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Discovery of a novel role for Sonic Hedgehog during the early stages of large-scale murine rib regeneration

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Discovery of a novel role for Sonic Hedgehog during the early stages of large-scale murine rib regeneration

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Content Discovery of a novel role for Sonic Hedgehog during the early stages of large-scale murine rib
regeneration
by
Maxwell Serowoky
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
(DEVELOPMENT, STEM CELLS, AND REGENERATIVE MEDICINE)
May 2022
Copyright 2022 Maxwell Serowoky
ACKNOWLEDGMENTS
Amy Merrill, PhD (Chair)
Gage Crump, PhD (Program Director)
Francesca Mariani, PhD (Mentor)
I am fortunate to consider my PhD training an experience full of growth and joy. This is in
large part due to the support of my mentor, Dr. Francesca Mariani. She is exactly the type of
mentor that PhD students dream of: helpful, patient, and unconditionally supportive. The positive
environment she has cultivated in her lab allows our group to thrive in ways that many labs don’t
allow for. She mentors humans, not just scientists, and her ability to connect on both personal
and professional levels with us trainees has made a powerful and lasting impression on me. I
hope to lead a life that embodies the kindness and empathy she showed me throughout my
training.
My training is only as complete as can be facilitated by my training environment. In that
thread, I am hugely indebted to the generosity and support provided by USC Stem Cell faculty,
my fellow BCC trainees, and the wider USC community. From oral presentation practice and F31
preparations to manuscript writing and networking support, I received much more time and
support than I ever deserved at every stage of my training. I’d especially like the thank the director
of the DSR PhD program and T32 program Dr. Gage Crump for the extraordinary amount of time
he has dedicated to supporting my training over the last 5+ years. I’d also like to thank my
committee chair Dr. Amy Merrill for the immense amount of feedback she has given me in our
Mariani/Merrill combined lab meetings. Drs. Crump and Merrill’s thoughtful perspectives from
outside of the Mariani lab have been instrumental in driving the direction of my research project
and my overall development as a scientist.
Most importantly I’d like to thank my wife Tina and daughter Palmer for their
encouragement. Coming home every evening to their beautiful faces makes every day special,
even after another day of failed in situ hybridization experiments.
ii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
LIST OF TABLES
LIST OF FIGURES
ABSTRACT
CHAPTER 1: Skeletal Stem Cells: Insights into maintaining and regenerating the skeleton
CHAPTER 2: The use of commercially available adhesive tapes to preserve cartilage and bone
tissue integrity during cryosectioning
CHAPTER 3: A murine model of large-scale bone regeneration reveals a selective requirement
for Sonic Hedgehog
CHAPTER 4: Transplantation of periskeletal tissues to improve bone regeneration
CHAPTER 5: Concluding remarks
REFERENCES
APPENDIX
ii
35
48
94
111
118
135
iii
iv
1
v
vii
LIST OF TABLES
Table 1: Comparative summary of reported skeletal stem cells
iv
25
LIST OF FIGURES
Chapter 1
Figure 1: Skeletal Stem Cell Niches
Figure 2: Redundant pathways to make bone during development and homeostasis
Figure 3: Redundant pathways to make bone during repair
Chapter 2
Figure 1- Tapes evaluated and method of storage before use
Figure 2 - Assessment of optical clarity, autofluorescence, and workability
Figure 3 - Use of commercially available tape for histology
Figure 4 - Use for fluorescence imaging
Chapter 3
Figure 1. Shh, but not Dhh or Ihh is upregulated during early callus formation.
Figure 1 - Supplement 1. Ihh is upregulated later in mature chondrocytes during callus
differentiation.
Figure 2. Shh is required for callus generation and subsequent large-scale rib regeneration.
Figure 3. The primary source of SHH is not Sox9-derived SSPCs.
Figure 4. Shh is expressed by Prrx1-expressing cells and is not dependent upon the presence
of Sox9+ lineage cells.
Figure 5. Shh is not required for large-scale rib regeneration after 5 dpi.
Figure 6. Smo is dispensable in callus cells after 4 dpi in a large-scale injury model.
Figure 7. Shh is not required for small-scale repair.
Figure 8. Smo is not required in Sox9+ lineage cells for femur fracture repair.
Figure 9. Activation of Sox9+ lineage cells by Hh signaling is required for Cxcl12 expressing
cells to populate the early callus.
Figure 9 - Supplement 1. Characterization of scRNAseq clusters
Figure 10 – Summary diagram
34
33
32
47
46
45
44
93
92
91
90
89
88
87
86
85
84
83
82
v
Chapter 4
Figure 1. Transplanted perichondrial tissue can serve as a very minor source of osteoblasts
during rib regeneration
Figure 2. Gene expression analysis of Hh-activate periosteal tissue
Figure 3. Evaluation of ScxCreER;tdT;Scx-GFP animals for tracing the activity of perichondrial
progenitor cells in costal cartilage
110
109
108
vi
ABSTRACT
Skeletal stem cells (SSCs) generate the progenitors needed for growth, maintenance, and
repair of the skeleton. Historically, SSCs have been defined as bone marrow-derived cells with
inconsistent characteristics. However, recent in vivo tracking experiments have revealed the
presence of SSCs not only within the bone marrow but also within the periosteum and growth
plate reserve zone. These studies show that SSCs are highly heterogeneous with regard to
lineage potential. During digit tip regeneration and in some non-mammalian vertebrates, the
dedifferentiation of osteoblasts may also contribute to skeletal regeneration. Here, I examine how
this research has furthered the understanding of the diversity and plasticity of SSCs that mediate
skeletal maintenance and repair.
vii
CHAPTER 1: Skeletal Stem Cells: Insights into maintaining and regenerating the skeleton
* Large portions of this work (Chapter 1) are published in Serowoky et al., Development
2020
1

Skeletal stem cells (SSCs) generate the progenitors needed for growth, maintenance, and
repair of the skeleton. Historically, SSCs have been defined as bone marrow-derived cells with
inconsistent characteristics. However, recent in vivo tracking experiments have revealed the
presence of SSCs not only within the bone marrow but also within the periosteum and growth
plate reserve zone. These studies show that SSCs are highly heterogeneous with regard to
lineage potential. During digit tip regeneration and in some non-mammalian vertebrates, the
dedifferentiation of osteoblasts may also contribute to skeletal regeneration. Here, I examine how
this research has furthered the understanding of the diversity and plasticity of SSCs that mediate
skeletal maintenance and repair.
Introduction
Identifying the cells that maintain and repair the skeleton has been an area of intense
recent investigation. A number of research groups have identified cell types that when engrafted
give rise to new skeletal tissue, as well as specific tissue-resident cells with varying multilineage
potential that participate in skeletal homeostasis and repair. It has also been shown that, in some
non-mammalian vertebrates that are capable of large-scale skeletal regeneration, osteoblasts
may dedifferentiate into a progenitor state to replace missing bone. Many of these cell populations
come under the umbrella of skeletal stem cells (SSCs) – a population of cells that can self-renew
and generate osteoblasts, chondrocytes, adipocytes and stroma – although only a few of these
populations have been rigorously tested.
SSCs from the bone marrow were initially defined by their ability to adhere to tissue culture
plastic, clonally expand, differentiate into multiple cell types in vitro, and generate skeletal tissue
1
upon subcutaneous transplantation into mice.
2,3
Later, fluorescence activated cell sorting (FACS)
was used to isolate subpopulations of marrow cells based on combinations of cell surface
markers.
4
Recently, genetic Cre-mediated lineage tracing experiments, primarily within the
postnatal mouse, have revealed diverse populations of cells with SSC properties. These putative
SSCs are located not only within the bone marrow but also within the periosteum (the connective
tissue surrounding bone) and in the resting zone of the cartilaginous growth plate (a cartilaginous
structure separating the primary and secondary ossification centers in growing bones) (Figure 1).
These populations vary in their lineage capabilities, their prevalence in embryonic through adult
stages, and their participation in repair, highlighting that there are likely multiple types of SSCs.
Several attempts to define SSCs have taken inspiration from studies of the hematopoietic
system, where a rare population of apex stem cells gives rise to lineage-committed intermediate
progenitors and eventually all blood cell types.
5
Recent investigations suggest a similar
hierarchical system may exist for SSCs,
6,7
yet other reports suggest that the skeletal system is
surprisingly plastic, more akin to the intestinal system where differentiated cells can adopt a stem
cell state when normal stem cells are ablated.
8
For example, osteoblasts have been observed to
dedifferentiate into a progenitor state during regeneration of the fin and skull bones of zebrafish
9–
11
(Box 1), as well as during murine digit tip regeneration.
12

While strategies to identify prospective SSCs are rapidly improving, current techniques
suffer from limitations in their interpretation. Cells can change their properties after tissue isolation
and prolonged culture, and thus multilineage differentiation and self-renewal after transplantation
does not necessarily mean that the cells originally isolated had these stem properties. Methods
to isolate cells can also vary from one lab to another, making it challenging to make direct
comparisons. As an alternative, researchers have used tissue-specific expression of Cre
recombinase to induce DNA recombination and drive permanent expression of fluorescent
proteins in Cre-expressing cells and all their descendants. However, determining the precise
temporal and spatial expression of Cre activity can be challenging. The regulatory sequences that
2
drive Cre expression can be active at multiple stages of development and/or activated in response
to injury. To obtain tighter control, inducible systems can be used to produce a pulse of Cre activity
at a defined time, typically relying on fusions of Cre to the Tamoxifen-dependent estrogen receptor
(CreER). However, non-Tamoxifen-treated controls are essential to ensure the absence of “leaky”
activation of CreER, which can create confusion when interpreting results (see (Song and
Palmiter, 2018
13
) for guidelines on proper Cre/Lox experiments). Even when tight temporal control
is achieved, CreER may still be expressed in more than one cell type. In addition, many CreER
lines used in skeletal research target regulators of major signaling pathways, such as WNT
(Axin2), BMP (Grem1), and HH (Gli1), and given the widespread activity of these pathways in
non-skeletal tissues, such CreER lines are likely not specific to a single cell population.
Fortunately, emerging technologies, such as single-cell RNA sequencing and cellular barcoding,
should allow creation of a complete catalog and lineage tree of cell types in the skeletal system.
Here, I review and critique recent studies that have identified various populations of SSCs
within vertebrates. A major challenge will be to understand the lineage relationships between
diverse SSCs, as well as the specialized functions of SSCs in homeostasis and repair. I also
discuss the role of niche factors in specifying and maintaining SSCs and examine how stem cell
plasticity may underlie the degree of skeletal repair in different contexts and organisms. Such
knowledge will allow the field to better develop targeted cellular therapies that enhance skeletal
repair in the clinic.
SSCs in bone development and homeostasis
Bone marrow-derived SSCs
The bone marrow is a common site for the extraction of “mesenchymal stem cells” (MSCs),
a very heterogeneous mixture of cells, only a fraction of which may have stem properties.
Unfortunately, these cells are being tested for treatment of a wide range of conditions, often in
non-scientific, exploitative, and potentially dangerous ways.
14–16
“Mesenchyme” is a broad term
3
referring to cells in the embryo with a connective tissue morphology and broad differentiation
capacity, as opposed to the more specialized connective tissue in the post-natal animal. In the
bone marrow, connective tissue has a prominent supportive or “stromal” function for
hematopoiesis and bone formation, and hence the connective tissue cells within bones are often
referred to as bone marrow stromal cells (BMSCs), a more accurate term than "MSCs". Initial
attempts to specifically isolate SSCs within the broader BMSC population in both mice and
humans have involved sorting cells based on the presence of a few cell surface makers (e.g.
CD146 and PDGFRa) and the absence of markers of hematopoietic (e.g. CD45) and endothelial
(e.g. CD31) lineages. The function of these populations is then often assessed by their ability to
differentiate into osteoblasts and other skeletal cell types in vitro, as well as in vivo upon
transplantation under the skin, intravenously, or into sites invested with substantial vasculature
such as the kidney capsule.
6,17–19

Cell properties can change when cells are isolated and placed in vitro, for example due to
selection biases during cell sorting or in response to the non-physiological conditions of cell
culture. In addition, the sorting procedure can often kill sub-populations of cells. Hence,
complimentary efforts have focused on defining endogenous SSC populations within skeletal
tissues in vivo. To do so, fate mapping using Cre-mediated DNA recombination is often employed.
In so doing, a number of markers of putative bone marrow SSCs have been identified in mice
(summarized in Table 1). One such marker is the Bmp antagonist Gremlin1.
20
Grem1+ cells are
located in the growth plate and marrow of long bones, primarily in the metaphysis (i.e. the region
just below the growth plate), and do not express markers of perivascular stromal cells such as
Nes and Cxcl12. Using a conditional Grem1-CreER transgenic line, it was shown that Grem1+
cells marked at postnatal day 1 (P1) give rise to growth plate chondrocytes, osteoblasts, marrow
stromal cells, and periosteal connective tissues by one month of age. Further, diphtheria-toxin-
mediated ablation of Grem1+ cells at P9 results in decreased bone formation by P23. Grem1-
mediated recombination at one month also labels stromal and periosteal cells in the femur at one
4
year, suggesting long-term self-renewal. Together, these data argue that Grem1+ cells are a type
of SSC, given their multilineage differentiation capacity in vivo, their requirements for post-natal
bone formation, and their capacity for long-term self-renewal.
In contrast to Grem1+ cells, cells expressing the Leptin receptor (LepR) and traced with
LepR-Cre exhibit SSC properties in later postnatal stages.
21
LepR+ cells also differ from Grem1+
cells in being concentrated near blood vessels (perivascular) and being able to generate marrow
adipocytes. Indeed, LepR+ cells contribute to ~75% of marrow adipocytes at 2 months of age and
~95% by 14 months, and can give rise to the majority of colony forming unit-fibroblasts (CFU-Fs),
an in vitro measure of stem cell activity. The contribution of LepR+ cells to osteoblasts is minimal
until about 6 months, at which point they constitute ~20% of osteoblasts in the tibia, and by 14
months ~90% of osteocytes in the femur. One caveat to these studies is that endogenous LepR
is abundantly expressed in chondrocytes, in contrast to the LepR-Cre transgene that appears
more specific for marrow cells, perhaps due to the insertion of Cre in a specific Ob-Rb splice form
of the LepR transcript.
21–23
LepR+ cells can also be found in the periosteum,
24
and recent single-
cell analyses of bone marrow have revealed distinct subpopulations of LepR+ cells that exhibit
differential lineage biases.
25,26
These findings indicate that LepR-Cre marks a broad and
heterogeneous population of BMSCs, of which only a subset may likely represent true SSCs. In
summary, Grem1 and the LepR Ob-Rb splice form appear to mark distinct early and late
populations of SSCs, respectively, with their timing of emergence perhaps underlying their
different lineage potentials.
It should be noted that BMSCs fulfill a dual role in the marrow, acting not only as a source
of skeletal-lineage cells but also supporting hematopoiesis.
27
In this context, many other surface
marker combinations and transgenic mouse lines have been used to label BMSCs. For example,
a Nestin-GFP transgene marks BMSCs that have been proposed to serve as osteoblast
precursors,
28
although a separate group found little contribution of Nestin+ cells to osteoblasts
during early mouse post-natal life.
20
Mx-1-Cre also labels a population of BMSCs with osteogenic
5
but not adipogenic or chondrogenic potential in vivo.
18
Recently, Ebf3-CreER was shown to mark
a subset of LepR+ BMSCs that express Cxcl12 and are self-renewing. When Ebf3 is deleted in
LepR+ stromal cells (using LepR-Cre), cells lose their HSC-supportive stromal function and
prematurely differentiate into osteoblasts, suggesting that Ebf3 functions to maintain an immature
“stromal” phenotype in a subset of LepR+ BMSCs.
29
The timing, contributions, and requirements
of these and other BMSCs marked by various genes are summarized in Table 1.
Growth plate SSCs
Longitudinal growth of long bones is accomplished by the growth plates where, slow-
cycling cells (which form a ‘resting zone’) give rise to columns of proliferating chondroblasts
(within a ‘proliferative zone), which then mature into hypertrophic chondrocytes (in a hypertrophic
zone). At the limit of the hypertrophic zone, the growth plate cartilage is eroded and replaced by
bone and marrow tissues via the process of ossification (Box 2). Marrow osteoblasts are derived
in part from progenitors from the perichondrium (the fibrous layer surrounding the cartilage
template) that migrate into the marrow space along with the newly forming vasculature.
30
It is also
now recognized that hypertrophic chondrocytes are another significant source of osteoblasts in
the post-natal animal. In mice
31–37
and zebrafish,
22
histological analysis and lineage tracing show
that a proportion of hypertrophic chondrocytes escape cell death and differentiate into osteoblasts
and, at least in zebrafish, marrow adipocytes. Hypertrophic chondrocytes also re-enter the cell
cycle in both mouse
34
and zebrafish,
22
and in zebrafish, also express lepr. As lepr is broadly
expressed outside of putative SSCs, including in chondrocytes of mouse and zebrafish,
22
whether
hypertrophic chondrocytes acquire progenitor characteristics needs to be tested more thoroughly.
Recent studies on growth plate chondrocytes have therefore been aimed at defining the stem
cells within the resting zone that fuel continued growth plate expansion, as well as the potential
transformation of hypertrophic chondrocytes into SSCs that will later reside in the marrow cavity.
6
Labeling of resting growth plate chondrocytes with Col2a1-CreER and a multicolor
fluorescent reporter reveals that columns of chondrocytes shift from being multiclonal in embryos
and neonates to being monoclonal at postnatal stages.
38
At postnatal stages, mTORC1 signaling
was found to fine-tune chondrocyte cell divisions to achieve the correct balance of asymmetric
divisions that optimally maintain chondrocyte stem cells in the resting zone.
38
A separate study
used PTHrP-CreER to label a subset of resting zone chondrocytes and found that they are a
major source of chondrocytes for several months after labeling.
33
In agreement with this
observation, diphtheria-toxin-mediated ablation of PTHrP+ cells disrupts bone elongation. In
addition, PTHrP-CreER-labeled cells were shown to contribute to Col1a1+ osteoblasts and
Cxcl12+ stromal cells in the marrow, consistent with the eventual transition of resting zone cells
into the hypertrophic chondrocytes that transdifferentiate into osteoblasts and stromal cells.
However, it cannot be ruled out that some or all PTHrP-CreER-labeled cells bypass the
hypertrophic state, as non-hypertrophic chondrocytes closest to the bone collar (“borderline
chondrocytes”) may selectively undergo transdifferentiation.
31,35,39
A salient feature of monoclonal
Col2a1+ and PTHrP+ cells is that they do not appear in the resting zone until after birth
(approximately P6). In addition, induction of Col2a1-CreER at P3 reveals a contribution of the
marked cells to marrow adipocytes in one-year-old animals,
40
whereas induction of PTHrP-CreER
at P6 and Col2a1-CreER at P28 does not label adipocytes at any time point assayed.
33,38
The
labeling of adipocytes by early Col2a1-CreER induction may be attributed to activity of Col2a1-
CreER outside the growth plate, such as in osteochondroprogenitors. Overall, these findings
suggest that embryonic growth plates are fueled by a distinct chondroprogenitor pool, with the
onset of PTHrP expression coincident with the acquisition of self-renewal ability within the post-
natal resting zone. Thus, resting zone SSCs do not appear to be simply remnants of a
developmental growth plate population.
Whereas the above studies focused on the in vivo potential of resting zone cells, a
separate study used a panel of surface markers (CD45-/TER119-/Tie2-/Thy-/6C3-/CD105-
7
/AlphaV+/CD200+) to purify cells from the growth plate of murine bones.
6
Isolated cells could be
serially passaged and induced to differentiate into bone, cartilage, and stromal cells upon
transplantation into the kidney capsule of recipient mice. Moreover, self-renewing cells could be
extracted from these ectopic ossicles and used for serial transplantation through several rounds
of recipient mice, while maintaining their multi-lineage differentiation capacity. The same group
subsequently reported that an analogous SSC could be isolated from the fetal and adult growth
plates of human bones, although a quite different set of surface markers were utilized compared
to the mouse study.
7
While these studies did not pinpoint where within the growth plate these
proposed SSCs might be located, it is possible that they correspond to cells similar to the
Col2a1+/PTHrP+ monoclonal resting zone cells found in vivo,
33,38
although transcriptomic
analysis did not reveal significant expression of PTHrP. A mystery is how these purified cartilage
cells acquire self-renewal, osteoblast and adipocyte differentiation capacity in culture. Might this
reflect that chondrocytes can normally transdifferentiate into osteoblasts and stromal cells within
the bone marrow? Or might this reflect selective sorting of rare, non-chondrocyte cells or the
induction of cell plasticity upon culture? Of note, it has long been appreciated that chondrocytes
can readily adopt a mesenchymal state upon in vitro culture,
41
and hence the culture conditions
could in theory apply selective pressure for self-renewal and multilineage differentiation capacity.
It will thus be important to understand how these isolated cells, which show clear SSC
characteristics upon serial transplantation, relate to endogenous SSCs within the growth plate.
Periosteal SSCs
The periosteum is a complex tissue that lines the outer surface of bones and is composed
of fibroblasts, blood vessels, nerves and, particularly in the inner layer, osteoprogenitors.
Whereas the growth plate plays a major role in longitudinal bone extension, cells in the periosteum
contribute to bone thickening and cortical maintenance during development and homeostasis.
42

The importance of the periosteum for bone growth and repair has been appreciated for over a
8
century, yet the identity and regulation of periosteal progenitor cells are just beginning to be
unraveled.
43–48

During development, the periosteum arises from the perichondrium as mesenchymal cells
become primed to generate osteoblasts. Gli1 is a transcriptional target and effector of the Hh
signaling pathway, which has well-known roles in bone and cartilage development. Several
groups have therefore used Gli1-based transgenes to mark cells with skeletogenic potential. Gli1-
CreER induction at E13.5 in mice results in labeling primarily in the perichondrium, with tracing of
these cells until 2 months of age showing major contributions to cortical and trabecular
osteoblasts, bone marrow adipocytes, and bone marrow stromal cells of the femur. Notably, ~75%
of Gli1-labeled cells in the marrow (traced from E13.5 to until 2 months old) express LepR,
suggesting that Gli1-expressing cells in the embryo give rise to most LepR+ SSCs in the postnatal
marrow.
49
However, Gli1-CreER-labeled cells are also found within the growth plates, making it
unclear whether lineage-traced LepR+ cells originate from periosteal progenitors, growth plate
chondrocytes, or some other Gli1+ source. Induction of Gli1-CreER at 4 months highlights a small
contribution of marked cells to the periosteum but decreased contribution to osteoblasts and
marrow cells, while induction at 12 months results in little to no labeling within the femur. Thus,
Gli1-CreER-labeled cells are similar to Grem1+ SSCs in being a transient SSC population
supplying cells for juvenile growth but less so for long-term bone homeostasis. As LepR+ SSCs
increase their contributions through adulthood, it may be that those LepR+ SSCs not derived from
embryonic Gli1-expressing cells have a preferential role in adult bone homeostasis. In addition, it
has been shown that Gli1 marks mesenchymal cells within postnatal cranial sutures, which
separate intramembranous bones of the skull and face (see Box 2), with Gli1-CreER-labeled cells
contributing to and being required for growth and repair of skull bones.
50
Similarly, Gli1-CreER-
labeled cells act as long-lived stem cells for the continuous growth of the mouse incisor.
51
Thus,
Hh activity, and in particular Gli1 expression, may generally mark stem cells within a range of
skeletal tissues including the developing perichondrium, the growth plate, the metaphysis bone
9
marrow compartment, and the periosteum. This broad contribution is similarly observed using
Axin2-CreER, suggesting that SSCs may also be Wnt-responsive, though it is unclear if these two
transgenes mark the same populations.
52–54

A recent study using a constitutive Prrx1-Cre line, which broadly labels mesenchymal cells
in the limbs and elsewhere, has provided more evidence that self-renewing, multipotent SSCs
exist within the periosteum.
55
In this study, Prrx1-Cre-marked cells were isolated from the
periosteum and shown to express a panel of markers associated with BMSCs, including Pdgfra,
Grem1, Cxcl12, and Nestin. In addition, following transplantation of Prrx1-Cre-labelled periosteum
into a fracture site, Prrx1+ periosteal cells re-establish a pool of progenitor cells after a second
injury, highlighting their self-renewing quality. Similar data to support the existence of a periosteal
SSC have been obtained using the broadly expressed transgene Acta2(aSMA)-CreER, whose
expression is highly enriched in the periosteum.
56,57
As these Cre lines label broad mesoderm-
derived populations, it has not been possible, however, to pinpoint which cells within the
periosteum behave as SSCs.
Another marker of periosteal SSCs is Ctsk,
58,59
which encodes the cysteine protease
Cathepsin K. Ctsk as a marker of periosteal cells came as a surprise given its historical use as a
marker of bone-resorbing osteoclasts (a hematopoietic lineage cell). Indeed, the majority of Ctsk-
Cre-marked cells in the marrow cavity express tartrate-resistant acid phosphatase (TRAP),
indicative of osteoclast identity. However, when isolated from the femur periosteum of juvenile
mice, cultured Ctsk+ cells exhibit self-renewing properties, with single cells able to form clones
with multilineage differentiation potential (bone, cartilage, adipocyte, but not stromal cells). A
similar Ctsk+ periosteal stem cell was also identified in human periosteal tissue, suggesting
conservation of Ctsk as a periosteal SSC marker across species (REF). In mice, in vivo deletion
of the critical osteogenic transcription factor Sp7 (Osterix) in Ctsk-Cre-marked cells results in a
profound loss of cortical bone, highlighting the requirement for Ctsk-Cre-marked cells in
osteoblast production. In addition, long-term lineage tracing revealed that Ctsk-Cre-marked cells
10
contribute to osteoblasts in the cortical bone but not osteoblasts or stromal cells in the marrow.
Thus, Ctsk-Cre appears to mark a population of SSCs distinct from those that migrate into the
marrow with the invading vasculature from the perichondrium.
30
However, it should be noted that,
similar to Prrx1 and aSMA, Ctsk-Cre marks a broad population of periosteal cells, only a subset
of which likely have SSC activity.
Sox9-CreER induction labels a smaller population of cells with stem cell like properties
within the periosteum of adult femur and rib bones in mice.
60–63
In the femur, induction of Sox9-
CreER at P42 with a two-day chase results in labeling of the growth plate, the adjacent
perichondrium, and cells in both the periosteal layer and the endosteal layer (a osteogenic
connective tissue lining the inner bone surface).
61,62
However, in the rib, induction at 3-4 months
results in labeling predominantly in the periosteum (~6% of cells), with many fewer cells labeled
in the endosteal compartment of the diaphysis – the shaft portion of the bone.
63
RNA sequencing
of Sox9+ periosteal cells shows enrichment for a panel of bone genes such as Bglap, Bglap2,
Col1a1, and Col1a2, and also cartilage genes such as Sox9 and Col2a1.
62
Osteochondral
progenitors have been previously shown to co-express Sox9, a major cartilage transcription
factor, and Runx2, a major bone transcription factor, potentially reflecting dual lineage potential
of these common bone/cartilage progenitors.
64
It is therefore possible that, within the periosteum,
Sox9 similarly marks cells that have retained multilineage skeletogenic potential from
development. Whereas these Sox9+ periosteal cells normally contribute only to bone during
homeostasis, they can be induced to form cartilage in response to injury, as discussed in more
detail below.
An integrated view of SSC populations
The above studies underscore the complexity of the SSC system throughout the lifetime
of the organism (summarized in Figure 1). The varying temporal emergence and extinction,
differing lineage potentials, and distinct spatial distributions of proposed SSC populations point to
11
heterogeneous pools of SSCs working together to construct and maintain the skeleton, rather
than the existence of a single apex SSC, as proposed for the hematopoietic system. Defining the
relationships between proposed SSC populations will be needed to clarify hierarchies and
plasticity in the skeletal system. Fortunately, gene expression data for many of the above
populations already exist and will allow us to begin formulating testable hypotheses of SSC
relatedness. For example, RNA sequencing of periosteal Ctsk-Cre+ cells shows high expression
of Sox9, suggesting Sox9-CreER may mark a subpopulation of Ctsk-expressing cells within the
periosteum. As marrow SSCs appear to be derived from both early periosteal cells that migrate
into the marrow and hypertrophic chondrocytes that dedifferentiate (and/or borderline
chondrocytes that escape hypertrophy), it will be interesting to examine heterogeneity in marrow
SSCs in relation to these two different developmental origins (Figure 2). For example, do marrow
SSCs of a growth plate origin have different lineage capacities (e.g. chondrocyte-biased) than
those from the invading periosteum? Reciprocally, osteoblasts of a growth plate chondrocyte
lineage can also be found in the periosteum of adult zebrafish,
22
suggesting that both the
perichondrium and growth plate may contribute to the mature periosteum. Clearly, the distinctions
between periosteal, marrow, and growth plate compartments are fluid. Elucidating how cells
transition between these compartments and how developmental origins influence later behavior
will be key to understanding the distinct roles of SSCs in maintaining and repairing adult skeletal
tissues.
Regulators of the SSC niche
Although PTHrP-CreER and Col2a1-CreER can be used to mark growth plate cells in the
embryo, it is not until postnatal stages that they clearly mark cells within the growth plate that
have distinct SSC properties. This suggests that these postnatal populations only acquire SSC
function under the appropriate niche conditions. Further, several groups have recently shown that
dysfunction in the niche can lead to skeletal dysfunction. For example, inflammation via enhanced
12
NF-kB and TNFa signaling reduces SSC abundance and function but can be counteracted
pharmacologically to rejuvenate SSCs to improve fracture healing in models of aging and
diabetes.
65,66
Thus, gaining a better understanding of the niche factors required for healthy SSC
biology is an important direction of research.
Using transcriptomic analyses to identify highly expressed morphogens and their cognate
receptors, it is possible to infer a potential role for these pathways in regulating SSC activity. For
instance, transcriptome analyses of Col2a1+ growth plate chondrocytes has revealed the
dynamic regulation of several key pathways.
38
Notably, negative regulators of the Wnt signaling
pathway are down-regulated, suggesting that Wnt signaling promotes SSC establishment in the
growth plate. This idea is supported by in vitro studies in which active Wnt signaling was shown
to support the undifferentiated state of SSCs; however, once these cells become committed to an
osteogenic fate, Wnt signaling enhances their differentiation.
67
Wnt signaling may also function to
maintain SSCs in the periosteum, as many Wnt ligands are enriched in Ctsk+ SSCs based on
RNA sequencing.
58
A role for Wnt signaling is further reflected by the ability of Axin2-CreER to
mark periosteal SSCs in the cranial and appendicular skeleton, as Axin2 is a direct transcriptional
target of Wnt signaling in many tissues.
52,53

One possibility is that interactions between SSCs and their downstream progeny serve to
maintain an appropriate pool of SSCs through feedback signaling. Bone morphogenetic protein
(BMP) signaling is one attractive candidate that may play a role in such feedback due to its well-
known roles in skeletal differentiation. As stated earlier, the BMP antagonist Grem1 is a marker
of SSCs in the marrow compartment.
20
Isolated AlphaV+/CD200+ cells also express several
BMPs and their receptors, whereas downstream progenitors express high levels of two other BMP
antagonists, Grem2 and Noggin.
6,7
In vitro, BMP2 addition enhances and Grem2 inhibits SSC
expansion, suggesting a feedback role for BMP signaling in maintaining SSCs. A recent study
has also shown that BMP2 is required in immature Prrx1+ periosteal progenitors, but not mature
osteogenic cells, to drive appositional bone growth during early life, with its upregulation in the
13
periosteum in response to injury accelerating fracture repair.
47
Given the widespread use of BMPs
in clinical application and trials, more studies are needed to tease out the specific roles of BMP
signaling in SSC biology.
68

The Hedgehog (Hh) signaling pathway likely also regulates the SSC niche. During the
time when Col2a1+ SSCs appear in the growth plate, high expression of Sonic hedgehog (Shh)
can be observed in the secondary ossification center along with Indian hedgehog (Ihh) expression
in growth plate cartilage cells.
38
Strong expression of the Hh-mediator Gli2, as measured by RNA
in situ hybridization, can be seen in these Col2a1+ cells; this is consistent with strong
recombination of Gli1-CreER in the region of the resting growth plate where Col2a1+ SSCs reside.
Pharmacological inhibition and/or activation of the Hh pathway, as well as genetic removal of the
obligate Hh mediator Smoothened, further support a role for Hh signaling in the regulation of
growth plate SSC proliferation.
38,69
Hh signaling promotes proliferation of growth plate SSCs
without pre-mature induction of differentiation,
38,69
which is somewhat surprising given a wide
body of literature describing a role for Hh signaling in driving the differentiation of skeletal lineage
cells in development and regeneration (reviewed in Alman 2015
70
). Moving forward, it will be
important to untangle the temporal and spatial role of Hh signaling in chondrogenesis from its
potential role in SSC maintenance and/or expansion.
Although many signaling pathways have been identified as potential niche regulators,
further research is needed to identify the precise ligands, receptors, and target genes involved.
In addition, it is likely that other non-skeletal cell types may modify the niche environment. For
example, studies have shown the importance of a perivascular niche for skeletal progenitor cells
during bone development and regeneration, including the action of endothelial-derived VEGF on
hypertrophic chondrocytes at the edge of the growth plate.
71,72
In a recent study, it was elegantly
shown that periosteal endothelial cells secrete PDGF ligands to attract PDGFRa+, Osterix+
osteoprogenitors, with PDGFa signaling keeping these progenitors in an undifferentiated,
14
proliferative state.
73
Nerve cells may also influence repair. For example, it has been shown that
Shh emanating from nerves maintains tooth incisor stem cells, and that Schwann cells from
nerves in the mandible potentially provide important paracrine factors for bone repair in the
jaw.
74,75
The influence of cells from the immune system may also be critical, with TRAP+
macrophages (derived from the monocyte lineage) driving periosteal osteoblast differentiation via
a PDGF-BB signal, with potentially other immune cell types and signaling pathways still to be
discovered.
24
It is likely that even moderate alterations to the niche environment could have major
impacts on SSC potency. Thus, identifying and characterizing niche regulators may help develop
new clinical strategies to combat skeletal degeneration in disease or aging.
SSCs in repair
In mammals, simple fractures of bone often exhibit scarless healing, while in certain non-
mammalian vertebrates, large pieces of missing bone and cartilage, and even entire skeletal
appendages, can undergo regeneration. Which cells fuel these regenerative processes? One
possibility is that the same SSCs involved in bone homeostasis function to repair bone after injury.
Alternatively, or in combination, skeletal injury may create an abnormal microenvironment (e.g.
inflammation, altered mechanical properties, cell death and migration) that results in certain cell
populations contributing to new bone that would not normally do so during homeostasis.
48
Current
evidence supports the local periosteum and bone marrow being the two primary sources of bone-
forming cells during repair; indeed, parabiosis experiments do not support significant contributions
from circulating cells.
6,21,44,60,76,77

Bone repair can occur either through a cartilage callus intermediate or through direct
ossification (Box 2). Recent data indicate that the cartilage callus during bone repair differs
substantially from the growth plate cartilage that builds endochondral bones developmentally.
During development, growth plate chondrocytes begin to express genes associated with
osteoblast differentiation (e.g. Runx2, Osx, Spp1, and Ocn) only after expression of Col10a1 at
15
the pre-hypertrophic stage. In contrast during bone regeneration in the zebrafish jaw and mouse
rib, callus chondrocytes co-express high levels of chondrocyte and osteoblast genes at much
earlier stages of differentiation.
63,78
For example, Sox9 and Runx2 are co-expressed at initial
stages of chondrogenesis in the callus, and the expression of Col1a1 precedes that of Col10a1,
a sequence never seen in normal development of zebrafish or mouse rib growth plates. This
acceleration of osteoblast gene expression relative to the chondrogenic program during repair
may reflect altered regulation in the wound setting and has led to callus cells being termed “hybrid
osteochondral cells". The mechanics of the fracture environment have also been shown to
influence the mode of bone repair. Whereas repair of unstabilized fractures typically involves a
robust cartilage callus, rigidly stabilized injuries, such as focal lesions in the intramembranous
bones of the skull or surgically fixated long bone fractures, heal primarily through direct
ossification.
79,80
Recent insights into the SSCs contained within the fibrous sutures separating the
intramembranous skull bones are providing interesting contrasts with the SSCs of endochondral
bones (Box 3). It may be that the same SSC populations react differently to mechanical stimuli
(i.e. preferentially forming osteoblasts over chondrocytes in a stiff environment), or that distinct
SSC populations with unique properties are activated depending on the injury type. SSC
heterogeneity may also be found across different bone types, as each bone has unique
biophysical requirements (i.e. weight-bearing versus non-weight-bearing), different
vascularization and innervation statuses, and distinct developmental histories. For example,
different bones express different Hox genes and, at least in the context of the ulna (but not the
humerus) and the tibia (but not the femur), paralogues of Hoxa11/d11 are required for normal
fracture healing.
81
In addition, bone healing could involve a significant contribution of cells that do
not fit the strict definition of a stem cell, as suggested by the observed dedifferentiation of
osteoblasts in zebrafish bone regeneration (Box 1
9,10,82
) and murine digit tip regeneration.
12

Probing the behavior of SSCs and other populations across a variety of injury types and bones
should help clarify the diversity of SSCs and repair mechanisms throughout the skeleton.
16
To date, the same transgenic tools used to track the contribution of SSCs to homeostasis
have been used to examine the contribution of these cells to repair. In 3-month old mice, Acta2
(aSMA)-CreER induction marks a broad population of periosteal cells that proliferate rapidly and
generate almost all chondrocytes and osteoblasts within the repair callus 6 days after tibial
fracture.
56
However, in these studies, tamoxifen induction was performed both before and after
tibia fracture, and thus the contributions of pre-existing aSMA+ cells and those that potentially
switch on Acta2(aSMA) expression after injury cannot not be distinguished. Similarly, Axin2-
CreER has been used to mark periosteal cells that participate in bone repair. For example,
periosteal-specific induction of Axin2-CreER (via local injection of tamoxifen) marks a small
population of periosteal cells in the tibia of 2-month-old mice.
53
Following a 1-mm drill injury,
Axin2-CreER-labeled cells are observed in the repair site and contribute to ~11% of callus
chondrocytes but to virtually no osteoblasts one week after injury. When Axin2-CreER is induced
systemically, a higher percentage of periosteal (~37%) and endosteal (~42%) cells are labeled,
with contributions now seen (presumably from endosteal cells) to the bony portions of the repair
callus. Axin2-CreER may therefore mark at least two populations of cells: a periosteal population
biased toward endochondral bone formation and an endosteal population biased toward direct
osteogenesis. Whereas Axin2+ cells are required for repair, diphtheria toxin-mediated ablation of
these cells prior to injury inhibits bony but not cartilage callus formation, suggesting that Axin2+
cells play only a minor role in cartilage callus formation. Another periosteal marker Ctsk-Cre marks
half of all chondrocytes in the femur fracture callus 6 days after injury.
58
However, as Ctsk-Cre is
constitutive, it is not possible to distinguish between repair cells derived from the periosteal
lineage and those that may have switched on Ctsk expression in response to injury.
Recent work in the mouse rib bone, which shows extraordinary regeneration capacity
compared to other bones, has revealed a role for periosteal cells in not only forming repair tissue
but also in organizing the regenerative response. Before injury, Sox9-CreER induction marks a
17
population of periosteal cells (~6% of the periosteum) in the rib diaphysis, with many fewer cells
labeled on the endosteal surface.
63
Following resection of 3 mm of rib bone, Sox9-CreER-labeled
cells contribute to ~20% of callus cells in both the cartilage and bony portions 10 days after injury.
In addition, upon deletion of the Hh co-receptor Smoothened in Sox9-CreER-labeled cells prior
to resection, progenitor cells fail to differentiate into the hybrid osteochondral cells that build the
cartilage callus. Strikingly, loss of Hh signaling in pre-injury Sox9+ cells blocks cartilage
differentiation in the 80% of cells not derived from the Sox9+ lineage. This suggests a particular
organizing function of periosteal Sox9+ cells in not only contributing to the cartilage callus but also
recruiting other cells into the callus. Hh signaling (mediated by Ihh) was shown to play a similar
role in promoting large-scale regeneration of the zebrafish jawbone, although in this context it was
not investigated whether Hh was acting on Sox9+ periosteal cells.
78

Markers for endosteal-specific populations in the marrow compartment can help determine
if periosteal versus endosteal cells have distinct roles during skeletal repair. In juvenile mice,
marrow cells below the growth plate (i.e. in the metaphysis) can be marked using a Gli1-CreER
line at P30.
49
Following femur fracture, these cells show substantial contributions to both the
cartilage and bone portions of the callus 10 days after injury. However, due to broad labeling by
Gli1-CreER beyond the marrow cavity, it remains unclear which of the P30 Gli1+ populations (or
their descendants) give rise to each the skeletal lineages observed. In addition, the number of
Gli1+ cells markedly decreases at four months, and disappears completely at 12 months. Thus,
in juvenile mice, marrow-resident Gli1+ cells serve as a reservoir for femur fracture repair, while
contribution in older animals is likely minimal. LepR-Cre labeling can also be used to mark
marrow-specific cells in the adult mouse tibia, although sparse labeling can be found in the
periosteum of other bones (e.g. the sternabrae).
50
LepR+ cells contribute substantially to both the
cartilage and bone callus two weeks after tibial fracture, with abundant osteocytes retaining label
even after 8 weeks. Due to the constitutive Cre, however, new induction of LepR expression after
injury could also explain the presence of labeled cells. The Grem1-CreER line, by contrast, is
18
conditionally inducible and can therefore be used to label metaphyseal cells prior to injury.
20
When
Cre is induced in 8 week old animals and followed one week later by femur fracture, Grem1+ cells
generate ~14% of Sox9-expressing chondrocytes and ~28% of Col1a1-expressing osteoblasts in
the one-week repair callus, providing the best evidence thus far for the contribution of marrow
cells to the early repair callus.
Together, these studies highlight a wide range of Cre-lines that can be used to observe
the contributions of cells during homeostasis and repair in the mouse (summarized in Table 1).
Cells from various compartments contribute during repair, through a cartilage-like intermediate,
via direct ossification, or both (Figure 3). Specific subpopulations may already be biased toward
one path versus another. Alternatively or in addition, specific niche factors, the unique properties
of specific bones (their developmental history), and the mechanical environment may influence
the lineage outcome. Further, osteoblasts may also dedifferentiate to repair bone in certain
contexts. While initially described in non-mammalian vertebrates (Box 1), recent data support
osteoblast dedifferentiation during murine digit tip regeneration.
12
When osteoblasts were pre-
labeled with Dmp1-CreER, which was confirmed to be osteoblast/osteocyte-specific via single-
cell RNA sequencing, and then subjected to digit amputation, Dmp1-lineage cells generated
proliferative blastema cells that lost the expression of differentiated osteoblast markers and
acquired a progenitor signature including expression of Grem1. These osteoblast-derived
blastema cells then contributed extensively to regenerated digit bone. In contrast, a separate
study found that Ocn-CreER-traced osteoblasts did not contribute in a murine calvarial
microfracture model,
18
suggesting osteoblast dedifferentiation may be specific to appendage
regeneration in mammals. In the future, it will be interesting to determine whether dedifferentiation
can be induced and used as a method to build new skeletal tissue clinically.
19

Conclusions and future directions
There has been an enormous effort in recent years to identify and characterize rare,
discrete populations of bona fide, self-renewing, multipotent SSCs. The search for SSCs has
undoubtedly been influenced by studies that have uncovered a hierarchy of lineage-restricted
stem cells in the hematopoietic system. While recent work lends support for such a hierarchy in
both the mouse and human skeletal systems,
6,7
it is also clear that diverse types of SSCs can be
isolated from distinct spatial locations. These heterogeneous populations likely coordinate their
activities to build, maintain, and repair the skeleton. Further, there appears to be considerable
plasticity in SSCs, which may help to ensure that the required skeletal cells are efficiently replaced
in response to different types of injuries.
In the future, a major challenge will be to relate diverse populations of SSCs to one
another. Do the distinct embryonic origins of SSC types prefigure their unique properties with
regards to building, maintaining, and repairing the skeleton? Emerging techniques should help to
better address the lineage relationships and potential of SSCs. As a complement to Cre/Lox-
based systems, it is now possible to rapidly generate new animal models using Flp/Frt or Dre/Rox
recombinases to expand the toolbox and enable in vivo lineage tracing of multiple cell populations
in parallel.
83
Genetic barcoding could potentially allow tracking of thousands of individual cells in
vivo.
84
Resolving SSC lineage relationships should help resolve major questions regarding the
relative contributions of cells from the bone marrow, growth plate, and periosteum in the skeletal
system. In parallel, identifying critical niche factors will help determine how SSC populations stay
quiescent, become activated due to injury, and undergo the specific transitions needed to build
new skeletal tissues. The field will need to complement studies in model organisms with those in
human tissues, as markers may differ between species. This will allow us to better understand
how cell populations and niche factors are altered in dysmorphology, injury, disease, and aging
of the human skeleton. Overall, these inquires will no doubt give us a greater understanding of
20
the plasticity of the skeletal system and ultimately will help bring innovative therapeutic
approaches to the clinic.
FIGURE LEGENDS
Figure 1. Skeletal stem cell populations and niches
Several populations of skeletal stem cells (SSCs) have been identified to date. (1) SSCs can be
identified in the marrow cavity (brown) with some populations being enriched in the metaphysis
region, particularly at early post-natal or juvenile stages. These populations can be identified using
various Cre lines for the genes indicated (with "J" indicating labelling at juvenile stages). (2) SSCs
can also be found in the resting zone (RZ) region of the growth plate (blue), expressing the genes
indicated. These cells contribute to more lineages than just cartilage. In a growing bone, the
chondrocytes of the growth plate proliferate (in the proliferation zone, PZ), and become larger and
hypertrophic (within the hypertrophic zone, HZ) near the juncture with the marrow cavity. Some
of these cells do not undergo apoptosis, but are ejected from the growth plate into the marrow
cavity (represented by blue arrow) where they contribute to osteoblasts, adipocytes, other marrow
cells, and potentially marrow SSCs. (3) The periosteum (indicated in dark red) is also known to
contain SSCs (marked by expression of the genes indicated) involved in homeostasis and repair.
During development, progenitor cells within the perichondrium (light red) translocate into the
marrow (represented by light red arrow) during initial vascularization of the bone.
Figure 2. Redundant pathways to make bone during development and homeostasis.
An SSC (red) in the growth plate resting zone is proposed to self-renew and give rise to
hypertrophic chondrocytes (blue) that can undergo transdifferentiation to give rise to osteocytes
(green)
34,36,37
and possibly to bone marrow SSCs (top row).
22,39
Bone marrow SSCs can then give
rise to osteocytes and adipocytes in the bone marrow compartment.
21
Better evidence for the
origin of bone marrow SSCs (middle row) comes from Maes et al.,
30
which shows that
21
periosteal/perichondrial SSCs contribute to the marrow compartment during development.
Whether this also happens postnatally is not clear. Osteocytes can also arise (via an osteoblast
intermediate) from periosteal SSCs at the periosteal surface.
58

Figure 3. Redundant pathways to make bone during repair.
In response to injury, SSCs (red) from the periosteum and/or the bone marrow compartment
generate bone through via an osteochondral intermediate (giving rise to cells with cartilage/bone
properties, i.e. hybrid osteochondral progenitors, purple) or through direct ossification (giving rise
to osteoprogenitors, green). In some contexts, such as the zebrafish fin and murine digit tip,
osteoblasts can dedifferentiate and re-differentiate to produce new bone.
BOXES:
Box 1. Osteoblast dedifferentiation during zebrafish bone regeneration
Several lines of evidence indicate that, in response to injury, osteoblasts in the adult
zebrafish fin and skull can revert to a progenitor state to generate new osteoblasts. For example,
following amputation of the bony fin skeleton or drill lesions of the calvarial bone, osteoblasts
downregulate expression of the mature osteoblast marker osteocalcin, upregulate expression of
the osteoprogenitor gene runx2b and the connective tissue marker Tenascin, re-enter the cell
cycle, and produce new osteoblasts.
9,10
Moreover, lineage tracing with an sp7(osterix)-CreER
transgene has shown substantial contributions of pre-existing osteoblasts to new osteoblasts in
the fin regenerate.
10,82
In order to rule out sp7(osterix)-CreER activity outside of osteoblasts,
40,85

Knopf and colleagues employed time-lapse imaging of CreER-converted cells, as well as
osteoblasts in which the entpdf5:Kaede transgene is photoconverted from green to red. In both
cases, osteoblast-derived cells are observed to migrate into the blastema, consistent with their
later differentiation into new osteoblasts at a distance.
9,10
However, even when pre-existing
osteoblasts are ablated, the fin bone still regenerates, suggesting that an additional reserve pool
22
of progenitors may also contribute to bone regeneration.
82
Good candidates for such a reserve
population are mmp9+ cells, as these are found in un-injured fin ray joints and contribute to new
osteoblasts after fin amputation.
86
In the future, it will be important to understand the mechanisms
by which osteoblasts dedifferentiate in response to injury in fish, and whether similar mechanisms
operate in mammals, such as during digit tip regeneration.
12
*This box was generated in
collaboration with Claire Arata.
Box 2. Endochondral vs direct ossification
Bone development mainly occurs via two pathways: endochondral ossification or direct
(or intramembranous) ossification. Endochondral ossification involves the formation of a transient
cartilage template in which skeletal progenitor cells condense, differentiate into chondrocytes,
and then progress through hypertrophy. Apoptosis of hypertrophic chondrocytes is thought to
create a marrow cavity, with osteoprogenitors from the perichondrium then migrating into this
cavity along with the vasculature. However, recent studies have shown that hypertrophic
chondrocytes are also a significant source of marrow osteoblasts and stromal cells,
32–34,36,37

although the percentage of marrow cells deriving from hypertrophic chondrocytes likely differs
depending on bone type. During the process of direct ossification, skeletal progenitors proliferate,
condense, and differentiate directly into osteoblasts without a cartilage template. In mammals,
most of the appendicular, spine, and thoracic skeleton forms via an endochondral pathway, while
most of the skullcap and facial skeleton form through direct ossification.
During repair, bone also forms through endochondral or direct ossification. In some
contexts (e.g. small injuries), repair appears to occur through direct ossification. Larger injuries
with more soft tissue trauma correlate with the formation of a cartilage callus – a healing tissue
that forms in response to injury. While this cartilage callus may simply provide a supportive role
before sufficient ossification has occurred, and may even help align the fracture,
87
some of the
callus cells appear to have hybrid cartilage/bone osteochondral properties. These hybrid cells
23
then mature into bone-producing osteoblasts, therefore actively participating in building new bone
tissue.
63,78

Box 3. Repair in cranial bones
Suture mesenchyme is a unique connective tissue that can be found at the junctures
between cranial bones. Complications of bone growth at the suture have been implicated in
craniofacial defects such as craniosynostosis. SSCs are concentrated in the suture region and
contribute extensively to new bone during skull growth and repair,
50
with efficiency of regeneration
decreasing as injury distance from the suture increases.
88
In mice, suture SSCs can be marked
using a Gli1-CreER line following induction at one month of age. Postnatal Gli1-CreER-traced
cells give rise to parts of the periosteum, dura, and osteocytes of the calvaria.
50
Ablation of this
population postnatally results in a complete loss of sutures, halted bone growth, and a malformed
skullcap.
50
Suture SSCs can also be labeled by Prrx1-CreER and Axin2-CreER lines.
52,89
While
both populations overlap with Gli1-expressing cells, it is unclear whether they overlap with each
other as the Prrx1 population does not express Axin2 unless stimulated with a WNT agonist.
89

As with the Gli1+ population, both the Axin2+ and Prrx1+ populations have been shown to
participate in repair. Ablation of the Prrx1+ population does not cause craniosynostosis,
suggesting differences from the Gli1 population. However, Axin2
-/-
mutants do display synostosis
of a subset of sutures.
90
Notably, markers for Axin2+ suture SSCs overlap with those for periosteal
SSCs in long bones in the appendicular skeleton, but not those of bone marrow SSCs (Gremlin1,
Nestin) with the exception of LepR which is highly enriched in suture SSCs.
52
This suggests
parallels between suture and long bone periosteal SSCs that will be interesting to pursue. *This
box was created in collaboration with Claire Arata.
Table 1: Comparative Summary of Reported Skeletal Stem Cells
24
Cre
Locatio
n
Conversion
Regimen
Contributions
(Cartilage, Bone,
Adipocyte,
Stroma)
Requirement Citation
Acta2(SMA)-
CreER
PO
BM
Tam at 3-5
months, day
before and
after fracture
Tibia callus (C-B) ND
(Matthews et
al., 2014)
Axin2-CreER
PC
Tam at P6,
chase to P9
Tibia (C)
Axin2-CreER;-
cat
fl/fl
–
ectopic cartilage
near PC (P13)
(Usami et al.,
2019)
PO
BM
Tam at 8
weeks,
1 week or 3
months before
injury
Tibia callus (C-B)
DTR ablation –
reduced cartilage
callus
(Ransom et al.,
2016)
Axin2-CreDox Su
Dox at P25-
P28,
chase for 1mo,
3mo, and 1
year
Calvarium (B) ND
(Maruyama et
al., 2016)
Dox at P25-
P28,
Calvarial injury
(B)
ND
25
2 days before
injury
Col2a1a-CreER GP
Tam at
perinatal and
juvenile stages
Tibia (C)
Humerus (C)
ND
(Newton et al.,
2019)
Tam at P3,
chase for 1
month
(B-S) or 2
months (A)
Femur (C-B-A-S) ND
(Ono et al.,
2014b)
Ctsk-Cre
PO
Constitutive
Kidney capsule
transplant (B)
Ctsk-Cre;Osx
fl/fl
–
reduced
mineralization of
calvarium and
femur
(Debnath et
al., 2018)
Femur callus (C-
B)
Ctsk-Cre;Osx
fl/fl
–
reduced repair and
bone volume
PC PC at P7
Ctsk-Cre;Ptpn11
fl/fl

–dwarfism,
scoliosis,
metachondromatosi
s
(Yang et al.,
2013)
26
Ebf3-CreER BM
Tam at 10
weeks, chase
for 13 months
Femur (B-A-S) ND
(Seike et al.,
2018)
Gli1-CreER
BM
Tam at E13.5,
chase for 2
months
Femur (C-B-A-S)
Calvarium (B)
DTA ablation –
reduced cancellous
bone
(Shi et al.,
2017)
Tam at 1
month,
fracture at 10
weeks
Femur callus (C-
B)
ND
Su
Tam at 1
month, chase
for 1–8 months
Calvarium (B) ND
(Zhao et al.,
2015)
Tam at 1
month,
5 days before
injury
Calvarial injury
(B)
DTA ablation –
suture fusion after 2
months
Grem1-CreER
BM
Tam at P1,
chase for 6
weeks
Femur (C-B-S)
DTA ablation –
reduced body size,
reduced bone
volume (Worthley et
al., 2015) Tam at >8
weeks
1 week before
fracture
Femur callus (C-
B)
ND
27
LepR-Cre BM Constitutive
Femur (B-A-S)
DTR ablation –
increased bone and
fat
(Zhou et al.,
2014a)
Tibia callus (C-B) ND
Mx1-Cre
BM
pIpC at 6-8
weeks, chase
for 20 days
Femur (B)
Calvarium (B)
ND
(Park et al.,
2012)
pIpC,
time unknown
Femur callus (B)
Calvarial injury
(B)
ND
BM
PO
pIpC at 4-6
weeks,
chase for 3
months
Tibia (B-S) ND
(Ortinau et al.,
2019)
pIpC and DT
before injury
Tibia callus (C-B)
Calvarial injury
(B)
DTR ablation –
reduced bone
repair
Nestin-CreER BM
Tam at 3
months, chase
for 8 months
Femur (C-B-S)
DTR ablation –
HSCs affected, not
bone
(Mendez-
Ferrer et al.,
2010)
Prrx1-CreER
PO
GP
Tam at E9,
E15.5-E16.5,
or P19-P23
with chase to
Radius, ulna, tibia
(C-B)
ND
(Kawanami et
al., 2009)
28
E17, E18.5 or
P26
Tam at P52
and P53,
fracture at P49
Ulna callus (C-B)
Femur callus (C-
B)
ND
Prrx1-Cre
Su
Constitutive
Calvarium (B)
DTA ablation –
no calvarial or limb
development
(Wilk et al.,
2017)
Calvarial injury
(B)
ND
Prrx1-CreER
Tam at P7 or
P28
ND
DTA ablation–
reduced femur/tibia
length
Tam at 8
weeks,
5 days before
and after injury
Calvarial injury
DTA ablation –
reduced repair
PTHrP-CreER GP
Tam at P6,
chase to P12 –
P36
Femur (C-B)
DTA ablation –
increased GP
hypertrophic zone
(Mizuhashi et
al., 2019)
Sox9-CreER PO
Tam at 12-16
weeks, chase
for 14 days
Femur (C-B) ND
(He et al.,
2017)
29
PO
Tam at 12-16
weeks, 2
weeks before
injury
Femur callus (C-
B)
Rib callus (C-B)
Sox9-CreER;Smo
fl/fl

– reduced1 callus
(He et al.,
2017;
Kuwahara et
al., 2019)
PO
GP
BM
Tam at P3,
chase to P30 &
P60
Tibia (C-B-A-S)
Sox9-
CreER;PTH1R
fl/fl
–
reduced Sox9-
derived osteoblasts
(Balani et al.,
2017)
Sox9-Cre
PO
GP
Constitutive
Entire limb (C-B)
E10.5-E17
Sox9-CreER;Osx
fl/fl

–
no bone
mineralization
(Akiyama et
al., 2005)
Marker
Locatio
n
Analysis Contributions Species Citation
αV+, CD200+ GP
Sorted,
transplanted
into mouse
kidney capsule
Ectopic (C-B-S) Mouse
(Chan et al.,
2015)
CD164+,
PDPN+, CD73+
GP
Sorted,
transplanted
into mouse
kidney capsule
Ectopic (C-B-S) Human
(Chan et al.,
2018)
Sca-1+,
PDGFR-α+
BM
Sorted,
transplanted
Differentiated (B-
A-S)
Mouse
(Morikawa et
al., 2009)
30
into mouse,
intravascular
CD146 BM
Sorted,
transplanted
into mouse,
subcutaneous
Ectopic (B-S) Human
(Sacchetti et
al., 2007)
Table 1. Comparative summary of mouse skeletal stem cells: For the listed Cre lines, the
following is indicated: the location shortly after labeling, the time of conversion if inducible, the
contributions during homeostasis or injury (indicated in blue), and required function of the marked
cell populations. For the listed cell surface markers the following is indicated: the location of
extraction, the type of analysis, the contribution upon transplantation, and the species of origin.
PO: periosteum, PC: perichondrium, BM: bone marrow, GP: growth plate, Su: suture, C: cartilage,
B: bone, A: adipocyte, S: stromal cell, DTA: diphtheria toxin, DTR: diphtheria toxin receptor, Tam:
tamoxifen, Dox: doxycycline, pIpC: polyinosinic:polycytidylic acid, ND: not determined.
31
Figure 1: Skeletal Stem Cell Niches
32
Figure 2: Redundant pathways to make bone during development and
homeostasis.
33
FIGURE 3: Redundant pathways to make bone during repair
34
CHAPTER 2: The Use of Commercially Available Adhesive Tapes to Preserve Cartilage and
Bone Tissue Integrity During Cryosectioning
* Large portions of this work (Chapter 2) are published in Serowoky and Patel et al.,
Biotechniques 2018.
The use of fluorescent tags to monitor protein expression and to lineage-trace cells has
become a standard complement to standard histological techniques in the fields of embryology,
pathology, and regenerative medicine. Unfortunately, traditional paraffin-embedding protocols
can substantially diminish or abolish the native emission signal of the fluorophore of interest. To
preserve the fluorescent signal, an alternative is to use cryosectioning, however, this can often
result in undesirable artefacts such as tearing or shattering—particularly for mineralized tissues
such as bone and cartilage. Here, in collaboration with my colleague Divya Patel, I present a
method of using a commercially available tape to stabilize murine femur tissue, thus allowing for
cryosectioning of cartilage and bone tissues carrying fluorescent tags without the need for
demineralization.
Introduction
The availability of genetic tools to identify and track cells within the skeletal system as well
as to manipulate gene expression has increased in the past 10 years.
91,92
The ability to rapidly
and cost-effectively analyze tissues is essential to in situ analysis of musculoskeletal tissue
histology and gene expression particularly when carrying out medium- to high-throughput
phenotyping analysis.
93
Generating high-quality histological sections of animal tissues that
preserve tissue integrity and morphology can be technically challenging. These challenges are
especially appreciated when sectioning mineralized tissues, such as cartilage and bone.
Standardized cryosectioning protocols typically provide an effective and rapid system for
visualizing tissue histology, however, when mineralized tissues are sectioned in this manner,
35
researchers frequently struggle with tissue distortion, tearing, wrinkling, and shattering of bone
and cartilage. To alleviate these issues, protocols have been developed that include
demineralization prior to sectioning. Indeed, tissue decalcification improves the ease of sectioning
mineralized tissues, however, decalcification protocols can distort tissue morphology (i.e.
demineralization by acidic solutions) or require long-term decalcification by chelating agents (i.e.
EDTA) for up to several weeks.
94,95
These caveats may be unacceptable for some experimental
analyses and disrupt fast-paced workflows in certain laboratory environments. In addition, many
protocols include paraffin embedding prior to sectioning. Paraffin embedding can be problematic
because the fixation and/or the embedding process can diminish or abolish the native emission
signal of fluorophores of interest.
96,97
Consequently, transgenic fluorescent proteins within the
tissue must be visualized by subsequent immunohistochemistry protocols, however due to the
high cost of antibodies, the additional time requirement, and potential diminished signal-to-noise,
this approach may not be optimal.
With these technical limitations in mind, Divya and I set out to develop a protocol for
inexpensive and effective cryosectioning of mineralized tissues. Our approach was to evaluate
the feasibility of using widely available commercial adhesive tapes to stabilize skeletal tissues
during cryosectioning. A product called Section-lab currently exists on the market (Section-lab
98
,
Hiroshima, Japan). Section-lab tape works by attaching an adhesive tape directly to the front of
the tissue block so that sectioned tissue adheres to the tape and remains in its natural
conformation and orientation during cryosectioning. Section-lab tape is excellent and has been
used with great success;
99–101
however, the cost of materials is considerable when sectioning a
large number of samples. Therefore, the focus of this project is to discover a more cost-effective
alternative for routine cryosectioning use. An optimal tape for our cryosectioning needs must be
inexpensive, easy to work with, non-autofluorescent, and adhere strongly to tissue samples.
Additionally, an ideal tape will retain these qualities during post-sectioning processing such as
H&E staining protocols, which utilize solvents that can disrupt the integrity of the adhesive.
36
From Divya’s analysis of 12 commercially available tapes, she discovered that several tapes
can indeed be used to stabilize skeletal tissues during cryosectioning, enabling rapid and cost-
effective generation of histological sections without the need for demineralization. Here, I show
that this method can reliably be used to generate high-quality sections of the murine femur that
preserves anatomical structures. Additionally, I show that these sections can be used to for
evaluation of native fluorescence and histochemical staining.
Materials and Methods
Divya procured 12 different tapes from standard stationary suppliers and from a tape
warehouse (FindTape.com) supplying tape for a wide range of applications including book repair,
greenhouse repair, packing, sealing, and construction (Figure 1A). These tapes were tested with
mouse muscle and femur tissue obtained from C57Bl/6 and double transgenic
Col2.3:GFP;ColX:mCherry mice.
102,103
Prior to sectioning, individual tape strips were prepared on
a cutting mat that had been cleaned of dust with 70% EtOH (Figure 1B). Tape strips of
approximately 30 cm were loaded onto the cutting mat, cut into ~3 cm segments with a razor
blade and tape ends were folded onto itself with forceps to create a non-sticky tab for subsequent
tape handling (Figure 1B).
Our protocol involved these steps:
1. Tissue Fixation
• Fix tissues in 4% paraformaldehyde overnight at room temperature.
• Cryo-protect samples in 20% sucrose overnight at 4°C.
2. Embedding
• Embed sample within OCT (Optimal Cutting Temperature) compound (Tissue-Tek, 4583),
within a cryomold (255608-925, VWR), ensuring that the entire sample (properly oriented)
is covered by OCT.
37
• Create a cold bath for sample freezing by adding dry ice to a bath of 95% EtOH.
• Gently place the cryomold into the EtOH bath so that the crymold floats on top of the EtOH.
• Leave sample floating in EtOH bath until the entire sample freezes (the OCT will change
from clear to white.)
NOTE: At this point the sample can be stored at -80°C for 2-3 months.
3. Cryo-sectioning
• Mount the sample onto the cryostat chuck using OCT.
• Begin sectioning according to the instructions for the device used. Leica Cryostat (CM
3050S).
• Set cryostat to section at 12 um thickness, however, success is possible at all thicknesses
between 8 um and 16 um.
• Peel tape off the mounting board (9952 6”x8” OLFA self-healing rotary mat) kept at room
temperature and press the adhesive side of the tape onto the sample block using a wide
and flat tool such as the end of a wooden paint stir-stick. Ensuring that the sample is entirely
covered by the tape.
• Section the sample in one slow, but continuous movement. If sectioning motion is uneven
the sample can fail to section cleanly and is unlikely to adhere to tape.
• Use forceps to lift the freshly-sectioned tissue by the tab on the tape and place the section
onto a glass slide (48311-703 Superfrost Plus, VWR), with the tissue adhered to the tape
and the tape placed adhesive side up on the slide (see Figure 1C).
• Using laboratory tape, secure the sections onto the slide by taping the tab of the sectioning
tape to the glass slide. Alternatively, the tape can be affixed to the glass with a chitosan or
UV-curing liquid adhesive.
100
NOTE: At this point, slides can be stored at -80°C for up to 6 months
38
4. Preparing slides for imaging
• If the tissue sample harbors native fluorescent markers, the following steps should be
conducted in a light-reduced environment such as an aluminum foil-covered chamber.
• Remove residual OCT from tissue sample by submerging the slide in 1x PBS for 20 minutes
at room temperature.
• Remove slide from 1x PBS bath and tip slide at an angle for 5 minutes to pour off residual
liquid.
• For immediate imaging, mount tissue with mounting media (Vectashield with DAPI, H1200,
Vector Laboratories) and apply the coverslip (16004-330, VWR) so that it rests on the tissue
sample with the adhesive-side of the tape face-up and the slide underneath (see Figure
1C).
• For Hematoxylin and Eosin staining, the typical initial steps (de-paraffin and rehydration)
are skipped and the slide can be immersed in the Hematoxylin stain step after removing
the OCT with 1x PBS (skip step1 and 2 and proceed to step 3 in ref
104
).
NOTE: At this point the sample can be stored at 4°C, protected from light
Images were collected using a Nikon AZ100 Macroscope and a Nikon DSFi1 camera with
brightfield illumination (see Figure 1A for the set-up). For fluorescence imaging, illumination was
provided by a Nikon Hg Intensilight (C-HGFI), while filter cubes were used to filter the emission
and excitation wavelengths (B-2E/C FITC, G-2E/C TRITC, and UV-2E/C DAPI, Nikon). To obtain
better depth of field, higher magnification images were collected and processed with the Nikon
Extended Depth of Focus (EDF) module.
39
Results and Discussion
Divya procured 12 types of widely available commercial tape for evaluation for use during
cryosectioning. These 12 tapes were chosen to represent a range of thicknesses, widths, and
adhesive materials (Figure 1A). Each tape was assigned a unique identifier during testing. Tape
thickness ranged from 10 to 140 microns and width ranged from 6.3 to 38.1 mm. All 12 tapes
were evaluated in comparison to the Cryofilm 3C tape produced by Section-lab, our “gold
standard” for stabilizing tissues during cryosectioning due to its optical clarity, lack of
autofluorescence, and workability. Importantly, all tapes tested cost less than $0.01 per cm
2
.
Prior to sectioning, individual tape strips were prepared on a cutting mat (Figure 1B) and
tape ends were folded onto itself with forceps to create a non-sticky tab for subsequent tape
handling (Figure 1B). After testing several cutting surfaces, Divya determined a cutting mat with
a rugged texture is desirable, as smooth highly adherent surfaces cause the tapes to curl upon
removal.
I suspect that the most important characteristics of a successful tape are 1) low auto-
fluoresce of the tape (both the base material and adhesive) and 2) with ability to adhere to frozen
tissues. Therefore, each tape was first assessed for optical clarity, autofluorescence, and
workability. To evaluate optical clarity, tapes were imaged by brightfield microscopy (see Figure
1C for slide arrangement). Tapes #1-11 were observed to be relatively optically clear, however,
tape 12 showed significant optical distortion (Figure 2A). Tapes were then tested for
autofluorescence in three commonly used fluorescent channels (DAPI, FITC, and TRITC). Tapes
1, 5, 7, 8, and 9 showed low autofluorescence in all three channels. Tapes 2, 3, 4, 6, 10, 11, and
12 all showed significant autofluorescence in at least one channel and were therefore not
evaluated further (Figure 2A). I next tested the workability of tapes 1, 5, 7, 8, and 9 during
cryosectioning of adult murine muscle tissue. Testing revealed that the physical properties of
tapes 5, 7, and 8 were suboptimal for use during cryosectioning due to tape curling or excessive
40
tape thickness, which made it difficult to mount tissue with a coverslip. I therefore selected tapes
1 and 9 for further testing (Figure 2B).
Next, I directly compared tapes 1 and 9 to the Section-lab tape (abbreviated “SL” in the
figures) during cryosectioning of a mineralized adult murine femur. I chose the murine femur
because it is a large, mineralized bone with a detailed ultrastructure that can reveal subtle
differences in the ability of a tape to produce high-quality histological sections.
105
I first compared
H&E-stained sections at several magnifications and found that the SL tape produced the highest-
quality images, followed by tape 1, and then tape 9 (Figure 3). The difference in section quality
is best observed in the epiphyseal trabecular bone (black arrows). These delicate structures are
mostly preserved by SL tape and tape 1, however, some are fragmentation is visible when using
tape 9. At high-magnification, I observed that all three tapes produce high-quality images of the
cartilaginous growth plate. The quality of section through bone marrow in tape 1 was comparable
to the SL tape. Additionally, I show excellent preservation of mineralized tissue as indicated by
alizarin red staining. Thus, these results demonstrate that the adhesives of tapes 1 and 9 tolerate
a variety of histological reagents, including: EtOH, Eosin Y stain, Hematoxylin stain, alizarin red
stain, Citrisolv and Cytoseal. These results suggest that tapes SL and 1 are best-suited for
preserving the integrity of delicate structures, however, all three tapes are acceptable for
preserving more robust skeletal structures such as cortical bone and growth-plate cartilage. Some
users may wish to try the alternative CryoJane tape transfer system which requires a specialized
instrument to be installed inside the Cryostat to preserve bone marrow integrity.
I additionally evaluated tapes during fluorescence imaging. I procured femora from mice
that express two transgenes: Col1(2.3):GFP
101
(GFP driven by osteoblast-specific Collagen I
promoter) and ColX:mCherry
102
(RFP driven by Collagen X promoter, specific to hypertrophic
chondrocytes). I found that tape SL and tape 1 are indistinguishable from each other at all
magnifications, while tape 9 produces an image plagued by air bubbles at low magnification (white
arrows). This is due to issues during coverslip mounting, where I observed that the adhesive used
41
in tape 9 prevents the mounting media from spreading evenly across the section, resulting in the
observed bubbles. These bubbles are not problematic if non-affected regions of tissue are imaged
at high-magnification (Figure 4, middle and right columns). All three tapes produced high quality
fluorescent image with no excessive autofluorescence in all three imaged channels. Together,
these results suggest that tapes SL and 1 are best suited for low-magnification fluorescence
imaging, however, all three tapes are suitable for high-magnification fluorescence imaging. After
fluorescence imaging, I found that the same sections can be subsequently processed for other
protocols including H and E or Alizarin Red staining. Indeed, the sections shown in Figure 4 were
processed to generate Figure 3 after imaging.
My work has demonstrated that at least two widely available and economical tapes can
be used to preserve murine femur tissue integrity during cryosectioning. Although the “gold
standard” cryotape developed by Kawamoto
98
(SL) was the easiest to work with and produced
the highest quality images of the tapes we tested, I show that at a Circuit Plating/Splicing tape
produced by JVCC (tape 1) can produce high-quality histological sections for H&E, alizarin red,
and fluorescent imaging at a reduced cost. Ultimately, this work has inspired our laboratory to
adopt a system where we use a combination of different tapes during sectioning. The SL tape is
reserved for every 3
rd
to 5
th
section to ensure the generation of publication quality images, while
the Circuit Plating/Splicing tape is used for intervening sections or for preliminary and pilot
experiments.
Figure Legends
Figure 1. Tapes evaluated and the method of storage before use
A. The tapes evaluated are listed and were assigned a unique identifier during testing. They
ranged in thickness from 10-140 microns (manufacturer dimensions often in 1/1000 of an inch).
The thickness, adhesives, and other characteristics for some tapes was not available from the
manufacturer.
42
B. Tape strips are shown loaded onto a cutting mat in preparation for sectioning. A mat with
texture is desirable as highly adherent surfaces cause the tape to curl upon removal. Dust is
removed with a 70% EtOH wash and tape strips are loaded in a dust free environment (left panel).
A tab can be created by folding over one side for easy pick-up (right panel).
C. Schematic diagram to illustrate the physical orientation of the tape system components. Note
that the tissue and tape is placed adhesive-side up and imaging should be carried out through
the coverslip.
Figure 2. Assessment of optical clarity, autofluorescence, and workability
A. Evaluated tapes are shown in brightfield to show transmitted light optical clarity and under
three different common epifluorescence imaging settings to demonstrate the qualitative level of
autofluorescence. (tape thickness is indicated in microns, scale bar = 100 microns)
B. Based on the images in A, five tapes were chosen to test workability in comparison with
the SL tape. A 12 micron section through DAPI-stained mouse muscle is shown, mounted with
Vectashield and a coverslip. Tapes 1 and 9 are the easiest to use and mount and were
comparable to the Section-lab (SL) tape. (scale bar = 50 microns)
Figure 3. Use of commercially available tape for histology
Sections of the proximal adult mouse femur which has a challenging ultrastructure including
cortical and trabecular bone, and bone marrow voids. While the SL tape produces a superior
tissue preparation, tapes 1 and 9 are acceptable with little shattering or loss of bone marrow
tissue. Dense tissue (growth plate, enlarged to the right) was well preserved. Alizarin-red stained
sections are shown on the far right to demonstrate tissue mineralization. For comparison, a
section obtained without the use of tape for tissue stabilization during cryosectioning is shown in
the bottom panel. Left panels, scale bar = 500 microns; Right panels, scale bar = 50 microns.
43
Figure 4. Use for fluorescence imaging
Femur tissue from mice carrying transgenic reporters was used for native fluorescence imaging.
Expression of the Col2.3:GFP reporter (green) can be seen in the developing trabeculae, while
the ColX:mCherry reporter (red) can be found in hypertrophic chondrocytes within the growth
plate. DAPI allows for visualization of the nuclei (blue). In this region of the mouse, SL tape, Tape
1, and Tape 9, all performed very well with respect to resolving the signal-to-noise for each
fluorophore. Left panels, scale bar = 500 microns; Middle panels, scale bar = 200 microns; Right
panels, scale bar = 100 microns.
44
Figure 1- Tapes evaluated and method of storage before use
ID Tape Type Company Catalog # Material Width
Thickness
in microns (in)
SL Cryofilm 3C (16UF) Kawamoto C-FUF303 Polyvinylidene chloride 1.5 - 4.5 cm 10
1 Circuit Plating/ Splicing JVCC PPT -25C Polyester/silicone 6.3 mm 64 (0.0025)
2 Book 3M Scotch 845 Polypropylene Film 1.5 in 86 (0.0034)
3 Sealing JVCC CELLO-1 Cellophane 0.5 in 46 (0.0018)
4 Cold Temperature Per-
formance Packaging
Shurtape HP-323 Synthetic Rubber-Resin Hot Melt 2.0 in 48 (0.0019)
5 Polyethylene Clean
Removal
Patco 560 Low Density Polyelthylene Film 0.5 in 140 (0.0055)
6 Film Packaging JVCC PES-32G Polyester Film 0.75 in 76 (0.003)
7 Greenhouse Repair Patco 5068 Low Density Polyelthylene Film 0.5 in 140 (0.0055
8 Removable Protective
Film
Patco 5560 Low Density Polyelthylene Film 0.5 in 140 (0.0055)
9 Crystal Clear Book
Repair
JVCC BOOK-
20CC
Polypropylene Film 0.25 in 63 (0.0025)
10 Utility 3M Scotch n/a n/a 0.5 in n/a
11 Transparent 3M Scotch n/a n/a 0.75 in n/a
12 Removable 3M Scotch n/a n/a 0.75 in n/a
A. Tapes evaluated
B. Mounted tape on cutting mat
C. Tape system
coverslip
tissue
tape
slide
45
Figure 2 - Assessment of optical clarity, autofluorescence, and workability
1
12
11
10
9
8
7
6
5
4
3
2
*
1
9
8
7
64
86
46
48
140
76
140
140
63
n/a
n/a
n/a
*
*
*
*
5
Poor workability
due to thickness
A
B
SL
Poor workability
due to thickness
Tape curled and
could not be
mounted alone
46
1
9
1
9
Figure 3 - Use of commercially available tape for histology
Figure 4 - Use for fluorescence imaging
SL
SL
No
Tape
H and E Alizarin Red
Col10mcherry;Col2.3GFP
47
CHAPTER 3: A murine model of large-scale bone regeneration reveals a selective
requirement for Sonic Hedgehog
* Large portions of this work (Chapter 3) are under revision for publication at npj
Regenerative Medicine.
Building and maintaining skeletal tissue requires the activity of skeletal stem and
progenitor cells (SSPCs). Following injury, local pools of these SSPCs become active and
coordinate to build new cartilage and bone tissues. While recent studies have identified specific
markers for these SSPCs, how they become activated in different injury contexts is not well-
understood. Here, using a model of large-scale rib bone regeneration in mice, I demonstrate that
the growth factor, Sonic Hedgehog (SHH), is an early and essential driver of large-scale bone
healing. Shh expression is broadly upregulated in the first few days following rib bone resection,
and conditional knockout of Shh at early but not late post-injury stages severely inhibits cartilage
callus formation and subesquent bone regeneration. Whereas the Hh co-receptor Smoothened
(Smo) is required in Sox9+ lineage cells for rib regeneration, I find that Shh is required in a distinct
Prrx1+ Sox9-negative mesenchymal population. Intriguingly, upregulation of Shh expression and
requirements for Shh and Smo may be unique to large-scale injuries, as they are dispensable for
both rib and femur fracture repair. In addition, single-cell RNA sequencing of callus tissue from
animals with deficient Hedgehog signaling reveals a depletion of Cxcl12-expressing cells, which
may indicate failed recruitment of Cxcl12-expressing SSPCs during the regenerative response.
These results reveal a mechanism by which Shh expression in the local injury environment
unleashes large-scale regenerative abilities in the murine rib.
INTRODUCTION
Some animals such as salamanders, zebrafish, and lizards are famously endowed with
the ability to regenerate exceptionally large and complex skeletal structures, however humans
48
and other mammalian vertebrates are typically only capable of healing minor skeletal injuries. For
example, bone lesions in humans greater than 1 cm
3
are generally defined as “critical-sized”
defects that will not repair without surgical intervention.
106
A notable exception is the human rib
cage, which is capable of regenerating astonishingly large skeletal segments.
99
Inspired by case-
reports of large-scale rib regeneration in adult humans,
107–109
the Mariani lab recently discovered
that ribs of adult mice are similarly capable of regenerating unusually large skeletal
segments.
63,99,110
Using this novel model, we identified the periosteum as a key source of
specialized skeletal stem/progenitor cells (SSPCs) that orchestrate large-scale bone
regeneration.
63,99,110
Additionally, my colleague Stephanie Kuwahara has shown that Sox9-
expressing periosteal SSPCs require the Hedgehog (Hh) signaling pathway co-receptor
Smoothened (Smo) for proper differentiation during the first few days of large-scale
regeneration.
63
These results are in stark contrast to recent data that have shown that inhibition
of Hh signaling does not affect fracture healing,
111,112
suggesting that the scale of the injury may
be a major component that determines the requirement for Hedgehog signaling.
It is now appreciated that skeletal regeneration is executed by a combination of multiple
SSPC populations located locally within the periosteum, bone marrow (BM), and skeletal muscle
interstitial compartments.
1,21,44,55,58,113,114
For example, a recent report highlights the importance of
BM-derived Cxcl12+ stromal cells for generating chondrocytes during bone repair.
114
Following
injury, most long bones regenerate through endochondral ossification whereby a cartilage callus
stabilizes and reduces the fracture prior to the conversion of the cartilage callus to bone.
87
The
earliest steps of injury-induced activation of SSPCs and their subsequent differentiation into a
cartilage callus are not completely understood. It is clear that SSPCs from multiple tissues
generate callus chondrocytes during repair,
21,55,63
however, it is unknown how cells within these
compartments coordinate their activities in different injury contexts.
Here, I show that Sonic Hedgehog (Shh) is required for the repair of large-scale bone
injuries in the rib, but not fractures in mice. I found that Shh expression is rapidly and broadly
49
upregulated at the site of injury in response to large-scale injury and that conditional knockout of
Shh severely inhibits regeneration. Hedgehog signaling is especially critical during the earliest
stages of regeneration, as conditional removal of Shh or Smo beginning at 5 days after injury
does not affect subsequent healing. In addition, I observed a decrease in the number of Cxcl12+
cells in the central region of the callus of Smo cKO mice, which may reflect impaired recruitment
of BM-derived Cxcl12+ SSPCs. Together, these data shed light on the selective expression and
requirement of Shh during large-scale regeneration.
RESULTS
Shh is expressed during early callus formation
Our previous work identified a population of periosteal resident SSPCs marked with a
Sox9:CreER transgene that requires the Hedgehog signaling co-receptor Smoothened (Smo) for
proper large-scale rib regeneration.
63
The Hh ligands that are responsible for activating Sox9+
SSPCs remain unclear. I therefore set out to characterize the expression of the three known
Hedgehog ligands, Desert Hedgehog (Dhh), Indian Hedgehog (Ihh), and Sonic Hedgehog (Shh),
during the early response to large-scale rib resection. Briefly, this injury model includes surgical
resection of a 3 mm segment of rib bone while leaving adjacent periosteal and muscle tissues
intact (Fig 1A). Repair of this injury model in control mice results with the formation a large
cartilage callus by 7 days post injury (dpi) marked by alcian blue, which is then mostly converted
to bone by 14 dpi marked by alizarin red, and subsequently remodeled to the original anatomy by
4-6 weeks, indicating successful regeneration (Fig 1A).
To characterize the expression of Hh ligands in response to injury I harvested rib tissue
from day 0 (uninjured), 3 dpi, and 5 dpi animals (Fig. 1B) and performed fluorescent RNA in situ
hybridization (RNA-ISH) for the expression of the three Hh ligands. At these early time points, the
resection gap has already been filled in with a mixture of presumed fibroblasts, SSPCs, and other
cell types, but chondrogenesis has not yet begun (Fig 1C), suggesting that prior to 5 dpi is a key
50
window of time for chondrogenic signaling. Similar to previous reports Dhh, Ihh and Shh are
expressed in a few sparse cells at low levels within the bone marrow compartment of the uninjured
rib
115,116
(Fig 1D) while expression in expected locations such as the testis and growth plate is
strong (Fig S1A,B).
117–119
However expression was not detected in the cortical bone, periosteum,
and adjacent muscle. These results suggest that uninjured animals have some baseline
expression of Dhh, Ihh and Shh ligands within the BM of uninjured ribs, whereas there is a minimal
baseline Hh expression in the cortical bone, periosteum, and skeletal muscle compartments. At
3 dpi, Dhh and Ihh are largely undetectable within the resection gap and adjacent tissues,
whereas Shh is prominently expressed in the central resection gap (Fig 1D). At 5 dpi, expression
of Ihh and Dhh remain undetectable within the injury site and adjacent tissues, whereas Shh
expression has expanded and increased in intensity, creating a domain of mesenchymal cells
with high Shh expression within the central resection gap and adjacent skeletal muscle interstitium
(Fig 1D). Notably, I did not detect substantial Shh expression in the areas of the callus
immediately adjacent to the cut bone ends.
I was surprised to find minimal expression of Ihh after injury, since the Crump lab’s recent
work previously identified ihha activity in large-scale regeneration of zebrafish facial bones.
78
My
colleague Stephanie Kuwahara therefore sought to verify the lack of Ihh expression in murine rib
regeneration by an independent method. To do so, she utilized Ihh;LacZ mice, which have a LacZ
reporter cassette knocked-into the first exon of the Ihh gene, allowing her to use LacZ activity as
a readout of Ihh expression (Fig 1 – Supplement 1C).
120
X-gal staining of these animals (Fig 1 –
Supplement 1D) showed expression in the growth plate as expected, but no detectable LacZ
expression at 3 dpi. At 7 dpi she did detect LacZ expression in large, maturing chondrocytes as
expected. However, since our previous work showed that Hh signaling is required prior to the
onset of chondrogenesis,
63,78
I determined that this Ihh expression in maturing chondrocytes is
outside the early window required for stimulation of initial cartilage callus generation. Thus, these
data provide an independent line of evidence to support the RNA-ISH data in Fig 1 demonstrating
51
that Ihh is not strongly expressed within the resection site prior to generation of the cartilage
callus.
Together these data indicate that Shh, but not Ihh or Dhh, is expressed during the early
response to a rib resection injury. Further, the domain of Shh expression lies within the central
region of the rib resection site, where a subset of mesenchymal cells begin to upregulate Shh
expression by 3 dpi and increase expression at 5 dpi. Thus, Shh is expressed at the right time
and place to activate SSPC chondrogenesis during early callus formation following rib resection
injury.
Shh is required for callus generation and subsequent large-scale rib regeneration
To test if Shh if is required for regeneration, I assayed rib repair in CAGG:CreER mice in
which I conditionally deleted (cKO) Shh globally in the animals prior to injury. To induce global
Shh cKO in adult animals, I administered Tamoxifen daily for 5 consecutive days prior to rib
resection (Fig 2A). I then assayed callus formation histologically at 10 and 28 dpi to assess
cartilage and bone callus production, respectively. At 10 dpi, control mice (CAGG:CreER;Shh
flox/+
)
generate a large, robust cartilage callus that fully spans the defect site, whereas Shh cKO mice
show cartilage formation near the cut ends but largely fail to generate cartilage in the most central
region of the injury site (Fig 2B). At 28 dpi, control animals generate a large bone callus that fully
spans the resection site, whereas the calluses of Shh cKO mice remain unbridged indicating a
non-union and show only a modest amount of newly generated bone near the cut ends (Fig 2B).
I then used Safranin-O stained sections to blindly score regeneration outcomes as Good,
Moderate, or Poor (see Methods for details regarding scoring criteria). Overall, controls largely
heal well (6/8 animals), whereas Shh cKO animals largely heal poorly (10/12 animals). Thus, Shh
is required for bridging cartilage callus formation and efficient bone regeneration.
52
Shh expression outside of Sox9+ SSPCs
Our previous work identified a population of periosteal resident SSPCs that can be marked
with a Sox9:CreER transgene, and that requires the Hedgehog signaling co-receptor Smo for
proper large-scale rib regeneration.
63
I next asked if Sox9+ lineage SSPCs are a cellular source
of SHH, which in combination with our previous results, would reflect an autocrine SHH-Smo
signaling mechanism. To test this hypothesis, I performed RNA-ISH for Shh on sections harvested
from Sox9:CreER;R26:tdTomato double transgenic animals. To induce lineage-tracing of Sox9+
SSPCs, I administered Tamoxifen to Sox9:CreER;R26:tdTomato animals for 3 consecutive days
beginning 7 days prior to surgery and harvested rib tissue at 7 dpi (Fig 3A). At this stage, most
Shh-expressing cells (green) were tdTomato (tdT) negative (Fig 3B). I was on occasion able to
detect a few tdT+ cells that were also expressing Shh, although almost all tdT+ cells were Shh-
negative. Thus, Shh is primarily expressed in tdT-negative cells, suggesting that the primary
source of SHH is not from the Sox9+ SSPC lineage.
To confirm that Sox9+ lineage SSPCs are not a critical source of SHH protein, my
colleague Shuwan Liu conditionally deleted Shh in Sox9-expressing cells using
Sox9:CreER;Shh
flox/flox
animals prior to injury and assayed rib regeneration. To induce Shh cKO
in the Sox9+ lineage, she administered Tamoxifen to Sox9:CreER;Shh
flox/flox
animals for 3
consecutive days beginning 7 days prior to surgery and harvested rib tissue at 7 and 14 dpi (Fig
3C). In both controls and Shh cKO animals, she observed large Safranin-O positive cartilage
calluses at 7 dpi and cartilage calluses with significant regions converted to bone at 14 dpi (Fig
3E). Blinded scoring of regeneration further corroborated a similar healing capacity of
experimental animals compared to controls (Fig 3D). These data reveal that cKO of Shh in Sox9-
lineage SSPCs has no major effect on large-scale rib regeneration, consistent with the
observation that the majority of Shh expression occurs outside of the Sox9+ lineage. Thus, Sox9+
lineage cells are not the major source of SHH protein.
53
Shh is expressed by Prrx1-expressing cells and is not dependent upon the presence of
Sox9+ lineage cells
To further characterize Shh-expressing cells, I next asked if Shh is expressed by
mesenchymal cells vs other cell types present after injury. Since Prrx1 expression broadly marks
mesenchymal cells following injury,
37,121
I performed double RNA-ISH for Shh and Prrx1 at 5 dpi
and observed cells with substantial overlap in expression (Fig 4A). Indeed, nearly all Shh-
expressing cells (red) are yellow due to co-expression of Prrx1 (green) (Fig 4A). In contrast, many
Prrx1-expresssing cells (green) are Shh negative. We can also on occasion, detect Shh+ Prrx1-
cells (magenta arrowheads), though they are quite rare. Together these data indicate that at 5
dpi, cells expressing Shh are largely constrained to a subset of Prrx1-expressing mesenchymal
cells. Together these data indicate that at 5 dpi, Shh expression is constrained within a subset of
Prrx1-expressing mesenchymal cells.
Although Shh does not appear to be expressed broadly by Sox9+ lineage SSPCs (Fig
3B), I wondered if Shh expression by Prrx1-expressing cells is dependent upon the presence of
Sox9+ lineage cells. I therefore assayed Shh expression in animals in which Sox9+ lineage cells
were selectively ablated with diphtheria toxin (DT) prior to injury.
122
To selectively ablate Sox9+
cells prior to injury, I generated Sox9:CreER;R26:DTR double transgenic mice. In this system,
Tamoxifen administration induces expression of the diphtheria toxin receptor (DTR) specifically
in Sox9+ cells, rendering Sox9+ cells susceptible to subsequent diphtheria toxin (DT) exposure.
To ablate Sox9+ cells prior to injury, I administered 3 daily injections of Tamoxifen to
Sox9:CreER;R26:DTR mice starting 7 days before injury. Mice were then treated with PBS
(Control) or Diphtheria Toxin (DT) daily for 3 days prior to injury (Fig 4A). Similar to our previous
results where Smo was removed from Sox9+ lineage cells,
63
DT-treated Sox9:CreER;R26:DTR
animals fail to generate a cartilage callus at 10 dpi, whereas PBS-treated animals generate large
cartilage calluses (Fig 4C). Blinded scoring of regeneration success further corroborated impaired
regeneration of DT-treated Sox9:CreER;R26:DTR animals vs PBS-treated controls (Fig 4D). To
54
determine if Shh is expressed even in the absence of these Sox9+ SSPCs, I harvested rib tissue
from DT-treated Sox9:CreER;R26:DTR mice at 5 dpi and performed RNA-ISH. At 5 dpi,
Sox9:CreER;R26:DTR animals broadly express Shh within the injury site (Fig 4E) similar to wild-
type animals (Fig 1), supporting that Shh expression in the local injury environment by Prrx1-
expressing cells is independent of Sox9+ lineage SSPC presence.
Together, these data provide multiple independent lines of evidence to support a model in
which Shh is broadly upregulated in the local injury environment by Prrx1+ cells following large-
scale rib injury and its expression is independent of Sox9+ lineage cells.
Shh is dispensable for large-scale rib regeneration after 5 dpi
My RNA-ISH analysis above (Fig 1D) demonstrates that Shh expression is detectable as
early as 3-5 dpi, however this does not rule out an important role for Shh during later stages of
repair, such as in promoting the proliferation and maturation of chondrocytes.
123
To test if Shh is
required at the later time points after injury, I genetically deleted Shh globally using Shh cKO mice.
In these experiments, I began 5 days of Tamoxifen administration at 5 dpi (Fig 5A). With this
Tamoxifen regimen, I aimed to induce a cKO of Shh expression after the initial wave described in
Fig 1D. I then assayed callus formation histologically at 10 and 28 dpi to assess cartilage and
bone callus production, respectively. At 10 dpi, both control and Shh cKO mice generate a large,
robust cartilage callus that fully spans the defect site (Fig 5B). At 28 dpi, both control and Shh
cKO animals display a bone matrix callus that fully spans the resection site (Fig 5B). Blinded
scoring of regeneration success further corroborated similar regeneration outcomes in control and
Late Shh cKO animals where the majority of animals were scored as Good and Moderate, in stark
contrast to the Early Shh cKO animals from Fig 2 where the majority of animals healed Poorly
(Fig 5C). Thus, Shh is not required for cartilage formation and subsequent large-scale rib
regeneration when removed at 5 dpi.
55
My colleague Stephanie Kuwahara also tested the timing of requirement of the Hh
receptor Smo in callus cells during healing using two alternative Tamoxifen regimens. In the early
KO approach, she treated Sox9:CreER;R26:tdTomato;Smo
flox/flox
animals with 3 doses of
Tamoxifen around the time of injury at -1, 1, and 2 dpi (Fig 6A), whereas in the late KO approach
she administered Tamoxifen at 4, 5, and 6 dpi (Fig 6B). Since Sox9:CreER is widely upregulated
in callus cells in response to local injury,
63
both of these Tamoxifen regimens broadly target many
cells in the injury milieu, as indicated by the broad expression of the tdTomato lineage trace (Fig
6C).
To monitor healing progress Stephanie harvested tissue at 14 dpi and analyzed H&E-
stained histological sections. She found that when Smo is removed in the early KO group, she
observed only a modest amount of new skeletal tissue generated near the cut ends, and the
central portion of the resection site fails to properly differentiate (Fig 6D), similar to global Shh
cKO animals (Fig 2B). In contrast, in the late Smo cKO animals she found that animals build a
large bony callus by 14 dpi suggesting a normal healing progression (Fig 6D).
Taken together, these results support the existence of a critical window for Hedgehog
activity during the first 5 days of large-scale rib regeneration and that loss of Shh or Smo after
this window does not significantly affect bone regeneration.
Hedgehog signaling is dispensable for small-scale bone repair
During the above experiments, I noticed that (1) Shh expression is largely restricted to the
most central portion of the resection gap, and (2) Shh and Smo cKO mice fail to regenerate this
central region, yet still generate callus tissue adjacent to the edges of the cut rib ends (Hh-
independent regeneration). I therefore hypothesized that Shh is uniquely required for regeneration
in large-scale contexts in which Shh-independent callus production at the cut ends is insufficient
to bridge the injury gap. To test this hypothesis, I created a surgical fracture in the rib bone in lieu
of resecting a 3 mm rib segment, leaving adjacent periosteal and muscle tissues intact (Fig 7A).
56
In this model, a small alcian blue-stained cartilage callus forms by 7 dpi indicating an
endochondral repair pathway (Fig 7A); bridging is complete by ~28 dpi.
I first asked if Shh is expressed in this small-scale injury context. To assay Shh expression,
I performed RNA-ISH for Shh at 3 and 5 dpi following surgical fracture. At both time points, Shh
expression was not detectable (Fig 7C). These data suggest that in contrast to a large-scale injury
context, Shh is not upregulated during early callus formation in a small-scale bone injury context.
To further determine if Shh is a critical mediator of small-scale bone repair, I conditionally
deleted Shh in Shh cKO mice prior to surgical fracture and then assayed repair. To induce global
cKO of Shh in adult animals, I administered Tamoxifen daily for 5 days prior to injury (Fig 7D). I
then assayed callus formation histologically at 10 and 28 dpi to assess cartilage and bone callus
production, respectively (Fig 7E). At 10 dpi, both CAGG:CreER;Shh
flox/+
controls and Shh cKO
mice generated small, but notable cartilage nodules within the periosteum adjacent to the injury
site. I also observed that the cut ends of both controls and Shh cKO animals abut each other and
are bridged by newly generated irregular skeletal tissue. At 28 dpi, both control and Shh cKO
animals generate a bone callus that fully spans the injury site, indicating successful healing in
both genotypes (Fig 7E). Blinded scoring of regeneration outcomes further corroborates similar
healing progression in control and Shh cKO animals (Fig 7F). Thus, Shh is dispensable for small-
scale rib repair.
To determine if Hh signaling is dispensable in other small-scale repair contexts, in
collaboration with my colleagues Stephanie Kuwahara and Dr. Venus Vakhshori of the Lieberman
lab, we tested if the Hh co-receptor Smo is required in Sox9+ SSPCs during femur fracture repair.
To induce Smo KO in Sox9+ lineage SSPCs, Stephanie and I administered Tamoxifen to
Sox9:CreER;Smo
flox/flox
animals for 3 consecutive days beginning 7 days prior to injury (Fig 8A).
Dr. Vakhshori then induced an impact femur fracture at day 0 and Stephanie harvested femur
tissue at 10 and 28 dpi to evaluate cartilage and bone production, respectively. At 10 dpi in both
Sox9:CreER;Smo
flox/+
controls and Sox9:CreER;Smo
flox/flox
animals, a large Safranin-O positive
57
cartilage callus of similar size is evident (Fig 8B). At 28 dpi, both controls and Smo KO animals
have a large bony callus (Fig 8C). Blinded scoring of regeneration outcomes further corroborates
similar healing progression in control and Shh cKO animals (Fig 8D). These results suggest that
Hh signaling in Sox9+ SSPCs is dispensable for small-scale femur fracture repair.
Together, these data suggest that the Hedgehog signaling pathway is not required for the
repair of small-scale rib or femur fracture injuries.
Single-cell RNA sequencing reveals a reduction in Cxcl12-expressing cells when Sox9+
lineage cells do not receive a Hh signal
The experiments above, as well as our previous work, demonstrate that activation of the
Hh pathway in periosteal cells is required for proper cartilage callus formation, yet how Hh activity
in periosteal SSPCs ultimately orchestrates repair is unknown. To investigate this question, I used
single-cell RNA sequencing (scRNAseq) to characterize the transcriptional landscape of the early
repair callus in controls (Sox9:CreER;Smo
flox/+
) vs. Sox9+ lineage Smo knockout (cKO) animals
(Sox9:CreER;Smo
flox/flox
) (Fig 9A). To induce Smo cKO, I administered Tamoxifen to
Sox9:CreER;Smo
flox/flox
for 3 consecutive days beginning 7 days prior to surgery and collected
fresh, dissociated callus cells at 4 dpi, and performed scRNAseq using the 10x Genomics
Chromium platform (Fig 9B). I chose 4 dpi as it is within the critical window for Hh activation.
UMAP visualization identified ~17 transcriptional cell states broadly representing the major cell
types expected, including connective, endothelial, hematopoietic, and muscle cells (Fig 9C, Fig
9 – Supplement 1).
To assess the activity of SSPCs, I specifically compared the cluster of connective-type
cells from control vs Smo cKO animals and revealed a dramatic shift in Cluster 0 cells upon Smo
deletion (Fig 9D, E). Most clusters overlapped between control and Smo cKO contexts,
suggesting that the shift in Cluster 0 grouping is biological and not a technical batch effect (Fig
9D). I next investigated the genes that best distinguish control vs Smo cKO Cluster 0 cells and
58
determined that Cxcl12 is expressed highly in large number of Cluster 0 cells in the control
context, but the number of cells expressing Cxcl12 within this cluster is severely reduced in the
Smo cKO context (Fig 9F, G). High Cxcl12 expression has been observed in bone-marrow
stromal cells that can adopt a SSPC-like state in response to injury and subsequently be recruited
to generate chondrogenic and osteogenic cells of the repair callus.
114
Cxcl12-high expressing
cells are notably lacking in Smo cKO mice, suggesting that Smo activity in Sox9+ periosteal
SSPCs may be required to recruit these Cxcl12-expressing bone marrow cells into the repair
callus.
To evaluate the expression of Cxcl12 within control and Smo cKO contexts, I performed
an immunofluorescence assay for CXCL12 at 4 dpi in control vs. Smo cKO animals. In control
animals, I observe a broad distribution of Cxcl12-expressing cells throughout the callus, with the
highest expressing cells located in the most central region of the repair callus (Fig 9H). In contrast,
in Smo cKO animals Cxcl12 expression was primarily localized to an area adjacent to the cut rib
bones with little to no expression in the most central region of the repair callus (Fig 9H). Together,
these data support a model of large-scale rib regeneration where Shh activation of periosteal
Sox9+ SSPCs through Smo ultimately leads to recruitment of Cxcl12-expressing cells into the
most central portion of the early repair callus to facilitate large-scale regeneration.
A similar but alternative interpretation of our scRNAseq data could be that Hh activation
of Sox9+ lineage cells stimulates their differentiation into a Cxcl12-high expressing population, as
described by Ono et al. 2014 during bone development. However, in our experiments Cxcl12-
expressing and Sox9+ lineage cell populations appear quite distinct, as we observed minimal
overlap between tdTomato fluorescence and CXCL12 protein expression (Fig 9I). In addition, a
UMAP for tdTomato expression reveals little correlation between where these cells cluster and
the cluster of high Cxcl12-expressing cells that is missing in the mutant (Fig 9J). Furthermore, a
sub-selection for tdTomato-expressing cells from our scRNAseq data did not express markedly
different levels of Cxcl12 in control and Smo cKO conditions (Fig 9J). Thus, these data support a
59
model where Cxcl12-high expressing cells are largely a distinct population from the Sox9+ lineage
cells and whose presence during repair is depending on Hh signaling.
DISCUSSION
Shh as an initiator of large-scale repair
Repairing a small vs large injury may not only necessitate distinct bone healing pathways
(direct and/or endochondral) but may also require distinct triggers to initiate these pathways. My
studies provide evidence for Shh as a required trigger to initiate a type of repair that creates a
bridging callus in the context of a large-scale injury. I was surprised to find Shh to be required
instead of Ihh, as SHH has been traditionally considered a ligand important for embryonic skeletal
patterning and discovering a role for SHH in adult skeletal biology was unexpected.
124–126
In
addition, it is IHH that has a long-appreciated role in endochondral bone growth,
123
and
furthermore, Dr. Crump’s previous work showed that ihha rather than shh is critical for large-scale
regeneration in the zebrafish jaw bone.
78
However, in the context of the mouse rib, Shh but not
Ihh or Dhh, is rapidly and broadly upregulated in the first few days following large-scale injury (Fig
1). Furthermore, genetic deletion of Shh prior to injury but not afterwards, dramatically impairs
regeneration (Fig 2, 5). It is possible that in mice, Shh may be playing a similar role to ihha in fish
- indicating that the particular type of Hh protein may not matter as much as when and where it is
expressed in these divergent species.
So as to prevent the accidental formation of ectopic cartilage or bone, the regulation of
these Hh ligand genes in response to injury is likely very tightly controlled, perhaps requiring
multiple inputs and/or chromatin alterations. How Shh in the mouse and ihha in the fish is
regulated or how Shh is regulated in the murine rib vs the femur will be a fruitful avenue of future
investigation. One approach may be to define regulatory elements that govern Shh expression
specifically in response to injury and determine if these elements are differentially regulated in
femur vs rib or large-scale vs small scale injury. In addition, since Shh is an important patterning
60
factor during both limb and rib development,
124–127
it is tempting to consider that large-scale
regeneration requires the redeployment of a developmental program involving Shh, whereas
small-scale injuries heal through in independent program of repair that does not mirror
developmental history. Whether or not Shh expression in large-scale regeneration represents a
bona-fide reactivation of a developmental program is still to be determined.
Hh-dependent vs Hh-independent regions of repair
My studies reveal that not only is Shh required to initiate large-scale rib repair but may be
dedicated to building new skeletal tissue within a specific region of the repair callus. My
observation that Shh cKO mice still produce some cartilage adjacent to the cut ends and later
convert that cartilage to bone supports a model where the regions near the cut ends repair via
endochondral ossification employing a Hh-independent mechanism, whereas repair in the central
region of the defect site with a bridging callus, also via endochondral ossification, is indeed Shh-
dependent. Notably, Shh cKO animals fail to form a cartilage callus in the most central region of
the injury site, while still producing cartilage immediately adjacent to the cut ends. The un-
differentiated central portion of the injury site in Shh cKO animals mirrors the domain of Shh
expression in control animals at 3-5 days post injury, further supporting a link between Shh
expression in the injury environment and subsequent callus differentiation. Together, these
observations support a model of large-scale bone regeneration where repair close to the cut bone
ends is Shh-independent, whereas repair in the most central region of the injury site is critically
dependent on early expression of Shh (Fig 10). Why there would be region-specific requirements
for Shh remains unclear. Perhaps the cells that occupy different regions of the callus are derived
from alternative lineages (i.e., periosteal enriched at the cut ends vs bone marrow enriched in the
central region) and therefore may have different required inputs for successful differentiation. It is
also worth noting that the injury environment is not uniform and there may be certain
61

environmental conditions (i.e., vascularization, biomechanics) that create “zones” of the callus
with different signaling requirements.
128–130

Intriguingly, a very similar regional requirement for Hh signaling has been observed in
regenerating lizard tails. When Hh-inhibiting drugs are applied to lizard injury site after tail
amputation, a new long tail fails to regenerate, yet the terminal vertebra that was injured during
amputation successfully repairs through an endochondral pathway that resembles fracture
healing.
131
Thus, lizards also exhibit a paradigm where bone healing at the site of injury repairs in
a Hh-independent manner, whereas the large-scale regenerative events are indeed Hh-
dependent.
The observation that Shh is generally not expressed in the region immediately adjacent to
the cut bone ends has important implications for how SSPC behavior is regulated in response to
injury. The most obvious is that a small-scale injury such as a simple fracture does not contain a
region of repair tissue distant from the injured bone ends and thus may not express Shh at all.
Indeed, I was unable to detect Shh expression within the first 5 day window following surgical
fracture, and global Shh cKO in these animals still resulted in complete healing. One prominent
outstanding mystery is which factors or conditions result in Shh being expressed following large-
scale, but not during small-scale injury. For example, a larger-scale injury may result in more
severe hypoxia,
132–134
increased immune response,
135
or an altered mechanical environment
which in turn induces Shh expression.
136–139
Indeed, biomechanical stability is known to have a
major impact on the lineage progression of SSPCs during healing and rigidly stabilize fractures
can heal via direct ossification without first building a cartilage callus.
80

When combined with my observations, these data provide similar lines of evidence in
several species and models of skeletal regeneration whereby a Hh ligand initiates repair in a
large-scale injury and has a regional role during building a bridging callus. These observations
point to a Hh ligand as the critical factor underlying the regeneration of large-scale, but not small-
scale injuries. Large- vs small-scale repair may require different pathways, mechanisms, and
62
regulatory controls. How conserved these paradigms may be in other bones and across other
species is yet to be determined.
Other roles for Hh signaling
While my colleague Stephanie Kuwahara’s previous work has shown that early Hh
signaling is required in Sox9+ lineage SSPCs to build a substantial bridging callus, other cell types
may also be responding to Hh signaling during repair. Indeed, her studies showed that the co-
receptors Ptch1 and Smo are also expressed outside the Sox9+ lineage.
63
For example, Hh
signaling may have additional roles on endothelial cells to stimulate angiogenesis
140,141
or may
act on cells that are part of the innate or adaptive immune system.
142,143
Whether the activity of
SHH on cell types outside of Sox9+ lineage is partially responsible for healing deficiencies in Shh
cKO animals is still to be determined.
Shh appears to be particularly critical during the early stages of regeneration, as genetic
deletion of Shh 5 days after injury still results in substantial healing (Fig 5). This is further
supported by Stephanie’s observation that deletion of co-receptor Smo at 4 days after injury also
results in complete healing (Fig 6). This, however, does not completely rule out a role for Hh
signaling during later stages. For example, growth plate chondrocytes express both Shh and Ihh
and this expression is important for their proliferation and maturation;
123
so it is possible that Hh
signaling could still have a role during later stages of regeneration by supporting chondrocyte
maturation and differentiation. For instance, there is a growing body of evidence describing non-
canonical, SMO-independent Hh signaling transduction (reviewed by Pietrobono et al., 2019
133
),
which would allow for Hh ligand activity later during the repair process, even in Smo cKO contexts.
Although evidence for SMO-independent Hh signaling is primarily observed in diseased
contexts,
132,133
it would be interesting to investigate if similar mechanisms may be active during
the late stages of normal bone healing.
63

A main function of Hh activated Sox9+ cells may be to recruit Cxcl12-expressing cells
Stephanie Kuwahara’s recent work has proposed that Sox9+ lineage SSPCs act as
“messenger” cells, since loss of Smo in this population (around 20% of the callus) also affects the
behavior of Sox9-negative cells in the callus.
63
How cells outside the Sox9+ lineage participate in
repair is still unclear. For example, SSPCs can be found in the growth plate, periosteum, and
bone marrow compartments.
1
One possibility is that periosteal-derived Sox9+ cells, once
activated by Hh signaling, facilitate the formation of a large bridging callus by recruiting other cells
types to the injury site. My scRNAseq data illuminated a population of Cxcl12-expressing cells
that is reduced in number within the repair callus of Sox9:CreER;Smo
flox/flox
animals at 4 dpi.
Perhaps the role of Sox9+ cells in large-scale repair is to recruit these Cxcl12-expressing cells?
While Cxcl12 expression has been observed in a number of cell types including endothelial cells,
periosteum, and cartilage (reviewed in Yellowley 2013
144
), interestingly, recent work from the Ono
group has shown that Cxcl12-expressing bone marrow stromal cells can adopt an SSPC-like state
in response to bone injury and subsequently contribute to cartilage and bone callus formation.
114

Linking this observation with my scRNAseq data suggests that the impaired large-scale healing
observed in these models of Hh inhibition, may be due to the failed recruitment of Cxcl12-
expressing bone marrow cells into the callus to participate in regeneration. Since Cxcl12-
expressiong and Sox9+ lineage cell populations appear quite distinct (as we observed minimal
overlap between the tdTomato and CXCL12 labels) the possibility that Hh activation of periosteal
Sox9+ lineage cells stimulates their differentiation into a Cxcl12-high expressing population,
similar as described by Ono et al. 2014 during bone development, seems unlikely. The idea that
periosteal and BM-derived cells may work in collaboration must be tested more thoroughly in the
future, as it implies crosstalk between periosteal and BM -derived SSPCs and the appropriate
recombinase mouse lines to investigate these relationships are much needed. Excitingly, the
recent emergence of Dre-based genetically modified mice unlocks the possibility of
simultaneously lineage tracing multiple cell populations in parallel.
145,146

64

In summary, together my data point to a model of skeletal regeneration where Shh
expression is uniquely required to stimulate cartilage callus formation following large-scale injury.
This revelation may inform future therapeutic strategies, especially in circumstances where large-
scale regeneration of skeletal elements is required, such as following traffic accidents, combat
wounds, and segmental resection due to bone cancer removal.

MATERIAL AND METHODS
Mice and animal housing
All procedures were approved by the University of Southern California Institutional Animal
Care and Use Committee (Protocol #: 11256, 20639). I used the following mouse
lines: Sox9:CreER2 (Sox9
tm1(cre/ERT2)Haak
),
147
Shh
flox/flox
(B6;129-Shh
tm2Amc
/J; JAX 004293),
R26R:tdTomato (B6;129S6-Gt(ROSA)26Sor
tm9(CAG-tdTomato)Hze
/J; JAX 007905), Smo
flox/flox

(Smo
tm2Amc
/J; JAX 004526), C57BL/6J (JAX 000664), Ihh:LacZ (Ihh
tm1.1Bhum
, MGI:5316284),
120

Cagg:CreER (B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J, JAX 004682). Both male and female mice
between 8–12 weeks old were used for experiments. Experimental Shh cKO mice crossed with
Caag:CreER carried either Shh
D/flox
or Shh
flox/flox
alleles. I did not detect any difference in outcome
in these two genotypes. Control mice were PBS treated (in lieu of Tamoxifen), uninduced Cre
positive, Tamoxifen-induced heterozygotes, or Tamoxifen treated Cre negative siblings. See each
figure legend for details. Tamoxifen was injected intraperitoneally as 100 uL of a 20 mg/ml stock
of Tamoxifen (Sigma-Aldrich: T5648-1G, dissolved in corn oil at 60°C for 2 hr).

Injury assays
Rib resections were performed as previously described.
63,110
Briefly, this injury model
includes surgical resection of a 3 mm segment of rib bone with care given to leave adjacent
periosteal and muscle tissues intact. Surgical fractures were performed under identical conditions
except that the rib bone was cut all the way through with surgical scissors in lieu of resecting a 3
65

mm segment. Post-operative pain was managed with one-time subcutaneous buprenorphine SR
(ZooPharm) at a dose of 0.5 uL/gram body weight. Impact femur fractures were performed as
previously described.
62

Histology tissue processing
For paraffin embedded samples: Skeletal tissue along with the adjacent muscle and
connective tissues was fixed with 4% PFA overnight at 4°C, decalcified with 20% ETDA at pH 7.5
for 10–14 days, and then processed for paraffin embedding. A microtome (Shandon Finesse Me+:
77500102) was used to cut paraffin sections 8 microns thick. The sections were mounted on
Superfrost Plus slides (VWR, 48311–703).
For cryo-embedded samples: Skeletal tissue along with the adjacent muscle and
connective tissues was fixed in 4% PFA at room temperature for 30 min and placed in 30%
sucrose overnight at 4°C. The samples were embedded in OCT and flash frozen in an EtOH dry
ice bath. 10 µM thick sections were cut using a Leica CM3050 S cryostat. Tape (cryofilm type
3C(16UF) C-FUF303) was used to preserve the histology of the bone.
98,148
OCT was removed
with a 1xPBS wash before mounting.
Hematoxylin and eosin (H&E), Safranin-O, and Xgal staining was carried out using
standard protocols. Whole mount samples were fixed in 95% EtOH overnight at 4°C and alizarin
red and alcian blue staining was performed as described by Rigueur and Lyons.
149

Regeneration scoring
Regeneration success of each animal was scored using a semi-quantitative method as
either poor, moderate, or good, based on the following rubric that considers histological features
similar to previously published
150
:
Poor Moderate Good
66

10 or 14 dpi Minimal/no cartilage,
and if cartilage does
exist, it is located
near cut ends and
does not fill resection
gap.
Moderate cartilage
production, incomplete
bridging, appears unclear
if injury will fully heal at
extended time points
Substantial and thick
cartilage production
bridging the injury
site.
28 dpi Little bone
production. Injured
bone end remains
very disconnected.
Moderate bone
production. Some
cartilage may still be
present. Cuts end are not
completely bridged, but
appears full thickness
healing is still possible.
Injury site fully
connected with thick
newly generate bone.

"Poor" was judged unlikely to heal, a "Moderate" score indicated uncertainty in healing outcome,
while "Good" was judged likely to exhibit successful regeneration. Samples were de-identified
and each animal was scored by two judges. If an animal was scored differently by the two judges,
it was settled by a third judge.

RNA in situ hybridization
Fluorescent RNA in situ hybridization (RNA-ISH) was performed on 8 µm paraffin sections
as previously described.
78
Complementary DIG or FL labeled RNA probes were generated
following kit instructions (Sigma-Aldrich: 11277073910 and 11685619910) and were detected
with Anti-Digoxigenin-POD (Sigma-Aldrich: 11207733910) and Anti-Fluorescein-POD (Sigma-
Aldrich: 11426346910). Slides with fluorescence were mounted with Vectashield with DAPI
(Vector Laboratories: H1200) and were imaged with a Nikon AZ100 Macroscope and
67
photographed (Nikon Digital sight DS-Fi1). Fluorescent images were edited for contrast and color
levels in Adobe Photoshop CS5.
Probes were made by RNA-transcription from plasmids containing 500-1000bp gene
fragments. The sequences used were:
Gene and ID Nucleotides Sequence ID Citation
Shh (Gene ID: 20423) 300-942 NM 009170.3 Echelard et al.,1993
151

Ihh (Gene ID: 16147) 1110–1684 NM 010544.3 Iwasaki et al.,1997
152

Dhh (Gene ID: 13363) 263-1208 NM 007857.5 Bitgood and McMahon,1995
119

Prrx1 (Gene ID: 18933) 2486-2987 NM 001025570.1 Parrilla et al., 2016
153

Immunofluorescence
Antibody staining was performed on paraffin sections as previously described.
7
Briefly,
slides were dewaxed for 60 min in Citrisolv and permeabilized with 0.1% Triton-X prior to antigen
retrieval 10 mM sodium citrate, 0.05% Tween 20, pH of 6.0 in a 95°C water bath for 30 min.
Antibody staining (1:200) was performed overnight at 4°C. Antibodies used were anti-CXCL12,
(Abcam: ab25117, used at 1:200); anti-mCHERRY, (Novus Biological: NBP2-25158SS, used at
1:200) and Alexa Fluor 568 goat anti-chicken (Abcam: ab175477, used at 1:500), Alexa Fluor 568
goat anti-rabbit (ThermoFisher: A-11011, used at 1:500).
scRNAseq preparation and analysis
Repair calluses were dissected from control (Sox9:CreER
;
Smo
flox/+
) and
Sox9:CreER;Smo
flox/flox
mice at 4 dpi. Care was taken to excise repair callus leaving the majority
of adjacent skeletal muscle behind. Calluses were digested at 37°C for 60 minutes on a shaker
at 50 rpm in digest buffer (3 mg/mL Collagenase II (Worthington: LS004176), 1 U/mL Dispase
(Corning: #354235), in a-MEM (STEMCELL Technologies: #36453)). Cells were then filtered
68
through 40 micron filter tips (Sigma: BAH136800040), spun at 300g for 5 min, and resuspended
for dead cell removal using the Dead Cell Removal Kit (Miltenyi Biotec: #130-090-101). Cells were
then resuspended in 1xPBS for loading into the 10x Chromium platform. Six calluses were
dissected, digested, and pooled by genotype (3 animals for each genotype) for generation of
single-cell libraries. Libraries were sequenced on an Illumina NextSeq machine.
Reads were aligned to mm10 genome using 10x Genomics’ Cell Ranger pipeline and
default parameters. Cell Ranger outputs were used for downstream analysis in Seurat. QC,
clustering, and analysis was performed using the code shown in supplemental methods. Cells
with less than 200 or more than 2000 unique genes were excluded from analysis as they may
represent poor quality cells or doublets. Cells with more than 10% of total reads mapping to
mitochondrial genes (defined as any gene beginning with “Mt-“) were also excluded from
downstream analysis. Clustering was done using Louvain method at resolution of 0.4. We used
UMAP dimensional reduction to visualize clusters and Wilcoxon Ran Sum test to identify
differentially expressed genes.
Acknowledgements
I would like to thank Divya Patel, Shifa Hossein, Abigail Leyva, and Sophia Bougioukli
for their technical assistance. I would additionally like to thank Gage Crump, D’Juan Farmer, and
Claire Arata for their thoughtful feedback on the manuscript. I also thank Andy McMahon and
Haruhiku Akiyama for sharing Ihh:LacZ and Sox9:CreER mouse lines. This work was supported
with funding from the Roy E. Thomas Graduate Fellowship (MAS), the National Institutes of Health
(T32 HD060549 to MAS, STK and SL, R01 AR069700 to FVM, R01 AR057076 to JRL), and a
University of Southern California Regenerative Medicine Initiative Award (FVM, JRL).
69

FIGURE LEGENDS
Figure 1. Shh, but not Dhh or Ihh is upregulated during early callus formation.

(A) Overview of resection procedure. Briefly, a 3 mm segmental resection of rib bone is performed
leaving adjacent periosteal and muscle tissues intact. Whole-mount samples are stained with
alizarin red and alcian blue as indicators of mineralized bone and cartilage respectively. 0 days
post injury (dpi) shows the empty resection gap. By 7 dpi, alcian-blue stained cartilage bridges
the gap. At 14 dpi, the cartilage callus has converted to alizarin red stained bone with some alcian
blue cartilage remaining adjacent to cut sites. By 28 dpi, the callus has substantially remodeled
back to the original anatomy.

(B) Experimental overview. Rib resections of C57BL/6J control mice were performed at day 0 and
tissues were harvested at 3 or 5 dpi for analysis in C.

(C) Hematoxylin and eosin (H&E) staining of uninjured, 3 dpi, and 5 dpi repair calluses shows
mesenchymal cells filling the resection gap prior to chondrogenesis which typically begins at 7
dpi. sm = skeletal muscle, cb = cortical bone, bm = bone marrow. Surgical cut sites are indicated
with yellow dotted lines. Scale bars: Uninjured = 100 microns, 3 dpi and 5 dpi = 200 microns.

(D) RNA-ISH for Dhh, Ihh, and Shh on histological sections harvested from an uninjured animal
or at 3 dpi and 5 dpi. In uninjured animals, Dhh and Ihh are detectable at low levels in the bone
marrow and in a few cells at 3 and 5 dpi. Shh is detectable at low levels in uninjured animals in
the bone marrow but not in mature osteocytes, periosteal tissue, or adjacent skeletal muscle. At
3 dpi, Shh is strongly detected within cells located in a central region of the resection gap and
modestly upregulated in the surrounding periosteal and skeletal muscle tissues. At 5 dpi the
central domain of Shh expression has expanded and intensified. sm = skeletal muscle, cb =
70
cortical bone, bm = bone marrow. N ≥ 2. Scale bars: Uninjured = 100 microns, 3 dpi and 5 dpi =
200 microns.
Figure 1 - Supplement 1. Ihh is upregulated later in mature chondrocytes during callus
differentiation.
(A) RNA-ISH for Dhh showing the expected expression pattern in murine testis tissue.
(B) RNA-ISH l for Ihh showing the expected expression in rib growth plate chondrocytes.
(C) Ihh;LacZ mice have a LacZ reporter cassette knocked into the first exon of the endogenous
Ihh gene, allowing LacZ expression to be used as a readout of Ihh expression. Rib resections
were performed at day 0 and tissues were harvested at 3 or 7 dpi. Uninjured rib growth plates
were also harvested as a positive control for the assay.
(D) As expected, LacZ expression is detectable with Xgal staining in mature chondrocytes of the
growth plate (black arrowheads). In injured samples, LacZ expression is not detectable at 3 dpi.
At 7 dpi, LacZ expression can be detected in many large, mature chondrocytes in regions of the
callus that have begun differentiating (black arrowheads). Surgical cut sites are indicated with
yellow dotted lines. N = 2. Scale bars = 100 microns. *Images courtesy of Stephanie Kuwahara.
Figure 2. Shh is required for callus generation and subsequent large-scale rib
regeneration.
(A) Experimental overview. To knock out Shh globally, Cagg:CreER;Shh
flox/+
controls and
Cagg:CreER;Shh
flox/flox
or Cagg:CreER;Shh
D/flox
experimental animals (Shh cKO) received
71
Tamoxifen injections on days -5, -4, -3, -2, and -1. Resections were performed on day 0 and tissue
was harvested at 10 or 28 dpi to assess regeneration outcomes.
(B) Safranin-O stained histological sections at 10 and 28 dpi. At 10 dpi, controls generate calluses
mainly composed of cells with chondrocyte morphology, whereas a large callus does not form in
Shh cKO mice. Only small amounts of Safranin-O stained cartilage can be seen adjacent to the
cut sites. At 28 dpi, controls have successfully converted the large cartilage callus to bone that
fully spans the defect site. Global Shh cKO

mice are similarly able to convert cartilaginous regions
adjacent to the cut sites to bone, however the central portion of the callus remains undifferentiated
and the injury site remains unbridged resulting in a non-union. Scale bar = 200 microns
(C) Blinded scoring of regeneration outcomes. Safranin-O stained sections were used to score
regeneration outcomes as Good, Moderate, or Poor (see Methods for details regarding scoring
criteria). Overall, controls largely heal well (6/8 animals), whereas Shh cKO animals largely heal
poorly (10/12 animals).
Figure 3. The primary source of SHH is not Sox9-derived SSPCs.
(A) Experimental overview. To lineage-trace Sox9+ lineage cells, Sox9:CreER;R26:tdTomato
animals received Tamoxifen injections on days -7, -6, and -5. All rib resections were performed
on day 0 and tissue was harvested at 7 dpi to assay Shh expression in B.
(B) RNA-ISH for Shh (green) on histological sections at 5 dpi. Sox9+ lineage cells express
tdTomato (tdT, red). Shh expression is strongly detected within cells located in the central
resection gap and in the surrounding periosteal tissues. Although a few tdT+ lineage-traced cells
72

express Shh (white arrowheads), the majority of Shh-expressing cells are tdT-negative. Surgical
cut sites are indicated with yellow dotted lines. N = 3. Scale bar = 200 microns

(C) Experimental overview. To knock out Shh in Sox9+ lineage cells, Sox9:CreER;Shh
flox/flox

animals received Tamoxifen injections on days -7, -6, and -5. All rib resections were performed
on day 0 and tissue was harvested at 10 or 14 dpi to assay regeneration outcomes.

(D) Blinded scoring of regeneration outcomes. Safranin-O stained sections were used to score
regeneration outcomes as Good, Moderate, or Poor (see Methods for details regarding scoring
criteria). Control animals largely healed well and scored as Good (4/9 animals) or Moderate (3/9
animals). Similarly, Sox9:CreER;Shh
flox/flox
animals largely healed well (3/8 animals) or Moderate
(2/8).

(E) At 10 dpi, Sox9:CreER;Shh
flox/+
controls and Sox9:CreER;Shh
flox/flox
mice both generate a large
Safranin-O stained callus composed largely of cells with chondrocyte morphology and with the
central-most portion beginning osteogenic differentiation. At 14 dpi, Sox9:CreER;Shh
flox/+
controls
and Sox9:CreER;Shh
flox/flox
mice have large Safranin-O-stained calluses with an expanded central
region undergoing bone conversion. Surgical cut sites are indicated with yellow dotted lines. Scale
bar = 200 microns. *Images courtesy of Shuwan Liu.

Figure 4. Shh is expressed by Prrx1-expressing cells and is not dependent upon the
presence of Sox9+ lineage cells.

(A) RNA-ISH for Prrx1 (green) at 5 dpi shows a wide distribution of cells throughout the callus.
Double RNA-ISH at higher magnification shows that Shh positive cells (red) co-express Prrx1
73

(yellow arrowheads). Some Prrx1-expressing cells are Shh negative and vice versa (white or
magenta arrowheads respectively). Surgical cut sites are indicated with yellow dotted lines. N =
2. Scale bars = 200 microns.

(B) Experimental overview of Sox9+ cell ablation regimen. All animals received Tamoxifen
injections on days -7, -6, and -5. Controls included Sox9:CreER;R26:DTR

animals that received
PBS injections on days -3, -2, and -1 and Cre negative R26:DTR animals that received DT
injections on days -3, -2, and -1. Sox9:CreER;R26:DTR experimental animals received DT
treatment on days -3, -2, and -1. All rib resections were performed on day 0 and rib tissue was
harvested at 10 dpi to assess healing progress in C.

(C) At 10 dpi, Sox9:CreER;R26:DTR control animals treated with PBS produce a large Safranin-
O stained cartilage callus. However, Sox9:CreER;R26:DTR animals treated with DT fail to build
a bridging callus. Scale bar = 200 microns.

(D) Blinded scoring of regeneration outcomes. Safranin-O stained sections were used to score
each animal’s regeneration outcome as Good, Moderate, or Poor (see Methods for details).
Control animals (early and late Tamoxifen) largely healed well and scored as Good (4/6 animals)
or Moderate (2/6 animals) whereas Sox9-ablation animals largely healed poorly (4/5 animals).

(E) To determine if the ablation of Sox9+ cells impacts the expression of Shh, RNA-ISH was
performed for Shh expression at 5 dpi. Shh was still strongly detected within cells located in the
central region of the resection and in the surrounding periosteal and skeletal muscle tissues.
Surgical cut sites are indicated with yellow dotted lines. N = 3. Scale bar = 200 microns.

74

Figure 5. Shh is not required for large-scale rib regeneration after 5 dpi.

(A) Experimental overview. To knock out Shh globally, Cagg:CreER;Shh
flox/+
controls and
Cagg:CreER;Shh
flox/flox
or Cagg:CreER;Shh
D/flox
experimental animals received Tamoxifen
injections on days 5, 6, 7, 8, and 9 post injury. All rib resections were performed on day 0 and rib
tissue was harvested at 10 or 28 dpi to assay regeneration outcomes.

(B) Controls and experimental

mice generate a large Safranin-O stained callus at 10 dpi. By 28
dpi, both genotypes have both successfully converted the cartilage callus to bone that spans the
defect site. Scale bar = 200 microns

(C) Blinded scoring of regeneration outcomes. Safranin-O stained sections were used to score
each animal’s regeneration outcome as Good, Moderate, or Poor (see Methods for details).
Control animals (Tamoxifen early and late) largely healed well and scored as Good (6/8 animals)
or Moderate (2/8 animals). Early Shh cKO animals healed poorly (10/12 animals). Late Shh cKO
animals mostly healed well and scored as Good (6/12 animals) or Moderate (3/12).

Figure 6. Smo is dispensable in callus cells after 4 dpi in a large-scale injury model.

(A) To broadly knock out Smo in callus cells at the time of injury ("Early Smo cKO"),
Sox9:CreER;R26:tdTomato;Smo
flox/flox
animals received Tamoxifen injections on -1, 1, and 2 dpi.
All rib resections were performed on day 0 and tissue was harvested at 14 dpi to assay
regeneration outcomes.

(B) To broadly knock out Smo in callus cells at 4 dpi ("Late Smo cKO"),
Sox9:CreER;R26
tdT
;Smo
flox/flox
animals received Tamoxifen injections at 4, 5, and 6 dpi. All rib
75

resections were performed on day 0 and tissue was harvested at 14 dpi to assay regeneration
outcomes.

(C) tdTomato (tdT) reporter expression shows that Sox9:CreER

targets the majority of the callus
when Tamoxifen is administered according to the scheme outlined in A and B. Scale bar = 200
microns.

(D) At 14 dpi, H&E staining reveals that Early Smo cKO mice generate some bone and cartilage
adjacent to the cut ends, but they largely fail to differentiate in the central region of the callus. Late
Smo cKO mice generate a substantial callus that is largely composed of bone. N= at least 3 for
each regimen. Surgical cut sites are indicated with yellow dotted lines. Scale bar = 200 microns.

Figure 7. Shh is not required for small-scale repair.

(A) Overview of surgical fracture procedure. Briefly, this injury model includes creating a surgical
fracture of the rib bone. At 7 dpi bridging of the injury site can be observed with an alcian blue
stained callus.

(B) Surgical fractures were performed at day 0 and tissues were harvested at 3 or 5 dpi for
analysis in C.

(C) RNA-ISH for Shh on histological sections of tissue harvested at 3 dpi or 5 dpi shows that Shh
is largely undetectable at both 3 and 5 dpi. Scale bar = 200 microns. N = 2

(D) To knock out Shh globally, Cagg:CreER;Shh
flox/+
controls and Cagg:CreER;Shh
flox/flox
or
Cagg:CreER;Shh
D/flox
experimental animals received Tamoxifen injections on days -5, -4, -3, -2,
76
and -1. All surgical fractures were performed on day 0 and tissue was harvested at 10 or 28 dpi
to assay regeneration outcomes.
(E) At 10 dpi, controls and Shh cKO experimental animals generate a Safranin-O stained callus
with cartilage morphology. Yellow arrowheads indicate areas where cartilage is forming while
white arrowheads indicate forming bone. At 28 dpi, both controls and Shh cKO animals have
successfully converted the cartilage callus to bone that fully spans the injury site. Scale bar = 200
microns.
(F) Blinded scoring of regeneration outcomes. Safranin-O stained sections were used to score
each animal’s regeneration outcome as Good, Moderate, or Poor (see Methods for details).
Control animals largely healed well and scored as Good (6/9 animals) or Moderate (3/9 animals).
Shh cKO animals also healed well and all scored as good (7/7 animals).
Figure 8. Smo is not required in Sox9+ lineage cells for femur fracture repair.
(A) To knock out Smo in Sox9+ lineage cells, animals received Tamoxifen injections on days -7,
-6, and -5. All femur fractures were performed on day 0 and tissue was harvested at 10 or 28 dpi
to assay regeneration outcomes.
(B) Safranin-O stained histological sections at 10 dpi show that both Sox9:CreER;Smo
flox/+
controls and Sox9:CreER;Smo
flox/flox
Smo cKO mice generate a Safranin-O stained cartilage
callus. N=4. Scale bar = 1 mm. *Images courtesy of Stephanie Kuwahara.
77

(C) H&E stained histological sections at 28 dpi. Both Sox9:CreER;Smo
flox/+
controls and
Sox9:CreER;Smo
flox/flox
Smo cKO mice exhibit an injury sited bridged with bone. N=4. *Images
courtesy of Stephanie Kuwahara.

(D) Blinded scoring of regeneration outcomes. Safranin-O (10 dpi) and H&E (28 dpi) stained
sections were used to score each animal’s regeneration outcome as Good, Moderate, or Poor
(see Methods for details). Control (Sox9:CreER;Smo
flox/+
) animals all healed well and scored as
Good (8/8 animals). Smo cKO (Sox9:CreER;Smo
flox/flox
) animals also healed well and scored as
Good (7/8 animals) or Moderate (1/8 animals).

Figure 9. Activation of Sox9+ lineage cells by Hh signaling is required for Cxcl12
expressing cells to populate the early callus.

(A) Mice used for single cell RNA sequencing (scRNAseq) included Sox9:CreER;Smo
flox/+

(controls) and Sox9:CreER;Smo
flox/flox
(Smo cKO) animals.

(B) To knock out Smo in Sox9+ lineage cells, animals received Tamoxifen injections on days -7,
-6, and -5. Rib resections were performed at day 0 and callus tissue was harvested for scRNAseq
at 4 dpi.

(C) UMAP visualization identified 17 transcriptional cell states broadly representing the major cell
types expected, including connective, endothelial, hematopoietic, and muscle cells.

(D, E) UMAP visualization reveals a shift in connective tissue cluster 0 cells between control and
Smo cKO animals.

78
(F) Feature plot identifies Cxcl12 as marker of cluster 0 that is depleted in Smo cKO compared to
control animals.
(G) Violin plot of control vs Smo cKO animals reveals decreased expression of Cxcl12 in cluster
0 of Smo cKO animals.
(H) IF for CXCL12 in control vs Smo cKO animals reveals a reduction of cells expressing CXCL12
in Smo cKO animals and their spatial restriction to the region adjacent to the cut ends (white
dotted lines). N= 2 for each genotype. Scale bar = 200 microns.
Figure 9 - Supplement 1. Characterization of scRNAseq clusters
(A) Heat map showing the expression the top 5 marker genes for all 17 clusters identified from
Figure 9C.
(B) Feature plot showing the expression of a top marker gene for each of 17 clusters identified in
Figure 9C.
(C) Feature plot showing the expression of 9 marker genes used to identify the large cluster as
“connective tissue” for further analysis in Figure 9E.
Figure 10 – Summary diagram
In response to large-scale injury in the rib, Shh is rapidly and broadly upregulated in Prrx1-
expressing mesenchymal cells in the central injury region at 3-5 days post injury (dpi), whereas
79
Shh expression is not detectable in response to small-scale injury. In animals where Shh is
conditionally removed (Shh cKO), large-scale injuries do not generate cartilage in the central
callus area and subsequently do not heal successfully. In contrast, the repair of small-scale injures
(both in rib and femur) does not require Hh signaling. Thus, bone repair is a process that can
involve region-specific signaling requirements depending on the injury type and Shh is selectively
required to build a centrally located bridging callus during large-scale repair.
Reagent Type Designation Source/Reference Identifiers Additional
Information
Genetic
Reagent
(M. musculus)
C57BL/6J JAX 000664 MGI:3028467 Bl6
Genetic
Reagent
(M. musculus)
Sox9
tm1(cre/ERT2)Haak
Soeda et al.,
2010
147

MGI:
4867441
Sox9:CreER
H Akiyama
Genetic
Reagent
(M. musculus)
Smo
tm2Amc
/J JAX 004526 MGI:2176256 Smo
flox/flox

Genetic
Reagent
(M. musculus)
Ihh
tm1.1Bhum
Fabian et
al.,2012
120

MGI:5316284 Ihh:LacZ
AP McMahon
Genetic
Reagent
(M. musculus)
B6.Cg-Tg(CAG-
cre/Esr1*)5Amc/J
JAX 004682 Cagg:CreER
80
Genetic
Reagent
(M. musculus)
Gt(ROSA)26Sor
tm9(CA
G-tdTomato)Hze
JAX 007909 MGI:3809523 Ai9
Genetic
Reagent
(M. musculus)
C57BL/6-
Gt(ROSA)26Sor
tm1(HB
EGF)Awai
/J
Jax 007900 R26:DTR
Genetic
Reagent
(M. musculus)
B6;129-Shh
tm2Amc
/J JAX 004293 MGI:1934268 Shh
flox/flox
AP McMahon
Antibody Chicken polyclonal
anti-mCherry
Novus Biological NBP2-25158SS 1:200
Antibody Rabbit polyclonal
Anti-Sdf1(Cxcl12)
Abcam ab25117 1:200
Antibody Alexa Fluor 568
goat anti-rabbit
ThermoFisher A-11011 1:500
Antibody Alexa Fluor 568
goat anti-chicken
Abcam ab175477 1:500
81
Dhh
Ihh
Uninjured
Shh
3 mm
periosteum
periosteum
surgical procedure
A
3 dpi
5 dpi
0 dpi
7 dpi
14 dpi
28 dpi
B
C
Harvest @
3 or 5 dpi
0
RR
53
rib
cb
cb
bm
sm
sm
cb
cb
bm
sm
sm
H&E
D
Figure 1
82
3 dpi 7 dpi Growth plate
C
D
Harvest @
3 or 7 dpi
0
RR
7 3
Testis Rib growth plate
Dhh Ihh
A B
Ihh;LacZ
Figure 1 - Supplement 1
83
28 dpi
Control
A
Harvest @
10 or 28 dpi
-5 -4 -3 -2 -1 0
Tamoxifen RR
B 10 dpi
Shh cKO
Figure 2
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Co n tr o l Ea rly K O La te K O
Ba d Mo d e ra te Go o d
n12 n8
Regeneration Score
Control Early cKO
20%
40%
80%
60%
100%
0%
Poor
Moderate
Good
C
Control: CaggCreER Shh
o/

Shh cKO: CaggCreERShh
o/o OR D/o
84
7 dpi
Sox9:CreER;
R26:tdTomato
A B
14 dpi 10 dpi
Sox9:CreER
Shh
flox/flox
C D
E
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Co n tr o l KO
Reg en era t i o n S co re
Go o d OK Po o r
n=8 n=9
Shh/tdTomato
Harvest @
7 dpi
-7 -6 -5 0
Tamoxifen RR
Harvest @
10 or 14 dpi
-7 -6 -5 0
Tamoxifen RR
Sox9:CreER
Shh
flox/+
Sox9:CreER;Shh
flox/flox
Figure 3
Sox9:CreER; R26:tdTomato
Regeneration Score
Control Shh cKO
20%
40%
80%
60%
100%
0%
Poor
Moderate
Good
85
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Co n tr o l Ea rly D T
Reg en era t i o n S co ri n g
Go o d OK Ba d
Harvest
10 dpi
-7 -6 -5 -2 -1 0
Tamoxifen RR
B
-3 -4
PBS or DT
DT PBS 10 dpi
C
D
5 dpi
E
Shh
5 dpi
A
Figure 4
Prrx1
Prrx1
merge
Shh
Shh
Sox9CreER
R26:DTR
Sox9CreER
R26:DTR
n5 n6
Regeneration Score
Control DT
20%
40%
80%
60%
100%
0%
Poor
Moderate
Good
Sox9CreER R26:DTR
86
28 dpi 10 dpi
A
Harvest @
10 or 28 dpi
0 5 67 8 9
Tamoxifen RR
B
C
Controls Shh cKO
0%
10%
20%
30%
40%
50%
60%
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80%
90%
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Co n tr o l Ea rly K O La te K O
Ba d Mo d e ra te Go o d
Figure 5
n12 n8
Regeneration Score
Control Early cKO
20%
40%
80%
60%
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0%
Poor
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n12
Late cKO
Control: CaggCreERShh
o/
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o/o OR D/o
87
Sox9CreERR26:Tdtomato;Smo
o/o
A B
Harvest @
14 DP
0 4 5 6
Tamoxifen
RR
Harvest @
14 DP
2
Tamoxifen
RR 1 -1
14 dpi
C
D
tdTomato tdTomato
14 dpi
Early Smo cKO Late Smo cKO
Figure 6
Sox9CreERR26:Tdtomato;Smo
o/o
88
10 dpi 28 dpi
periosteum
periosteum
surgical procedure
A
7 dpi
B
C
Harvest @
3 or 5 dpi
0
SF
53
rib
D
5 dpi 3 dpi
Control Shh cKO
Harvest @
10 or 28 dpi
-5 -4 -3 -2 -1 0
Tamoxifen SF
E
Figure 7
Shh Shh
n9
Regeneration Score
Control
20%
40%
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60%
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0%
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Good
n7
Shh cKO
F Control: CaggCreER Shh
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o/
89
A
Harvest @
10 or 28 dpi
-7 -6 -5 0
Tamoxifen FF
28 dpi 10 dpi
Sox9CreER
Smo
o/o
B
Sox9CreER
Smo
o/
C
Figure 8
n8 n8
Regeneration Score
Control Smo cKO
20%
40%
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0%
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D
90
Control:
Smo cKO:
Sox9CreERSmo
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o/o
A
Harvest 4 dpi
for scRNAseq
-7 -6 -5 0
Tamoxifen RR
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C D
E F G
H
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Cxcl12
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Endothelial
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Epithelial
Hematopoetic
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Figure 9
CCL12 CCL12
Connective Endothelial
Hematopoietic
Muscle
Epithelial
Control
Smo cKO
Expression level
Identity
0
4
2
CTRL
cKO
Control Smo cKO
91
A
B C
Figure 9 - Supplement 1
92
Figure 10
Figure 10. Summary diagram
n response to large-scale inury in the rib Shh is rapidly and broadly upregulated in Prrx1-expressing
mesenchymal cells in the central inury region at 3-5 days post inury (dpi) whereas Shh expression is not
detectable in response to small-scale inury. n animals where Shh is conditionally removed (Shh cKO) large-
scale inuries do not generate cartilage in the central callus area and subseuently do not heal successfully.
n contrast the repair of small-scale inures (both in rib and femur) does not reuire Hh signaling. Thus
bone repair is a process that can involve region-specific signaling reuirements depending on the inury
type and Shh is selectively reuired to build a centrally located bridging callus during large-scale repair.
93
CHAPTER 4: Transplantation of periskeletal tissues to improve bone regeneration
The Mariani lab and others’ work has clearly demonstrated that periskeletal tissues are a
potent source of skeletal stem and progenitor cells (SSPCs) that rebuild skeletal tissue in
response to injury
63,99,110
. Thus, one approach to improving bone healing in patients could be to
transplant healthy, harvested periosteal tissues from one location into the injury site of severe
bone injuries. I set out to enhance this approach using two alternative strategies. First, in lieu of
transplanting periosteal tissue, my colleagues Armando Garcia, Shifa Hossein and I harvested
and transplanted costal cartilage perichondrial tissue into large-scale rib injuries and assessed
the integration and differentiation of transplanted cells. We found that perichondrial tissue has the
capacity to integrate into the repair callus and differentiate into Col2.3GFP-expressing
osteoblasts, although their participation in the repair process was very limited and did not appear
to improve callus formation. Second, given the newly discovered role for Hedgehog signaling
during rib regeneration, my colleague Jason Hsieh compared the behavior of transplanted
wildtype rib periosteum vs Hh-activated periosteal tissue. He found that transplanted Hh-activated
periosteal tissue stimulated a robust chondrogenic response in the adjacent rib bones of recipient
animals, demonstrating that Hh-activated periosteum is a potent source of chondrogenic signals.
I then performed gene expression analysis of Hh-activated periosteum and identified many genes,
including Postn and Clec11a as potential paracrine mediators of this phenomenon. Together,
these results suggest that although transplanted costal perichondrial tissue does not augment rib
regeneration, Hh-activation of transplanted rib periosteal tissue may be a potent booster of bone
regeneration. Going forward it will be important to directly test the ability of Hh-activated periosteal
tissue to rescue bone healing in large-scale and complex bone injuries which are unlikely to heal.
94
RESULTS
Transplanted perichondrial tissue integrates into rib injuries long-term, but is not a major
source of mature skeletal cells
The Mariani lab’s recent work has demonstrated that costal cartilage perichondrial tissue
is a potent source of chondrogenic progenitor cells which rebuild the underlying costal cartilage
in response to injury.
99
I therefore asked if transplanted perichondrial tissue can augment cartilage
callus formation and subsequent healing of rib bone injuries. To test this, I worked in collaboration
with Armando Garcia and Shifa Hossein to harvest rib perichondrial tissue from globally and
constitutively expressing tdTomato animals and transplanted the tissue into the rib resection void
in Col2.3GFP (osteoblast-GFP+) mice. We then euthanized animals at 5, 7, and 28 dpi (days post
injury), harvested rib tissue, and assessed transplant integration by fluorescence imaging of
histological sections.
At 5 dpi, we observed significant tdTomato+ expression in the central resection site,
indicating the presence of the transplanted tissue (Fig 1A). Adjacent to the tdT+ signal we
observed expression of Col2.3GFP, indicating the presence of host-derived osteoblasts to begin
the construction of a reparative callus (Fig 1A). The close physical association of tdT+ cells and
Col2.3GFP+ cells within the callus suggests that transplanted perichondrial tissue may be working
together with host-derived cells to establish an environment conducive for repair. Since 5 dpi is
prior to the onset of chondrogenesis, it is unclear from this data if the transplanted tissue can
differentiate into chondrocytes or osteoblasts to augment the repair process.
At 7 dpi, we still detected tdT+ cells within the resection void indicating that the
transplanted tissue persists at least until 7 dpi (Fig 1B). The tdT+ tissue is still in close association
with Col2.3+ osteoblasts, suggesting that the transplanted tissue may be participating in the repair
process. However, at this time point I can observe the initial stages of differentiation and callus
formation as indicated by the mineralizing regions of the callus seen in H&E stained tissue (Fig
1B) and I noticed that the tdT+ signal is excluded from the regions of mineralizing tissue,
95
suggesting that although the tdT+ tissue is still present and in close association with host-derived
skeletal tissue, the transplanted tdT+ tissue itself is not acting as a source of skeletogenic
progenitor cells for callus construction and mineralization.
At 28 dpi, we still observed tdT+ tissue at the injury site, suggesting long term integration
of transplanted perichondrial tissue to the injury site. Interestingly, all tdT+ tissue is located
adjacent to new bone formation and the reestablished periosteum (Fig 1C) and does not directly
contribute bone regeneration, as all new bone formation is produced by host-derived Col2.3GFP+
osteoblasts, consistent with the results at 5 and 7 dpi. The transplanted tissue remains excluded
from the repair site and appears to be primarily integrated into the adjacent myotendinous junction
and skeletal muscle.
Together, these results suggest that transplanted perichondrial tissue can integrate with
host animals for at least 28 days, however at all time points observed, the transplanted tissue did
not generate many mature skeletal cells and remained an “adjacent observer” to construction of
a reparative callus by host cells.
Transplanted perichondrial tissue can serve as a very minor source of osteoblasts during
rib regeneration
The data above support a model of rib regeneration where host-derived cells generate
most of the reparative skeletal tissue, whereas transplanted perichondrial tissue only integrates
long-term into the skeletal-adjacent tissues and is not a major source of skeletogenic cells. To
further test this model, we evaluated the ability of transplanted perichondrial tissue to generate
Col2.3GFP+ osteoblasts. To do this, I again collaborated with Shifa Hossein and Armando Garcia
to transplant rib perichondrial tissue from Col2.3GFP mice into the rib bone resection sites of Bl6
hosts. We then euthanized animals at 14 and 21 dpi to assay Col2.3GFP expression in
histological sections.
96
At 14 dpi we were able to detect Col2.3GFP expression within the callus, indicating that
the transplanted cells have integrated into the host animal and that the transplanted cells have
initiated an osteogenic program. Although we detect Col2.3GFP expression, analysis of H&E-
stained sections reveals that there was only very little, if any mineralized tissue in the region of
the callus occupied by Col2.3GFP+ cells (Fig 1D). These data suggest that the transplanted
perichondrial tissue may retain osteogenic activity and activate expression of Col2.3GFP,
although their contribution to mineralized callus formation is minimal.
At 21 dpi we a observed a large callus comprised primarily of new woven bone (Fig 1E)
and did detect a few Col2.3GFP+ cells lining the newly generated bone (Fig 1E – yellow arrows),
suggesting full-fledged differentiation of transplanted perichondrial cells into osteoblasts. As
previously observed, however, their contribution to reparative skeletal tissue is sparse, suggesting
they are only a minor contributor to bone callus formation, but these data provide evidence for an
osteogenic capacity of perichondrial cells.
These data support the model above suggesting that transplanted perichondrial tissue can
integrate long-term following transplantation, yet are only very minimal contributors to the cartilage
and bone callus formation. Together, these data suggest that perichondrial tissue is likely not a
useful source of progenitor cells for repair and that perichondrial transplantation is unlikely to be
of therapeutic use.
Gene expression analysis of Hh-activate periosteal tissue
My colleague Jason Hsieh’s recent work showed that Hh-activated periosteal tissue
generated large osteochondral bone nodules when transplanted into costal muscle (Fig 2B),
whereas wildtype transplanted periosteum does not (Fig 2A). In addition, transplanted Hh-
activated periosteal tissue stimulated a robust chondrogenic response in the adjacent rib bones
of recipient animals (Fig 2B – arrowheads), demonstrating that Hh-activated periosteum is a
97
potent source of chondrogenic signals. I therefore set out to characterize gene expression
changes of periosteal tissue in response to Hh activation.
All animals used for this experiment ubiquitously expressed CreER (Cagg:CreER) and the
SmoM2 allele.
154
SmoM2 is a constitutively active form of the Hh co-receptor Smo. In these
animals, administration of Tamoxifen induces global expression of the SmoM2 allele. To identify
dose-dependent gene expression changes, I used 3 treatment groups: control (no Tamoxifen),
Low Tamoxifen, and High Tamoxifen. Animals received tamoxifen doses (or PBS sham control)
for 5 consecutive days. On day 6, periosteal tissues were harvested from ribs, lysed, and RNA
collected for RNA sequencing. I then performed differential gene expression analysis to identify
genes that are differentially expressed between the 3 groups.
Since the SmoM2 allele contains a GFP tag, I first evaluated GFP expression and
observed a dose dependent response to Tamoxifen administration (Fig 2C). These results
validate that my Tamoxifen dosage scheme indeed induced dose-dependent SmoM2 activation.
I then looked at the expression of canonical osteogenic genes across groups and consistently
observed a dose-dependent response of increasing expression of osteogenic genes such as Sp7,
Col1a1, Bglap, Bglap2, and Spp1 (Fig 2 D+E). Together, these results are consistent with the
increased osteogenic activity of Hh-activated periosteal transplants that Jason previously
observed.
Since Hh-activated periosteal transplants also stimulated a robust chondrogenic response
in the rib bones neighboring the transplantation location in the intercostal muscle, I next looked in
my RNA sequencing data for the expression of secreted molecules that could be responsible for
this phenomenon. I noticed the differential and dose-dependent expression profile of several
known skeletogenic molecules, such as Postn and Clec11a (Fig 2F). These results suggest that
the secreted skeletogenic factors Postn and Clec11a are expressed by periosteal cells in a Hh-
dependent manner and are good candidates for explaining the mechanism by which Hh-activated
periosteal transplant can induce chondrogenesis in the adjacent ribs of host animals.
98
Analysis of ScxCreER as a potential marker of perichondrial progenitor cells
One outstanding challenge in tracing the fate and activity of perichondrial progenitor cells
is the lack of tools that are specific to perichondrial progenitor cells. With this is mind, we set out
to evaluate if the expression of Scx (Scleraxis, the master transcriptional regulator of the tendon
differentiation program) can be used as a perichondrial progenitor-specific marker in collaboration
with Ryan Roberts of the Merrill lab. Ryan injected ScxCreER;tdT;Scx-GFP adult animals with
tamoxifen for 3 days, and then sacrificed the animals for analysis. In these animals, administration
of tamoxifen induced a TdTomato lineage trace of all ScxCreER-expressing cells. In addition,
these animals carry a Scx-GFP allele which labels all cells that currently express Scx with GFP.
Therefore, in these animals, all cells currently expressing Scx will be GFP+ and all cells that were
expressing Scx at the time of tamoxifen injection will be TdTomato+.
I found that in rib costal cartilage, almost all tdT+ cells are located in the outer fibrous layer
of the perichondrium, or are located within the adjacent skeletal muscle compartment and exhibit
a clear tenocyte morphology (Fig 3A+B). Since the progenitor cells within the perichondrium are
largely located within the inner chondrogenic layers of perichondrial tissue, I concluded that this
ScxCreER transgene largely lineage traces mature tenocytes and presumed tendon progenitor in
the outer fibrous layer, but not chondrogenic progenitors. The labeled cells are unlikely to
generate chondrocytes during costal cartilage growth and development. It remains possible that
these tendon-primed cells could be convinced to generate chondrocytes in response to injury of
the underlying costal cartilage tissue, but this remains to be tested directly. Intriguingly, Scx-GFP
seems to mark the inner layer of perichondrium more clearly, but since this is not a CreER allele,
it should not be used to trace the fate of these cells and is therefore not of use as a lineage tracing
tool. Further CreER alleles must be evaluated to find a tool more useful for specifically tracing the
fate of perichondrial cells during the costal cartilage regenerative process.
99
DISCUSSION
Since the Mariani lab’s recent work has shown perichondrial and periosteal tissues to be
a rich source of skeletal progenitor cells,
99
it is logical to ask if these tissues could be transplanted
into the sites of bone injuries to augment skeletal regeneration. One major advantage of this
approach is that periskeletal tissues could be harvested from patients and immediately implanted
into the bone injury site during the same procedure. This eliminates the need for cell isolation,
culturing, and expansion, as well as avoiding the need for FDA approval. Unfortunately, the data
above suggest that simply transplanting periskeletal tissues is not a miracle solution for
regenerating skeletal tissues. Although cells from transplanted tissue can integrate into host
animals long term, they very rarely generated chondrocytes or osteoblasts for callus formation. In
addition, repair calluses in these animals did not appear regenerate any better than normal.
It is still unresolved why exactly transplanted perichondrial cells would be reluctant to
produce chondrocytes, whereas they happily generate chondrocytes in response to injury in their
native environment.
99
One possibility is that the injury environment of injured rib bone is very
different than the environment following rib cartilage injury. Indeed, the vascularization, presence
of bone marrow, and nervous tissue input is all markedly different in the context of bone
injury
44,51,128
, and these differences may account for how the signaling environment following bone
injury is more optimized for directing the behavior of native periosteal cells vs. transplanted
perichondrial cells. In addition, it is possible that the process of transplantation may be
handicapping the ability of transplanted cells to contribute to callus formation. For example, the
process of stripping them from their native environment and structure could very well influence
their behavior such that their transplanted environment is not conducive of skeletogenic
differentiation. In theory, some of these limitations can be addressed by augmenting cells during
the transplantation process, but this approach would need significant trouble shooting,
optimization, and FDA approval, and is thus not likely a desirable approach.
100
In lieu of manipulating cells for transplantation, one approach could be to learn from the
activities of manipulated cells and devise a pharmaceutical intervention that would mimic the
activity of transplanted cells. For example, the my colleague Jason Hsieh’s work has shown that
Hh-activation of transplanted periosteal tissue greatly increased the osteogenic capacity of the
periosteal tissue and turns the periosteal tissue into a potent source of chondrogenic signals (Fig
2 A+B). Within that framework, I identified a number of candidate genes that are upregulated in
periosteal cells in response to a dose-dependent Hh-activation scheme. For example, Postn and
Clec11a are secreted molecules with known skeletogenic functions,
55,155
are upregulated in
periosteal cells in response to Hh-activation, and thus may be beneficial as a therapeutic agent.
Going forward it will be important to evaluate the effectiveness of enhancing skeletogenesis with
these molecules. Indeed, my colleagues in the Mariani lab are developing an in vitro model of
chondrogenesis and are testing the ability of Postn and Clec11a to enhance differentiation. If
these efforts prove fruitful it will be exiting to test the ability of these molecules to enhance skeletal
healing in an in vivo animal model of skeletal regeneration, such as rib repair.
Together, these experiments provide evidence that transplantation of unmanipulated rib
periskeletal tissue is unlikely to act as a potent source of skeletal progenitor cells to boost bone
repair. In addition, I have identified several Hh-dependent skeletogenic factors secreted by
periosteal cells that have the potential to boost repair callus formation. Future experiments are
required to further validate the pro-regenerative effects of these candidate molecules.
MATERIAL AND METHODS
Perichondrial Transplantation
Perichondrial tissues were micro-dissected from costal cartilages and placed in PBS on
ice. Within ~30 minutes, perichondrial tissue was transplanted into the resection void following
large-scale rib bone resection in a recipient mouse. Perichondrial tissue was added to the
resection void until the entire void was full of transplanted tissue. Skeletal muscle superficial to
101
the injured bone was sutured together to trap the transplanted tissue in place underneath the
muscle. Animals were then euthanized at the appropriate time point, and rib tissue was harvested
for cryosectioning and analysis.
Periosteal Transplantation
Periosteal tissues were micro-dissected from costal rib bones and then placed in PBS on
ice. Periosteal tissues were then transplanted into the intercostal muscle of a recipient mouse.
The periosteal transplants were sutured in place by suturing the transplant to the adjacent skeletal
muscle tissue. Recipient mice were then euthanized at 7 days following transplantation and tissue
was harvested for cryosectioning and histological analysis.
Harvesting RNA for gene expression analysis of periosteal transplants
Cell/ Tissue Lysis and Homogenization:
• Extract tissue from animal and flash freeze in liquid nitrogen.
• Use pestle and mortar to grind down frozen tissue section into a fine powder.
Do not let tissue section thaw during grinding process.
• Once tissue section has been broken down, add up to 0.6 mL of Lysis Buffer
(depending on amount of tissue) with 2-Mercaptoethanol to the tissue
sample.
• Vortex until the sample appears to be dispersed and tissue appears lysed.
• Pass the lysate 5-10 times through an 18 to 21-gauge syringe needle.
Cells/Tissue should appear dispersed with little to no visible sample present.
RNA Purification ->
1. Once lysate has been homogenized, add one volume of 70% ethanol to each
volume of cell homogenate.
102
2. Vortex to mix thoroughly and disperse any visible precipitate that may form
after adding ethanol.
3. Transfer up to 700uL of sample to the spin cartridge (with collection tube).
4. Centrifuge at 12,000 x g (~10,000 RPM) for 15-30 seconds at room
temperature
5. Repeat steps 3-4 until the entire samples has been processed.
6. Add 700uL of Wash Buffer I to the Spin Cartridge. Centrifuge at 12,000 x g
(~10,000 RPM) for 15 seconds at room temperature. Discard Flow through
and place spin cartridge in a new Collection tube.
7. Add 500uL of Wash Buffer II with ethanol to the spin cartridge. Centrifuge at
12,000 x g (~10,000 RPM) for 15 seconds at room temperature. Discard flow-
through.
8. Repeat Step 8 once.
9. Centrifuge at 12,000 x g (~10,000 RPM) for 1 minute 30 seconds to dry the
membrane with bound RNA. Discard collection tube and insert the spin
cartridge into a Recovery Tube.
10. Add 30-100uL RNase-free water to the center of the spin cartridge. Incubate
at room temperature for 1 minute.
11. Centrifuge at >12,000 x g (>12,000 RPM) for 2 minutes at room temperature.
If expected RNA yield is >100ug total RNA, perform 3 sequential elution’s of
100uL each.
12. Store purified RNA long-term at -80°C for downstream application.
13. Send samples to Loma Linda for sequencing if RIN is acceptable.
103
Histology tissue processing
For cryo-embedded samples: Skeletal tissue along with the adjacent muscle and
connective tissues was fixed in 4% PFA at room temperature for 30 min and placed in 30%
sucrose overnight at 4°C. The samples were embedded in OCT and flash frozen in an EtOH dry
ice bath. 10 µM thick sections were cut using a Leica CM3050 S cryostat. Tape (cryofilm type
3C(16UF) C-FUF303) was used to preserve the histology of the bone.
58,59
OCT was removed with
a 1xPBS wash before mounting.
Hematoxylin and eosin (H&E) and Safranin-O staining were carried out using standard
protocols.
FIGURE LEGENDS
Figure 1. Transplanted perichondrial tissue can serve as a very minor source of
osteoblasts during rib regeneration
(A) Fluorescence and H&E imaging of rib repair callus at 5 dpi. tdT+ perichondrium transplant into
Col2.3GFP. Transplanted perichondrial tissue (red) can be found in the central resection site,
indicating the presence of the transplanted tissue. Adjacent to the tdT+ signal we observed
expression of Col2.3GFP+, indicating the presence of host-derived osteoblasts to begin the
construction of a reparative callus. Cut bone ends indicated with dotted yellow lines.
(B) Fluorescence and H&E imaging of rib repair callus at 7 dpi. tdT+ perichondrium transplant into
Col2.3GFP. Detectable tdT+ cells within the resection void indicating that the transplanted tissue
persists at least until 7 dpi. The tdT+ tissue is still in close association with Col2.3+ osteoblasts,
suggesting that the transplanted tissue may be participating in the repair process. Initial stages of
differentiation and callus formation evident by the mineralizing regions of the callus seen in H&E
stained tissue. tdT+ signal is excluded from the regions of mineralizing tissue, suggesting that
104
although the tdT+ tissue is still present and in close association with host-derived skeletal tissue,
the transplanted tdT+ tissue itself is not acting as a source of skeletogenic progenitor cells for
callus construction and mineralization. Cut bone ends indicated with dotted yellow lines.
(C) Fluorescence and H&E imaging of rib repair callus at 28 dpi. tdT+ perichondrium transplant
into Col2.3GFP. Detectable tdT+ tissue at the injury site, suggesting long term integration of
transplanted perichondrial tissue to the injury site. tdT+ tissue is located adjacent to new bone
formation and the reestablished periosteum and does not directly contribute bone regeneration,
as all new bone formation is produced by host-derived Col2.3GFP+ osteoblasts, consistent with
the results at 5 and 7 dpi. The transplanted tissue remains excluded from the repair site and
appears to be primarily integrated into the adjacent myotendinous junction and skeletal muscle.
Cut bone ends indicated with dotted yellow lines.
(D) Fluorescence and H&E imaging of rib repair callus at 14 dpi. Col2.3GFP perichondrium
transplanted into Bl6. Detectable Col2.3GFP expression within the callus, indicating that the
transplanted cells have integrated into the host animal and that the transplanted cells have
initiated an osteogenic program. Analysis of H&E-stained sections reveals very little, if any
mineralized tissue in the region of the callus occupied by Col2.3GFP+ cells suggesting the
transplanted perichondrial tissue may activate expression of Col2.3GFP, although their
contribution to mineralized callus formation is minimal. Cut bone ends indicated with dotted yellow
lines.
(E) Fluorescence and H&E imaging of rib repair callus at 21 dpi. Col2.3GFP perichondrium
transplanted into Bl6. Large callus comprised primarily of new woven bone and detectable
Col2.3GFP+ cells lining the newly generated bone (yellow arrows), suggesting full-fledged
differentiation of transplanted perichondrial cells into osteoblasts. Contribution to reparative
105
skeletal tissue is sparse, suggesting they are only a minor contributor to bone callus formation.
Cut bone ends indicated with dotted yellow lines.
Figure 2. Gene expression analysis of Hh-activate periosteal tissue
(A) Safranin-O stained histology of WT periosteal transplant into intercostal muscle 7 days
following transplantation. Transplanted tissue (outlined with black dotted lines, identified by
fluorescent analysis of adjacent section, transplanted tissue is tdT+) fails to generate large repair
callus and only small amounts of mineralization are detected.
(B) Safranin-O stained histology of SmoM2-activated periosteal transplant into intercostal muscle
7 days following transplantation. Transplanted tissue (outlined with black dotted lines, identified
by fluorescent analysis of adjacent section, transplanted tissue is YFP+) generated large
osteochondral skeletal nodule. In addition, adjacent rib bones of transplantation site exhibit robust
chondrogenic response (black arrows). Fluorescent of adjacent sections confirms that
chondrogenic response is host derived. Images in A and B are courtesy of Jason Hsieh.
(C) RNA sequencing analysis of harvested periosteal tubes. Administration of Tamoxifen induces
global expression of the SmoM2 allele. To identify dose-dependent gene expression changes, we
used 3 treatment groups: control (no Tamoxifen), Low Tamoxifen, and High Tamoxifen. SmoM2
allele contains a GFP tag – therefore we observed a dependent response to Tamoxifen
administration (Fig 2C). These results validate that our Tamoxifen dosage scheme indeed
induced dose-dependent SmoM2 activation.
(D+E) RNA sequencing analysis of harvested periosteal tubes. Data reveal a dose-dependent
response of increasing expression of key osteogenic genes such as Sp7, Col1a1, Bglap, Bglap2,
106
and Spp1 (Fig 2 D+E). Together, these results are consistent with the increased osteogenic
activity of Hh-activated periosteal transplants observed in A + B.
(F) RNA sequencing analysis of harvested periosteal tubes. Dose-dependent expression profile
of several known secreted skeletogenic molecules, such as Postn and Clec11a. These results
suggest that the secreted skeletogenic factors Postn and Clec11a are expressed by periosteal
cells in a Hh-dependent manner and are good candidates for explaining the mechanism by which
Hh-activated periosteal transplant can induce chondrogenesis in the adjacent ribs of host animals.
Figure 3. Evaluation of ScxCreER;tdT;Scx-GFP animals for tracing the activity of
perichondrial progenitor cells in costal cartilage
(A) Fluorescence imaging of costal cartilage section from ScxCreER;tdT;Scx-GFP. In rib costal
cartilage, almost all tdT+ cells are located in the outer fibrous layer of the perichondrium, or are
located within the adjacent skeletal muscle compartment and exhibit a clear tenocyte morphology.
Scx-GFP allele broadly labels perichondrial tissue.
(B) Fluorescence imaging of costal joint section from ScxCreER;tdT;Scx-GFP. In rib costal joint,
almost all tdT+ cells are located in the outer fibrous layer of the perichondrium, or are located
within the adjacent skeletal muscle compartment and exhibit a clear tenocyte morphology. Scx-
GFP allele broadly labels perichondrial tissue around the costal joint.
Animals received 3 tamoxifen injections on consecutive days and then were euthanized for
cryosectioning and analysis.
107
A
5 dpi 7 dpi
B
tdTomato/Col2.3GFP tdTomato/Col2.3GFP
H&E H&E
tdTomato/Col2.3GFP
C
H&E
28 dpi
D
21 dpi
Col2.3GFP Col2.3GFP
H&E H&E
14 dpi
E
Figure 1
108
A B
C D
E
F
Control Low
Tam
High
Tam
Control Low
Tam
High
Tam
Control Low
Tam
High
Tam
Control Low
Tam
High
Tam
Control Low
Tam
High
Tam
Control Low
Tam
High
Tam
Control Low
Tam
High
Tam
Control Low
Tam
High
Tam
Figure 2
GFP Sp7
Col1a1
Bglap Bglap2
Postn
Clec11a
109
Figure 3
A
B
tdTomato/ScxGFP
tdTomato/ScxGFP
110
CHAPTER 5: Concluding remarks
Introduction
The unfortunate clinical reality is that patients with complex bone injuries have limited
treatment options, particularly when the scale of injury is large. One potential approach to these
patients is to transplant skeletogenic cells into the injury site to provide the progenitors required
to rebuild the injured bone. Unfortunately, simply providing skeletogenic cell populations, or even
whole skeletogenic tissue such as periosteum, have had limited success in the clinic. Therefore,
it is imperative that these approaches are improved and augmented. My view is that this is best
accomplished using the following 2 steps: (1) developing a more thorough understanding of the
biology underlying natural skeletal regeneration, and (2), applying the lessons learned from 1 to
develop bio-inspired approaches to better facilitate the proper expansion and differentiation of cell
therapy approaches.
Identifying the cells that maintain and repair the skeleton has been an area of intense
recent investigation. A major challenge will be to understand the similarities and differences
across populations of SSPCs. For example, do different embryonic origins of certain SSPCs
influence and poise them with unique abilities with regards to building, maintaining, and repairing
the skeleton? Emerging technologies should help improve our understanding of the specific
lineage relationships and potential of different pools of SSPCs. Determining SSPC lineage
relationships should help resolve major questions regarding the relative contributions of cells from
the bone marrow, growth plate, and periosteum in the skeletal system. In parallel, identifying
important niche factors will help determine how SSPC populations stay quiescent, become
activated due to injury, and undergo the specific transitions needed to build new skeletal tissues.
Importantly, the field will need to complement studies in model organisms with those in human
tissues, as markers, cell populations, and regenerative abilities may differ between species. This
will allow us to better understand how cell populations and niche factors are altered in
dysmorphology, injury, disease, and aging of the human skeleton. Overall, these investigations
111
will give us a greater understanding of the plasticity of the skeletal system and ultimately will help
bring novel therapeutic approaches to the clinic.
Shh as a critical regulator of bone regeneration
Whereas many efforts seek to understand the signaling pathways that promote the
differentiation of skeletogenic cells once they have arrived in the right place and in the right
quantity, a unique aspect of my research is that I ask a slightly different question: what are the
signaling mediators which recruit skeletogenic cells to the right location in the first place? By
taking a close look at the first few days after injury, I discovered a novel role for the Hedgehog
signaling pathway in coordinating the early recruitment and subsequent regenerative function of
SSPCs derived from multiple sources, including the periosteum and bone marrow. This is
important since investigative approaches often look at either bone marrow or periosteal derived
SSPCs in isolation. To my knowledge, this is the first report describing the molecular drivers
coordinating SSPCs from multiple sources.
Repairing a small vs large injury may not only necessitate distinct bone healing pathways
(direct and/or endochondral) but may also require distinct triggers to initiate these pathways. My
studies provide evidence for Shh as a required trigger to initiate a type of repair that creates a
bridging callus in the context of a large-scale injury. I was surprised to find Shh to be required
instead of Ihh, as SHH has been traditionally considered a ligand important for embryonic skeletal
patterning and discovering a role for SHH in adult skeletal biology was unexpected.
124–126

However, in the context of the mouse rib, Shh but not Ihh or Dhh, is rapidly and broadly
upregulated in the first few days following large-scale injury and genetic deletion of Shh prior to
injury but not afterwards, dramatically impairs regeneration.
To prevent the accidental formation of ectopic cartilage or bone, the regulation of Shh in
response to injury must be tightly controlled. Discovering how Shh expression is regulated in the
mouse will be a fruitful avenue of future investigation. One approach may be to define regulatory
112

elements that govern Shh expression in response to injury and determine if these elements are
differentially regulated in large-scale vs small scale injury. In addition, since Shh is an important
patterning factor during both limb and rib development,
124–127
it is tempting to consider that large-
scale regeneration requires the redeployment of a developmental program involving Shh,
whereas small-scale injuries heal through in independent program of repair that does not mirror
developmental history. Whether or not Shh expression in large-scale regeneration represents a
bona-fide reactivation of a developmental program is still to be determined.
My studies reveal that not only is Shh required to initiate large-scale rib repair but may be
dedicated to building new skeletal tissue within a specific region of the repair callus. My
observation that Shh cKO mice still produce some cartilage adjacent to the cut ends and later
convert that cartilage to bone supports a model where the regions near the cut ends repair via
endochondral ossification employing a Hh-independent mechanism, whereas repair in the central
region of the defect site with a bridging callus is indeed Shh-dependent. The un-differentiated
central portion of the injury site in Shh cKO animals mirrors the domain of Shh expression in
control animals, further supporting a link between Shh expression in the injury environment and
subsequent callus differentiation. Together, these observations support a model of large-scale
bone regeneration where repair close to the cut bone ends is Shh-independent, whereas repair
in the most central region of the injury site is critically dependent on early expression of Shh. Why
there would be region-specific requirements for Shh remains unclear. Perhaps the cells that
occupy different regions of the callus are derived from alternative lineages (i.e., periosteal
enriched at the cut ends vs bone marrow enriched in the central region) and therefore may have
different required inputs for successful differentiation? It is also worth noting that the injury
environment is not uniform and there may be certain environmental conditions (i.e.,
vascularization, biomechanics) that create “zones” of the callus with different signaling
requirements.
128–130

113

The observation that Shh is generally not expressed in the region immediately adjacent to
the cut bone ends has important implications for how SSPC behavior is regulated in response to
injury. The most obvious is that a small-scale injury such as a simple fracture does not contain a
region of repair tissue distant from the injured bone ends and thus may not express Shh at all.
Indeed, I was unable to detect Shh expression within the first 5-day window following surgical
fracture, and global Shh cKO in these animals still resulted in complete healing. One prominent
outstanding mystery is which factors or conditions result in Shh being expressed following large-
scale, but not during small-scale injury. For example, a larger-scale injury may result in more
severe hypoxia,
132–134
increased immune response,
135
or an altered mechanical environment
which in turn induces Shh expression.
136–139
Indeed, biomechanical stability is known to have a
major impact on the lineage progression of SSPCs during healing and rigidly stabilize fractures
can heal via direct ossification without first building a cartilage callus.
80

When combined with my data, these observations point to a Hh ligand as the critical factor
underlying the regeneration of large-scale, but not small-scale injuries. Large- vs small-scale
repair may require different pathways, mechanisms, and regulatory controls. How conserved
these paradigms may be in other bones and across other species is yet to be determined.

A main function of Hh activated Sox9+ cells may be to recruit Cxcl12-expressing cells
Stephanie Kuwahara’s recent work has proposed that Sox9+ lineage SSPCs act as
“messenger” cells, since loss of Smo in this population (around 20% of the callus) also affects the
behavior of Sox9-negative cells in the callus.
63
How cells outside the Sox9+ lineage participate in
repair is still unclear. For example, SSPCs can be found in the growth plate, periosteum, and
bone marrow compartments.
1
One possibility is that periosteal-derived Sox9+ cells, once
activated by Hh signaling, facilitate the formation of a large bridging callus by recruiting other cells
types to the injury site. My scRNAseq data illuminated a population of Cxcl12-expressing cells
that is reduced in number within the repair callus of Sox9:CreER;Smo
flox/flox
animals at 4 dpi.
114

Perhaps the role of Sox9+ cells in large-scale repair is to recruit these Cxcl12-expressing cells?
Interestingly, recent work from the Ono group has shown that Cxcl12-expressing bone marrow
stromal cells can adopt an SSPC-like state in response to bone injury and subsequently contribute
to cartilage and bone callus formation.
114
Linking this observation with my scRNAseq data
suggests that the impaired large-scale healing observed in these models of Hh inhibition, may be
due to the failed recruitment of Cxcl12-expressing bone marrow cells into the callus to participate
in regeneration. The idea that periosteal and BM-derived cells may work in collaboration must be
tested more thoroughly in the future, as it implies crosstalk between periosteal and BM -derived
SSPCs and the appropriate recombinase mouse lines to investigate these relationships are much
needed. Excitingly, the recent emergence of Dre-based genetically modified mice unlocks the
possibility of simultaneously lineage tracing multiple cell populations in parallel.
145,146

Transplantation of skeletogenic tissues
Since the Mariani lab’s recent work has shown perichondrial and periosteal tissues to be
a rich source of skeletal progenitor cells,
99
it is logical to ask if these tissues could be transplanted
into the sites of bone injuries to augment skeletal regeneration. One major advantage of this
approach is that periskeletal tissues could be harvested from patients and immediately implanted
into the bone injury site during the same procedure. This eliminates the need for cell isolation,
culturing, and expansion, as well as avoiding the need for FDA approval. Unfortunately, the data
above in Chapter 4 suggest that simply transplanting periskeletal tissues is not a miracle solution
for regenerating skeletal tissues. Although cells from transplanted tissue can integrate into host
animals long term, they very rarely generated chondrocytes or osteoblasts for callus formation. In
addition, repair calluses in these animals did not appear regenerate any better than normal.
It is still unresolved why exactly transplanted perichondrial cells would be reluctant to
produce chondrocytes, whereas they happily generate chondrocytes in response to injury in their
native environment.
99
One possibility is that the injury environment of injured rib bone is very
115

different than the environment following rib cartilage injury. Indeed, the vascularization, presence
of bone marrow, and nervous tissue input is all markedly different in the context of bone
injury
44,51,128
, and these differences may account for how the signaling environment following bone
injury is more optimized for directing the behavior of native periosteal cells vs. transplanted
perichondrial cells. In addition, it is possible that the process of transplantation may be
handicapping the ability of transplanted cells to contribute to callus formation. For example, the
process of stripping them from their native environment and structure could very well influence
their behavior such that their transplanted environment is not conducive of skeletogenic
differentiation. In theory, some of these limitations can be addressed by augmenting cells during
the transplantation process, but this approach would need significant trouble shooting,
optimization, and FDA approval, and is thus not likely a desirable approach.
In lieu of manipulating cells for transplantation, one approach could be to learn from the
activities of manipulated cells and devise a pharmaceutical intervention that would mimic the
activity of transplanted cells. For example, my colleague Jason Hsieh’s work has shown that Hh-
activation of transplanted periosteal tissue greatly increased the osteogenic capacity of the
periosteal tissue and turns the periosteal tissue into a potent source of chondrogenic signals.
Within that framework, I identified a number of candidate genes that are upregulated in periosteal
cells in response to a dose-dependent Hh-activation scheme. For example, Postn and Clec11a
are secreted molecules with known skeletogenic functions,
55,155
are upregulated in periosteal cells
in response to Hh-activation, and thus may be beneficial as a therapeutic agent. Going forward it
will be important to evaluate the effectiveness of enhancing skeletogenesis with these molecules.
Indeed, my colleagues in the Mariani lab are developing an in vitro model of chondrogenesis and
are testing the ability of Postn and Clec11a to enhance differentiation. If these efforts prove fruitful
it will be exiting to test the ability of these molecules to enhance skeletal healing in an in vivo
animal model of skeletal regeneration, such as rib repair.
116

Together, these observations provide evidence that transplantation of unmanipulated rib
periskeletal tissue is unlikely to act as a potent source of skeletal progenitor cells to boost bone
repair. In addition, I have identified several Hh-dependent skeletogenic factors secreted by
periosteal cells that have the potential to boost repair callus formation. Future experiments are
required to further validate the pro-regenerative effects of these candidate molecules.

Closing
Ultimately, I believe that therapeutic approaches tailored towards coordinating the
activities of multiple pools of SSPCs are more likely to succeed than those directed at an individual
population. It is my hope that the lessons learned from my work will inform future therapeutic
strategies and that bone regeneration will someday become the poster child for safe and effective
cell therapy.

117
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APPENDIX
Maxwell A. Serowoky, Stephanie T. Kuwahara, Shuwan Liu, Venus Vakhshori, Jay R.
Lieberman, and Francesca V. Mariani. A murine model of large-scale bone regeneration reveals
a selective requirement for Sonic Hedgehog. npj Regenerative Medicine. 2022. Under Review.
Stephanie T. Kuwahara, Shuwan Liu, Andrew Chareunsouk, Maxwell Serowoky and Francesca
V. Mariani. On the horizon: Hedgehog signaling to heal broken bones. Bone Research. 2022. In
Press
Roelofs AJ, Kania K, Rafipay AJ, Sambale M, Kuwahara ST, Collins FL, Smeeton J, Serowoky
MA, Rowley L, Wang H, Gronewold R, Kapeni C, Méndez-Ferrer S, Little CB, Bateman JF, Pap
T, Mariani FV, Sherwood J, Crump JG, De Bari C. Identification of the skeletal progenitor cells
forming osteophytes in osteoarthritis. Ann Rheum Dis. 2020 Dec;79(12):1625-1634. PubMed
Central ID: PMC8136618.
Serowoky MA, Arata CE, Crump JG, Mariani FV. Skeletal stem cells: insights into maintaining
and regenerating the skeleton. Development. 2020 Mar 11;147(5) PubMed Central ID:
PMC7075071.
Kuwahara ST, Serowoky MA, Vakhshori V, Tripuraneni N, Hegde NV, Lieberman JR, Crump JG,
Mariani FV. Sox9+ messenger cells orchestrate large-scale skeletal regeneration in the
mammalian rib. Elife. 2019 Apr 15;8 PubMed Central ID: PMC6464605.
135
Serowoky MA, Patel DD, Hsieh JW, Mariani FV. The use of commercially available adhesive
tapes to preserve cartilage and bone tissue integrity during cryosectioning. Biotechniques. 2018
Oct;65(4):191-196. PubMed Central ID: PMC6642614.
136

Abstract (if available)

Abstract Skeletal stem cells (SSCs) generate the progenitors needed for growth, maintenance, and repair of the skeleton. Historically, SSCs have been defined as bone marrow-derived cells with inconsistent characteristics. However, recent in vivo tracking experiments have revealed the presence of SSCs not only within the bone marrow but also within the periosteum and growth plate reserve zone. These studies show that SSCs are highly heterogeneous with regard to lineage potential. During digit tip regeneration and in some non-mammalian vertebrates, the dedifferentiation of osteoblasts may also contribute to skeletal regeneration. Here, I examine how this research has furthered the understanding of the diversity and plasticity of SSCs that mediate skeletal maintenance and repair.

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Asset Metadata

Creator Serowoky, Maxwell (author)

Core Title Discovery of a novel role for Sonic Hedgehog during the early stages of large-scale murine rib regeneration

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Development, Stem Cells and Regenerative Medicine

Degree Conferral Date 2022-05

Publication Date 05/02/2022

Defense Date 05/01/2022

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

Tag hedgehog signaling,OAI-PMH Harvest,skeletal regeneration,stem cells

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Merrill, Amy (committee chair), Crump, Gage (committee member), Mariani, Francesca (committee member)

Creator Email maxwell.serowoky@gmail.com,serowoky@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111160252

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