cells is being
activated fits into the macroenvironmental stem cell regulation hypothesis. Are
30
the dermal fat and follicular epidermis interacting in a reciprocal manner, or is
one governing the other? Because so little is known regarding the development
of subcutaneous fat it is my goal to describe the development of these two
distinct compartments in relation to each other.
31
Macroenvironmental regulation of hair follicle stem cells:
understanding extra-niche signalling phenomena
Taken together this wealth of information poises the field of stem cell
research at a new frontier, one which moves beyond the niche and into a larger
environment. It is my hypothesis that hair follicle stem cells are regulated, in
part, by the macroenvironment. To explore this new environment the manner in
which hair follicle stem cells are regulated to impart survival advantages on the
individual will be evaluated in the following chapters. How subcutaneous
adipose tissue signalling regulates hair follicle activity will demonstrate signalling
from adjacent tissue as a regulator of follicular stem cells. Hair follicle
quiescence will be investigated during pregnancy illustrating the role of remote
neuroendocrine signalling in follicular stem cell maintenance. Finally, circadian
rhythms as a modulator of follicular stem cell activity will elucidate the integration
of signals from the external environment by stem cells. In the field of hair
biology there are questions that remain regarding the macroenvironment. It is
my goal to elucidate how light cues from the external environment may play a
role in regulating the hair cycle, explore how the hair wave reset to telogen is
initiated during pregnancy and understand interactions between the hair follicle
and subcutaneous adipose tissue during development.
32
Figures
Figure 1: Hair Cycle
33
Figure 2: Biosynthetic pathways of steroid hormones
34
Figure 3: Steroid hormone signal transduction
35
Figure 4: Prolactin signal transduction
36
Figure Legends:
Figure 1: Hair Cycle
The regenerative hair cycle. Anagen is the growth phase, proliferative matrix
cells (red) are seen at the base of the follicle. The bulge stem cells are
highlighted in green. Catagen, is the resting phase. Telogen is the regression
phase. Anagen initiates after telogen. (Adapted from Goldstein and Horsley
2012).
Figure 2: Biosynthetic pathways of steroid hormones
Biosynthetic pathway for the synthesis of steroid hormones. The left panel
illustrates the synthesis of estrogens from testosterone and androstenedione via
aromatase (Adapted from Mayer et al. 2004). The right panel illustrates the
synthesis of progesterone fro pregnenolone via the enzyme 3-beta hydroxysterid
dehydrogenase (Adapted from Burger 2002).
Figure 3: Steroid hormone signal transduction
Illustration of steroid hormone signalling. The lipophilic hormone passes through
the lipid bilayer of the cell membrane and binds its cognate receptor, here the
prolactin receptor. Once bound the receptors dimerize and translocate to the
nucleus to initiate transcription (adapted from Mesiano et al. 2011).
37
Figure 4: Prolactin signal transduction
Prolactin signalling via the short long and short receptor. The signal cascade is
illustrated. The primary cascade involves the tyrosine kinase Jak2 (adapted from
Bouilly et al. 2012).
38
Chapter 2: The Macroenvironment
Background
The concept that the macroenvironment is influential in stem cell
homeostasis is a prevailing concept in the laboratory of Cheng-Ming Chuong. In
the past, stem cells were considered to interact predominantly with the
environment in which they resided, the microenvironment. By either interacting
directly with the supporting cells in the immediate environment or integrating the
signals from supporting cells, stem cell activity was regulated (Schofield, R.
1978) (Li et al., 2005). The concept that stem cell activity can also be regulated
by extra-niche signalling and input does not contest or diminish earlier concepts,
yet adds to the understanding of these specialized cells on a more global basis.
Hair follicle development is fairly well understood, during embryonic
development the dermal mesenchymal layer and overlying epithelial layer
interact using the morphological signals Fgf(s) and Bmps (Fuchs 2007), a
placode is the result. Follicular spacing is determined by Wnt and Dkk signals
(Sick et al. 2006).
Hair growth is cyclic in nature. This means that over a lifetime follicular
stem cells go through periods of quiescence and activity. The hair cycle is
primarily thought of in four phases: anagen, catagen, telogen, and exogen. Hair
fibers are produced and elongated during the anagen growth phase. During this
39
phase proliferative cells at the base of the hair shaft differentiate from matrix cells
causing an elongation of the hair fiber that can be understood and visualized as a
growing hair. After the growth period catagen ensues, the regression period. In
this phase matrix cell proliferation has ceased and the follicle regresses. Telogen
is the resting phase of the hair cycle, at this point follicular stem cells are
quiescent. Exogen is marked by the loss of hair, shedding. Importantly, anagen
and telogen can be visualized in mice (Figure 5). Visualization allows one to
define which follicular stem cells are active, marked by the dark growing anagen
hairs, and which are quiescent, the pink areas of telogen (Goldstein et al. 2012).
Interestingly a propagating anagen signal results in what appears as a “hair
wave”. Because the anagen period is shorter than the telogen period (Figure 6)
a wave of growing hair appears on the skin. Subsequently, over the dorsal
domain there can be both areas of anagen and areas of telogen (Figure 5).
The hair cycle has been studied extensively and is fairly well understood. When
a hair is in anagen the stem cells are activated and during telogen stem cells are
quiescent. Several activating molecules have been implicated in anagen, Noggin
(Botchkarev et al. 2001), Follistatin (Nakamura et al. 2003) Wnt/Beta-catenin
(Fuchs et al. 2004), and Fgf7 (Fibroblast growth factor) (Plikus 2012). Several
members of the transforming growth factor family, generally considered inhibitory
molecules, have been implicated in telogen including activin and Bmp2/4 (Bone
Morphogenic Protein) (Nakamura et al. 2003; Plikus 2012). It has also been
observed that at times of stress the hair wave can “reset” and lock in a telogen
40
state (Figure 7) (Fraser and Nay 1953 and Ebling 1964) . What remains to elude
hair biologists is the force that drives the hair cycle, what initiates it and what
concludes it.
41
Results
Cyclic Dermal BMP Signalling Regulates Stem Cell Activation
During Hair Regeneration -Nature Jan 2008
The paper, “Cyclic Dermal BMP Signalling Regulates Stem Cell Activation
During Hair Regeneration” added a new dimension to hair biology research and
ultimately stem cell biology. In a pioneering technique that allowed visualization
of a propagating hair wave it was possible to use current staining techniques to
elucidate when signalling molecule were on in relation to populations of hair
follicles. In this technique a sagittal whole mount section was collected from the
entire dorsal aspect of mice with a propagating hair wave. Sections collected in
this manner were termed “Skin Strips”. The result was a section that provided
spacio-temporal information regarding hair follicle activity (Figure 8). Figure four
illustrates how a skin strip allows investigation of a population of hair follicles,
some in anagen and some in telogen. Skin strips differed from conventional
whole mounts by exposing the all compartments of the pilosebaceous unit, the
inner and outer root sheath, the matrix, and importantly the stem cell containing
bulge and, it did the same for neighboring follicles. Importantly, when samples
like this were stained with molecules that had already been elucidated as
important in the hair cycle new clues emerged regarding wave propagation.
42
Utilizing the skin strip technique mouse skin with hair waves were stained
with riboprobes for morphogens that were important in the hair wave. One
molecule stood out from the rest, Bmp2. Already known for its role as an
inhibitor it was out of phase with Wnt, a known activator. Interestingly Bmp2
staining was observed when there was a lack of wave propagation, in fact when
the hair follicles were refractory to activation signals. Surprisingly the refractory
Bmp2 signal was present in the subcutaneous adipose tissue (Figure 8) that was
underlying the hair follicles. This was novel, signalling from a tissue
compartment that although adjacent to the hair follicle it was distinct from the hair
follicle.
Results from this study provided momentum to the idea of
macroenvironmental stem cell research. Subsequent groups have begun to
examine the interaction of the dermal fat layer relative to the superficial hair
follicles. Research has elucidated that a number of intrinsic molecular
mechanisms gate the transition of follicular stem cells from quiescence to
activation, additionally numerous cell types influence the activity and
characteristics of hair follicle stem cells (Festa et al. 2011; Goldstein et al. 2012).

43
Self-Organizing and Stochastic Behaviors During the
Regeneration of Hair Follicles- Science April 2011
Carrying concepts from the previous paper forward we dug deeper into the
idea that follicular cycling is mediated through inputs that are both intrinsic and
extrinsic to the hair follicle, thereby building upon the macroenvironmental
regulation theme. Expanding on the four functional phases of the hair cycle from
the previous paper that were defined based on the ability to transmit an activating
signal (propagating), the inability to transmit a propagation signal (autonomous),
the ability to receive a propagation signal and become activated (competent) or
being refractory to propagation signals (refractory) a mathematical model was
developed to test the functional states. Parameters to the model were based on
the functional states described earlier and inputs were the activation or inhibition
signals discussed previously. It was hypothesized that the mathematical model
could be applied to different systems by altering the parameters, inputs or both.
Soundness of the model was tested against he rabbit hair wave. Similar to mice,
rabbit hair grows in a semi-coordinated fashion (Figures 5 and 9). The rabbit hair
wave differs from the mouse in that it is more fractal. Presumably this is based
on different thresholds of responsiveness to signals from neighboring follicles.
When parameters representative of the rabbit were inputted into the
mathematical model a more fractal pattern was achieved when compared to the
mouse patterns. This confirmed the soundness of the model.
44
Summary
The results of these studies alter the understanding of both hair and skin
biology. The novel finding here is an expansion stem cell regulation beyond the
niche to a larger, more systemic, macroenvironment. Morphogenic signals from
the dermis, specifically the subcutaneous adipose tissue, are important to
maintaining a quiescent state in hair follicles (Figure 8). Periodic, and out of
phase, Beta-catenin and Bmp2/4 signaling sheds light on the coordinated
regulation of a population of hair follicles. It was also shown that stem cells can
be manipulated in an organ wide manner. Confirmation of these observations
have been made across species supporting the idea that mechanisms that drive
this coordination are evolutionarily conserved.
Although not the primary author on the above studies I was involved with
both. For the Cyclic Dermal BMP Signalling Regulates Stem Cell Activation
During Hair Regeneration paper I was responsible for protein bead experiments,
pSmad immunostaining, oil o red staining and some photography. For the Self-
Organizing and Stochastic Behaviors During the Regeneration of Hair Follicles
paper I was responsible for hair wave tracking on the rabbits, rabbit tissue
collection and some immunostaining of rabbit tissue for cell proliferation markers.
45
Figures
Figure 5: A propagating hair wave
Figure 6: Days of Anagen/Telogen
Figure 7: Pregnancy induced hair wave reset
46
Figure 8: Bmp signalling in the subcutaneous adipocytes
Figure 9: Hair wave propagation in rabbit
47
Figure Legends:
Figure 5: A propagating hair wave.
The black areas are pigmented hair follicles that are in the growing phase,
anagen. In these follicles the stem cells are active. The grey areas are in early
anagen. The areas that are pink are in telogen, the hair follicles are not growing
therefore the hair follicle stem cells are quiescent. The open arrow is pointed in
the direction of the “hair wave”. The second panel illustrates how some areas of
the skin are competent and some are refractory to the growth signals of the
spreading wave. Adapted from Plikus et. al., 2008.
Figure 6: Days of Anagen/Telogen
Quantification of the time length of telogen and anagen. Adapted from Plikus et.
al., 2008.
Figure 7: Pregnancy induced hair wave reset
Prior to pregnancy areas of activated stem cells are observed as growing hair
follicles. During pregnancy and lactation the hair wave is reset and is maintained
in the telogen phase. At the conclusion of lactation the hair wave enters anangen
in a synchronized fashion. Adapted from Plikus et. al., 2008.
48
Figure 8: Bmp signalling in the subcutaneous adipocytes
Bmp2 transcript expression in the subcutaneous adipocytes during telogen.
Expression of this inhibitory signalling molecule from the dermal fat layer
influences the homeostasis of the hair follicle stem cells. Oil Red O staining
denotes lipid filled subcutaneous adipocytes. Scale Bars 200um Adapted from
Plikus et. al., 2008.
Figure 9: Hair wave propagation in rabbit.
This Figure illustrates that the hair wave dynamics observed in mice can be
applied to other species. In this set of images, taken over 14 days, the
propagating signal of the proceeding hair wave can be observed to have similar
effects on quiescent stem cell population as previously observed in mice. The
propagating signals manifest as a spreading hair wave. The more complex
“fractal” patterns can be understood as a difference in threshold requirements for
activation that exists at the species level. Also illustrated in this Figure is the
concept that in addition to the single niche stimulation, populations of stem cell
can be activated over an area. Adapted from Plikus et. al., 2011.
49
Chapter 3: Circadian Rhythms as a stem cell modulator
Background
Circadian Rhythms are endogenous clocks inherent to all living things,
examples are apparent across phyla. Studies have been published on , turtles,
whales, bears,d eer and birds to name a few examples (Wibbels et al. 1990;
Stevick, et al. 2010; Platner et al. 1971; Goss 1984; Thornton 2001; Gwinner
2003). Coordination with the external environment is maintained by an internal
clock that regulates circadian gene expression (Bell-Pendersen 2005).Circadian
clocks are conserved across species and impart survival by enabling organisms
make behavioral and physiological changes by anticipating daily environmental
changes (Panda et al. 2002).
In mammals there exists a central clock that governs all other peripheral
clocks. This clock is located in the suprchiasmatic nucleus (SCN), in the
hypothalamus. (Welsh et al. 1995; Bell-Pendersen 2005).The current model of
the mammalian circadian oscillator is comprised of four clock genes. The
negative aspects of the feedback loop, Period (Per1,2) and Cryptochrome
(Cry1,2) are activated early in circadian day by heterodimers of CLOCK and
BMAL1. As nuclear levels of PER and CRY elevate transcription of Clock and
BMAL1 cease. Eventually levels of PER and CRY in the nucleus decrease and
CLOCK and BMAL1 are again transcribed (Figure 6) (Hastings et al.2004). In
50
addition to the central clock there are peripheral clocks in multiple systems such
as the liver lung and muscle (Yamazaki et al. 2000). Limited Circadian Rhythm
studies have been performed on skin to investigate the role of circadian
oscillation in dermal homeostasis. Several studies, performed in humans, clearly
illustrated that there was a distinct peripheral clock in the skin that operated
independently from the SCN (Bjarnason et al. 1999,2001). In mouse studies a
differential expression of mPer1 was observed in skin when mice were subjected
to different lighting conditions,suggesting differential regulation of mPer1 mRNA
SCN and peripheral tissues (Oishi et al. 2002). Another study performed on the
hair follicle suggested circadian regulation at the cell cycle level in hair follicles
(Lin et. al., 2009).
The basis of the hair cycle is a perplexing question to hair biologists. What
drives the cycle, is it a consequence of molting, a means to control the length of
the hair, a function to cleanse the surface of the body, even to adapt to social
conditions and to allow flexibility to adapt to the surrounding environment ( i.e.
seasonal changes); the force that perpetuates the cycle are unknown (Stenn and
Paus 2011).
It was hypothesized that a peripheral circadian clock may be governing
the hair cycle. To test this theory two transgenic mouse lines were created using
the cre/lox system. In the cre-lox system two mouse lines are crossed, one line
houses a Cre-recombinase gene that catalyzes the reciprocal site-specific
recombination between two specific 34- base pair sites called “Lox-p”. The Lox-p
51
sites are artifically inserted into the genome of the second mouse flanking a gene
of interest. The intramolecular recombination of the two Lox-p sites results in
deletion (Araki et al. 1997) of the gene of interest. In our system it was important
to knock out the circadian clock in the skin only to see if the hair cycle would be
altered. To achieve this a mouse in which cre recombinase was driven by the
Keratin 14 promoter was selected. Keratin 14 a intermediate filament that is
present in the basal layer of all stratified squamous epithelia (Vassar et al. 1998).
This mouse was crossed with a second mouse which had the eighth exon of the
Bmal1 gene floxed (Stroch et al. 2007). As second mouse line was generated
using a similar system with the additional ability to regulate the activity of Cre
recombinase. Similar to the first mouse line Cre was still under the direction of
the keratin 14 promoter however, in this model cre was fused to the tamoxifen
responsive hormone binding domain of the estrogen receptor. In this manner
Cre can be activated by topical administration of tamoxifen (Vasioukhin et al.
1999) and therefore be controlled temporally. The two mice were termed
K14cre,Bmal1 and K14creEr
tam
, Bmal1 respectively. Figure eleven is graphical
representation of the two K14cre transgenes. After the mice were generated and
genotyped, cre activity would be turned on, if not already active, and the mice
would be monitored for a period of no more than 6 months to determine if the hair
wave propagation was altered when compared to that of control littermates.

52
Results
K14creER
tam
,Baml1 mice were treated with tamoxifen to excise the 8th
exon of the Bmal1 gene to produce Bmal

-/-
mice (Figures 11,12). To confirm
tissue specific expression of Cre mediated excision K14cre lines were crossed
with a rosa 26 reporter mouse line (Figure 13). Specific expression was
observed in cells expressing Keratin 14 promoter as noted by LacZ staining.
Initially the beta-galactosidase activity is observable in the basement layer of the
epidermis, the signal is maintained by the progeny cells that migrate upward from
the basement membrane to the surface of the skin. Because the transgene of
the K14creER
tam
,Baml1 mice was regulatable at both time and tissue level the
study was primarily done using this line. Mice carrying the mutant allele were
generated and maintained until 2 months old, mice were then shaved and the
hair cycle was monitored for roughly 150 days in 12 hour light/dark cycles. At the
end of the 150 days no phenotype was observed in the null mice compared to
mice treated with vehicle only (Figure 14). Further studies elicited that although
no overt phenotype was observed there was some misregulation of the cell cycle
in the Keratin 14 expressing cells of the null mice.
In a Per1,2 luciferase reporter mouse vibrissae cultures oscillated for 2
days until after which the signal was lost due to culture limitations, no shown.
Histomophrometric analysis of Phosphohistone 3 was used to study the rate of
the G2/M transition. Compared to wildtype mice it was observed that the number
53
of mitotic cells oscillated over time. The highest number was observed at CT50
and the lowest at CT62, with a difference of roughly 70%. This additional data,
not generated by me, suggests that although on the K14creER
tam
,Baml1 mice
there was not an obvious phenotype there was circadian control at the cell cycle
level suggesting there is a peripheral clock in the skin but it may be overridden by
the central clock mechanism.
54
Summary
Circadian gene expression as a factor in hair follicle stem cell
maintenance is a novel concept. Here using a mouse model with tissue specific
circadian gene knockouts did not render a phenotype. However, subsequent
work investigating the cell cycle progression of this model system has shown that
at the subcellular level there is some cell cycle misregulation, unpublished data.
This is corroborated by recently published data from several labs. In a
fluorescent mouse model engineered to report the activity of circadian genes it
was observed that there exist two populations of epidermal stem cells in the hair
follicle, each active at different phases of the clock (Janich et. al., 2011). The
authors report that deletion of Bmal1 modulated an accumulation of stem cells in
a progressive manner. It was further reported that in isolated bulge stem cells of
Bmal1 null mice there was a lower expression of Wnt related genes as well as
lower levels of Tgf-beta related genes.
Another group investigated the interaction of circadian genes and hair
follicle stem cells using a genomics approach (Lin et. al., 2009). The study was
preformed on mice with a synchronized hair wave, at post-natal day 21. In this
study there was a delay of first anagen in Bmal1 mutant mice. Although this
study provides clues to the role that circadian genes may play in hair follicle stem
cell regulation the model differed from our study in that the mouse model we
used the hair wave on the dorsal aspect of the mice was not synchronized. In
55
our system the mice were an average age of ~2months, as opposed to 21 days.
At the cell cycle level they report modulation during proliferation, this is consistent
with our studies using the Bmal1 null and other transgenic systems system.
Taken together, our study is consistent with current literature. The lack of
a significant phenotype can be understood by the accumulation of Wnt
associated proteins and Tgf-beta proteins observed by the Janich group. These
two morphogens act in opposition, therefore an accumulation of both could
conceivably result in a lack of phenotype. Further, the phenotype observed by
the Lin group doe not negate a lack of phenotype in mice with an asynchronous
hair wave. Consistently all the groups have reported a modulation of the cell
cycle in systems with ablated clock genes. Importantly, in nature seasonal
changes have been observed to impact molting, mating and shedding among
other physiological changes. In our system animals are confined to a indoor
environment where seasonal changes are not experienced. Specifically our
animals are exposed to a 12 hour light cycle, with no shortening of light and
elongation of dark. Accompanying this idea, animals are housed in a vivaria
where a specific temperature is maintained, in the wild temperatures vary.
Because mouse hair follicles are important in thermo-regulation, these
environmental cues could be a major factors that could drive circadian clocks to
have a larger impact on stem cell homeostasis in the hair follicle regeneration
cycle in the wild that is lost on domesticated animals.
56
Figures
Figure 10: Circadian rhythm core loop
Figure 11: K14cre transgene
57
Figure 12: Mouse genotype
Figure 13: K14cre, R26R cross

58
Figure 14: Bmal1 null mouse hair wave
59
Figure 14, Continued
60
Figure legends:
Figure 10: Circadian rhythm core loop
At the heart of the circadian clock are interlocking, autoregulatory feedback
loops. In the core loop of the clock, positive elements CLOCK and BMAL1
(orange and yellow circles) heterodimerize and initiate transcription of their
negative regulators PER and CRY (rounded purple squares). As the levels of
PER and CRY accumulate, these proteins form multimers, are phosphorylated,
and translocate to the nucleus where they repress their own transcription by
interacting with CLOCK-BMAL1 heterodimers. Phosphorylation induced
degradation of PER and CRY decreases their concentrations, which reactivates
the positive elements, allowing the cycle to start again. Core loop components
also activate multiple ccgs (clock-controlled genes) to form interlocking positive-
and negative-feedback loops that are important for maintaining robustness of the
clock. In mammals, CLOCK-BMAL1 heterodimers activate transcription of RORα
and REV-ERBα (green square), which provide additional positive and negative
feedback, respectively, to transcription of BMAL1. Image from Hamilton and Kay-
Cell 2008
Figure 11: K14cre transgene
Graphical representation of the K14cre and K14creERtam transgenes. Adapted
from Vasioukhin et. al.
61
Figure 12: Mouse genotype
Image of agarose gel used to genotype Bmal1
f/f
mice. Wildtype mice lacking the
floxed allele band at 327bp, mice homozygous for the floxed allele band at
431bp. Heterozygotes had bands at 327bp and 431bp.
Figure 13: K14cre, R26R cross
K14cre and K14creER
tam
mice were crossed with Rosa 26 reporter mice to
confirm tissue specificity. In the images beta galactosidase activity is apparent
and restricted to the cells expressing the K14 promoter in the epidermis.
Figure 14: Bmal1 null mouse hair wave
Mice were shaved to observe the hair cycle. Hair waves were monitored for
roughly 5 months. No significant phenotype was observed in the transgenic mice
when compared to the control mice. Tamoxifen was diluted in ethanol, control
mice were treated with ethanol vehicle only.
62
Chapter 4: Pregnancy Telogen Hair Wave Reset
Background
Procreation allows for the transmission of genetic material and is inherent
to all living. In mammals, pregnancy is marked by myriad of physiological and
biochemical changes to the individual such as alterations to protein metabolism,
immune function, neuroendocrine signalling(Figure1) and food consumption
(King 1975, Roberts et al., 1996, Neville et al. 2002, Douglas et al. 2007). These
hormonal fluctuations can manifest physiologically in several ways including
increased adipose accumulation, immunological fluctuations and epithelial tissue
remodeling. To accommodate lactation in the postpartum period the mammary
gland goes through a significant remodeling from a prepregnant branched ductal
system to a lobuloalveolar expansion during pregnancy, post lactation the
mammary gland involutes to a prepregnant branched ductal system
(Hennighausen and Robinson 2001). Epithelial reorganization is observed in
other animals, examples are development of the brood patch in avia, mediated
by a combination of prolactin, estrogen and progesterone (Hutchison et al. 1967)
and hair plucking during nest building in the lagomorph mediated by prolactin
(Gonzales-Mariscal et al. 1996).
63
In addition to mammary remodeling other epithelial organs are modified
during pregnancy; it was observed in the past that pregnancy can also affect hair
stem cell homeostasis. In humans, pregnancy related modifications to the hair
cycle have been reported(Lynfield 1960; Ebling 1964,Winton and Lewis 1982).
Modulation to the mouse hair cycle during pregnancy has also been observed. In
classical literature studies done on sexually dimorphic hair growth on mice
included a brief description of the hair growth of pregnant mice as “naked”
(Fraser and Nay 1953).Recently work from the Chuong lab reported that the hair
wave is reset to telogen during pregnancy and anagen resumes after lactation
has ceased (Plikus et al. 2008). These time point parameters have only been
loosely defined and have by no means been formally investigated.
Modifications to hair follicle epithelium as a result of fluctuations in
circulating levels of hormones is not surprising. Several receptors for pregnancy
related hormones are reported to be expressed in some compartment of the hair
follicle compartment (Ohnemus et al. 2004, Pelletier and Ren, 2004, Craven et
al. 2006). Because circulating levels of hormones important in pregnancy differ
from a nonpregnant state in conjunction with the fact that, according to the
literature, receptors for these hormones are expressed in hair follicles it was
hypothesized that one or a combination of these hormones might be responsible
for the hair wave reset observed during pregnancy and lactation. Based on the
literature, hormones that could be implicated were estrogen, prolactin,
progesterone.
64
Estrogen plays an important role in the pilosebaceous unit. It has been
reported that estrogen can regulate collagen content and quality, increase skin
thickness and enhance vascularization (Thornton et. al., 2007). There are two
distinct estrogen receptors present in the skin and hair follicle, ER alpha and ER
beta. The two receptors have similar but unique binding qualities and have been
observed to heterodimerize in vitro (Cowley et. al., 1997). In the human skin ER
beta is predominantly in the hair follicle and ER alpha is predominantly in the
sebaceous gland (Thornton et. al., 2003). Circulating estradiol levels are elevated
during pregnancy and increase dramatically post pregnancy and during lactation.
Classical work in the 1950s had shown that estrogen increases the mitotic rate in
the epidermis but decreases the size of the sebaceous gland (Ebling, 1957).
Prolactin is a pituitary hormone known for a role in lactation and
reproduction in mammals (Craven et al. 2006)and development of the brood
patch in avians (Ohkubo, et. al., 1998). Recently this role has been expanding as
the number tissues expressing the receptor for this hormone has been growing.
Specifically, the role of prolactin in modulating hair growth has been observed.
This is not surprising considering that the mammary gland is an epithelial
appendage as is the hair follicle (Foitzik et. al., 2009). Additionally prolactin
receptors are present in the skin and prolactin expression elongates telogen and
shortens anagen (Craven et. al., 2006).
65
Progesterone levels elevate during pregnancy (McCormack et. al., 1974).
Progesterone receptors were localized to the the sebaceous gland, outer root
sheath and epidermis using immunohistochemistry (Pelletier and Ren 2004;
Kariya et. al., 2005).
It was hypothesized that the hair wave reset was driven by hormones that
were present during pregnancy. In an effort to better define the hair wave as a
physiological and molecular phenomena the hair waves of pregnant mice
pregnant mice were tracked over time to elucidate the time parameter at which
the wave reset, and the point at which the follicles re-entered anagen. Molecular
analysis of pregnant tissue for morphogens that are known to have an inhibitory
effect on the hair wave to determine a potential pathway through which the hair
wave might be coordinated was performed. Ectopic hormone administration to
non pregnant mice via affi-gel beads was utilized in an effort to recapitulate the
hair wave phenomena. Finally, non pregnant tissue with ectopic hormone
expression was interrogated for transcripts of inhibitory molecules to further
delineate the molecular pathway through which the hair wave reset was
occurring.
66
Results
The pregnancy associated hair wave reset timing was interrogated by
allowing mice to become pregnant, and monitoring hair growth throughout
pregnancy and lactation. Although the reset of the hair wave was observed to
initiate at different times over several mice it consistently occurred late in
pregnancy and the hair cycle was always in the telogen state during lactation. It
was reported that anagen will initiate once lactation ceases. In mice that were
allowed to go to term and nurse anagen was not observed until after nursing
halted, approximately 20 days post-natally. To confirm lactation as a telogenic
factor, lactation was prematurely halted via the removal of pups from lactating
mice. Anagen was observed to initiate within an average of six days after the
premature halt of lactation (Figure 16 n-3).
In situ hybridization was used to interrogate the skin of pregnant and
lactating mice for the presence of morphogens reported to maintain a quiescent
state in hair follicle stem cells. Bmp2 and SFRP4 known to be expressed during
refractory telogen (Plikus et al. 2008, unpublished data). Additionally expressed
are SFRP1 and Follistatin, are known to be expressed during competent telogen
(unpublished data). Follistatin and Sfrp1 mRNA were localized to the
subcutaneous adipose tissue during late pregnancy and throughout lactation
noted by the positive blue alkaline phosphatase signal in the dermal layer. Sfrp4
and Bmp2 mRNA expression were also observed in the dermal fat layer but
expression was throughout the entire duration of pregnancy and lactation
67
(Figure 17). The presence of these inhibitory morphogens suggests that the
pregnancy induced hair wave reset encompasses an elongated refractory
telogen phase. The presence of morphogens was detected via whole-mount
sections collected at various time points during pregnancy and lactation. After
the staining was completed the sections were mounted in paraffin and sectioned
using a microtome. This allowed for higher magnification of the stained tissue.
At higher resolution it was obvious that positive signal for the transcripts was
localized to the subcutaneous adipocytes. This finding is consistent with data
published regarding these morphogens and the hair wave in a telogen state.
This suggests that the hair wave telogen reset during pregnancy is mediated
through the subcutaneous adipocytes.
To examine if hormones associated with pregnancy and lactation might
play a role in the hair wave reset affi-gel beads were coated with Prolactin,
Estradiol Alpha, Estradiol Beta, or Progesterone. Control beads were coated
with BSA. Beads were implanted at the wave front of a propagating hair wave.
The advancing hair waves were monitored for five days. At the conclusion of the
monitoring period the hair waves of the mice in which estradiol alpha, estradiol
beta, and progesterone exhibited little to no inhibition on the advancing hair
wave. Conversely, in the skin of the mouse treated with prolactin the hair wave
appeared to be inhibited where the beads were implanted but moved normally
lateral to the site of the bead implantation. Further analysis indicated that in mice
treated with prolactin two (2) anagen hair follicles were counted in a one
68
millimeter proximity to the bead implantations. In mice treated with Estradiol
Alpha 21 anagen follicles were counted, in mice treated with Estradiol beta 14
anagen follicles were present. In mice treated with progesterone 12 anagen
follicles were counted within one millimeter of the bead implantation site.
Hematoxilyn and eosin staining confirmed that in tissue treated with prolactin the
area surrounding the bead implantation was void of anagen hair follicles.
Contrary to this, in tissue treated with the other hormones anagen follicles can be
observed in the tissue sections (Figure 18).
Because the expression of inhibitory morphogen mRNA was observed in
the subcutaneous adipose tissue and prolactin coated beads were observed to
inhibit the progressing hair wave the subcutaneous adipose tissue was
interrogated for prolactin receptors. Figure 19 confirms that as pregnancy
progresses into lactation and towards the later stages of natural lactation an
increase in prolactin receptor mRNA expression is observed in subcutaneous
adipose tissues.
Because several inhibitory signalling molecules were expressed within the
subcutaneous adipose tissue during pregnancy and levels of the prolactin
receptor were observed to elevate throughout pregnancy and lactation it was
important to determine if prolactin expression might be upregulating the
expression of one of the inhibitory molecules. Using in situ hybridization it was
determined that Bmp2 expression was upregulated relative to the other signalling
morphogens( Figure 20).
69
Summary
The modulation of the hair cycle during pregnancy has been observed and
reported in the literature in the past. However, other than a brief description of
the phenomena, no formal study has been published. Here is provided a
detailed study of the hair wave reset during pregnancy. A time point at which the
wave is reset during pregnancy is provided and hormones that might contribute
to the reset are also offered.
Although the reset of the wave was observed to initiate at different times it
consistently occurred late in pregnancy and the hair cycle was always in the
telogen state during lactation. Anagen has been described in the literature to
return in an “explosive” fashion once lactation ceases. These strongly implicated
a pregnancy related hormone or multiple pregnancy associated hormones to be
involved in this phenomena. By observing the hair wave of pregnant mice it
appeared that the reset consistently occurred during lactation. To confirm if
lactation was a factor in the hair wave reset the duration of lactation was
artificially truncated to determine the amount of time necessary for anagen to
initiate (Figure16 n-3). In three trials anagen initiated an average of 6 days after
the removal of pups. This strongly implicates hormonal cues that are expressed
during lactation promote the telogen phase.
70
It has been previously been determined that several molecules can
maintain hair follicle stem cells in a quiescent state (unpublished data) as well as
act as Wnt antagonists (Park et. al.2008). This information provided a candidate
pool from which were selected Follistatin, Sfrp1, Sfrp4, and Bmp2 as
downstream factors that might be upregulated during the pregnancy/lactation hair
wave reset. In situ hybridization studies exhibited mRNA transcripts from the
above candidates to be expressed at varying degrees during the pregnancy and
lactation period in the subcutaneous adipose tissue. Subcutaneous adipocytes
have previously been shown to express inhibitory factors associated with telogen
(Plikus et. al., 2008). It can be hypothesized that this redundancy in the
expression of inhibitory morphogens functions to ensure a telogen state during
pregnancy potentially to allow for hair loss to provide material for nest building. A
similar phenomena has been observed in pregnant rabbits; telogen results in hair
loss, this fallen hair is subsequently used in nest building for kits (Gonzalez-
Mariscal et. al., 1996).
During pregnancy several hormones are known to fluctuate relative to non
pregnant physiological levels (Soares et. al., 2004). Several of these hormones
are known to interact with hair follicles on multiple levels. Using immuo-assays
progesterone has been localized to the dermal papilla of hair follicles but not the
epithelial cells of the hair follicles (Pelletier et. al., 2004). Both estrogen
receptors have been localized to the mouse hair follicle and are expressed
through out the hair cycle. Topical estrogen has been observed to arrest hair
71
follicles in telogen, and topical E2 potentates catagen. Estrogen receptor
antagonists were not observed to induce anagen (Ohnemus et. al., 2005).
Finally, prolactin has been reported to elongate telogen (Craven et al., 2006). To
identify if one or more hormones that are associated with pregnancy might be
important in the hair wave reset observed the application of hormone coated
beads was utilized. In this assay affi-gel blue beads were soaked in a protein of
question then inserted intradermally into the skin of a non-pregant wildtype
female mouse. By exogenously expressing hormones in the skin, adjacent to the
propagating hair wave it is possible to observe the interaction of the hormone
and hair follicle stem cell. In the assay performed in this study beads were
coated with progesterone, estradiol alpha and beta, and prolactin. Based on
observations made over several mice the beads coated with prolactin exerted the
most significant, telogen-like effect on the hair wave.
A literature search regarding a relationship between telogen like effects of
progesterone yielded little information. This fact in conjunction with the results of
the ectopic bead assay suggested that progesterone does not interact with hair
follicle stem cells during the hair wave reset seen in Figure 18.
Similarly, the results of the estrogen receptor agonist coated beads do not
conflict with what is previously published. The results of the assay preformed
here did not show an induction of telogen by estrogen receptor agonists (Figure
18). The estrogen induced duration of the telogen state previously observed was
preformed on wild type mice not enduring pregnancy. However, the role of an
72
estrogen maintained telogen was not found in the literature.
The results of the ectopic application of prolactin is consistent with
previous reports, an elongation of telogen. This implicates prolactin as a key
hormone in the hair wave reset. It has been previously reported that prolactin
elongates telogen in non-pregnant mice (Craven et. al., 2006). Affi-gel blue
beads are implanted into telogen state skin. As the hair wave progressed
towards the beads, the area adjacent the beads was not coerced into anagen as
seen in Figure 18. Instead the hair follicles remained in telogen. As lactation
begins receptor expression levels of prolactin elevate in the subcutaneous
adipose tissue(Figure 19). This coincides with time point at which telogen
initiates during the pregnancy lactation reset. This fact further implicates
prolactin as a major player in the pregnancy telogen phenomena.
To further investigate how circulating levels of prolactin might be exerting a
modularity role on hair follicle stem cells, skin tissue was interrogated for
prolactin receptor expression. Because there is an increase in the number of
adipocytes expressing prolactin mRNA (Figure 19) throughout the pregnancy and
lactation period it can be inferred that to some degree prolactin signalling may
induce the expression of the inhibitory molecules responsible for the hair wave
reset that is observed during lactation.
Subcutaneous Bmp2 expression has been implicated as a significant
factor in normal telogen (Plikus et. al., 2008). It was observed here that during
the pregnancy telogen state, several inhibitory molecules are expressed in the
73
dermal fat layer. Because prolactin was strongly implicated in reseting the hair
wave it was important to see if one or more of the inhibitory molecules were
being expressed downstream of prolactin signalling. Here we ectopically
expressed prolactin then using in situ hybridization investigated the skin for
mRNA of inhibitory molecules. Figure 20 illustrates that while Bmp2 expression
is high transcripts for the other inhibitory signalling molecules are not.
In summary, although there are a multitude of differentially expressed
hormones and morphogens during pregnancy it is shown here that an increase of
prolactin signalling late in pregnancy and maintained through lactation is a strong
candidate in the hair wave reset. Additionally based on results of ectopic
prolactin expression it can be inferred that Bmp2 is operating downstream of
prolactin to maintain the quiescence of hair follicle stem cells, and subsequently
the telogen state of the hair wave through the subcutaneous adipocytes. In
classical literature the hair wave reset was described as a telogen state that
endured throughout lactation with anagen returning in an explosive manner at the
conclusion of lactation. Here is a plausible path in which the telogen portion of
the reset has been explained, the route in which anagen returns is a candidate
for future studies.
74
Figures
Figure 15:Hormone signature during pregnancy
75
Figure 16: Hair wave during pregnancy
76
Figure 16, Continued
77
Figure 17:Signalling morphogens in the skin during pregnancy
78
Figure 18: Bead implantation experiment
79
Control Prolactin Estradiol alpha Estradiol Beta Progesterone
0
5
10
15
20
25
30
Number of Anagen Hair Follicles
Hormone
Number of Anagen Hair Follicles
Figure 19: Prolactin receptor expression in skin compartments during
pregnancy/lactation
Figure 20: Morphogens downstream from Prolactin signalling
80
Figure Legends:
Figure 15: Hormone signature during pregnancy
A schematic representation of lactogenic hormone profiles during pregnancy.
Early pregnancy is dominated by twice daily surges of PRL (Prolactin) produced
by the maternal anterior pituitary. Midgestation is marked by the products of
three placental lactogen genes and the second half of gestation by PL-II
(placental lactogen). The fetal anterior pituitary begins to produce PRL at the
end of pregnancy. PRL-Prolactin, PL-Is Placental lactogen- alpha, beta gamma,
PL-II placental lactogen II (Soares et al., 2004)
Figure 16: Hair wave during pregnancy
In mice that had recently given birth pups were removed to truncate lactation. In
these mice anagen was observed to initiate in five to seven days after pups were
removed (n=3). This is in contrast to the mice that nursed pups for a full term
days in which anagen did not initiate until the cessation of nursing (not shown).
Figure 17: Signalling morphogens in the skin during pregnancy
In situ hybridization was performed on skin tissue of pregnant mice collected at
day ten during pregnancy and days one, five, fourteen and nineteen during
lactation for mRNA expression of signalling morphogens that are known to have
a role in maintaining a quiescent stem cell state in hair follicles. In situ
81
hybridization was also preformed on non pregnant telogen skin as a control.
Follistatin, Sfrp1, Sfrp4 and Bmp2 mRNA were observed to have variable
expression throughout pregnancy and lactation. Follistatin and Sfrp4 expression
were observed during nursing minimal levels. Sfrp1 and Bmp2 were both
expressed throughout pregnancy and lactation. Orange arrows denote blue
alkaline phosphatase signal in the subcutaneous adipose tissue positive for
mRNA transcripts. Further analysis included sectioning the previously stained
whole-mount sections. At a high resolution morphogen transcripts are localized to
the subcutaneous adipose tissue. Orange arrowheads denote positive
adipocytes.
Figure 18: Bead implantation experiment
Affi-gel blue beads were coated with either Prolactin( n-3), Estradiol alpha (n-2),
Estradiol beta or Progesterone(n-3). Control beads were coated in BSA only.
Beads were implanted in the dermis proceeding the propagating hair wave. Of
the proteins used in the experiment prolactin seemed to modulate the hair wave
most significantly. Size bar, 0.5mm. Anagen hairs were counted in an area
measuring 1.0mm x 0.5mm. In control mice 28 anagen hairs counted in the
measured area. In mice treated with prolactin 2 anagen hair follicles were
counted in the most distal part of the measurement area. In mice treated with
Estradiol Alpha 21 anagen follicles were counted, in mice treated with Estradiol
beta 14 anagen follicles were present. In mice treated with progesterone 12
82
anagen follicles were counted. Analysis using hematoxilyn and eosin staining
revealed that while anagen hair follicles (orange arrowheads) were present in
close proximity to the implanted beads in the control, Estradiol alpha, estradiol
beta and progesterone treated tissue there was an absence of hair follicles in the
skin tissue treated with prolactin. green arrows: direction of the propagating hair
wave, red T bars: zone of inhibition, black arrows: affi-blue beads orange
arrowheads: anagen hair follicles. Size bars, 1mm, 1mm, and 200um.
Figure 19: Prolactin receptor expression in skin compartments during
pregnancy/lactation
Using in situ hybridization subcutaneous adipocytes in the mouse skin were
interrogated for prolactin receptor expression. Samples were stained then
positive cells were counted at different stages of pregnancy and lactation. During
pregnancy no adipocytes expressed prolactin receptors. At the initiation of
lactation prolactin receptor mRNA was observed within the subcutaneous
adipocyte depot. The number of positive cells increased throughout the lactation
period and decreased as lactation concluded.
Figure 20: Morphogens downstream from Prolactin signalling
83
Prolactin coated affi-gel beads were implanted intradermally as previously
described. After 5 days of treatment with prolactin, tissue was collected and,
using in situ hybridization, interrogated for inhibitory morphogen signalling. Of
the four morphogens observed during pregnancy only Bmp2 mRNA was detected
in the tissue where the beads were inserted. Size bars 1mm, .25mm
84
Chapter 5: Subcutaneous Adipose Development
Background
Traditionally adipose tissue was thought to be static, acting only as a
support tissue that offered thermoregulation and physical protection. Much of the
information regarding adipocyte differentiation and proliferation has been
obtained from in vitro studies, subsequently depot specific information is limited.
Even less is understood about adipocyte differentiation in relation to hair follicle.
There are two types of adipocytes, brown and white. Brown adipocytes
store less lipid and have more mitochondria than white adipocytes. Both cell
types express similar genes but brown adipose tissue expresses some unique
genes that allow for energy metabolism as heat. Generally brown fat is found at
the interscapular region while white fat is found throughout the organism.
Additionally, brown fat is found in mammals during infancy but is lost as age
progresses. Conversely white fat persists through the life of the organism.
Recently the prevailing perception of adipose tissue as static has shifted.
Work by several groups has shown that adipose derived signalling plays an
important role in stem cell homeostasis. Findings by our lab delineated that
Bmp2 signalling from the subcutaneous adipocyte layer helps maintain a
quiescent state in hair follicle stem cells (Plikus et. al., 2008). This led to the
novel concept that the stem cells residing in the hair follicle niche can, at least in
part, interact with signals from adjacent tissues or even remote tissues.
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Interestingly the thickness of the subcutaneous adipose has been
observed to fluctuate. During activation of hair growth, the expansion of the
intra-dermal adipocyte layer in the skin doubles the skin’s thickness(Butcher,
1934; Chase et al., 1953; Hansen et al., 1984). Expansion of this deposit could
occur from hypertrophy (an increase in cell size) or hyperplasia (an increase in
cell number). Recently it was delineated that PDGF is secreted by dermal
adipocytes to stimulate hair growth (Festa et. al. 2011). Using the information
from this study another group showed that dermal adipocyte precursor cells are
restricted to the lower dermis during postnatal stages one and two.
Adipocyte commitment and differentiation
Adipocytes are derived from multipotent stem cells. Due to the inherent
nature of adipocytes it has been difficult to describe intermediate stages of
adipogenesis at the molecular level (Rosen et. al., 2006). For practical purposes
adipogenesis is thought of in two phases, determination and terminal
differentiation. The first stage, determination, is understood as the commitment
of pluripotent stem cells to the pre-adipocyte. These two cell types are difficult to
distinguish molecularly, however pre-adipocytes no longer have the potential to
differentiate into other cell types. Terminal differentiation is marked by the pre-
adipocyte taking on the characteristics of the mature adipocyte. The terminal
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differentiation phase is more thoroughly understood due to the fact that cells that
have transitioned into the determination phase have been primarily used in
research. The breadth of the information available in the literature has
delineated several markers that are important in adipocyte differentiation. These
markers include C/EBPa, FABP-4, Dkk-1, Leptin and Pref-1. According to the
literature these markers are expressed at various stages during adipocyte cell
line commitment.
Adipocyte Markers
C/EBP- CCAAT enhancer binding protein (C/ebp)is a family of
transcription factors comprised of several members including C/EBPa, C/EBPb,
C/EBP gamma and C/EBP delta. Early expression of C/EBPb and C/EBP delta
is transient and initiates the expression of C/EBPalpha. C/EBP alpha induces
many adipocyte genes directly, in vivo research using C/ebp a null mice has
shown that this factor is important in adipose development. C/EBPa has been
observed in p4 differentiated hair follicles (Lopez et.al. 2009). However, no
epidermal phenotype has been observed in tissue specific ablation models.
Fabp4- Fatty Acid binding protein 4 is one of nine members of a the Fatty
acid binding protein family. This protein is important in lipid uptake and unlike the
other Fabps is unique to adipose tissue, subsequently Fabp4 expression has not
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been observed in the developing hair follicle. The structure of the protein is a
barrel conformation comprised of beta sheets lined with water molecules allowing
for the uptake and transport of lipid moieties. Fabp4 is expressed only in mature
adipocytes.
Dickkopf-related protein 1(Dkk-1) is a protein involved in development and
is known to be an antagonist of the WNT/beta catenin pathway. Dkk1 levels
have been observed to be upregulated during adipogenesis by adipocytes
(Christodoulides et. al. 2006, Park et. al., 2008). This idea consistent with recent
unpublished data suggesting that elevated levels of beta catenin inhibit adipocyte
differentiation in subcutaneous adipocytes. Consistent with the known roles of
Dkk-1 in hair development, ectopic expression of Dkk-1 inhibited the formation of
hair placodes (Andl et. al. 2002).
Pref-1 is a transmembrane molecule present on pre-adipocytes and is
known to function as an inhibitor of adipogenesis (Wang et. al., 2006) . Pref-1
mRNA and protein expression are abolished during adipocyte differentiation
indicating that pref-1 is regulated during this process (Wang et. al. 2010). Pref-1
has not been associated with hair follicle development or maintenance.
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Results
Adipocyte development in vitro is not well understood, even less is know
of subcutaneous adipocyte development. In an effort to delineate subcutaneous
adipocyte development Oil Red O staining was first utilized to describe adipoctye
maturation in relation to hair follicle development. In Figure 21a it is apparent
that sin post natal day one mice, hair follicles are present and adipocytes are Oil
Red O negative. Samples were collected and stained daily for oil o red from post
natal day one until post natal day nine. Adipocytes were not positive for lipid
accumulation until day five and increased in size until day 9. Figure 21b is a
graphical representation of the adipocyte maturation relative to hair follicle
development. Taken together this data implies that the development and
maturation of adipocytes occurs after the overlying hair follicles.
Adipocyte differentiation markers are well known, however much of this
work has been performed in vitro. This information provided a pool of candidate
molecules with which to interrogate the developing adipocytes. C/ebp alpha
expression was high at days 1-3 and absent thereafter. Conversely, Fabp4
expression was not observed in the first 3 days but became apparent from post
natal days 4 to 9. Dkk-1 expression was absent during the first three days,
minimal expression was observed at the fourth day. The expression of DKK-1 at
this time frame suggests inhibition of Wnt signalling which is important to hair
follicle development but inhibitory to adipocyte differentiation. Interestingly, Pref-
1 was minimal at p1 and decreases by day p4 (Figure 22), this suggests that at
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p1 and later adipocytes are predominantly committed.
A summary Figure (Figure 23) illustrates expression of adipogenic markers
as subcutaneous adipose tissue develops in the skin as observed in Oil Red O
and in situ hybridization studies performed here. Prior to adipocytes
accumulating lipids there is a high expression of C/ebpa in the lower dermis. As
the levels of C/ebpa decrease, levels of adipocyte maturation markers, Fabp4,
increases. Additionally, Dkk-1 expression increases at the same time as the
adipogenic maturation markers. Taken together this data suggests that a at post
natal day 4 adipocytes begin to differentiate and begin to accumulate lipids.
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Summary
Recently subcutaneous adipocytes have gained the interest of several
researchers, due to the fact that signalling from these cells was implicated in
maintaining a quiescent state in stem cells of the hair follicle (Plikus et.al., 2006).
This suggests that hair follicles and adipocytes communicate at some level and
one may contribute to the development and or homeostasis of the other.
Additionally, it has been suggested that there is a there is a subcutaneous
adipocyte cycle similar to the hair cycle due to the expansion and contraction of
the dermal fat layer (Festa et al., 2011) observed during the hair cycle. Because
little is understood about adipocytes in vivo this was an effort to describe the
development and identify markers for these cells. The Oil Red O staining (Figure
21) suggests that although adipocytes may be present in the dermis from post
natal day 1 these cells are not taking up lipids until the fourth or fifth postnatal
day. This would imply that these adipocytes are not mature. Taken with the fact
that there minimal minimal Pref-1 positive cells implies that the adipocytes that
are present are committed to the adipocyte lineage, but not mature (Gregoire et.
al., 1998). This idea is buttressed by Fabp4 in situ hybridization data where
expression of this mature adipocyte marker is absent on the first through third
days but is thereafter present. To further support this idea is the expression of
Dkk-1 that is present after at day 4 but not at earlier stages.
Dkk-1 is a know inhibitor of the Wnt/Beta catenin pathway. Recent
unpublished data using beta catenin null mice has shown an expansion of dermal
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adipocytes, conversely overexpression of beta catenin resulted in a decreased
fat layer. The observed expression of Dkk-1 at post natal day 4 is in correlation
with the time frame in which we see Oil Red O expression, C/ebpA turns off and
FABP4 expression turns on indicating that Dkk-1 is inhibiting Beta Catenin
expression. Beta Catenin is a known morphogen expressed during hair follicle
development. Prior to the time Dkk-1 expression is turned on the hair follicles are
developing, at the time Dkk-1 is on the hair follicles appear morphologically
mature. It is at this time that the adipocytes begin to maturate.
C/ebpA expression is a marker for committed but immature adipocytes
(Lopez et. al., 2009). C/ebpA is observed day one through 3 then is absent at day
4, this is consistent with the oil red o staining and the expression of the markers
for mature adipocytes. Throughout the samples Pref-1 expression was absent.
This is consistent with the idea that Pref-1 is expressed by adipoctyes that are
not committed and immature and indicates that at postnatal day 1 the adipocytes
in the skin are committed and immature.
All of this information taken together suggests that in the developing
mouse skin C/ebpA is a marker for committed but immature adipocytes, Fabp-4
is a marker for committed and mature dermal fat, and subcutaneous adipocyte
Dkk-1 expression is effectively the switch between hair follicle maturation and
adipocyte maturation.
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Figures
Figure 21: Adipocyte maturation relative to Hair follicle development in mouse
skin
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Figure 22: In Situ Hybridization of adipocyte markers in developing skin
94
Figure 22, Continued
95
Figure 22, Continued
Figure 23: Summary of signalling molecule expression
96
Figure legend:
Figure 21: Adipocyte maturation relative to Hair follicle development in mouse
skin
A. Mouse pup skin was collected at days 1-9. Skin was then stained with
Oil Red O to visualize adipocytes that possess lipids. At days one through three
there was no positive staining in the tissue. At day 4 there was minor staining
and by day 5 adipocytes were staining positive for lipids throughout the skin.
Size bars: 1mm,.25mm.
B. A schematic for the time frame illustrating the temporal differences
between hair follicle development and adipocyte maturation.
Figure 22: In Situ Hybridization of adipocyte markers in developing skin
Whole mount in situ hybridization for adipocyte marker mRNA. Probes for
C/EBP alpha, Fabp4, DKK-1, Leptin, Leptin Receptor (ObRb), and Pref 1 were
used to interrogate pup skin for mRNA expression. While Leptin and Obrb were
negative C/EBPa, Fabp4 and DKK exhibit interesting expression patterns. C/ebp
alpha expression is high during days one through three. Expression is not
apparent after day 4. Fabp4 expression is the inverse of C/ebp alpha. No
expression is observed in days one through three but after day 4 expression is
high. DKK expression is not present at day 1 but is faint at day 4. Minimal of
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Pref-1 mRNA expression indicates that pre-adipocytes are differentiating. Size
bars, 200um.
Figure 23: Summary of signalling molecule expression
C/EBP alpha signalling is observed in committed but immature adipocytes.
As expression of Dkk-1 increases, thereby decreasing Beta Catenin,
subcutaneous adipocytes mature. This maturation is marked by an increase in
FABP4 expression, followed by an uptake in lipids.
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Chapter 6: Discussion and Future Directions
Does the macroenvironment influence stem cell homeostasis? If we pan
out from the information gathered here we see that on three levels stem cell
activity can be influenced by the external environment, circulating hormones and
tissues that although adjacent to the stem cell niche are not directly in contact
with the niche we can see that undeniably stem cells integrate inputs from
macroenvironment.. Examining the regenerative hair cycle and its propagation
qualities that have been defined (Goldstein and Horsely 2012; Plikus et al.,
2008 ), it is possible to see that circadian rhythms, pregnancy and adipose
development influence stem cell activity and the propagating hair wave.
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Circadian Rhythms as a Stem Cell Modulator
Circadian gene expression as a factor in hair follicle stem cell
maintenance is a novel concept. Here using a mouse model with tissue specific
circadian gene knockouts did not render a dramatic phenotype. However,
subsequent work investigating the cell cycle progression of this model system
has shown that at the subcellular level there is some cell cycle misregulation,
unpublished data. Additionally, in Period 1,2 luciferase reporter mice vibrissae
cultures were observed to oscillations over a 48 hour period. This is corroborated
by recently published data from several labs. In a fluorescent mouse model
engineered to report the activity of circadian genes it was observed that there
exist two populations of epidermal stem cells in the hair follicle, each active at
different phases of the clock (Janich et. al., 2011). The authors report that
deletion of Bmal1 modulated an accumulation of stem cells in a progressive
manner. It was further reported that in isolated bulge stem cells of Bmal1 null
mice there was a lower expression of Wnt related genes as well as lower levels
of Tgf-beta related genes.
Another group investigated the interaction of circadian genes and hair
follicle stem cells using a genomics approach (Lin et. al., 2009). The study was
performed on mice with a synchronized hair wave, at post-natal day 21. In this
study there was a delay of first anagen in Bmal1 mutant mice. Although this study
provides clues to the role that circadian genes may play in hair follicle stem cell
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regulation the model differed from our study in that the mouse model we used the
hair wave on the dorsal aspect of the mice was not synchronized. In our system
the mice were an average age of approximately 2months, as opposed to 21
days. The goal was to investigate the role of the external environment on the
hair cycle as it propagates in adult mice. At the cell cycle level they report
modulation during proliferation, this is consistent with our studies using the
Bmal1 null and other transgenic systems system.
Taken together, our study is consistent with current literature. The lack of a
significant phenotype can be understood by the accumulation of Wnt associated
proteins and Tgf-beta proteins observed by the Janich group. These two
morphogens act in opposition, therefore an accumulation of both could
conceivably result in a lack of phenotype. Further, the phenotype observed by the
Lin group does not negate a lack of phenotype in mice with an asynchronous hair
wave. Consistently all the groups have reported a modulation of the cell cycle in
systems with ablated clock genes. Importantly, in nature seasonal changes have
been observed to impact molting, mating and shedding among other
physiological changes. In our system animals are confined to a indoor
environment where seasonal changes are not experienced. Specifically our
animals are exposed to a 12 hour light cycle, with no shortening of light and
elongation of dark. Accompanying this idea, animals are housed in a vivaria
where a specific temperature is maintained, in the wild temperatures vary.
Because mouse hair follicles are important in thermo-regulation, these
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environmental cues could be a major factors that could drive circadian clocks to
have a larger impact on stem cell homeostasis in the hair follicle regeneration
cycle in the wild that is lost on domesticated animals. To further investigate how
peripheral clocks may play a role in the propagating hair wave it may be
beneficial to design an experiment where mice were housed in an environment
that incorporate fluctuations of light and temperature that more closely resembled
those in nature. A more “natural” environment may give a phenotype in wildtype
mice that when compared to a transgenic system may yield significant data. The
novel finding of this study is that peripheral clocks in the skin and hair follicle are
regulated at the cell cycle level and that although in our system a phenotype was
not obvious a mechanism in which circadian rhythms can, to a larger degree,
influence the regenerative hair cycle.
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Pregnancy Telogen Hair Wave Reset
The modulation of the hair cycle during pregnancy has been observed and
reported in the literature in the past (Fraser and Nay 1953; Ebling 1964; Winton
and Lewis 1982; Plikus et al. 2008). However, other than a brief description of the
phenotype, no formal study describing the molecular mechanisms that drive the
hair wave reset has been published. Here is provided a detailed study of the hair
wave reset during pregnancy. A time point at which the wave is reset during
pregnancy is provided, hormones that might contribute to the reset, and a
molecular pathway in which morphogens may hold the hair cycle in refractory
telogen are also offered.
Although the reset of the wave was observed to initiate at different times
during mouse pregnancy it consistently occurred late in pregnancy and the hair
cycle was always in the telogen state during lactation. Anagen was observed to
initiate after lactation ceases. These strongly implicated a pregnancy related
hormone or multiple pregnancy associated hormones to be involved in this
phenomena. By observing the hair wave of pregnant mice it appeared that the
reset consistently occurred during lactation. To confirm if lactation was a factor in
the hair wave reset the duration of lactation was artificially truncated to determine
the amount of time necessary for anagen to initiate (Figure16 n-3). In three trials
anagen initiated an average of 6 days after the removal of suckling pups
(standard deviation 1.1547). This strongly implicates hormonal cues that are
expressed during lactation promote the telogen phase, or inhibit anagen.
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Additionally the suckling may invoke the telogen response. To examine this, a
successful foster mouse system would provide some clues. Unfortunately, I was
not able to successfully get a nonpregnant female mouse to feed pups in a foster
fashion. This experiment would have certainly answered the question of the
timing of the hair wave reset being either a factor of pregnancy or lactation.
It has been previously been determined that several molecules can
maintain hair follicle stem cells in a quiescent state (Plikus 2012) as well as act
as Wnt antagonists (Park et. al.2008). This information provided a candidate pool
from which were selected Follistatin, Sfrp1, Sfrp4, and Bmp2 as downstream
factors that might be upregulated during the pregnancy/lactation hair wave reset.
In situ hybridization studies exhibited mRNA transcripts from the above
candidates to be expressed at varying degrees during the pregnancy and
lactation period in the subcutaneous adipose tissue. The subcutaneous
adipocytes have previously been shown to express inhibitory factors associated
with telogen (Plikus et. al., 2008). It can be hypothesized that this redundancy in
the expression of inhibitory morphogens functions to ensure a telogen state
during pregnancy potentially to allow for hair loss to provide material for nest
building. A similar phenomena has been observed in pregnant rabbits; telogen
results in hair loss, this fallen hair is subsequently used in nest building for kits
(Gonzalez-Mariscal et. al., 1996). Mice are diligent nest builders. Again in our
system with “domesticated” mice that are housed in climate controlled
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environments and provided excess material with which to build nests the hair
plucking and nest building phenomena may be lost.
During pregnancy several hormones are known to fluctuate relative to non
pregnant levels (Soares et. al., 2004). Several of these hormones are known to
have cognate receptors that have been localized to one or more hair follicle
compartment. Using immuo-assays progesterone has been localized to the
dermal papilla of hair follicles but not the epithelial cells of the hair follicles
(Pelletier et. al., 2004). Both estrogen receptors have been localized to the
mouse hair follicle and are expressed throughout the hair cycle. Topical estrogen
has been observed to arrest hair follicles in telogen, and topical E2 potentates
catagen. Estrogen receptor antagonists were not observed to induce anagen
(Ohnemus et. al., 2005). Finally, prolactin has been reported to elongate telogen
(Craven et al., 2006). To identify if one or more hormones that are associated
with pregnancy might be important in the hair wave reset observed the
application of protein coated beads was utilized. In this assay affi-gel blue beads
were soaked in a hormone of question then inserted intradermally into the skin of
a non-pregant wild type female mouse. By exogenously expressing hormones in
the skin, adjacent to the propagating hair wave it is possible to observe the
interaction of the hormone and hair follicle stem cell. In the assay performed in
this study beads were coated with progesterone, estradiol alpha and beta and
prolactin. Based on observations made over several mice the beads coated with
prolactin exerted the most significant, telogen-like stall effect on the hair wave.
105
Although progesterone receptors have also been localized to the skin and
some follicular compartments(Pelletier and Ren 2004) limited information has
been published regarding the influence of this hormone on the hair cycle. This
fact in conjunction with the results of the exogenous bead assay suggested that
progesterone has minimal interaction with hair follicle stem cells during the hair
wave reset seen in Figure 18. Similarly, the results of the estadiol alpha and beta
coated beads do not conflict with what is previously published. The results of the
assay performed showed an induction of telogen by estradiol alpha and estradiol
beta(Figure 18). This finding is supported by the published finding regarding
estrogen elongation of telogen (Ohnemus et al 2005).
The results of the exogenous application of prolactin is consistent with
previous reports, an elongation of telogen. It has been previously reported that
prolactin elongates telogen in non-pregnant mice (Craven et. al., 2006). This
implicates prolactin as a key hormone in the hair wave stall. Previous studies
report a prolactin induced elongation of telogen in non-pregnant mice but no work
implicating prolactin in the hair wave stall during pregnancy has been done. In
our functional study as the hair wave progressed towards the prolactin coated
beads, the area adjacent the beads was not coerced into anagen, but rather
telogen persisted in what appeared to be a zone of inhibition (Figure 3).Affi-gel
blue beads are implanted into telogen state skin. As the hair wave progressed
towards the beads, the area adjacent the beads was not coerced into anagen as
seen in Figure 18. Instead the hair follicles remained in telogen. As lactation
106
begins there is a up regulation of prolactin receptors in the subcutaneous
adipose tissue (Figure 19). This coincides with time point at which telogen
initiates during the pregnancy lactation reset. This fact further implicates prolactin
as a major player in the pregnancy telogen phenomena.
To further investigate how circulating levels of prolactin might be exerting a
modulatory role on hair follicle stem cells, skin tissue was interrogated for
prolactin receptor expression. Because there is an increase in the number of
adipocytes expressing prolactin mRNA (Figure 19) throughout the pregnancy and
lactation period it can be inferred that to some degree prolactin signalling may
induce the expression of the inhibitory molecules responsible for the hair wave
reset that is observed during lactation.
Subcutaneous Bmp2 expression has been implicated as a significant
factor in refractory telogen (Plikus et. al., 2008). It was observed here that during
the pregnancy telogen state, several inhibitory molecules are expressed in the
dermal fat layer. Because prolactin was strongly implicated in reseting the hair
wave it was important to see if one or more of the inhibitory molecules were
being expressed downstream of prolactin signalling. Here ectopic prolactin
expression was followed with in situ hybridization investigated the skin for mRNA
of inhibitiory molecules. Figure 20 illustrates that while Bmp2 expression is high
transcripts for the other inhibitory signalling molecules are not.
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In summary, although there are a multitude of differentially expressed
hormones and morphogens during pregnancy it is shown here that an increase of
prolactin signalling late in pregnancy and maintained through lactation is a strong
candidate in the hair wave reset. Additionally based on results of exogenous
prolactin expression it can be inferred that Bmp2 is operating downstream of
prolactin to maintain the quiescence of hair follicle stem cells, and subsequently
the telogen state of the hair wave through the subcutaneous adipocytes. In
classical literature the hair wave reset was described as a telogen state that
endured throughout lactation with anagen returning in an explosive manner at the
conclusion of lactation. Here is a plausible path in which the telogen portion of
the reset has been explained, the route in which anagen returns, possibly due to
Wnt/beta catenin or noggin, is a candidate for future studies.
Because hormonal signals are important to the hair wave an interesting
future direction would be castration to ablate either ovarian derived estrogen in
the female, or testosterone in the male. Although the hair cycle is not generally
considered to be sexually dimorphic in mice studies such as these may show
sexually dimorphic trends. Additinaly, the estrus cycle has not been investigated
as a factor in the hair cycle. Although the estrus cycle is four days and anagen
and telogen periods are approximately 20, and 60 respectively, there may be
some differential hair growth observed over the the phases of the estrus cycle as
a response to circulating hormones. Both the castration and estrus cycle studies
are good candidates for future investigations.
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This study provides clues to how the hair wave reset occurs during
pregnancy. On a larger scale the results from this study provide clues as to how
hormonal signaling can influence stem cell activity. The applications for the
results here can have implications in cancer research and regenerative medicine.
The novel finding here is that late in pregnancy subcutaneous adipocytes
express prolactin receptors and appear to mediate systemic prolactin signaling
the result of which is the expression of inhibitory morphogens that act on the hair
cycle to suspend the hair cycle in a resting state while the organism endures
pregnancy. Undoubtedly, phenomena like these are conserved evolutionarily. In
this case it can only be speculated that this mechanism is retained as a nest
building strategy or a means to restrict energy output. In any case, it appears
there is a redundancy in the mechanism. In this study it has been observed that
at least two inhibitory molecules are expressed during pregnancy indicating that
there is a biological importance in the pregnancy related telogen state.
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Subcutaneous Adipocyte Development
Recently subcutaneous adipocytes have gained the interest of several
researchers due to the fact that signalling from these cells was implicated in
maintaining a quiescent state in stem cells of the hair follicle (Plikus et.al., 2006).
This suggests that hair follicles and adipocytes communicate at some level and
one may contribute to the development and or homeostasis of the other.
Additionally, it has been suggested that there is a there is a subcutaneous
adipocyte cycle similar to the hair cycle due to the expansion and contraction of
the dermal fat layer (Festa et al., 2011) observed during the hair cycle. Because
little understood about adipocytes in vivo this was an effort to describe the
development and identify markers for these cells. The Oil Red O staining (Figure
21) suggests that although adipocytes may be present in the dermis from post
natal day 1 these cells are not taking up lipids until the fourth or fifth day post
natally. This would imply that these adipocytes are not mature. Taken with the
fact that there minimal minimal Pref-1 positive cells implies that the adipocytes
that are present are committed to the adipocyte lineage, but not mature (Gregoire
et. al., 1998). This idea is buttressed by Fabp4 in situ hybridization data where
expression of this mature adipocyte marker is absent on the first through third
days but is thereafter present. To further support this idea is the expression of
Dkk-1 that is present after at day 4 but not at earlier stages.
110
Dkk-1 is a know inhibitor of the Wnt/Beta catenin pathway. Recent
unpublished data using beta catenin null mice has shown an expansion of dermal
adipocytes, conversely overexpression of beta catenin resulted in a decreased
fat layer. The observed expression of Dkk-1 at post natal day 4 is in correlation
with the time frame in which we see Oil Red O expression, C/ebpA turns off and
FABP4 expression turns on indicating that Dkk is inhibiting Beta Catenin
expression. Beta Catenin is a known morphogen expressed during hair follicle
development. Prior to the time Dkk-1 expression is turned on the hair follicles are
developing, at the time Dkk-1 is on the hair follicles appear mature. It is at this
time that the adipocytes begin to maturate.
C/ebpA expression is a marker for committed but immature adipocytes
(Lopez et. al., 2009) C/ebpA is observed day one through 3 then is absent at day
4, this is consistent with the oil red o staining and the expression of the markers
for mature adipocytes. Pref-1 expression was minimal at day 1 and by day 4
appeared absent. This is consistent with the idea that Pref-1 is expressed by
adipocytes that are not committed and immature and indicates that at postnatal
day 1 the adipocytes in the skin are committed and immature.
All of this information taken together suggests that in the developing mouse skin
C/ebpA is a marker for committed but immature adipocytes and subcutaneous
adipocyte Dkk-1 expression is effectively the switch between hair follicle
maturation and adipocyte maturation.
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This study was descriptive in nature and provide some clues for further
studies on the interplay between the developing hair follicles and adipose tissue.
Missexpression studies using Fabp4 and Dkk1 would be particularlly interesting.
By overexpressing FABP4 it could be speculated that the dermal adipoctyes
would accumulate lipids at either an earlier stage or at a faster rate. Because the
hair follicle development appears to be slightly advanced compared to the fat
layer a premature accumulation of lipids by the fat layer may truncate hair follicle
development. Conversely, blocking FABP4 lipid transport activity it can be
surmised that the subcutaneous fat layer would not develop properly, this could
potentially in turn disrupt the robustness of the hair follicle as a result of improper
signalling or trophic supply. It would be interesting to misexpress DKK1 in mouse
skin. It could be surmised from the studies here that if DKK1 was overexpressed
there would be an inhibition of Wnt/beta catenin signalling resulting in an
increased dermal fat layer and a decrease in hair development. Because the
Wnt/beta catenin path is so important in development these studies would have
to be done in a time and tissue specific manner. Currently adipocytes research
has gained momentum based on the fact that there are an abundant amount of
stem cells housed in adipose tissue. This research provides clues to the
development of the subcutaneous fat layer in relation to hair follicle development.
This research also provides information for the application of the dermal fat layer
as a potential candidate in regenerative medicine. What I learned here is that a
delicate interplay between hair follicle and subcutaneous adipocyte tissue exists.
112
This study provided evidence that beta catenin siganling, which is important to
hair follicle development is inhibitory to the development of the adjacent dermal
fat. Beta-catenin signaling is inhibited by Dkk-1 at a point when hair follicles are
maturing, at this time point adipocytes begin to mature. The interaction of the
morphogens governing tow tissues in tandem is novel.
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Future Directions
The studies contained here provide novel insight to the
macroenvironmental influence on hair follicle stem cell homeostasis. Closely
examining the role of circadian rhythms, hormonal signaling, and adipose tissue
as modulators of follicular stem cell activity has provided some clues but has left
also some novel questions.
Life is influenced by light cycles. While the studies here shed some
enlightenment on the influence of light on stem cell activity more work needs to
be done expand on the information presented here. These studies were limited
to mice housed in a controlled environment that included static lighting
conditions. Understanding how hair follicle specific peripheral circadian clocks
influence the hair cycle in natural conditions is necessary to better understand
how light cues can influence hair follicle stem cells. Information from studies
such as these will have applications in human health and disease. To better
understand the influence of light cues on stem cells, studies that utilize a more
natural setting would provide insight. Molecular studies using tissue samples
from mice housed in a natural environment would elucidate cell proliferation
differentials between light and dark cycles.
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A relationship exists between physiological states and stem cell
maintenance. Observed here is how pregnancy can influence quiescent stem
cell activity of hair follicles via hormonal signaling. How stem cell activity is
initiated in the post lactation period will provide clues that will aid in elucidating
the role of hormonal regulation of stem cells. How the reciprocal quiescence and
activity is regulated by hormonal signaling will have an impact in the fields of
cancer research and human health care. The hair wave initiates at the
conclusion of lactation. A similar model system to the one utilized here, modified
to investigate the onset of anagen would be used. Molecular studies
investigating morphogen signalling would provide insight as to which molecular
pathway the initiation follows.
The emerging recognition of adipose tissue in the fields of regenerative
medicine and stem cell research is going to expand. The research presented
here suggests that a dynamic relationship exists between dermal adipocytes and
the hair follicles. Understanding the reciprocal signaling that occurs between
these two tissues to maintain homeostasis is necessary. Discerning how two
tissues communicate has applications in stem cell research and regenerative
medicine. To investigate the role adipose plays in follicular stem cell
maintenance a mouse models with aberrant adipose would be useful.
Taken as a whole these studies provide significant evidence that hair
follicle stem cells are regulated, to some degree, by the macroenvironment.
Undoubtedly there is an integration of signals that are inputted at both the
115
macroenvironmental and microenvironmental level. How these signals sets are
integrated and ultimately influence how stem cell homeostasis is a future goal for
stem cell research.
116
Materials and Methods
Animals
Animals were housed in the University of Southern California vivaria in
accordance with Institutional Animal Care and Use Committee and with the
Principles of Laboratory Animal Care.
Oil Red O staining:
Whole back tissue samples were collected and washed in 1X Phosphate
buffer solution. Following a tap water rinse samples were washed in Propylene
glycol twice for five minutes. Samples were then immersed in Oil Red O stain for
7 minutes and incubated at 60°C. After the incubation period samples were
washed in 85% Propylene glycol for three minutes. then rinsed in cold tap water.
Samples were stored in 50% glycerol/diH2O containing 3% Sodium Azide. Oil
Red O Solution was prepared per the manufacturer's protocol. Oil Red O (.7gm)
was slowly dissolved in propylene glycol (100mL) with stiring at 100°C. The
solution was then filtered and stored at 60°C.
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RNA probes (adipose):
RNA probes were generated based in the following primer sets: C/EBP: 5’-
TGGAACCCCGAGGCTTTAT-3’,5’-GTGGACAAGAACAGCAACGA-3’
(Differential gene expressions in subcutaneous adipose tissue pointed to a
delayed adipocytic differentiation in small pig fetuses compared to their
heavier siblings-F. Gondreta ,M.H. Perruchota, S. Tachera,J.Be´rard, G. Bee)
FABP4:5’-TCGACTTTCCATCCCACTTC-3’, 5’- CATCAGCGTAAATGGGGATT-3’,
Leptin 5’-TCAAGCAGTGCCTATCCA-3’, 5’CATTCAGGGCTAACATCCA-3’,
Leptin Receptor: 5’TGACTGGAGTTCACCTCA-3’, 5’-
GACCACATAGACTGCACA-3’ PPar gamma 2 5’-TCACAAGAGCTGACCCAA-
3’, 5-’CTCTGAGATGAGGACTCCA-3’.
RNA probes (neuroendocrine signalling):
RNA probes were generated based in the following primer sets: Prolactin
receptors: prlr1-T7 5’-TAATACGACTCACTATAGGGAGA
-AAGCCAGACCATGGATACTGGAG-3’, pL-T35’-
AATTAACCCTCACTAAAGGGAGA-AGCAGTTCTTCAGACTTGCCCTT-3’, pS1-
T35’-AATTAACCCTCACTAAAGGGAGA-AACTGGAGAATAGAACACCAGAG-3’,
pS2-T35’-AATTAACCCTCACTAAAGGGAGA-
TCAAGTTGCTCTTTGTTGTGAAC-3’, pS3-T35’-
AATTAACCCTCACTAAAGGGAGA-TTGTATTTGCTTGGAGAGCCAGT-3’.
118
Follistatin
5’-GAAACTAATACGACTCACTATAGGGTTTTGCCCAAAGGCTATGTC-3’, 5’-
GAAACTAATACGACTACTATAGGGCCTCCTCTTCCTCCGTTTCT-3’, Sfrp1 5’-
GAAACTAATACGACTCACTATAGGGCGTTCTTCAGGAACAGCACA-3’, 5’-
GAAACTAATACGACTCACTATAGGGCGTTCTTCAGGAACAGCACA-3’, Sfrp2
5-’GAAACTAATACGACTCACTATAGGGACCAGATACGGAGCGTTGAT-3’, 5’-
GAAACTAATACGACTCACTATAGGGACCAGATACGGAGCGTTGAT-3’ Sfrp4
5’-GAAACTAATACGACTCACTATAGGGATCATCCTTGAACGCCACTC-3’
5’-GAACTAATACGACTCACTATAGGGATCATCCTTGAACGCCACTC-3’ Sfrp5
5’- GAAACTAATACGACTCACTATAGGGTTCAGCTGCCCCATAGAAA-3’
5’-GAAACTAATACGACTCACTATAGGGTTCAGCTGCCCCATAGAAA-3’
The plasmid containing the DKK1 probe was a gift from Sarah Miller.
BMP2 probes were previously generated by the Chuong laboratory (Plikus et. al.
2008.)
Circadian Rhythms:
Animals were generated using the cre-lox system. Mice were either tissue
specific or tissue specific and regulatable K14cre mice were crossed with Bmal 1
floxed mice to create tissue specific lines. To regulate both time and tissue
specificity Bmal 1 floxed mice were crossed with K14cre ER mice. Adult
K14creER;Bmal1-/- homozygous mice were treated with tamoxifen (Sigma
Aldrich T5648). Treatment was as follows: Tamoxifen was diluted in 100% EtOH
119
at 25mg/ml. For the first three days of treatment 200 uL was applied to the back
of shaved mice. For the subsequent 8 days of treatment 100uL of the working
solution was applied to the back of the mice. Control, also positive for the both
Cre and lox genes, mice were treated with EtOH vehicle only with the same
volumes as the experimental mice. At the conclusion of the tamoxifen treatment
mice were housed in a room with strict 12 hour light and 12 hour dark cycles to
synchronize the central circadian clock. Mice were sacrificed at 50 hours and 62
hours after they were released into constant darkness, dorsal skin containing hair
cycle wave(s) was collected. The skin samples were fixed in 4%
paraformaldehyde at 4°C overnight and processed for histology as described
previously (Plikus MV et al., 2004).
Prior to being housed in constant dark, dorsal fur on the mice was clipped
as previously described so that the hair cycle stage of the mice could be
monitored.

Genotyping of Bmal1 mice:
Mice were anesthetized using ketamine and xylazine (7:3). DNA was
isolated from tail clippings using the DNeasy Blood and Tissue Kit (Qiagen,
Catalog number:69504). Isolated DNA was amplified using standard PCR
techniques. Primers sequences for conditional Bmal1-/- allele were provided by
Jackson laboratory. Forward primer: 5'-ACTGGAAGTAACTTTATCAAACTG-3',
120
and reverse primer: 5'-CTGACCAAC TTGCTA ACA ATTA-3'. The expected
results are illustrated in Figure 12 . Mutant mice homozygous for the floxed Bmal
allele had a single band at 431bp. Heterozygous mice had two bands, one with
the floxed allele at 431bp, and another band 327bp for the wild type allele. Wild
type mice that did not carry the floxed allel had a single band at 327bp. Oligos
for Cre recombinase were, forward 5’- TTGCCCCTGTTTCACTATCCAG-3’ and
reverse 5’-ATGGATTTCCGTCTCTGGTG-3'. The expected product of the PCR
reaction using these primer was 335bp. Bmal1 (B6.129S4(Cg)-Arntl
tm1Weit/
J) were
purchased from Jackson Laboratories. K14creER mice were a gift from
Krzysztof Kobielak. K14cre mice were a gift from the lab of Yang Chai.
RNA Probe Synthesis:
Probe synthesis was performed as follows, briefly primes based on the above
design were amplified using mouse pup cDNA. The amplification product was
then incubated with Dig-RNA labeling mix and RNA polyemerase for 2 hours at
37°C. The labeled RNA product was then purified and suspended in
prehybridization solution.
121
In Situ Hybridization:
Whole mount in situ hybridization was performed as follows. Whole back
sample were collected and washed in 1X Phosphate Buffered Solution with 1%
Tween 20 containing DEPC water. Samples were then dehydrated through a
methanol gradient and stored at -20 degrees until required. Samples were
rehydrated by washing for ten minutes in a graded methanol series in PBT. To
minimize endogenous peroxidase signal tissue samples were treated with 6%
H2O2 for 20 minutes. The tissue was then treated with 10ug/ml of Proteinase K
for 10 minutes at room tempurature. Samples were post fixed with fresh 0.2%
glutaraldehyde with 4% paraformaldehyde in PBT solution for 20 minutes.
Samples were then incubated for two hours in prehybridization solution at 70°C.
During the two hour incubation the RNA probes were denatured at 70°C for two
minutes. Samples were incubated with RNA probes overnight at 70°C with
gentle rocking. The next day the samples were washed three times with 2X SSC
followed by three washes in 0.2X SSC. Following a PBT rinse samples were
blocked for 2 hours at room temperature in 20% goat serum in PBT. During the
blocking period anti-Dig-AP antibodies (Roche) were preabsorbed in 20% goat
serum in PBT for 2 hours. Samples were then incubated overnight at 4°C. The
following day the samples were washed in PBT containing 1mM levamisole for 1
hour five times. Samples were then washed in NTMT and developed in a
solution of NTMT containing NBT and BPIC. Samples were stored in 50%
glycerol/diH2O containing 3% Sodium Azide.
122
Protein bead administration:
Intracutaneous administration of exogenous protein was performed as
follows. Affinity chromatography Affi-gel blue beads were obtained from Biorad.
Beads were washed in 1X PBS, followed by drying overnight at room
temperature. The beads were then re-suspended in 5µl protein solution, either
control (0.1% BSA) or experimental (recombinant mouse Prolactin, 100ug/ml;
recombinant mouse Progesterone, 100ug/ml, Estradiol alpha 100ug/mL or
Estradiol beta 100ug/mL), at 4 °C for 30 min. Both recombinant Prolactin was
from R&D Systems, Estrogen receptor alpha and beta agonists and
Progesterone were obtained from Tocris bioscience. Reconstitution of the protein
was as per the manufacturer's guidelines. Approximately 100 beads were
introduced to the telogen skin of adult mice female by means of a single puncture
made by a 30G syringe needle. To replenish proteins, subsequent doses of 1.5 µl
protein solution were microinjected to the site of the bead implantation every 24
hrs by means of a glass micro-needle. Subsequently, skin was collected and
inverted for whole-mount analysis of hair regeneration patterns around the
control beads and beads with Prolactin, Progesterone, and the estradiol alpha
and estradiol beta.
123
Hair clipping of mice:
Mice were anesthetized using a 7:3 mixture of Ketamine to Xylezine.
Approximately 100 uL was delivered intramuscularly to the mice. Mice were held
in an inverted fashion and using an Oster A5 the back hair of the mice was
shaved, moving against the grain of the hair, to 0.5mm (Plikus MV and Chuong
CM, 2008).
124
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Abstract (if available)

Abstract The burgeoning field of stem cell research is expanding beyond the initial studies that involved localizing, isolating and culturing of the cells. How stem cell homeostasis is regulated is now the focus of current research. Even more nascent is the idea of how homeostasis is regulated from signals outside the defined stem cell niche or microenvironment. The extra-niche area is defined as the Macroenvironment. This includes adjacent tissue, distant organ systems and even the external environment. To investigate the macroenviroment as a stem cell regulator, hair follicle stem cell interactions with 1)adjacent tissues (subcutaneous adipose tissue), 2)remote signalling (neuroendocrine signalling), and 3)the external environment (circadian rhythms) were studied. Capitalizing on recent work that highlighted inhibitory Bmp2 signalling from the subcutaneous adipose tissue to be in phase with quiescent telogen and non-propogating autonomous anagen portions of the hair cycle we investigate the post-natal development of the dermal fat layer in relation to hair follicle development. During pregnancy in humans and mice modulation to the hair wave, hair cycle and ultimately hair follicle stem cells has been recorded in both classical and current literature yet no studies have been recorded that investigate this phenomena in detail. Using a murine system it is shown show that the hair wave is reset to a telogen resting phase as a consequence of prolactin signalling late in pregnancy and throughout lactation. Additionally, it is observed using in situ hybridization that the prolactin signalling is mediated via the increased expression of prolactin receptors associated with the dermal fat layer and subsequent expression of Bmp2. In the murine model hair grows in a cyclic fashion. Although much of the cycle has been elucidated the source of cycle timing still eludes hair biologist. Using current mouse technologies from the field of circadian rhythms we investigate the possible role of peripheral clocks in the skin by ablating the core component Bmal1 from the circadian clock system in a skin specific knock out model, K14creERtam, Bmal1 mice. These studies have impact on not only the field of hair biology but on a larger scale stem cell research, regenerative medicine and pathologies involved with adipose tissue, hormonal signalling involved with cancers, and circadian rhythms.

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Asset Metadata

Creator de la Cruz, Damon Nino (author)

Core Title Macroenvironmental regulation of hair follicle stem cells

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Pathobiology

Publication Date 07/23/2012

Defense Date 04/14/2012

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

Tag circadian rhythms,hair follicle,OAI-PMH Harvest,Pregnancy,subcutaneous adipose tissue

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chuong, Cheng-Ming (committee chair), Coetzee, Gerhard (Gerry) A. (committee member), Dubeau, Louis (committee member), Widelitz, Randall B. (committee member)

Creator Email dndelacr@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-61471

Unique identifier UC11288858

Identifier usctheses-c3-61471 (legacy record id)

Legacy Identifier etd-delaCruzDa-970.pdf

Dmrecord 61471

Document Type Dissertation

Rights de la Cruz, Damon Nino

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...

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