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The role of Hedgehog signaling in Sox9 expressing progenitors during large scale bone repair

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The role of Hedgehog signaling in Sox9 expressing progenitors during large scale bone repair

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Content i

The role of Hedgehog signaling in Sox9 expressing
progenitors during large scale bone repair

By Stephanie T. Kuwahara

A dissertation presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
Development, Stem Cell, and Regenerative Medicine
Doctor of Philosophy
August 15, 2019

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ACKNOWLEDGEMENTS
Thesis committee members:
Amy E. Merrill, PhD (Chair)
Francesca V. Mariani. PhD (Mentor)
Gage Crump, PhD
Denis Evseenko, MD, PhD
Baruch Frenkel, PhD

My PhD career has been a wonderful experience thanks to my mentor Dr. Francesca
Mariani. She has been everything I wanted in a PhD mentor: supportive, brilliant, and
encouraging. Under her mentorship I was able to grow, learn, and become a better scientist. I
know that I have been very fortunate to have had Francesca as my mentor and role model.
Thanks to her I feel ready to take the next step in my career.
I’d like to thank my thesis committee for continually giving me encouragement,
constructive criticism, and support. Their suggestions kept my research project moving forward
and in the right direction. I’d like to thank the Development, stem cell, and regenerative
medicine department at USC. I’d especially like to thank the director of the program Gage
Crump, who helped guide my project and was always willing to give advice and help with
edits/writing. Also thanks for working with me on the osteophyte project. I’d like to thank the Eli
and Edythe Broad Center at USC as well, being a part of BCC truly made my PhD experience
better.
My labmates have been another system of support for me throughout my PhD. Thanks
so much for putting up with me when experiments did not work. Doing lab work would not have
been nearly as enjoyable without you guys. Thanks for sitting in on practice presentations,
giving me support, bouncing ideas around with me, and for becoming my friend.
I’d also like to thank all my friends and family for supporting me. My family has always
encouraged me to do well in academics and pursue a career I enjoy. My friends have kept me
sane. They were always willing to go out to happy hour or go dancing with me. And Porter,
thanks for being loving, patient, supportive, and making me laugh after a long day in lab.
Without my friends and family, I would not have been able to make it through this journey.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF FIGURES
ABBREVIATIONS
CHAPTER 1: Introduction
CHAPTER 2: Characterization of murine rib regeneration
CHAPTER 3: Role of Hedgehog signaling during large scale rib
regeneration
CHAPTER 4: Role of Hh signaling in the Sox9 progenitor population in
other injury contexts
CHAPTER 5: Conclusions
FUNDING
REFERENCES

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LIST OF FIGURES
Figure 2.1: Schematic of murine rib resection model
Figure 2.2: Regeneration involved skeletal cells with dual cartilage-bone
properties
Figure 2.3: Sox9-CreERT2 marks cells that participate in repair
Supplementary Figure 2.1: Analysis of hybrid skeletal cells
Supplementary Figure 2.2: Colorimetric RNA-ISH
Supplementary Figure 2.3: Removal of periosteum and tamoxifen control
Figure 3.1: Hh signaling during rib repair
Figure 3.2: Requirement of Hh signaling for rib callus formation
Figure 3.3: Hh signaling is required for bone formation
Figure 3.4: Hh signaling is required for full bone regeneration
Figure 3.5: SmoM2 increased size of the cartilage callus
Figure 3.6: Model for large-scale bone repair
Supplementary Figure 3.1: Characterization of tdTomato+ cells during repair
Supplementary Figure 3.2: Characterization of Smo knock-out calluses
Supplemental Figure 3.3: Progenitors retain Sox9 and Runx2 so-expression
Supplementary Figure 3.4: Late KO of Smo
Figure 4.1: Sox9-CreERT2 marks cells that participate in femur fracture repair
Figure 4.2: Callus formation is not affected by the Smo KO
Figure 4.3: Hybrid cells are not affected by the Smo KO
Figure 4.4: Smo KO does not affect the ability for a femur fracture to fully repair
Figure 4.5: Sox9-CreERT2 marks cells in the periosteum near the knee joint
Figure 4.6: Characterization of osteophyte
Figure 4.7: Smo is not required in the Sox9 expressing progenitors for
osteophyte formation
Supplemental Figure 4.1: Safranin-O staining of osteophytes

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ABBREVIATIONS
Hh: Hedgehog
Ihh: Indian Hedgehog
Shh: Sonic Hedgehog
Ptch1: Patched 1
Smo: Smoothened
tdTom: tdTomato
KO: Knock-out
RNA-ISH : RNA in situ hybridization
IF: immunofluorescence
DPR: days post resection
WRP: weeks post resection
DPF: days post fracture
OA: osteoarthritis
DMM: destabilization of the medial meniscus

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CHAPTER 1: Introduction
While many bone fractures can repair on their own, non-unions and critical sized defects
are still major clinical problems. Between 5-10% of fractures become non-unions, which are
fractures that will not fully healing without additional medical involvement, such as surgery. Large-
scale bone injuries from trauma or bone resections due to cancer are examples of critical sized
defects. Current treatment options for bone repair are very limited, with the current gold standard
being autologous bone grafting, which involves some risks (Toosi et al., 2018). To improve clinical
treatment options the process of bone repair needs to be better understood. The goal of this
project was to determine the role of Hh signaling in large-scale bone repair, as well as determine
its role in a specific (Sox9 expressing) periosteal subpopulation.
Skeletal development
The skeleton has various developmental origins. The axial skeleton, which includes the
bones of the head, vertebrae column, and rib cage, are derived from the paraxial mesoderm,
while the appendicular skeleton, which includes the remaining bones of the body such as the
limbs and pelvis, are derived from the lateral plate mesoderm. Neural crest cells give rise to the
face and anterior skull, while the prechordal mesoderm gives rise to the posterior skull. Bones
can develop directly through a process known as intramembranous ossification or indirectly
through a cartilage intermediate, also known as endochondral ossification. Bone formation begins
with the migration and condensation of mesenchymal cells. During intramembranous ossification
the mesenchyme will differentiate directly into osteoblasts, while in endochondral ossification the
mesenchyme differentiates into chondrocytes which act as a scaffold for the bone to form on
(Berendsen and Olsen, 2015).
Flat bones, such as craniofacial bones, form through intramembranous ossification. The
mesenchymal cells proliferate, aggregate, and start to differentiate into osteoblasts. The
osteoblasts produce an extracellular matrix rich in collagen-proteoglycans that bind to calcium,
allowing for mineralization. Many osteoblasts continue to produce bone matrix on the surface of
the developing bone, but some become trapped within the bone matrix and become osteocytes
(Hall and Miyake, 2000) (Opperman, 2000) (Berendsen and Olsen, 2015).
Long bones form through endochondral ossification. Upon condensation of the
mesenchymal cells, the cells in the center start to differentiate into chondrocytes. This initial
differentiation starts with the expression of Sox9. While it is dispensable for initial condensation,
SOX9 is required for maintaining condensation and for the subsequent steps towards complete
chondrocyte differentiation (Akiyama et al., 2002). These cells then start to express and secrete
cartilage matrix proteins such as aggrecan, collagen type II, IX, and XI. The cells at the edge of
2

this condensing mass form the perichondrium, a connective tissue that surrounds chondrocytes.
The perichondrium will later respond to IHH signaling produced by the chondrocytes and become
osteoblasts. These osteoblasts will make up the bone collar which is characterized by the
expression of collagen type I (Col1a1) (Long and Ornitz, 2013). The chondrocytes of the
condensation will proliferate, and the central ones will start to mature into hypertrophic
chondrocytes. Hypertrophic chondrocytes exit the cell cycle and synthesize the extracellular
matrix protein collagen type X (COL10a1). They will later mineralize and undergo apoptosis.
Hypertrophic chondrocytes also secrete angiogenic factors, allowing for vascularization. Up until
this point the condensation was avascular. This vascularization brings in osteoprogenitors and
establishes the primary ossification center, which is typically located in the diaphysis of long bones
(Maes et al., 2010).
Ossification starts at the primary ossification center and extends outward towards the
epiphysis ends of the bones. Incoming osteoprogenitors differentiate into osteoblasts and produce
bone matrix on top of the mineralized matrix left by the hypertrophic chondrocyte (Maes et al.,
2010). The active, matrix producing osteoblasts will eventually either undergo apoptosis, become
quiescent bone lining cells, or become embedded in bone matrix as osteocytes (about 5-20% of
osteoblasts become osteocytes). The osteoblasts that differentiate into osteocytes are first
engulfed in an unmineralized lacunae, known as an osteoid. Once the bone matrix is mineralized
and they have formed dendritic-like processes, these cells are considered to be osteocytes. The
dendritic-like cytoplasmic processes run through canaliculi connecting the osteocyte to one
another, as well as the periosteal and endocortical bone surfaces. This is important for osteocytes
to sense changes in the skeletal environment and respond to these changes (reviewed in
(Bonewald, 2011) (Noble, 2008)). There has been evidence that some chondrocytes can
transdifferentiate into osteoblasts and directly participate in bone formation as well. Multiple
lineage tracing experiments have indicated that some chondrocytes will transdifferentiate during
endochondral ossification, postnatal growth, and the repair process (Yang et al., 2014b) (Bahney
et al., 2014) (Zhou et al., 2014c) (Park et al., 2015).
Bone growth and homeostasis
A secondary ossification center will form in the epiphyses of long bones. Once it is formed
the growth plate can be established in the area between the primary and secondary ossification
centers. The growth plate allows for continual longitudinal growth and is made up of organized
zones of chondrocytes: resting, proliferating, pre-hypertrophic, and hypertrophic. Lineage tracing
studies have shown that after the establishment of the secondary ossification center, some
chondroprogenitors in the resting zone are able to self-renew and form large, stable monoclonal
3

columns of chondrocytes that make up the growth plate. Additionally, some of the
chondroprogenitors will start to express stem cell markers (Newton et al., 2019). Similar results
have shown that in the resting zone, there is a subset of PTHrP-positive chondrocytes that have
been shown to act as a skeletal stem cell. These cells can give rise to the rows of chondrocytes
in the growth plate, osteoblasts, and marrow stromal cells (Mizuhashi et al., 2018). Similar to
endochondral ossification, blood vessels invade the hypertrophic chondrocytes bringing in
osteoblast precursors that can differentiate and form new bone (Kronenberg, 2003).
In addition to growth, bone continually remodels itself throughout an organism's life. During
the first step of remodeling, known as initiation, osteoclast precursors are recruited to the surface
of the bone. Osteoclasts are of hematopoietic lineage and are multinucleated. Before the
hematopoietic precursors are recruited to the bone they are found in the bone marrow and in
circulation. Upon recruitment of the bone, the precursors will proliferate, differentiate, and fuse
into mature osteoclasts. Two critical factors for osteoclastogenesis are macrophage colony-
stimulating factor 1 (M-CSF) and RANKL. Once resorption by the osteoclasts is complete they
will undergo apoptosis (Boyle et al., 2003). During resorption, the osteoclasts dissolve bone
mineral and degrade the organic bone matrix, which causes the release of factors that stimulate
the recruitment, differentiation, and activity of osteoblasts. This phase is known as reversal and
allows for new bone formation to be initiated. Next, during the terminating phase of remodeling,
osteoblasts produce more bone matrix and will eventually either undergo apoptosis, become an
osteocyte, or bone lining cell (reviewed in (Blair and Athanasou, 2004) (Reddy, 2004)).
Hedgehog signaling
The Hedgehog family of proteins are secreted signaling molecules. In vertebrates there
are three variants: Sonic (Shh), Indian (Ihh), and Desert (Dhh) (Echelard et al., 1993). All Hh
ligands bind to the Patched (PTCH1) receptor and inhibit it. Normally PTCH1 inhibits the activation
of another receptor, Smoothened (SMO), but when a Hh ligand is bound to PTCH1, SMO is free
to activate the GLI transcription factors which leads to changed gene expression. Hh can also
bind to Hh-interacting protein (HIP), which functions to reduce the Hh ligands’ range of movement
(Briscoe and Thérond, 2013).
The precursor of the Hh protein undergoes some post-translational modifications before it
is functional and can be secreted. The Hh precursor is cleaved into two parts: the C-terminal,
which gets degraded, and the N-terminal, which contains the signaling domain. This processing
results in the addition of a cholesterol to the N-terminal, allowing for the Hh protein to become
associated with the plasma membrane. The acyltransferase skinny hedgehog (SKI) then adds a
palmitic acid moiety, resulting in a fully active Hh ligand. Dispatched (DISP) and SCUBE2 must
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bind the cholesterol moiety for the Hh ligand to be released from the cell membrane (Petrova and
Joyner, 2014).
Shh, Ihh, and Dhh all have highly conserved sequences and can induce the same
physiological impact. It seems that the differences in their expression patterns (between SHH,
IHH, and DHH) are responsible for the various roles (Varjosalo and Taipale, 2008). Although they
appear to bind PTCH1 and HIP with comparable affinities, in vitro differentiation assays have
indicated that SHH is the most potent. It is not completely clear why there are different potencies
(Pathi et al., 2001).
When Hh is not bound to PTCH1, PTCH1 can cause either the confinement of SMO to an
intracellular compartment or its quick degradation. Other cell surface proteins, CAM-
related/downregulated by oncogenes (CDO), Brother of CDO (BOC), and growth arrest-specific
1 (GAS1), make a complex with PTCH1, allowing for high-affinity Hh binding. Upon Hh binding,
PTCH1 becomes internalized for degradation (Briscoe and Thérond, 2013) (Varjosalo and
Taipale, 2008). When PTCH1 is degraded, SMO is able to become phosphorylated, which
induces a conformational change, allowing for SMO to move into the primary cilium. Here it
interacts with KIF7 and with the SUFU-GLI complex, abolishing its ability to be
phosphorylated. The SUFU-GLI complex is dissociated and SUFU is unable to interact with the
GLI proteins. There are three GLI transcription factors: GLI1, GLI2, GLI3. All contain activator
domains, while GLI2 and GLI3 also contain repressor domains. Hh signaling activation changes
the response from repressor to activator, although GLI2 is mainly responsible for the activator
function while GLI3 is the main repressor. When Hh signaling is not active, GLI2 and GLI3 are in
a complex with SUFU, which causes them to become phosphorylated. This phosphorylation leads
to the partial degradation of their active domain. When Hh signaling is activated, GLI proteins are
able to move into the nucleus where they are converted into the activator form (reviewed in
(Briscoe and Thérond, 2013) (Ryan and Chiang, 2012).
Hh signaling, specifically IHH, is involved in both endochondral and intramembranous
bone formation. Hh signaling is required for osteoblast differentiation during bone formation. Ihh
null mice have a failure of osteoblast development in endochondral bones (St-Jacques et al.,
1999). IHH is directly required for the perichondrial cells to initiate osteoblast differentiation and
form the bone collar during endochondral ossification (Long et al., 2004) (Razzaque et al., 2005).
The removal of Smo from perichondrial cells using Cre3 and Cre10 during development resulted
in improper bone collar formation. The bone collar fails to form in some areas and separated by
from the marrow cavity by cartilage in others. Due to a lack of mature osteoblasts, these mice
also lacked primary spongiosa. In embryos with mixed WT and Smo null cells, only the WT cells
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were able to differentiate into osteoblasts, while the Smo null cells became chondrocytes. These
results indicate that Hh signaling is directly required for osteoblast differentiation (Long et al.,
2004). Ihh was also conditionally knocked out of chondrocytes using the Col2a1Cre. This caused
not only chondrocyte proliferation and differentiation defects, but also caused osteoblast
differentiation defects. In these mutants the perichondrial cells failed to differentiate into
osteoblasts and form the bone collar (Razzaque et al., 2005). Taken together these results have
determined that IHH released from the chondrocytes of the cartilage template signal to the
perichondrial cells to differentiate into osteoblasts.
During endochondral bone development IHH is produced from prehypertrophic and
hypertrophic chondrocytes. It signals to the surrounding cells, specifically the proliferating
chondrocytes and the overlying perichondrial cells. In response to IHH the perichondral cells
produce parathyroid related hormone (PTHrP) (Bitgood and McMahon, 1995) (Kindblom et al.,
2002). PTHrP is received by the proliferating chondrocytes which inhibit expression of Ihh,
promotes proliferation, and suppresses the maturation into hypertrophy. As the chondrocytes
continue to proliferate and move farther from the source of PTHrP they begin to differentiate. This
negative feedback loop allows for proper differentiation and proliferation control of chondrocytes
(Vortkamp et al., 1996b) (Zhao et al., 2002). Double and single knockouts (KOs) of Ihh and PTHrP
have shown that Ihh has an additional PTHrP-independent role inducing the proliferating
chondrocytes to continue to proliferate (St-Jacques et al., 1999). In the growth plate, IHH has also
been shown to stimulate the differentiation of the periarticular chondrocytes to differentiate into
the columnar chondrocytes (Kobayashi et al., 2005). Postnatally the IHH/PTHrP feedback loop
described above continues to regulate and maintain the growth plate, allowing for proper bone
growth. It also continues to be involved in osteoblast differentiation. When Ihh was knocked out
of chondrocytes postnatally, this not only affected the growth plate but also caused a loss of
trabecular bone, indicating a decrease in osteoblast formation (Maeda et al., 2007).
Ihh is also involved in intramembranous ossification. During intramembranous ossification
Ihh expression is seen at the osteogenic front of the growing bones, in the cells surrounding bone,
and in the epidermis. Ihh null embryos have significantly reduced cranial bone formation with an
increase in undifferentiated mesenchyme. This loss of Hh signaling did not affect proliferation of
the osteoprogenitors but did causes a decrease in osteogenic differentiation markers. These
results indicate that Ihh positively regulates intramembranous ossification and is a pro-osteogenic
factor. This is also supported by the decrease in BMP expression in Ihh null mice (Lenton et al.,
2011) (Jacob et al., 2007). Although Shh is expressed in the midline suture mesenchyme during
intramembranous ossification, it was not able to compensate for the loss of Ihh (Pan et al., 2013).
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Hh signaling also has a role in homeostasis by indirectly inducing osteoclast
differentiation. Hh signaling produced by osteoblasts upregulates expression of PTHrP. This
promotes RANKL expression, which is required for osteoclast development (Mak et al., 2008).
Other roles of Hh signaling
As previously mentioned, Hh signaling has multiple roles during development. Although
the different Hh ligands are functionally interchangeable, the expression of the ligands are
associated with specific functions. While Ihh has a well characterized role in bone development,
Shh plays a role in limb patterning. Dhh is expressed mainly in the gonads and is not required for
embryo survival. Dhh null mice are able to survive to adulthood with no distinguished phenotype
(Varjosalo and Taipale, 2008).
During limb development SHH works in a concentration and time dependent manner to
set up anteroposterior patterning. During limb growth FGF signaling induces SHH signal from the
posterior region of the apical ectodermal ridge. SHH signaling causes a shift from GLI3’s
repressor function (GLI3R) to its activator function (GLI3A) in the posterior region, while in the
anterior region there is still high GLI3R and low GLI3A due to lower SHH. The levels of GLI3R
and GLI3A set up the anteroposterior axis (Singh et al., 2015).
Shh is also involved in the patterning of the nervous system during the development of
the neural tube. As the notochord develops, moving ventral to dorsal, SHH is released and a
gradient is established. This gradient of SHH patterns the neural tube by inducing ventral cell
types and specifying the identity of neurons (Patten and Placzek, 2000). This SHH signal from
the notochord is also involved in vertebrae formation by promoting both the survival of somatic
cells, the epithelial mesenchymal transition to sclerotome, and its subsequent differentiation into
cartilage. SHH induces the expression of SOX9 and allows the cells to be competent to respond
to BMP, which induces chondrocyte differentiation (Murtaugh et al., 1999).
Hh signaling has been shown to be involved in angiogenesis and vasculogenesis in the
yolk sac of mice and during vascular injury. In response to Hh signaling support cells express
vascular-specific growth factors (Byrd and Grabel, 2004). Interestingly, IHH signaling was shown
to not affect vasculogenesis during intramembranous ossification (Lenton et al., 2011).
Chondrogenesis
As previously mentioned, mesenchymal cells condense and differentiate into
chondrocytes to start endochondral ossification. In the limb bud, the core is competent to
differentiate into chondrocytes due to FGF signaling, while the periphery tissue receives WNT
signaling which inhibits chondrogenesis (ten Berge et al., 2008) (Rudnicki and Brown, 1997). The
competency to differentiate into chondrocytes is due to Sox9 expression. WNT signaling blocks
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Sox9 expression, while FGF signaling allows cells to have the ability to express Sox9 by inhibiting
DNA methylation of the Sox9 promoter (Kumar and Lassar, 2009). Sox9 is a transcription factor
that is required for the chondrogenic fate. It has various roles throughout chondrogenesis, in both
the differentiation and maturation of chondrocytes. For example, it is needed for chondrocyte
survival and is a direct activator of chondrocyte differentiation markers (Bi et al., 1999) (reviewed
in (Kozhemyakina et al., 2015).
Other signaling molecules such as BMP, protein kinase A (PKA), and hypoxia-induced
factor 1a (HIF1a) also have a role in chondrogenesis. Knockout experiments of various BMP
receptors and downstream proteins in the BMP pathway have indicated that BMP signaling is
required for the differentiation, maturation, and maintenance of chondrocytes (Yoon et al.,
2005)(Yoon et al., 2006) (Retting et al., 2009). PKA has been shown to mediate the
phosphorylation of SOX9, increasing its transcriptional activity, and therefor increasing the
expression of other chondrogenic genes (Huang et al., 2002). Hif1a allows chondrocytes to
differentiate and adapt to the hypoxic environment by promoting the expression of Sox9 and
glycolytic enzymes and glucose transporters (Amarilio et al., 2007) (all reviewed in
(Kozhemyakina et al., 2015).
Transdifferentiation
Although chondrocytes and osteoblasts share an early common progenitor, they
eventually differentiate down separate lineages. During development, hypertrophic chondrocytes
are thought to undergo apoptosis, but recent studies have shown that some do not. Instead these
chondrocytes transdifferentiate and become osteoblasts, directly contributing to bone. Several
groups have shown this ability through lineage tracing studies. Both an inducible and non-
inducible version of a Col10a1-Cre has been used to lineage trace hypertrophic chondrocytes.
Lineage traced cells, that morphologically looked like osteoblasts and osteocytes, were found in
bone. Further experiments confirmed that the lineage traced cells did express bone markers such
as Col1a1, Runx2, Osx, Bglap, and SOST(Yang et al., 2014a) (Bahney et al., 2014) (Zhou et al.,
2014c) (Park et al., 2015). Col2a1-CreERT (Yang et al., 2014b) and Agc1-CreERT2 (Zhou et al.,
2014c), two chondrocytes specific driven Cre lines, were also used for lineage tracing
experiments. Similar results of chondrocytes becoming osteoblasts were seen. There are still
many unanswered questions about these transdifferentiating chondrocytes. It is unknown if they
are a unique population from the chondrocytes that undergo apoptosis or if differential signaling
is responsible for the transdifferentiation. Although some groups have shown that chondrocytes
can transdifferentiate into osteoblasts during bone repair (Zhou et al., 2014c) (Yang et al., 2014b),
much remains unknown about how they fit into the context of repair.
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Bone repair
When a bone is injured the periosteum, blood vessels, and endosteum are also injured.
The most common bone injury is a fracture, which repairs through a series of phases:
inflammatory, soft callus, hard callus, remodeling. Directly after injury a hematoma forms along
with the initiation of the inflammatory response. This response includes the release of TGF-β1
and PGDF, which recruit inflammatory cells such as macrophages to the injury. The inflammatory
cells release pro-inflammatory cytokines, such as TNF-α and Interleukin-1 (Xing et al., 2010) (Kon
et al., 2001), which recruit mesenchymal osteochondral skeletal progenitors, the majority of which
come from the periosteum. The factors released during the inflammation phase may allow for the
proper initiation of the repair response (Einhorn et al., 1995). Many of the skeletal progenitors will
proliferate and start to differentiate into cartilage, forming the soft callus around the fracture site.
The chondrocytes will continue to proliferate, mature, and will become mineralized. While the soft
callus is forming, intramembranous ossification also occurs directly at the ends of the fracture.
Later, osteoblasts start to replace the chondrocytes that built the soft callus with bone, resulting
in a hard callus made of woven bone. This will later be remodeled into lamellar bone, which is
more organized and stronger (reviewed in (Colnot et al., 2012) (Marsell and Einhorn, 2011)).
Much like bone homeostasis, remodeling is orchestrated by osteoblasts and osteoclasts.
Although most fractures will repair through a combination of both intramembranous and
endochondral ossification, some fractures will repair solely through intramembranous ossification.
This is known as direct fracture healing and typically only occur when there are no gaps between
the two fractured ends and the fracture is stably fixed (Thompson et al., 2002).
The traditional method of improving bone repair has been inducing osteogenic
differentiation directly, but recently some studies have determined that inducing a large cartilage
callus may be beneficial to the repair process. For example, the vascular system is damaged
during a bone injury, usually creating a lower oxygen environment, which chondrocytes are more
suited for. Cartilage is an avascular tissue and can more readily survive in low oxygen
environments. Another possibility is that the cartilage callus provides the break with stability.
Additionally, chondrocytes release growth factors beneficial to the repair process that stimulate
bone formation and vascularization. Some studies have supported the idea that enhancing
chondrogenesis can help improve the bone repair process. This was shown by chondrogenically
priming cells or implanting chondrocytes into defects (Bernhard et al., 2017) (Farrell et al., 2009)
(van der Stok et al., 2014), resulting in improved bone repair or bone formation. A comparison
between intramembranous and endochondral ossification also supports the importance of a
cartilage callus. Scaffolds with either osteogenic or chondrogenic potential were placed into
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critical sized calvarial defects and compared to one another. The defects implanted with the
chondrogenic scaffolds had enhanced bone repair compared to controls and the osteogenic
scaffolds (Thompson et al., 2016). As mentioned previously, chondrocytes have the ability to
transdifferentiate into osteoblasts and directly participate in bone formation (Yang et al., 2014a)
(Bahney et al., 2014) (Zhou et al., 2014c) (Park et al., 2015) (Yang et al., 2014b). It is possible
that chondrocytes of the repair callus directly contribute to new bone formation and that by
stimulating chondrogenesis there could be an increase in the transdifferentiation into bone.
The repair process is very dynamic and complex, involving multiple cell types and
signaling pathways (reviewed in (Einhorn and Gerstenfeld, 2015) (Marsell and Einhorn, 2011)).
Although the basic steps of repair have been characterized, little is known about the roles of
specific signaling pathways, progenitor populations, or how they converge with each other to
facilitate bone repair.
Skeletal progenitors
The identity of the skeletal stem cell has not been clearly defined, but recent studies have
started to elucidate potential skeletal stem cell candidates. The Longaker lab identified a mouse
skeletal stem cell by isolating and analyzing cells from the growth plate using the differential
expression of surface markers. The subpopulation CD45
-
Ter119
-
Tie2
-
AlphaV
+
Thy
-
6C3
-
CD105
-
CD200
+
was determined to be a mouse skeletal stem cell based on its ability to form bone,
cartilage, and marrow when transplanted beneath the renal capsule in mice and for its ability to
be able to generate all other subpopulations (Chan et al., 2015). Using these markers, another
study determined that there is a population of skeletal stem and progenitor cells located in the
resting zone of the growth plate that could be marked by PTHrP-mCherry (Mizuhashi et al., 2018).
Lineage tracing showed that these cells could become columnar chondrocytes, bone, and marrow
stromal cells, as well as be maintained in the resting zone. Another study showed similar results,
using the Col2-CreERT mouse line. A chondroprogenitor population in the resting zone was
shown to self-renew and form long, stable columns of chondrocytes in the growth plate (Newton
et al., 2019). Although these studies identify skeletal stem cells involved in development and
growth, none determined if they participate in bone repair. Recent results have indicated that
skeletal progenitors may be divided into separate subsets some responsible for growth and others
for repair.
One follow-up study to the Chan et al., 2015 did find a progenitor population that
participates in repair: the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) (CD45
-
Ter119
-
Tie2
-
AlphaV
+
Thy
-
6C3
-
CD105
+
). Upon injury, this population of multipotent skeletal
progenitors is activated, which leads to changes in transcription. These transcriptional changes
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correlate with increased proliferation, cell survival, and osteogenic potential (Marecic et al., 2015).
Although this study is a start to elucidating skeletal progenitors involved in repair, there is still
much to learn. For example, this study only used the femur fracture repair model, but it is unclear
if the same progenitor populations participate in all type of repair or even exist in all bones. Bones
have different developmental origins and it is possible that the progenitors can vary in different
skeletal tissues. In the future, additional injury models should be investigated. The originating
location of the f-BCSPs or how they are activated is still yet to be determined as well. Additionally,
the f-BCSPs were not the only cell type to participate in bone repair, yet little is known about the
remaining cells and their relationship to the f-BCSPs.
Lineage tracing experiments have been performed to determine the main source of cells
that mediate repair. These experiments have shown that the periosteum is the main source of
skeletal progenitors that mediate bone repair (Murao et al., 2013) (Duchamp de Lageneste et al.,
2018). Although progenitors from the bone marrow (Zhou et al., 2014a), endosteum (Park et al.,
2012), and muscle (Liu et al., 2011) (Abou-Khalil et al., 2015)can also contribute to the bone repair
process, Murano et al., 2013 used a periosteum specific Cre line, Prx-1-Cre, crossed with R26R
mice to show that most cells in the early repair callus, the later remodeled bone, and reestablished
periosteum are from this periosteal lineage. Although the Prx-1-Cre also labeled cells in the
cortical bone and on the endosteum prior to bone injury, other lineage tracing experiments ruled
out this contribution. The Col1a1(2.3kb)-Cre line was used to label mature osteoblasts that reside
in the endosteum and cortical bone and were shown to not give rise to chondrocytes during repair.
Together these lineage tracing experiments indicate that main source of progenitors that form the
repair callus, specifically the soft callus, come from the periosteum (Murao et al., 2013). Similar
results were seen when Duchamp de Lageneste et al., 2018 used the Prx1-Cre;mTmG line to
study fracture calluses. This study also determined that periosteal cells have higher regenerative
capacity, based on clonogenicity, growth, and differentiation, when compared to bone marrow
derived cells (Duchamp de Lageneste et al., 2018). Additional experiments have shown that the
progenitors from the periosteum and bone marrow/endosteum have different cell fates capacities
during bone repair. Progenitors from the periosteum were able to give rise to chondrocytes, while
progenitors from the bone marrow/endosteum were limited to making osteoblasts (Colnot, 2009).
In addition, rib resection studies have also shown that bone repair completely fails when the
periosteum is removed with the bone (Tripuraneni et al., 2015).
Although the periosteum has been shown to be the main source of progenitors involved
in bone repair, much remains unknown about the periosteal cells themselves. Subpopulations of
periosteal skeletal progenitors that participate in bone repair have been identified using Cre mice
11

crossed to reporter lines. This has allowed for specific subpopulations to be marked and lineage
traced throughout the repair process. In addition to Prx1, Sox9 (He et al., 2017), Gli1 (Shi et al.,
2017), Axin2 (Ransom et al., 2016), αSMA (Matthews et al., 2014), Cathepsin K (Ctsk) (Debnath
et al., 2018) and Gremlin1 (Grem1) (Worthley et al., 2015) were all identified as periosteal skeletal
progenitor markers that could contribute to both bone and cartilage formation during fracture
repair. Unfortunately, little is known about each subpopulation, apart from their participation. For
example, it is unknown if these subpopulations overlap with one another, interact, respond
differently to signals, are required for repair, or if they have specific roles during repair.
Sox9 is expressed in all chondroprogenitors and is essential for chondrogenesis, although,
during development Sox9-expressing precursors can also give rise to other tissues, including
bone, intestine, pancreas, and testis (Akiyama et al., 2005). Sox9creERT2;tdTom mice were
used to show that, upon Tamoxifen injection, labeled cells could be found in the periosteum of an
uninjured femur. While some cells in the endosteum were also labeled particularly near the
epiphysis, the majority were found in the periosteum especially in the diaphysis. These cells were
lineage-traced after femur fracture and co-localization of the tdTomato reporter with cartilage
marker SOX9 and osteoblast markers COL1a1 and SP7 have confirmed that the lineage-traced
cells can give rise to both bone and cartilage during femur fracture repair (He et al., 2017).
Alpha smooth muscle actin ( α S M A) is a known marker of mesenchymal progenitors that
have been shown to proliferate and differentiate into multiple lineages in vitro. α S M A-CreERT2
mice were used to determine that α S M A expression marks a subpopulation in the periosteum that
could give rise to both chondrocytes and osteoblasts/osteocytes during fracture repair. It was also
determined that the activation of the Notch pathway inhibits the differentiation of the α S M A cells
in vitro (Matthews et al., 2014).
Axin2 expression marks Wnt-responding cells, a signaling pathway known to be involved
in multiple aspects of skeletal development and repair. Axin2CreER;R26mTmG mice were used
to demonstrate that an Axin2-expressing subpopulation could be found in the periosteum and
endosteum. Lineage tracing experiments indicated that these cells could contribute to cortical
bone formation. Additionally, upon injury these cells could contribute to both cartilage and bone
formation in the repair callus. Further investigation into the role of the Axin2-expressing
progenitors during repair was done by ablating these cells using diptheria sensitive mice. The
experimental mice showed impaired repair and a decrease in overall bone formation, indicating
that Axin2 expressing cells have a functional role in bone repair (Ransom et al., 2016).
Gli1 expression was investigated as a skeletal marker because it is a readout of Hh
signaling, which is required for proper bone formation and induces osteoblast differentiation. Gli1-
12

CreERT2 mice were used to show that Gli1 expression could be found in the articular cartilage,
the growth place, the perichondrium, and chondro-osseous junction immediately below the growth
plate. Upon injury these cells were also found in the periosteum and could participate in the repair
callus making both bone and cartilage (Shi et al., 2017).
Ctsk-Cre has been shown to label some periosteal mesenchyme. Although during injury
these cells contribute to both bone and cartilage, in the absence of injury these cells will only form
bone. When these cells are isolated and transplanted under the kidney capsule to form bone in
vivo, the bone formed through intramembranous ossification, which is unique from many other
skeletal mesenchymal stem cells that form bone through endochondral ossification (Debnath et
al., 2018).
Gremlin1, which is important for skeletal development and homeostasis, was found to
mark a population of osteochondro-reticular stem cells in the bone marrow and
periosteum. Through clonogenicity assays and lineage tracing assays it was determined that this
population can self-renew and give rise to osteoblasts, chondrocytes, and reticular marrow cells.
Femur fractures in Grem1-CreERT2 mice show that Grem1+ cells could differentiate into Col1a1
and Bglap positive osteoblast and SOX9 positive chondrocytes of the repair callus (Worthley et
al., 2015).
Other Cre lines have also been used to mark progenitors that participate in bone repair,
but do not reside in the periosteum. Leptin Receptor (LepR) is highly enriched in mesenchymal
stromal cells in the bone marrow. Lineage tracing of LepR+ cells using the LepRcre;tdTomato
mice has shown that these cells are the main source of bone that is formed by adult bone marrow.
They can also contribute to both bone and cartilage during fracture repair (Zhou et al., 2014a).
Mx1 expression was also found to mark an endogenous bone marrow MSC population in Mx1-
Cre/YFP mice. After induction, many of the endosteal bone cells were YFP positive. These cells
showed the potential to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro, but
in vivo experiments showed that during bone homeostasis and in microfracture repair, they could
only form osteoblasts (Park et al., 2012). The contribution of myogenic progenitors has also been
investigated during fracture repair. In the absence of injury, MyoD expressing cells are only able
to form skeletal muscle, but in certain injury context these cells can differentiate into both
chondrocytes and osteoblasts. Lineage tracing studies in MyoD-Cre mice have shown that in
open tibial fractures cells from the MyoD expressing lineage can contribute to the repair callus,
forming both chondrocytes and osteoblasts (Liu et al., 2011). Satellite cells, which are precursors
to skeletal muscle cells, have also been shown to contribute during bone regeneration. Pax3Cre
13

and Pax7CreERT2 have both been used to lineage trace satellite cells that contribute to the repair
callus of murine tibial fractures (Abou-Khalil et al., 2015).
Although these studies have determined many of the cell populations that are involved in
bone repair, much remains unknown about how these populations relate to one another and how
they interact with one another to facilitate repair. Their specific requirements and functions during
repair remain unclear as well. To fully understand the repair process these subpopulations need
to be study in greater depth during repair and during homeostasis.
Hh signaling in bone repair
Hh signaling has been investigated during bone repair due to its requirements during
embryonic bone development. In both intramembranous and endochondral ossification, it is
required during development for osteoblast differentiation. During intramembranous ossification
Ihh is expressed at the osteogenic front of the growing bones inducing osteogenesis (Jacob et
al., 2007). In this situation, IHH induces differentiation but not proliferation of the osteoprogenitors
(Lenton et al., 2011). During endochondral ossification, IHH is released from chondrocytes and
has been found to be directly required for the perichondral cells to initiate osteoblast differentiation
and form the bone collar. This was shown through the inability of perichondrial cells to form a
bone collar when those cells were null for Smo (Long et al., 2001).

Additionally, a Col2-Cre line
was used to ablate Ihh from chondrocytes during development, resulting in a lack of bone marker
expression in the perichondrium, indicating an absence of osteoblasts (Razzaque et al., 2005).
Hh signaling is also produced by osteoblasts postnatally, which promotes osteoblast proliferation,
survival, and differentiation (Mak et al., 2008).
The osteogenic potential of Hh signaling has been investigated in vitro using various cell
types and conditions, all of which have indicated that Hh signaling can induce osteogenesis. The
overexpression of IHH in bone marrow MSCs and C3H10T1/2 cells, by flag–tagged human Ihh
cDNA retroviral constructs, caused an increase in ALP staining and an upregulation of Bglap,
indicating an increase in osteogenic differentiation (Zou et al., 2014a). SAG, a Hh agonist, was
also used to treat C3H10T1/2 cells in culture for 7 days. This resulted in an increase in both Alp
and Gli1 expression, demonstrating that SAG stimulates osteogenic differentiation through the
Hh pathway (Maeda et al., 2013). Baht et al., 2014 showed that Hh signaling can not only increase
osteogenic differentiation in vitro but can also increase osteoblast activity, such as matrix
production. Bone marrow stromal cells isolated from Col1-Cre;Smo
Stab
and control mice were
cultured under osteogenic conditions. The osteoblasts from the Col1-Cre;Smo
Stab
mice expressed
a constitutively active version of the Smo receptor, causing constitutively active Hh signaling. The
osteoblasts isolated from Col1-Cre;Smo
Stab
mice had increased alkaline phosphatase and Von
14

Kossa staining, as well as increased transcription levels of Col1 and Alp when compared to
controls, indicating an increase in osteoblast activity and matrix production (Baht et al., 2014).
It has previously been shown that the periosteum is a major source of progenitors involved
in the repair callus (Murao et al., 2013). With the context of repair in mind, some in vitro studies
have used periosteal cells to determine if Hh signaling can induce the osteogenic differentiation
specifically in periosteal progenitors in vitro. Periosteal derived mesenchymal progenitors cells
(PDMPCs) (Huang et al., 2014) and periosteum callus derived mesenchymal stem cells
(PCDSCs) isolated from a segmental bone graft (Wang et al., 2010) were both manipulated to
over express the N terminal Shh peptide. This overexpression caused an increase in osteogenic
differentiation, as seen through increased ALP and BGLAP expression when compared to
controls (Wang et al., 2010)

(Huang et al., 2014). In addition, purmorphamine, a Hh agonist, was
added to osteogenic differentiation media and was found to strongly induce osteogenic
differentiation and mineralization of the PCDSCs when compared to cultures under only
osteogenic differentiation media (Wang et al., 2010). Additionally, during an injury, more than just
the bone needs to be repaired. There are other aspects of the repair process, such as
reestablishing the vasculature system, that Hh signaling may be involved in. In vitro co-culture
studies using osteoblasts and endothelial cells have indicated that Shh promotes both
angiogenesis and osteogenesis (Dohle et al., 2009).
In vitro experiments have also shown the effects of inhibiting Hh signaling. Cells that are
unable to respond to Hh signaling, due to the ablation of Smo, were isolated from mice and
cultured to determine their osteogenic potential. PCDSCs isolated from Smo
f/f
;CreER mice, which
had an 80% reduction of Smo gene expression when compared to controls, were cultured in
osteogenic differentiation media and compared to control PCDSCs under the same culture
conditions. The Smo null cultures had decreased osteogenic potential as seen by reduced ALP
staining and decreased expression of Alp and Osx by RT-PCR analysis. Similar results were seen
when control PCDSCs were cultured with cyclopamine, a Hh inhibitor (Wang et al., 2010). These
results show that Hh signaling has a role in the differentiation of periosteal cells into osteoblasts.
Consistent with the Smo
stab
results, Baht et al showed that Hh signaling also has a role in
osteoblast activity and matrix production by culturing bone marrow stromal cells from Col1-
Cre;Smo
Null
mice. The osteoblasts, which are null for Smo, showed decreased alkaline
phosphatase and Von Kossa staining when compared to controls indicating a decrease in
osteoblast activity and matrix production (Baht et al., 2014). All of these studies have shown that
Hh signaling has a potent osteogenic effect in vitro. These together with studies using in vivo
approaches as described below, indicate that Hh signaling can improve bone repair.
15

In vivo studies
A variety of bone repair models have been used to investigate the osteogenic potential of
Hh signaling in vivo. Although the studies did not determine the exact requirements of Hh
signaling, they indicate that Hh signaling has a positive effect on bone repair, possibly through
inducing osteogenesis and promoting osteoblast matrix production. Both the addition of
exogenous Hh and the inhibition of Hh signaling was investigated in vivo.
Cells overexpressing a Hh ligand were implanted into various types of bone defects to
determine the effect during bone repair (Huang et al., 2014)

(Zou et al., 2014a)

(Edwards et al.,
2005). Although these studies show that additional exogenous Hh leads to increased bone
formation during repair, how Hh signaling promotes this and specifically which cells were affected
remains unclear. Allographs of devitalized bone with PDMPCs overexpressing ShhN were
implanted into 4-mm segmental defects in the femur by Huang et al., 2014. When compared to
controls with only PDMPCs the ShhN expressing PDMPCs had increased bone formation and
increased cell survival within the allographs (Huang et al., 2014). Edwards et al., 2005, implanted
cells overexpressing ShhN with matrix into a cranial defect in rabbit, while control defects were
either empty, had matrix alone, or unenhanced cells. The controls had minimal bone formation,
but the defects with ShhN had substantial bone formation, seen by histological and radiographic
analysis. Unfortunately further analysis was not done to determine if the increase in bone
formation was due to an increase in proliferation, cell survival, or differentiation (Edwards et al.,
2005). Zou et al., 2014a implanted scaffolds with MSCs that express IHH and EGFP or EGFP
alone into rabbit tibia defects. These defects were made by drilling a 5 mm wide by 2 mm deep
hole. Increased bone formation was seen in the IHH expression group when compared to the
control groups, as determined by CT imaging and histology. IHH did not affect cell survival or
proliferation of the MSCs on the scaffolds, indicating that IHH specifically enhanced osteogenesis
(Zou et al., 2014a). Although it cannot be ruled out that Ihh expression from the MSCs influenced
the proliferation or cell survival of other cells involved in repair.
Agonists of Hh signaling have also been used. For example, the use of SAG has shown
that Hh signaling could improve bone repair in vivo (Lee et al., 2017)

(Maeda et al., 2013). Lee et
al placed scaffolds with and without SAG in a 4 mm calvarial defect in mice. The results showed
an increase in bone formation in the scaffolds that had SAG. In addition to the claim that SAG
could promote osteogenic differentiation, they also state that SAG suppress adipogenic
differentiation. Although they did not have data supporting this, they cited multiple papers that
indicate that Hh signaling has anti-adipogenic properties (Lee et al., 2017). SAG was also loaded
onto calcium phosphate granules and implanted into a femur defect in rats. There was increased
16

bone formation with SAG when compared to controls with no SAG. Since SAG was released only
during early repair, they argue that Hh signaling has a role early in the repair process, possibly in
the initial specification of cells down the osteoblast lineage (Maeda et al., 2013). Daily systemic
administration of the Hh agonist, Hh-Ag1.5, was used to treat femur fractures of aged mice (18
months old). When compared to controls, the treated groups had earlier callus bridging, greater
callus and bone volume, and mechanical testing indicated greater strength (McKenzie et al.,
2018). These results indicate that the Hh agonist improves the osteogenic repair response.
Although these in vivo repair experiments determined that the addition of Hh signaling can have
a positive effect on bone repair increasing bone formation, they did not determine the
requirements of Hh signaling or which cell populations were being affected.
The requirements of Hh signaling during bone repair were tested by genetically knocking
out different components of the Hh pathway. Ihh was ablated from chondrocytes (Col2a1-
CreERT2;Ihh
fl/fl
mice) during tibia fracture repair. This resulted in decreased cartilage formation
but did not affect overall bone formation. Micro-CT and histomorphometry analysis showed similar
amounts of bone formation. Similar results were seen when the Hh inhibitor, cyclopamine, was
used (Li et al., 2018). Although these results indicate that IHH signaling from chondrocytes does
not affect bone formation, it is possible that the bone formation is due to another Hh ligand, such
as SHH, or IHH signaling from a non-Col2 expressing cell type. The low levels of Hh signaling
that were expressed in the KO and cyclopamine treated mice could have been enough to induce
normal bone formation.
Other studies knocked out Smo, the required receptor for Hh signaling. Wang et al., 2010
knocked out Smo using an inducible, ubiquitous Cre during a bone autograft surgery, resulting in
a decrease in total bone callus formation, an increase in cells with an undifferentiated morphology,
and decreased proliferation in the periosteal callus. These results indicate that Hh plays a role in
the proliferation and differentiation of the mesenchymal lineages at the early stages of bone
repair. This is also supported by the expression pattern of Hh signaling during repair. In situ
hybridization (ISH) showed Ihh expression in the early prehypertrophic chondrocytes of the repair
callus. Additionally, the Ptch1-LacZ mouse was used during femur fractures to determine the
responding population. At 3 and 7 days post injury the periosteal cells above the bone, the
surrounding the prehypertrophic chondrocytes, the chondroprogenitors, proliferating
chondrocytes, vascular progenitors, mesenchymal cells and osteoblastic cells forming the new
bone were found to be the responding population (Wang et al., 2010). Smo was also globally
knocked out by the inducible Cre Esr-1 in a murine tibial fracture, causing a reduction in total bone
callus, although not a statistically significant one. To more clearly determine how Hh signaling
17

was affecting bone formation, an osteoblast specific Cre (Col1-Cre) was used. This resulted in a
more dramatic decrease in total bone callus. Consistently, when a constitutively active version of
the Smo receptor was crossed to the Esr1-Cre and Col1-Cre there was an increase in total bone
callus and matrix when compared to controls by radiographic analysis and histologically.
Additionally, RT-PCR of Ptch and Gli expression showed highest expression at 14 days post
fracture, a time point when the callus is mostly composed of woven bone. Since Ptch and Gli are
direct readouts of Hh signaling, this suggests that Hh signaling is most active during bone
formation (Baht et al., 2014). Interestingly, although there was a reduction in bone callus, bone
formation was still able to occur and these fractures did not result in non-unions. Since the bones
were still able to repair these results suggest that Hh signaling is not absolutely required for repair
but, instead it seems to moderate the quality of repair through the enhancement of osteoblast
differentiation, as well as osteoblast maturation and matrix production at later stages of repair.
Both Baht et al. 2014 and Wang et al., 2010 determine that Hh signaling has a role relating
to osteoblasts during bone repair, but it is unclear what specific requirements it has. Unfortunately,
there was no confirmation of the KO and lineage tracing studies were not done to determine which
specific population was affected since Cre lines do not always behave as expected. To truly
understand the requirements of Hh signaling in osteogenesis during bone repair Cre lines specific
for only osteoprogenitors must be utilized. Although it is unclear specifically how Hh signaling
promotes bone repair, some possibilities are that progenitors and/or osteoblasts respond to Hh
signaling by either differentiating, maturing, or producing matrix. As mentioned previously, many
of these repair studies focused on bone formation at late time points and did not thoroughly
investigate the entire repair process. It is possible that Hh signaling could directly affect an early
step in the repair process, with changes in bone formation being a secondary effect.
Hh and role in chondrocyte proliferation and chondrogenesis
The cartilage callus is one of the early steps in the repair process that Hh signaling could
be involved. Hh signaling has a role in chondrocyte proliferation during bone development and in
the growth plate of post-natal mice. During endochondral bone development, the IHH ligand is
produced by prehypertrophic and hypertrophic chondrocytes. It signals to the surrounding cells,
including the chondrocytes and the overlying perichondrial cells. In response to IHH, the
perichondrial cells produce parathyroid related hormone (PTHrP) (Bitgood and McMahon, 1995)

(Kindblom et al., 2002). PTHrP is received by the chondrocytes where it promotes proliferation
and suppresses the maturation into hypertrophy. As the chondrocytes continue to proliferate and
move farther from the source of PTHrP they begin to differentiate. This negative feedback loop
allows for proper differentiation and proliferation control of chondrocytes (Vortkamp et al., 1996a)

18

(Zhao et al., 2002). It is possible that Hh signaling affects the cartilage callus of the bone repair
process as it does during bone development.
Although most of the previous studies have focused on the osteogenic potential of Hh
signaling, some studies also investigated how Hh signaling affected the cartilage callus. Recently
it has been suggested that improving the cartilage callus will lead to better repair overall. A major
issue with bone injuries, especially large-scale ones, is the hypoxic nature of the injury due to the
disruption of blood vessels. Since cartilage is an avascular tissue it is better suited for a low
oxygen environment. Chondrocytes are also known to release VEGF and MMPs and other growth
factors that are beneficial to the repair process (Bernhard et al., 2017)

(Dennis et al., 2015)

(Farrell
et al., 2009). In addition to contributing indirectly, recent studies have indicated that chondrocytes
have the ability to transdifferentiate into matrix producing osteoblasts and therefore may directly
contribute to new bone formation (Yang et al., 2014b)

(Zhou et al., 2014c)

(Hu et al., 2017).
Although bone formation is the end goal, the entire process of bone repair should be studied. The
above reasons highlight why the cartilage callus is significant and why it should also be
investigated during bone repair.
In vitro
In vitro studies have shown that Hh signaling can enhance chondrogenesis, while
inhibiting it decreases chondrogenic potential. PCDSCs overexpressing ShhN in micromass
culture showed enhanced chondrogenesis when treated in synergy with BMP-2 (Wang et al.,
2010). Similarly bone marrow derived MSCs from rabbit femurs and tibias, C3H10T1/2, and
ATDC5 cells that overexpress Ihh had increased alcian blue staining when compared to controls
(Zou et al., 2014a)

(Amano et al., 2014). In vitro experiments have also shown that Ihh positively
regulates chondrocyte maturation and calcification. This was demonstrated in several ways. Ihh
signaling was shown to induce the expression of Col10a1, a marker of mature hypertrophic
chondrocytes. ATDC5 cells overexpressing Ihh were cultured with or without cyclopamine, a Hh
inhibitor. Cultures without cyclopamine showed an increase in Col10a1 expression, as well as
chondrocyte mineralization when compared to controls. Culturing with cyclopamine and β-
glycerophosphate resulted in decreased Col10a1 expression and completely blocked
mineralization, when compared to cultures with only β-glycerophosphate added (Amano et al.,
2014). Similarly limb mesenchymal cells treated with the Hh inhibitor, HhAntag, had decreased
alcian blue positive nodules, as well as a decrease in the amount of cartilage marker genes when
compared to controls (Mundy et al., 2016). Like the pro-osteogenic effect in vitro, these
experiments indicate that Hh signaling has the ability to induce chondrocyte differentiation and
maturation.
19

In vivo
While most of the studies focused on bone formation, some in vivo studies also
investigated how the cartilage was affected by Hh signaling during the repair process. From these
studies there is evidence that Hh signaling is not required in chondrocytes during repair, that it
induces chondrocyte proliferation during repair, and that is required for the induction of
chondrogenesis during repair. It is possible that some of the conflicting results may be due to
differences in injury model. For example, in fracture studies, although some studies indicated that
Hh signaling affects the cartilage callus, none showed that it is required. In contrast large-scale
injuries have showed that Hh signaling is required for chondrogenesis and for complete repair.
The conflicting conclusions between these studies will be discussed in detail below and highlight
the importance of determining the specific timing and cell types that require Hh signaling during
bone repair.
Some studies have indicated that Hh signaling is not required in chondrocytes during bone
repair. Older mice, 18 months old, that had undergone femur fractures were given a Hh agonist
(Hh-Ag1.5) orally every day from fracture until sacrifice. These mice did not show any increase in
cartilage formation when compared to controls, indicating that Hh signaling, at least through the
Hh agonist Hh-Ag1.5, does not affect chondrogenesis (McKenzie et al., 2018). Murine tibia
fracture experiments done by Baht et al., 2014 also indicated that Hh signaling is not required for
chondrocyte differentiation or proliferation during repair. WT and Ers-1Cre;SmoKO calluses were
compared at 1 week post fracture. The relative area of cartilage was measured and found to be
similar, indicating that Hh signaling is not involved in the formation of the cartilage callus. A
chondrocyte specific cre (Col2-Cre) was also used to KO Smo specifically in chondrocytes to
further determine the role of Hh signaling in cartilage. The fracture calluses as well as bone
formation were compared at 28 days post fracture and were found to be similar between the Col2-
Cre;SmoKO and WT. From these results Baht et al., 2014 concluded that Hh signaling is not
required in chondrocytes during repair (Baht et al., 2014). Although overall repair was unaffected,
the effect that Hh signaling has on chondrocytes during repair cannot be determined from these
experiments. By 28 days post fracture there may not be enough cartilage to analyze. It has
previously been shown that the cartilage callus may not by required for fracture repair, so the
analysis that overall repair was unaffected does not necessarily indicate if formation of cartilage
was affected (Thompson et al., 2002). It is best to measure the amount of cartilage and
proliferation at an earlier time point when the cartilage callus was most prominent (Baht et al.,
2014). Additionally, Baht et al., 2014 did not include lineage tracing to determine what cells were
being affected or confirm the Smo KO. These results support that during fracture repair, Hh
20

signaling is mainly involved in osteogenesis and bone matrix production rather than building the
cartilage callus.
In contrast to these results, Wang et al., 2010 concluded that Hh signaling does affect
chondrocytes during the repair process. The inducible ubiquitous deletion of Smo during a bone
autograft surgery caused reduced proliferation in chondroprogenitors and chondrocytes when
compared to Cre-negative mice (Wang et al., 2010). Although the formation or amount of cartilage
was not quantified in this study, the results indicate that Hh signaling induces chondrocyte
proliferation during repair, as it does during development and at the growth plate. These mice had
decreased total bone formation as well, (Wang et al., 2010) but the question remains if Hh
signaling has a direct role in that bone formation or if it is secondary to the reduction in cartilage.
Similar results showed that the deletion of Ihh from chondrocytes during repair caused a decrease
in cartilage during tibia fracture repair. The decrease in cartilage formation could be seen by
Safranin-O staining at 7 and 14 days post fracture. Additionally, RT-PCR of the repair callus
showed a significant decrease in Col2 transcription. Although these results show that Hh signaling
in involved in chondrocytes during repair, it is unclear if it plays a role in chondrocyte proliferation
or differentiation (Li et al., 2018). It is possible that Hh signaling produced by the chondrocytes
induces either proliferation or additional chondrocyte differentiation, unfortunately without
proliferation assays, the answer is not known.
Results from Zou et al., 2014a suggested that overexpression of IHH could induce
chondrogenesis. MSCs overexpressing IHH were implanted into a bone defect and cartilage
formation was detected, while in the control group using MSCs that did not over-express IHH, no
cartilage was detected. In addition to cartilage formation, only in the IHH experimental group was
complete healing achieved (Zou et al., 2014a). These results not only indicate that Ihh has a role
in chondrocyte induction but also indicate that the addition of cartilage can improve repair.
Genetic knockout experiments have also indicated that Hh signaling is required for
chondrocyte differentiation during repair. Paul et al., 2016 has shown that Hh signaling is required
for the formation of the cartilage callus during zebrafish jawbone repair. The loss of Ihha in the
zebrafish jawbone repair model lead to a lack in cartilage callus formation and a decrease in bone
formation, with no significant decrease in proliferation. This study suggested that Ihh’s role in
repair is to induce the formation of the cartilage callus and that the decrease in bone may be a
secondary effect due to the lack of cartilage. During normal jawbone repair Ihh expression is seen
by ISH in the early stages of repair, produced by the prehypertrophic chondrocytes, while
expression of its receptor Ptch can be seen early in the resected area before cartilage
differentiation as well as in the chondrocytes of the callus (Paul et al., 2016). It is important to
21

note that the a required role of inducing the formation of the cartilage is not seen during bone
development, where Hh signaling is required for chondrocyte proliferation and osteoblast
differentiation. Although, these results to do not exclude the possibility that Hh signaling can
induce chondrocyte proliferation and osteoblast differentiation, they indicate that its primary role
is inducing chondrocyte differentiation during large-scale jawbone repair in zebrafish.
Overall, these studies have determined that Hh signaling has a positive effect on bone
repair and is a potential therapeutic target for improving bone repair in the clinic. To determine
how to best target Hh signaling additional in-depth analysis must be done to answer some
remaining unanswered questions. The specific role that Hh signaling has and which cell
population it targets during bone repair is unknown. Although most studies indicated that Hh
signaling contributes to bone repair by inducing osteoblast differentiation, some indicate that it
can induce chondrogenesis as well. The large-scale regeneration of the zebrafish jawbone
showed a requirement for Hh signaling in the induction of the cartilage callus. None of the
mammalian bone repair studies were performed on a large-scale like this. To determine if Hh
signaling has a similar role in mammals, a large-scale regeneration model in mammals must be
used.

22
1
Large portions of this work (Chapter 2 and 3) are published in eLife (Kuwahara et al., eLife
2019;8:e40715)
CHAPTER 2: Characterization of the murine rib resection model
1
2.1 Introduction
Bone repair has been studied using fracture and drill defect models in mammals, but there
is a lack of large-scale mammalian bone repair models. Whereas amphibians regenerate large
portions of their skeletons after injury or amputation, natural large-scale skeletal repair in
mammals is more limited. A notable exception is the rib. Craniofacial surgeons often extract large
segments of rib cartilage and bone for autologous repair of skeletal defects in other parts of the
body, and in post-operative visits have noted extensive regeneration at the donor rib site
(Kawanabe and Nagata, 2007) (Munro and Guyuron, 1981) (Srour et al., 2015). To better
understand the unique regenerative potential of the rib, the Mariani lab recently developed
analogous large-scale rib cartilage and bone regeneration models in the mouse (Srour et al.,
2015) (Tripuraneni et al., 2015).
In most mammalian bones, repair involves the formation of a callus via a mixture of direct
and endochondral ossification through a cartilage callus intermediate (Gerstenfeld et al., 2003)
(Marsell and Einhorn, 2011) (Hall, 2015). The identity and regulation of the adult skeletal
progenitors that build the callus remain incompletely understood. While markers for a number of
different skeletal stem cells have been reported (He et al., 2017), (Shi et al., 2017), (Ransom et
al., 2016), (Matthews et al., 2014), (Park et al., 2012), (Balani et al., 2017), (Bianco and Robey,
2015), their relative roles during bone repair are less clear, particularly in cases of large-scale
bone regeneration such as in the rib. During skeletal repair, studies have shown that cells from
the periosteum, a heterogeneous connective tissue sheath covering the bone, are major
contributors to the callus (Colnot, 2009) (Murao et al., 2013) (Duchamp de Lageneste et al., 2018).
Accordingly, the Mariani lab has previously shown that the periosteum is essential for
regeneration of the rib bone (Tripuraneni et al., 2015). Additionally, the cellular basis of the repair
callus has not been investigated. It is unknown if the chondrocytes and osteoblasts that form the
repair callus follow the same differentiation pathway as they do during development.
During normal bone homeostasis, periosteal stem cells generate bone-producing
osteoblasts but not cartilage-producing chondrocytes (Roberts et al., 2015). How injury stimulates
periosteal stem cells to generate chondrocytes is unclear. Although some fractures can heal in
the absence of a cartilage callus, for example when the fracture is rigidly stabilized (Thompson et
al., 2002), the formation of a large cartilage callus appears to be required in large-scale bone
regeneration. Unfortunately, little is known about the specific periosteal
23

progenitor population that drives the formation of the cartilage callus nor the signaling pathways
required. Recent studies have used Cre-based lineage tracing experiments to show that cells
marked by expression of Gremlin1 (Worthley et al., 2015), Axin2 (Ransom et al., 2016), Gli1 (Shi
et al., 2017), Act2a (Matthews et al., 2014), Periostin (Duchamp de Lageneste et al., 2018) and
Sox9 (Balani et al., 2017) (He et al., 2017) can be found in the periosteum and contribute to the
fracture callus during repair. Other than participation, the specific role of any of these progenitor
population remains unclear. In this study, I focus on the role of one subpopulation within the
periosteum and its specific role in driving callus formation and bone regeneration. As Sox9 has a
well-known function in promoting chondrogenesis during embryonic development (Akiyama et al.,
2002) (Lefebvre et al., 1997), I postulated that Sox9-expressing cells in the adult periosteum may
possess a potent ability to form cartilage in response to bone injury. In addition, a requirement for
Sox9-expressing cells in repair has not yet been established.
In this study I show that large-scale mammalian rib regeneration occurs through a cartilage
intermediate. Unlike in development, the repair callus cells with chondrocyte morphology express
high levels of both chondrocyte and osteoblast associated genes. I also show that there is a
subpopulation of progenitors that can be marked by the expression of Sox9 in the periosteum and
that these progenitors are able to contribute to the repair callus, forming both bone and cartilage.
2.2 Materials and methods
Rib resection
Mice between 6-8 weeks of age are used. Mice are weighed to determine the amount of
pain medicine to administer then placed in an induction chamber with 4% isoflurane. Once
unconscious, mice are moved to the dissecting scope, provided a warming pouch, a nose cone
is placed over their nose to continue the administration of isoflurane. The isoflurane is turned
down to 2.5%. The fur is shaved around the area of interest and iodine and isopropanol (70%)
are used to clean the area (3 times each). Above the desired rib, a 2 cm incision is made through
the skin and then subsequently through the fat and muscle. The intercostal muscle and
periosteum directly connected to the rib are cut with a 5.0 mm scalpel. This is done by running
the scalpel longitudinally down the rib. Once the bone is exposed, the periosteum is peeled from
the bone with forceps, making sure to not give the mouse a pneumothorax, but to also peel all
the periosteum from the bone. Next micro scissors are used to cut through the bone in cross-
section manner to the left and right of the area being resected. The resected piece of the rib is
pulled out, being careful to leave the periosteum intact. If bleeding occurs at any time during the
surgery, light pressure with a clean cotton swab is applied until the bleed stops. Then 9-0 sutures
are used to close the intercostal muscle and the overlying layer of fat/muscle (typically 1-2 sutures
24

in each location). Administer the slow release Buprenorphine sub-cutaneously (0.5 microliter per
gram). To close, 7-0 sutures (between 4-7 sutures) are used. Skin glue (3M Vetbond tissue
adhesive) is then added to secure the site. The mouse is placed under a heat lamp and monitored
during recovery. For analysis, Mice are sacrificed between 3 days post resection to 10 weeks post
resection. Also as described in (Tripuraneni et al., 2015).
Histological analyses
Samples were fixed with 4% PFA overnight at room temperature, 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 7 microns thick. The
sections were mounted on Superfrost Plus slides (VWR, 48311-703). After deparaffinizing slides
in Citrisolv and rehydrating, H and E or Safranin O staining was carried out using standard
protocols. Skeletal staining was performed on EtOH-fixed samples (Rigueur and Lyons, 2014).
To visualize native tdTomato fluorescence, samples were fixed in 4% PFA on ice for 30 min and
placed in 30% sucrose overnight at room temperature. 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 (Kawamoto, 2003). OCT was removed with a 1xPBS wash before mounting.
Immunofluorescence
Detection of Tdtomato proteins was carried out on paraffin sections. Slides were de-waxed
and cells were permeabilized with 0.1% Triton-X. Slides were blocked in 20% serum for 1 hour
and then incubated with the primary antibodies overnight at 4°C (anti-mCherry which also detects
tdTomato, Novus Biological: NBP2-25158SS, 1:200). The secondary antibody Alexa Fluor 568
goat anti-chicken (Abcam: ab175477, 1:500) was used.
RNA in situ hybridization
Fluorescent and colorimetric RNA in situ hybridization (RNA-ISH) was performed on 7 µm
paraffin sections as previously described (Paul et al., 2016), with some changes. The incubation
in PK was increased to 8 minutes and the incubation with the fluorophore was decreased to 10
minutes. 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). For
double fluorescent RNA-ISH the TSA Cyanine 3 and Fluorescein system from Perkin Elmer was
used as directed (NEL753001KT). For colorimetric RNA-ISH Anti-Digoxigenin-AP (Sigma-Aldrich:
11093274910) was used to detect the probes.
Probes were generated to the following sequences:
25

Slides with fluorescence were mounted with Vectashield with DAPI (Vector Laboratories:
H1200) and were imaged with a Nikon AZ100 Macroscope and photographed (Nikon Digital sight
DS-Fi1). Fluorescent images were edited for contrast and color levels in Adobe Photoshop CS5.
2.3 Results
Ribs regenerate through a cartilage intermediate made up of hybrid skeletal cells
Like appendicular long bones, the bony portion of the rib develops via an endochondral
process including growth plates at either end and a central hollow bone marrow cavity. Both
human and murine rib bones display remarkable regenerative potential (Tripuraneni et al.,
2015)(Srour et al., 2015), however the cellular basis for such large-scale repair remains unknown.
To better understand the cellular sequence of events during regeneration, 3 mm rib bone defects
at sequential time points up to 10 weeks post-resection (wpr) were analyzed (Figure 2.1).
Histology at 5 days post-resection (dpr) revealed cells with a mesenchymal-like morphology filling
the entire resected region (Figure 2.2A). Formation of a substantial alcian-blue positive callus
spanning most of the defect by 1 wpr (Figure 2.1) was observed, with many of these cells
displaying a cartilage-like morphology at 10 dpr (Figure 2.2B). Histology revealed increasing bone
formation by 10 and 14 dpr (Figure 2.2B, C), with extensive alizarin-positive mineralization across
the defect at 4 wpr and full remodeling to pre-injury organization by 10 wpr (Figure 2.1).
Next, double fluorescent RNA in situ hybridization (RNA-ISH) were used to characterize
the molecular identity of cells during the regeneration process. At 5 dpr, mesenchymal cells within
the lesion co-expressed Sox9, a master regulator of the chondrocyte lineage (Akiyama et al.,
2002) (Lefebvre et al., 1997), and Runx2, a master regulator of the osteoblast lineage (Lian and
Stein, 2005) (Figure 2.2A). While co-expression of chondrocyte and osteoblast markers has been
observed during early bone development, normally as differentiation proceeds a cell will express
only chondrocyte or only osteoblast markers. Surprisingly, I observed co-expression of genes
26

associated with chondrocyte and osteoblast differentiation within single cells throughout the repair
process. For example, co-expression of Sox9 with the major osteoblast collagen gene Col1a1
was seen (Supplemental Figure 2.1A). As the callus matures bone and cartilage markers continue
to be co-expressed at high levels. The major chondrocyte collagen gene Col2a1 was observed to
be co-expressed with the late osteoblast marker Bglap (also known as Osteocalcin) (Figure 2.2B
and Supplemental Figure 2.1C), and with Col1a1 at 10 and 14 dpr (Figure 2.2C and Supplemental
Figure 1B). I observed co-expression of chondrocyte and osteoblast markers in cells of both
cartilage and osteoblast morphology, including cells on the surface of newly formed trabecular
bone (Figure 2.2B, C, Supplemental Figure 2.1C).
Similar co-expression results were obtained using mice double transgenic for a reporter
of hypertrophic chondrocytes (Col10a1-mCherry) and osteoblasts (Col1(2.3)-GFP) as well as
using double immunofluorescence for COL1 and COL2 protein (Figure 1D, Supplemental Figure
1D). In contrast, co-expression of Col2a1 and Col1a1 in the rib growth plate under the same assay
conditions was not observed (Figure 2.1E). Single colorimetric RNA-ISH assays of near-adjacent
sections at 7 dpr confirmed co-expression of Sox9, Runx2, Col2a1, Col1a1, Bglap, and the
hypertrophic cartilage collagen gene Col10a1 in cells of cartilage morphology within the callus
(Supplementary Figure 2.2). Together, these findings indicate that, in marked contrast to the
growth plate, cells within the rib repair callus co-express cartilage and bone markers while
displaying either a chondrocyte or osteoblast morphology. I therefore refer to these cells as
“hybrid” osteochondral skeletal cells.
Lineage tracing of Sox9-expressing cells to the regenerating rib callus
I next examined the source of the hybrid skeletal cells that mediate repair. These cells are
likely derived from the periosteum, since removal of the periosteum along with the bone, results
in a failure of cartilage callus formation (Supplemental Figure 2.3) and subsequent bone repair
(Tripuraneni et al., 2015). Sox9 is a master regulatory gene of chondrogenesis, and a previous
study indicated that Sox9-positive cells in the femur periosteum can contribute to callus formation
after fracture (He et al., 2017). I therefore examined whether Sox9-expressing periosteal cells
contribute to the repair callus during rib regeneration. To do so, tamoxifen was administered for
three consecutive days to Sox9-CreERT2; ROSA26-loxP-stop-loxP-tdTomato mice, followed by
a 4-day chase to allow clearance of residual tamoxifen (Figure 2.3A, “Pre” regimen). In the
absence of injury, I observed that Sox9-expressing cells were predominantly found in the
periosteum and constituted 6 0.3% of the population, with almost no labeled cells seen in the
marrow compartment (Figure 2.3B). After rib resection, 22 1.3% of callus cells were tdTomato+
by 10 dpr with many of these cells having a typical chondrocyte morphology (Figure 2.3C). At 14
27

dpr (Figure 2.3C), tdTomato expression in both chondrocyte-like cells, as well as osteoblast-like
cells lining new trabecular bone was observed. Fewer tdTomato+ cells were seen in the callus at
14 dpr suggesting that as the callus matures, Sox9+ lineage cells are remodeled out and replaced
by non-Sox9+ lineage cells. These data indicate that pre-existing Sox9-expressing cells, likely
from the periosteum, contribute to only a minority of the cells that form the initial repair callus,
including only a subset of the callus cells with hybrid osteochondral characteristics. Thus, most of
the cells in the callus are derived from a lineage that did not express Sox9 prior to injury.
Since only a minority of the callus is derived from the Sox9-expressing periosteal
subpopulation, as opposed to the majority of the callus that expresses Sox9 mRNA at 5 dpr
(Figure 2.2A), tamoxifen was applied at the time of injury plus 2 days following (“Post” regimen,
Figure 2.3D). This regimen will capture both the subpopulation that expresses Sox9 prior to injury
as well as any cells that turn on Sox9 in response to injury. Consistent with detection of
endogenous Sox9 mRNA expression in early callus cells (e.g. Figure 2.2A), this later tamoxifen
regimen labeled the majority of callus cells by 10 dpr (85.5 2.8%) and most of the osteoblast-like
cells lining newly forming trabecular bone at 14 (Figure 2.2E). Thus, depending on when
tamoxifen is administered (Pre vs. Post), either a minority or majority of the callus cells are
labelled. At early time points, 7 dpr, control mice (CD-1 background) injected with Tamoxifen
repaired on the same timeline that lineage traced mice did (Supplemental Figure 2.3).
2.4 Discussion
High-level co-expression of genes typically associated with cartilage (Sox9, Col2a1,
Col10a1) or bone (Col1a1, Bglap) were observed in callus cells during murine rib bone
regeneration. This is in marked contrast to the largely exclusive expression of cartilage versus
bone genes in the developing growth plates, although hypertrophic chondrocytes do express low
levels of many bone-associated genes (Gerstenfeld and Shapiro, 1996). The trajectory of hybrid
cells is also fundamentally different from the subpopulation that is proposed to “transdifferentiate”
from chondrocytes to osteoblasts in the growth plate (Bahney et al., 2014) (Shimomura et al.,
1975)(von der Mark and von der Mark, 2018) (Yang et al., 2014a) (Zhou et al., 2014c). In the
growth plate, these transdifferentiating cells express chondrocyte-associated genes first and then,
through a process that remains mysterious, turn off chondrocyte-associated genes and turn on
osteoblast-associated genes. Here, I confirm a similar process of “first cartilage and then bone”
in the rib growth plate. In contrast, in the regenerating rib, I observe that callus cells co-express
cartilage- and bone-associated genes at the earliest stages. I propose that they then maintain this
hybrid osteochondral identity as they shift from making a cartilaginous and then a bone-like matrix.
Moreover, similar hybrid cells have been reported during zebrafish jawbone repair (Paul et al.,
28

2016) suggesting that skeletal cells with dual chondrocyte/osteoblast properties may be critical
for bone regeneration across vertebrate species.
Co-expression of bone and cartilage programs in the same cell is certainly not unique to
the regenerating callus. For example, Col1a1 and Col2a1 are also highly expressed in
fibrocartilage cells, however this tissue is morphologically quite different in terms of its high fiber
to cell ratio when compared to the regenerating rib callus (Benjamin and Ralphs, 2004). Of note,
a rare developmental skeletal type has been described, historically referred to as “chondroid
bone” or “secondary cartilage”, that does share many features with the hybrid osteochondral cells
observed during regeneration (Goret-Nicaise, 1984) (Shibata and Yokohama-Tamaki, 2008).
Therefore, it may be that these cells are not unique to regeneration, but rather are selectively
utilized due to their ability to rapidly proliferate in a relatively avascular environment (a property
of cartilage (Dennis et al., 2015)) while directly producing mineralized matrix (a property of bone).
The periosteal origin of the callus cells may also be the reason for the high co-expression.
Periosteal cells have been shown to express Col1a1 (Kawanami et al., 2009). In homeostasis,
periosteal cells will contribute to new bone formation and may be primed to differentiate into
osteoblasts. In response to injury many of these cells will differentiate into chondrocytes. It is
possible that because these cells normally differentiate into bone and already express Col1a1,
they may simply start to express cartilage genes while also expressing osteoblast associated
genes.
The lineage tracing results under both tamoxifen induction schedules showed that the
Sox9-CreERT2 line can be used to mark either a minority or majority of cells in the repair callus.
These results provided me with an interesting tool that could be used to study a majority of the
repair cells, or just one of the periosteal progenitor subpopulations based on when tamoxifen is
administered. The increase in labeling when Tamoxifen was injected after injury (Post) indicates
that, in response to injury, many cells involved in forming the repair callus upregulate Sox9
expression. This was not surprising since Sox9 is an early osteochondral marker. It was also not
surprising to find Sox9 expression progenitors in the periosteum of the uninjured rib bone or that
they contribute to the repair callus, similar to what has been previously reported in the femur (He
et al., 2017). The Sox9 expressing periosteal progenitors did not appear to have a preferential
ability to differentiate into cartilage over bone but were able to contribute to both during the repair
process. These cells seem to be remodeled out as the callus matures, this could indicate that
these cells have an early role during repair but are not required for later stage, such as
remodeling. In the future it will be important to determine if some of the other progenitors such as,
Gremlin1 (Worthley et al., 2015), Axin2 (Ransom et al., 2016), Gli1 (Shi et al., 2017), and Act2a
29

(Matthews et al., 2014), are also found in the rib and contribute to the repair callus. It remains
unclear how these populations relate to one another and how they interact to facilitate the repair
process.

2.5 Figures

Figure 2.1 Schematic of the murine rib resection model.
A 3 mm bone segment is resected from one rib (8-11), while the periosteum is carefully released and
left in the mouse. Alizarin red and alcian blue whole mount staining indicates that repair occurs
through a cartilage intermediate. The figure shows an image of a rib immediately after resection, with
an empty space at 0 wpr. At 1 wpr alcian blue positive material is evident between the cut ends, by 4
wpr the lesion is fully-spanned by a mineralized callus, and by 10 wpr remodeling has occurred.
30

31

Figure 2.2 Regeneration involved skeletal cells with dual cartilage-bone properties
Histological sections stained with hematoxylin and eosin (H&E) show the morphology, while near
adjacent double fluorescent RNA in situ hybridization (RNA-ISH) assays confirm the presence of a
cartilage intermediate and show expression patterns in the repair callus. (A) At 5 dpr mesenchymal-like
progenitor cells have moved into the resected region and are positive for the expression of both Sox9
(green) and Runx2 (red). The enlarged boxes with the separated color channels show co-expression in
many of the cells within the resected region (overlap is yellow). (B) By 10 dpr bone and cartilage
formation spans the resected region. Many cells that mediate repair express both the chondrocyte-
associated gene Col2a1 (green), as well as the osteoblast-associated gene Bglap (red). The enlarged
boxes show cells with chondrocyte morphology expressing both Col2a1 and Bglap. (C) At 14 dpr
trabecular bone spans almost the entire resected region with only a small amount of cartilage at the cut
ends, cells expressing both Col2a1 (green) and the osteoblast-associated gene Col1a1 (red) are
widespread. The enlarged boxes show the surface of newly formed trabecular bone where cells can be
found that co-express Col1a1 and Col2a1. (D) At 10 dpr, animals double transgenic for Col1(2.3)-
GFP;ColX-mCherry have mCherry (red) positive cells that are also expressing the osteoblast specific
reporter for Col1 (green). (E) Expression of Col2a1 (green) and Col1a1 (red) of the rib growth plate from
an uninjured animal does not show a high degree of overlap. Col2a1 is highly expressed in chondrocytes
of the growth plate but not in osteoblasts forming new bone, while Col1a1 is highly expressed in the
osteoblasts below the growth plate but not in cartilage cells.
32

Figure 2.3 Sox9-CreERT2 marks cells that participate in repair
(A) To target a sub-population of callus cells that arise from the periosteum, Sox9-
CreERT2;tdTomato mice were injected for 3 consecutive days starting 7 days before
analysis/surgery. This tamoxifen induction schedule is referred to as “Pre”. (B) In uninjured Sox9-
CreERT2;tdTomato mice, Sox9+ cells can be observed within the periosteum of the diaphysis by
immunofluorescence (IF) for the tdTomato protein (red). (C) To determine if these cells participate in
repair, rib resections were performed. At 10 dpr, lineage tracing of the Sox9+ cells show that the
tdTomato+ expressing cells contribute to both cartilage and bone. The enlarged images shows
chondrocytes, some of which are positive for tdTomato along with a near-adjacent section showing
the histology of the area. At 14 dpr, tdTomato-expressing cells can be seen contributing to the
trabecular bone. The enlarged image shows cells lining the trabeculae that are positive for
tdTomato. (D) To activate Cre in a larger percentage of cells that build the repair callus, Sox9-
CreERT2;tdTomato mice were injected with tamoxifen for 4 consecutive days, starting the day
before surgery took place, the day of surgery, and for 2 more days. This tamoxifen induction
schedule is referred to as “Post”. (E) At 10 dpr, tdTomato+ cells are present in developing cartilage
and bone, as well as in the periosteum surrounding the callus (native tdTomato fluoresence from a
cryosection). The enlarged panel shows cells with chondrocyte morphology that are tdTomato+. At
14 dpr, tdTomato+ cells can be seen building new trabecular bone. In comparison to the Pre-
induced mice, these mice have significantly more tdTomato+ cells within the repair callus.
33

Supplemental Figures

Supplemental Figure 2.1 Analysis of hybrid skeletal cells
(A) At 7 dpr the early progenitors not only express Sox9 (green) but also express the osteoblast-
associated gene Col1a1 (red). (B) At 10 dpr, most of the callus cells co-express the chondrocyte-
associated gene Col2a1 (green) and osteoblast-associated gene Col1a1 (red), especially those
cells with chondrocyte morphology (n=4). (C) At 14 dpr, cells in the newly formed trabecular bone
co-express Col2a1 (green) and Bglap (red). (D) At 10 dpr, cells can be found that co-express
COL1 (red) and COL2 (green) protein.

34

Supplemental Figure 2.2 Colormetric RNA-ISH
Near-adjacent sections of H&E staining and colormetric RNA-ISH of a 7 dpr repair callus suggests
that similar cell populations express genes associated with cartilage (Sox9, Col2a1, Col10a1) as well
as bone (Runx2, Col1a1, Bglap). The cartilage genes Sox9, Col2a1, and Col10a1 are strongly
expressed in cells with chondrocyte morphology with Col10a1 expression most prevalent in the more
mature, hypertrophic chondrocytes. The expression of Runx2, Bglap, and Col1a1 (genes associated
with bone production) could also be found in these populations along with their expected expression
in cells located in newly formed bone.
Supplemental Figure 2.3 Removal of periosteum and Tamoxifen control
(A) Resections were performed in which both the bone and periosteum (PO) were removed. No
cartilage is evident at 7 dpr. Sox9 (red) and Runx2 (green) expression is not readily detectable.
Scale bar = 100 microns; enlarged box = 50 microns (B) Control mice (CD-1) injected with Tamoxifen
show that they heal along the same timeline as experimental mice.
35
1
Large portions of this work (Chapter 2 and 3) are published in eLife (Kuwahara et al., eLife
2019;8:e40715)
Chapter 3 The role of Hh signaling in murine rib regeneration
1
3.1 Introduction
The signaling pathways required for generating a cartilage callus from the periosteum in
response to injury are also not well-characterized. The formation of the cartilage callus in fractures
has been thought to involve a recapitulation of endochondral bone development (Bahney et al.,
2014) (Maes et al., 2010) (Marsell and Einhorn, 2011) (Yang et al., 2014b) (Zhou et al., 2014c).
One of the most well-known signaling pathways during endochondral bone development is the
Hh pathway, activation of which promotes chondrogenic proliferation and osteocyte maturation
(Long et al., 2001) (Shi et al., 2017) (St-Jacques et al., 1999). Studies have investigated Hh
signaling during fracture repair, but due to conflicting results it remains unclear if Hh signaling has
the same role in bone repair as it does during bone development. Genetic ablation of the obligate
Hh co-receptor Smo in mice, using two different ubiquitously inducible Cre lines, resulted in
reduced bone formation during fracture repair, yet was not reported to disrupt initial cartilage
callus formation (Baht et al., 2014) (Wang et al., 2010). Forced activation of Hh signaling
throughout the mouse during fracture repair, using an inducible constitutively active Smo allele,
resulted in increased bone formation (Baht et al., 2014), similar to what was seen upon
engraftment of cells overexpressing Hh or treatment with an Hh agonist (Edwards et al., 2005)
(Wang et al., 2010) (Zou et al., 2014a). However, on which cell types Hh acts upon, and whether
it regulates the decision of periosteal cells to build the cartilage callus and/or other aspects of
bone repair in mammals, has remained unknown.
In this study I examine the role of the Sox9+ periosteal subpopulation during large-scale
rib repair. This subpopulation appears to be a key player in orchestrating the formation of a large
repair callus that consists of an unusual hybrid osteochondral cell type with properties of both
chondrocytes and osteoblasts. Loss of Smo in Sox9+ periosteal cells prior to injury results in a
near-complete failure of cartilage callus formation and bone regeneration. This Sox9+
subpopulation must be able to respond to Hh signaling in order to initiate this process, indicating
that Hh signaling’s role in bone repair is distinct from its role in bone development. Additionally,
since Sox9+ periosteal cells contribute to only a minority of callus cells, suggesting that Sox9+
periosteal cells act as “messenger” cells and orchestrate repair by inducing the differentiation of
neighboring callus cells through non-autonomous signals. Over all these results indicate that bone
regeneration does not fully recapitulate bone development, and that the periosteum consists of
subpopulations that may have different roles/responses during repair.
3.2 Materials and methods
Mice and animal housing
36

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-
CreERT2 (Sox9
tm1(cre/ERT2)Haak
(Soeda et al., 2010)), R26R-tdTomato (B6;129S6-
Gt(ROSA)26Sor
tm9(CAG-tdTomato)Hze
/J; JAX 007905), Smo
fl/fl
(Smo
tm2Amc
/J; JAX 004526, Col1a1(2.3-
GFP) (B6.Cg-Tg; Col1a1*2.3-GFP)
1Rowe/J
;JAX 013134), Col10a1-mCherry (Maye et al., 2011),
SmoM2 (Jeong et al., 2004).To generate Sox9-CreERT2;tdTomato;Smo
fl/fl
mice, Sox9-
CreERT2;tdTomato males were crossed to Smo
fl/fl
females and Sox9CreERT2;tdTomato;Smo
fl/+
offspring males were back-crossed to Smo
fl/fl
females to generate Sox9-
CreERT2;tdTomato;Smo
fl/fl
offspring. Both male and female mice between 6-8 weeks old were
used for experiments. Control mice were uninduced siblings or tamoxifen-induced Sox9-
CreERT2;tdTomato mice.
Murine rib resections, histology, In situ hybridization, and immunofluorescence
These were carried out as previously described in chapter 2. Additional antibodies were
used (anti-pHH3, Millipore: 06-570, 1:200; anti-mCherry which also detects tdTomato, Novus
Biological: NBP2-25158SS, 1:200; anti-Col1, Abcam: ab34710, 1:250; anti-Col2, Southern
Biotech: 1320-01, 1:200). Secondary antibodies used were: Alexa Fluor 568 goat anti-rabbit
(ThermoFisher: A-11011, 1:250), Alexa Fluor 568 goat anti-chicken (Abcam: ab175477, 1:500),
and Alexa Fluor 488 donkey anti-goat (Abcam:ab150129, 1:500).
TUNEL
To detect apoptosis, the In Situ Cell Death Detection Kit, Fluorescein (Sigma-Aldrich:
11684795910) was used as directed.
ISH in combination with immunofluorescence
To detect if the lineage traced cells were also expressing certain mRNA, ISH were
combined with immunofluorescence. The ISH was performed first to completion, up until the
tissue is to be mounted with DAPI. The immunofluorescence is then carried out starting with
permeabilization with 0.1% Triton-X. Once the immunofluorescence is completed the slides are
mounted with DAPI.
Periosteal implants
The periosteal implants were done as described in (Srour et al., 2015), with some
modifications. A rib bone was removed, and the periosteum was striped and implanted in the
intercostal muscle, instead of the costal cartilage and periochondrium that was described in the
paper.
Quantification and Statistical Analysis

37

For quantification, all images were taken at the same magnification for each data set.
Student’s t-test was used to compare groups. A probability value of 0.05 or less was considered
significant. Each data point was plotted on the scatter plot and the mean was defined on the
graph, unless the data set was normalized. Statistical tests were performed using GraphPad.
To determine the amount of cartilage, a mid-sagittal section through both mutant and
control sample was stained with Safranin O and quantified in ImageJ. In brief, the image was
thresholded for the Safranin O color (orange) and the area of the color was measured. To
determine bone area, samples were stained with H&E and analyzed with the BioQuant image
analysis program. The bone area was compared to the entire resected area. The values were
normalized within each time point.
Quantification of the number of cells expressing pHH3 was carried out using the analyze
particle function in ImageJ with the repair callus defined as the area of interest. The red channel
was used to count the number of cells positive for pHH3 while the blue channel was used to count
the total number of cells (nuclei stained with DAPI). The ratio of pHH3 positive cells to total cells
was used to calculate the percentage of cells in mitosis. This method was also used to quantify
the number of tdTomato+ cells within the callus. To quantify apoptosis, Adobe Photoshop CS5
was used to analyze the green channel. The number of green pixels compared to the total number
of blue pixels (DAPI) in the region of interest was calculated. Analysis of pHH3 and TUNEL
positivity was done on de-identified images by several laboratory members.
3.3 Results
Expression pattern of Hh signaling
Since I hypothesized that the Hh pathway may be important for large-scale repair, I first
determined the expression of Hh ligand after rib resection. As expected, based on the expression
of Ihh in the growth plate, callus cells with cartilage morphology expressed Ihh and Ptch1 (Figure
3.1A, C). Interestingly the related ligand, Shh was also detected in these cells. At earlier stages
Ihh expression was undetectable until 7 dpr and then only at very low levels in developing
chondrocytes. However, Shh expression was readily detectable at 5 dpr and 7dpr (similar to
(Matsumoto et al., 2016)) although at lower levels than found in cells with cartilage morphology
(Figure 3.1A, 3.1C). At early stages (3 dpr) Ptch1 expression, a read-out of the Hh pathways, was
also observed in tdTomato+ periosteal cells, supporting the idea that these cells are responsive
to a Hh signaling (Supplemental Figure 3.1).
Requirement of Hh signaling in Sox9-expressing cells for callus formation
The requirement for Hh signaling in large-scale bone regeneration was tested by deleting
Smo, the required Hh co-receptor, using the Sox9-CreERT2 transgene and employing either the
38

Pre or Post tamoxifen treatment regimens. Using Smo RNA-ISH, I observed that the Post
tamoxifen treatment resulted in deletion of Smo broadly (Figure 3.1B) as predicted given that Cre
is active in most cells (Figure 2.3E). Pre tamoxifen treatment resulted in deletion of Smo in the
Sox9-positive lineage subpopulation which were marked by including the tdTomato reporter in
the cross (Figure 3.1B). This was expected, since deletion of Smo occurred prior to surgery and
thus only a minority of the cells in the repair callus are expected to be null for Smo (Figure 2.3C).
Next the expression of Hh pathway members was examined. To do this I decided to
examine the callus at 7 dpr while there is a mix of both immature and differentiating cells. In
control Pre tamoxifen treatment animals, tdTomato+ cells could be found scattered across the
developing callus and were not preferentially found in clusters of differentiating chondrocytes
(Supplemental Figure 3.1B). In regions of the control callus where cells appeared immature and
in Pre tamoxifen treatment calluses, there was no strong correlation between the tdTomato trace
and the expression of Shh, or read-outs of Hh signaling such as Ptch1 or Gli2 (Pak and Segal,
2016) (Figure 3.1C and Supplemental Figure 3.1C). In addition, in those cells neighboring
Tdtomato+ cells, enrichment of Ptch1 or Gli2 expression was not seen suggesting that they were
not receiving a potent Hh signal.
The consequence of Smo deletion on cartilage callus formation was investigated.
Surprisingly, Smo deletion using either the Pre or Post tamoxifen treatment resulted in a similarly
near-complete loss of the cartilage callus at both 7 and 10 dpr (Figure 3.2A), despite the Pre
treatment only deleting Smo in a small subset of callus progenitors. After Smo deletion, although
cells did not form a mature callus, they still co-expressed Sox9 and Runx2 at 5 dpr (Supplemental
Figure 3.2A). At 10 dpr, in contrast to co-expression of Col2a1 and Col1a1 throughout the control
callus, the few Col1a1-expressing cells that formed after Smo deletion lacked high levels of
Col2a1 and vice versa, demonstrating a lack of hybrid cells (Figure 3.2B).
The lack of callus formation and hybrid cells following Smo deletion was not reflected by
altered numbers of pHH3+ proliferative or TUNEL+ apoptotic cells at either 7 or 10 dpr, and
lineage tracing with the tdTomato reporter revealed similar numbers of cells within the resection
site at 10 dpr (Supplemental Figure 3.2B-D). Thus, Hh signaling is likely not required for the early
proliferative expansion in response to injury or for cell survival. Instead, Hh is essential in the
Sox9+ cells for promoting the differentiation of non-Sox9 lineage cells into hybrid cells of the
callus. Thus, in the absence of Smo in the Sox9+ Pre population, non-Sox9 lineage cells are still
unable to differentiate even though they can respond to Hh signaling. Further, the near complete
failure of callus formation points to a critical initiating role for this small population of Sox9+
periosteal lineage cells in callus formation.
39

Requirement of Hh signaling in Sox9+ lineage cells for rib bone regeneration
Next the consequence of a defective cartilage callus formation on subsequent
regeneration of the rib bone in Smo-deleted animals was determined. In contrast to control
animals showing robust Col1a1 and Col2a1 co-expression in cells lining new trabecular bone at
14 dpr, deletion of Smo using either the Pre or Post regimens resulted in a near complete absence
of co-expressing cells. Instead, only small numbers Col2a1-only cells were observed, primarily
near the cut ends of the bone, while cells with osteoblast morphology expressed predominantly
only Col1a1(Figure 3.3A). Analysis of H&E-stained histological sections confirmed a marked
decrease in bone formation at 14 dpr, despite substantial mesenchyme still observed in the
resection site, with the magnitude of the bone defect similar in both regimens (Figure 3.3B). At 21
dpr the cells in the callus still have mesenchyme progenitor morphology. Double RNA-ISH for
Runx2 and Sox9 showed that the cells still co-express these markers (Supplemental Figure 3.3).
These results further indicate that the cells in the Smo KO calluses fail to differentiate. “Late”
removal of Smo (injection of tamoxifen at 3-5 dpr targeting the whole callus) only causes a slight
delay in repair with a fully bridged callus evident at 14 dpr (Supplemental Figure 3.4) suggesting
that the decrease in bone formation seen in both the Pre and Post regimens is largely related to
reduced cartilage callus formation. Whole-mount staining with alizarin red and alcian blue
confirmed a failure of bone union at 6 wpr in both Pre KO and Post KO conditions and at 8 wpr in
the Pre KO condition (Figure 3.4). These findings demonstrate that the Sox9+ subpopulation
requires Hh signaling not only to build the repair callus but that a substantial cartilage callus may
be needed to efficiently regenerate bone.
Constitutive activation of Hh signaling
Since the ablation of Hh signaling prevents the periosteal cells from differentiating into
chondrocytes during large scale repair, I wanted to determine if overactivation of the Hh pathway
could induce chondrogenic differentiation of the periosteal cells. A ubiquitous inducible Cre line
(CAGG-CreERT) was crossed to the SmoM2 mouse line. Upon tamoxifen injection these mice
would express a constitutively active version of the Smo receptor, therefor having continual
activation of the Hh pathway. Mice were injected with tamoxifen and later had their periosteum
from the ribs stripped. These experiments were performed and analyzed by Jason Hsieh, another
member of the Mariani Lab. Keeping the structural integrity of the periosteum intact as much as
possible, the periosteum was implanted in the intercostal muscle between the ribs. When
examined 7 days after implantation both bone and cartilage formation could be by H&E staining
and Safranin-O staining. In contrast, when control periosteum, which did not have overactivation
of the Hh pathway, was implanted no cartilage formation could be seen and there was overall less
40

bone formation. While the SmoM2 implants appear to undergo endochondral ossification to form
bone, the control implants go through intramembranous ossification (data not shown, personal
communication).
To determine how the overactivation of the Hh pathway could affect bone repair, I used
Sox9CreERT2;SmoM2 mice. These mice were injected with Tamoxifen using the “Post” schedule
(Figure 2.3 D), causing the SmoM2 receptor to be expressed in a majority of the callus cells.
When compared to controls the SmoM2 callus appears more organized and mature at the early
time points 5 dpr, as seen by H&E staining (Figure 3.5 A). A near adjacent section was used for
a double RNA ISH of Col1a1 and Col2a1 which showed an increase in hybrid cells (Figure 3.5
B). At 7 dpr the cartilage callus appears to be much larger when compared to controls (Figure 3.5
C).
3.4 Discussion
These results show that building an extensive callus of a unique type of hybrid
osteochondral skeletal cell is essential for successfully bridging large gaps in adult mammalian
rib bone. During this process, Hh signaling plays a critical role, distinct from that in the developing
growth plate, in promoting the differentiation of these hybrid osteochondral skeletal cells. Further,
in response to Hh signaling a rare periosteal subpopulation of Sox9 expressing cells, acts as a
messenger cell. This Sox9+ periosteal subpopulation stimulates, through a yet to be determined
signal, their neighboring non-Sox9+ lineage cells, which constitutes a majority of the callus, to
differentiate and build new callus and bone (summarized in Figure 3.6).
Whereas some bone does form in the Smo-deleted mice, this is not sufficient to bridge the
lesion and healing fails, resulting in a persistent non-union. Cells within this bone do not display
hybrid chondrocyte/osteoblast character, consistent with residual bone forming by direct
ossification rather than ossification through a callus intermediate. These findings suggest that
during large-scale bone regeneration, Hh signaling in Sox9-expressing periosteal cells is
selectively required for the formation of a hybrid osteochondral callus. Hh signaling may play an
important but subtler role in osteocyte maturation. Production of bone can be seen in the rib
resection model with a Late KO of Smo, although it is delayed (Supplemental Figure 3.4).
Similarly, fractures still heal in the absence of Smo with decreased or delayed bone formation
(Baht et al., 2014) (Wang et al., 2010). However, in contrast to the rib model, loss of Smo in these
fracture assays still resulted in the formation of a cartilage callus. It is unclear why these results
contrast from what I see where cartilage callus formation is dramatically compromised. One
possibility is that the efficacy of Smo removal in these fracture experiments was not efficient during
cartilage callus stages, alternatively, there could be different requirements for Hh signaling
41

depending on the type of injury (resection vs. fractures). Interestingly, the formation of a cartilage
callus may not be as critical during fracture repair, as fractures can repair solely through direct
ossification (Colnot, 2003). While in contrast, during large-scale repair, the process of direct
ossification is not sufficient to build large pieces of bone and instead, building a hybrid
osteochondral callus may be particularly important for bridging large bone gaps.
Hh signaling is known to promote chondrocyte proliferation and osteoblast differentiation
in the developing growth plate (Long et al., 2001) (Long et al., 2004). Surprisingly, loss of Hh
signaling did not affect the early proliferation of callus cells or the differentiation of osteoblasts in
the residual directly ossifying bone. Instead, these results provide evidence that Hh signaling has
a distinct and essential role in inducing the differentiation of Sox9/Runx2 expressing progenitors
into the hybrid osteochondral skeletal cells that form the repair callus. These results indicate that
the regeneration of the rib bone, including its dependence on Hh signaling, does not simply the
recapitulate developmental processes seen at the growth plate.
While the heterogeneity of the cells in the periosteum and their lineage relationships
remain incompletely understood, I propose that Sox9+ periosteal cells or a subpopulation within
them, play an essential instructive role for callus formation during large-scale bone regeneration
that has not been previously described in the context of repair. This is the first study to show a
specific role for a repair progenitor population beyond participation. Although the Sox9+ cells fall
within the periosteum, they could have very different expression patterns or epigenetic
landscapes from the other periosteal cells, either of which could allow them to respond to repair
signals uniquely from other cells. How this Sox9+ population relates to, differs from, and interacts
with other progenitor populations during repair will be important to determine. The Sox9+
periosteal subpopulation may be distinct from the previously described subpopulations that are
Gli1+, aSMA+, Gremlin1+, or Axin2+, as RNA-seq analysis of the periosteum from an uninjured
femur indicated that Gli1, aSMA, Axin2, and Gremlin1 were not highly expressed in the Sox9+
cells (He et al., 2017). Future studies tracing the lineage of these other populations and
determining their dependence on Hh signaling will be needed to resolve whether Sox9+ cells
represent a subset of one of these other more abundant populations, or alternatively a distinct
progenitor subpopulation. In addition, efforts to delineate potential differences that may exist in
periosteal populations from different bones, may explain why some bones regenerate well, while
others do not.
One of the most striking findings from this study is that Sox9+ cells are essential for
efficient callus formation and rib bone regeneration, despite contributing to only a minority of cells
within the callus and regenerated bone. Based on this observation, I propose that the Sox9+ cells
42

act as “messenger” cells by releasing a yet to be determined signal that promotes the
differentiation of neighboring Sox9-negative lineage cells. Without this signal the neighboring
progenitors remain Sox9/Runx2 positive but are unable to further differentiate. One possibility is
that Sox9+ progenitors differentiate early in response to the initial Hh signal (potentially Shh, since
strong expression is evident early) and then propagate another wave of Hh signaling to
neighboring Sox9-negative cells, similar to the role of Hh in the morphogenetic furrow during
Drosophila eye development (Domínguez and Hafen, 1997) (Kumar, 2011) (Ma et al., 1993). The
failure of Sox9+ cells to differentiate and thus express Shh and Ihh, could then lower the total Hh
signal in the callus to below a critical threshold need for cartilage differentiation. However, these
results suggest that Sox9+ cells are not the first to differentiate and while they likely respond to a
Hh signal early in the periosteum, they are likely not strong propagators of a further Hh signal as
cells nearby Sox9+ lineage cells do not display a strong upregulation of Hh pathway read-outs as
the callus differentiates. While a response to Hh signaling may require more sensitive assays, the
expression of both Ptch1 and Gli2 was found to be very low in undifferentiated cells at 7 dpr in
both control and Pre Smo KO calluses (Figure 3.1C and Supplemental Figure 3.1C) suggesting
that they were not receiving a potent secondary Hh signal. In addition, the expression of Shh was
not particularly strong in the tdTomato+ cells at this stage (Figure 3.1C). Thus, I instead favor a
hypothesis, that in response to Hh signaling Sox9+ cells emit another to-be-identified relay signal.
This signal then orchestrates repair by promoting the differentiation of neighboring cells, from a
non-Sox9+ lineage, into mature matrix-producing hybrid osteochondral skeletal cells that bridge
the lesion (Figure 3.5). Future investigations into factors produced by Sox9-lineage cells that
promote callus formation may lead to better strategies of boosting bone repair in other parts of
the body that do not heal as effectively.
While this study supports a critical role for Hh signaling early in large-scale bone repair, it
is possible that Hh signaling may have additional roles in later stages of repair. Hh signaling could
also affect chondrocyte proliferation or osteoblast differentiation, like it does during bone
development. It is not possible to assess the role in chondrocyte proliferation since there is
minimal chondrocyte formation in the Smo KO. The “late” KO of Smo experiments support Hh
signaling having an additional role in repair. In this KO a majority of the cells in the callus become
null for Smo 4 days after injury. Although the repair callus can form and fully bridge the resected
region, it is smaller and appears to be delayed. While most cells are unable to respond to Hh
signaling, bone and cartilage formation can still be seen. It is unclear how the “Late” KO of Smo
is affecting the repair cells but there are several possibilities. One possibility is that although the
43

repair cells can differentiate into chondrocytes, they are unable to proliferate properly, resulting in
a smaller cartilage callus and later a smaller and delayed boney callus.
One of the main unanswered questions is the expression pattern of Hh signaling during
repair. Although these and previous results have begun to elucidate this question (Figure 3.1 and
Supplemental Figure 3.1), the results were incomplete. Which specific cells are producing the
ligand, which Hh ligand is being used, and which cells are responding still need to be determined.
IHH is the ligand known to have a critical role in bone development for chondrocyte proliferation
and osteoblast differentiation, but my results have indicated that only the Shh transcript can be
seen in the early repair callus when Hh signaling appears to be critical. The strongest read out of
Hh signaling, by Ptch1 and Gli expression, was seen in chondrocytes, but the KO results show
that the critical role of Hh signaling occurs prior to chondrocyte formation. Understanding the
dynamics of the Hh signaling pathway during repair could lead to a more effective therapeutic
option.
As previously mentioned studies have shown that there is an increase in bone formation
with the addition of Hh signaling during bone repair (Huang et al., 2014)

(Zou et al., 2014b)

(Edwards et al., 2005) (Lee et al., 2017) (Maeda et al., 2013) (Baht et al., 2014). While most
conclude that Hh signaling induces bone formation, the proper analysis, such as investigating
early repair, was not done to definitively determine of Hh signaling might have an earlier role. The
results presented here indicate that stimulating Hh signaling can improve bone repair through
improvement of the cartilage callus. The SmoM2 expression experiments indicate that Hh
signaling can induce cartilage formation from periosteal cells and can increase the size of the
cartilage callus during repair. Although the KO data indicates that Hh signaling induces
chondrogenic differentiation, the possibility that Hh signaling induces chondrocyte proliferation
when constitutively active cannot be ruled out. Additionally, the consequences of the increased
cartilage callus need to be further investigated. It will be important to determine if and how
inducing a larger cartilage callus improves bone repair. It was difficult to determine if SmoM2
expression improved rib bone regeneration since the rib resections naturally repair. In the future,
SmoM2 expression should be induced in a bone injury model that will not naturally repair, such
as a femur resection, to determine if it can rescue the non-union.
It will also be important to determine if it is sufficient to induce SmoM2 expression only in
the Sox9 expressing periosteal population to increase the cartilage callus. The “Pre” tamoxifen
induction schedule (Figure 2.3 A) should be used in both rib and femur resection models to
determine if that is sufficient to cause an increase in cartilage formation and rescue repair
44

respectively. These results could further support the use of Hh signaling as a potentially
therapeutic treatment, as well as the use of the Sox9 expressing progenitors.
45

3.5 Figures

Figure 3.1. Hh signaling during rib repair
(A) Expression of Ihh (red) and Shh (green) is evident in differentiating cartilage cells. At earlier
stages, prior to cartilage formation, Ihh is hard to detect even at 7 dpr, while Shh is expressed at 5 dpr
in many cells across the lesion. (B) Many cells express Smo (red) in the control callus at 7 dpr, while
in the Post KO, Smo expression is not detectable. Fluorescent RNA-ISH for Smo (green) combined
with IF for tdTomato (red) shows that when using the Pre induction schedule, most Tdtomato+ cells
are negative for Smo expression, while many non-Sox9+ lineage cells still express Smo. White arrow
heads indicate tdTomato+ cells that are negative for Smo expression. (C) IF for the tdTomato protein
(red) in combination with RNA-ISH for Shh and Ptch1 (green) at 7 dpr. In control mice, Shh and Ptch1
expression can be seen in many cells across the callus but most strongly in the chondrocytes. Cells
neighboring tdTomato+ cells do not have strong Ptch1 expression. In Pre KO mice, tdTomato+ cells
can be seen throughout the callus. Ptch1 expression is strongest in the small regions of cartilage that
form at the cut ends.
Scale bar A = 200 microns, enlarged boxes = 50 microns; B = 200 microns, enlarged boxes = 50
microns; C = 100 microns
46

Figure 3.2 Requirement of Hh signaling for rib callus formation
(A) Safranin O staining was used to visualize cartilage formation. At 10 dpr, the control callus has
significantly more cartilage then both the Pre KO and Post KO repair calluses. In the graph showing
the quantification of cartilage based on Safranin O staining at both 7 and 10 dpr, data is presented
as compared to the average of controls, normalized to 1. When comparing both the Pre and Post KO
to the control, the difference in the amount of cartilage is statistically siginificant, but between the Pre
and Post KO, there is no statistically significant difference. (B) Double fluorescent RNA-ISH of
Col1a1 (red) and Col2a1 (green) expression shows that there are fewer hybrid cells that mediate
large scale repair in the KO calluses when compared to the control. Most of the cells in the control
callus express high levels of both Col1a1 and Col2a1, while in both Pre and Post KO calluses many
of the chondrocytes only express Col2a1. The enlarged boxes show cells with chondrocyte
morphology with color channels merged and separated.
Data are mean + SEM. Statistical differences were determined using the unpaired t test. * p value <
0.005 ** p value < 0.001
47

Figure 3.3 Hh signaling is required for bone formation
(A) Double fluorescent RNA-ISH of Col1a1 (red) and Col2a1 (green). The enlarged boxes show an
area of newly formed trabecular bone in separate channels. In both KO contexts, the cells building
the trabecular bone expresses Col1a1 at high levels but are largely negative for the expression of
Col2a1. While in the control, cells lining the trabecular bone express high levels of both. White
arrowheads point to differentiating osteocytes that still express Cola1. (B) H&E staining at 14 dpr
shows the histology of the repair callus in control, Pre, and Post KO animals. Both KO mice have
much less bone than in controls and many of the cells that have entered the lesion have a
progenitor-like morphology. Bone formation was quantified based on histology and the data is shown
compared to the average of controls which has been normalized to 1 in the graph. At both 10 and 14
dpr, both the Pre KO and the Post KO have significantly less bone when compared to the controls.
No statistically significant difference is seen when comparing the Pre to the Post KO. Whiskers show
mean + SEM. Statistical differences were determined using the unpaired t test. * p value < 0.0002
48

Figure 3.4 Hh signaling is required for full bone regeneration
Alizarin red and alcian blue whole mount staining show that at 4 wpr, the resected region is fully
spanned by mineralized material in control mice (n=3), while both the Pre KO and Post KO animals
fail to heal at 6 wpr. Similar results can be seen at 8 wpr in the Pre KO.
Figure 3.5 A constitutively active Smo receptor increases the size of the cartilage callus
Sox9CreERT2;SmoM2 mice were injected with Tamoxifen for 4 consecutive days starting the day
before surgery. The SmoM2 expressing repair calluses (left) were compared to control mice (right) at
the same time points. (A) H&E staining of 5 dpr calluses show that the SmoM2 expressing calluses
appear to be more organized and advanced when compared to the controls. (B) Double RNA-ISH of
Col2a1 (green) and Col1a1 (red) on a near adjacent sections of (A) show that there is an increase in
the number of cells expressing both markers in the SmoM2 expressing callus. (C) H&E staining of 7
dpr calluses show that the cartilage callus is larger in the SmoM2 expressing mice when compared to
the control. The chondrocytes also appear to be more mature overall in the SmoM2 expressing callus.
49

Figure 3.6 Model for large-scale bone repair
In wildtype animals, represented by the left side of the diagram, extensive gaps in the mouse rib (as
indicated by the hatched lines) can naturally regenerate. A Sox9-expressing periosteal
subpopulation (indicated in red) along with other skeletal progenitors (yellow) proliferate and migrate
into the lesion. These Sox9+ lineage cells require Hh signaling (green arrows) to be able to signal
via a yet-to-be identified mechanism to neighboring cells (purple arrows). This signal induces
neighboring cells to differentiate into a reparative callus with hybrid osteochondral qualities, leading
to complete bridging and bone repair. Sox9+ lineage cells ultimately contribute to the callus and
regenerated bone (indicated in brown) although they are represented in the minority. The right side
of the diagram represents the outcome of a Pre regimen KO of Smo (tamoxifen administered prior to
injury). When Smo is removed from the Sox9+ periosteal subpopulation prior to resection, the Sox9+
lineage cells can still contribute to the callus but are not activated and therefore do not relay a
differentiation signal to neighboring cells. Thus, the entire callus fails to differentiate and healing
fails.
50

Supplemental Figures

Supplemental Figure 3.1. Characterization of tdTomato+ cells during repair
(A) RNA-ISH for Ptch1 (green) shows strong expression in tdTomato+ cells in the thickened
periosteum (bracket) at 3 dpr close to the cut end. (B) Near adjacent sections of IF for tdTomato
(red) and H&E straining. At 7 dpr there are tdTomato+ cells seen throughout the callus, a majority
of them have not yet differentiated into chondrocytes. The enlarged boxes of the IF and H&E on the
left show a region of differentiated chondrocytes with no tdTomato+ cells. The enlarged boxes on
the right show a region with many tdTomato+ cells that are less mature. (C) IF for the tdTomato
protein (red) in combination with RNA-ISH for Gli2 (green) at 7 dpr. In control mice Gli2 expression
can be seen most strongly in areas where chondrocytes are differentiating. Cells neighboring
tdTomato+ cells do not have strong Gli2 expression. In Pre KO calluses, tdTomato+ cells can still
be seen throughout the callus. Some Gli2 expression can be seen in the small regions of cartilage
that form at the cut ends.
Scale bar A= 100 microns, enlarged boxes = 50 microns; B = 200 microns, enlarged boxes = 100
microns; C = 100 microns, enlarged boxes= 25 microns
51

Supplemental Figure 3.2 Characterization of Smo knock-out calluses
(A) At 5 dpr, in Post KO mice, the progenitors that fill the resected region express both Sox9 (green)
and Runx2 (red), as seen in the control animals. (B) The percentage of tdTomato+ cells within the
callus in Pre KO and Post KO mice is compared to controls at 10 dpr. In Pre-induced control mice
22.8 + 1.3% of the cells in the callus were tdTomato+ while in the Pre KO mice 20.6 + 0.44% of the
cells were tdTomato+. In Post induced control mice 85.5 + 2.8% of the callus cells were tdTomato+
while in the Post KO 87 + 3.9% of the cells were tdTomato+. There was no statistically significant
difference between controls and KOs. (Pre-induction p = 0.262 and Post-induction p = 0.757). Panels
show lineage tracing of tdTomato+ cells from Pre KO and Post KO mice demonstrating that
the majority of cells null for Smo are still present and participate. See Figure 4-supplement 1-source
data 1. (C) IF against pHH3 was used to mark cells undergoing proliferation. The percentage of
positive cells in the callus was calculated vs. the total number of callus cells. No statistically significant
difference between the control (n=6, 5) and the Sox9-CreERT2; Smofl/fl (n=7, 5) mice at 7 or 10 dpr
(p = 0.460 and 0.210 respectively) was evident. Representative panels are shown. (D) TUNEL
staining was used to detect apoptotic cell death at 7 and 10 dpr. The graph shows the percentage of
green pixels, in comparison to total pixels in the callus area. There is no statistically significant
difference between the control (n=4,3) and Sox9-creERT2;Smofl/fl (n=6,6) mice at 7 and 10 dpr (p =
0.243 and 0.141 respectively). Representative panels are shown.
52

Supplemental Figure 3.3 Progenitors retain Sox9 and Runx2 co-expression
In a Pre KO at 21 dpr the cells within the resected region still look like mesenchymal progenitors
based on H&E histology. Double RNA-ISH for Runx2 and Sox9 was performed on a near adjacent
section and showed that many cells still co-express both markers.
53

Supplemental Figure 3.4 Late KO of Smo
“Late” tamoxifen induction schedule. Sox9-CreERT2;tdTomato;Smo
fl/fl
mice were injected with
tamoxifen days 3-5 post resection. H&E staining at 14 dpr shows ample trabecular bone and
cartilage formation. Although repair is delayed when compared to control mice, there is much more
bone and cartilage formation when compared to either the Pre or Post KO calluses at 14 dpr.
54

Chapter 4 Role of Hh signaling in the Sox9 progenitor population in other injury contexts
4.1 Introduction
Based on the strong phenotype seen in the Pre Smo KO rib resection repair calluses, I
decided to investigate additional in vivo models. As previously mentioned, there have been
conflicting conclusions when it comes to the specific role of Hh signaling during bone repair (Wang
et al., 2010) (Li et al., 2018) (Baht et al., 2014) (Murao et al., 2013) (Zou et al., 2014a) (Huang et
al., 2014) (Edwards et al., 2005) (Thompson et al., 2002) (Paul et al., 2016). It is possible that
some of the differences between the studies caused these conflicts. For example, different injury
models, methods of inducing and inhibiting Hh signaling, and different analysis of the repair
process were used between the previous studies. With the new understanding of how Hh
signaling specifically functions in the Sox9+ population, inducing the differentiation of other cells,
I wanted to determine if this function is conserved in other injury models. By keeping the progenitor
population (Sox9 expressing), tamoxifen induction schedule, and mouse line consistent between
the models, the requirements for Hh signaling within the Sox9 expressing progenitor population
in other injury contexts may be able to be determined.
In order to help elucidate this mystery, the role of Hh signaling in Sox9 expressing
periosteal progenitors during femur fracture repair was investigated. Although Sox9+ progenitors
have been shown to participate in femur fracture repair (He et al., 2017), the effect that Hh
signaling has in this context has not been determined. Previous studies have shown that Hh
signaling does not appear to be required for fracture repair, but only modulates it. A critical role
for Hh signaling in cartilage formation was observed during rib regeneration, which may not be
required for fracture repair since some are able to repair through direct ossification. Based on
previous studies and the results seen during large-scale repair, I predicted that the KO of Smo
prior to injury in the Sox9 expressing population would lead to a reduction in the cartilage callus
but would not affect the ability of the fracture to repair through intramembranous ossification.
Destabilization of the medial meniscus (DMM) is a surgical model often used to reproduce
human osteoarthritis (OA) in mice (Glasson et al., 2007). In OA, along with cartilage degradation
and subchondral bone turnover, osteophytes form. Osteophytes, also known as bone spurs, are
bony projections that develop along the edge of a bone. Formation of osteophytes are a clinical
problem, due to the pain and dysfunction they cause. Much about osteophytes remain unknown,
including the originating cell source and the signals that cause them. Although the cell source is
thought to be periosteal cells or synovial mesenchymal stem cells (van der Kraan and van den
Berg, 2007), the proper experiments to determine the specific population that gives rise to them
have not been performed. Recent work has begun to determine the cell population that
55

contributes to osteophyte formation by lineage tracing analysis of different Cre lines. LepR-Cre,
which marks bone marrow stem cells, and Prg4-CreER, which marks the articular cartilage, have
both been shown to not contribute to the osteophyte formation (Personal communication with the
De Bari lab and with Gage Crump). Gdf5-Cre did show contribution, although this Cre marks a
very wide population of cells including all the cells involved in the joint development. Currently
Nestin-CreER, which marks another bone marrow derived population, and Col2-CreER, which
marks chondrocytes, are also being tested (Personal communication with the De Bari Lab). Since
Sox9-CreERT is known to mark periosteal cells involved in the bone repair process, it is possible
that cells from the Sox9 expressing lineage can be found in osteophytes.
It is known that osteophytes form through endochondral ossification and are composed of
fibroblasts, chondrocytes, and osteoblasts, but how OA leads to osteophyte formation is not
completely understood (van der Kraan and van den Berg, 2007). Although some studies have
indicated that mechanical factors are dispensable for their formation, it is possible that
osteophytes are formed as a repair mechanism to help stabilize joints that have developed OA.
Osteophyte formation may be a consequence of the signaling involved in OA, such as BMP and
TGF-β. Macrophages are also thought to contribute to osteophyte formation due to their
production of growth factors (van der Kraan and van den Berg, 2007). Although the signaling
pathways involved are not fully elucidated, many pathways involved in cartilage and bone
development are hypothesized to also be involved in osteophyte formation. Due to the role of Hh
signaling in cartilage proliferation and osteoblast differentiation during bone development it is
possible that it plays a similar role during osteophyte development. Increased expression of Hh
signaling has already been associated with arthritic chondrocytes and previous studies have
shown that inhibiting Hh signaling reduces the severity of OA (Lin et al., 2009) (Zhou et al., 2014b).
Taken together, these results and the results I observe during rib regeneration, it is possible that
Hh signaling may have a role in osteophyte development. Furthermore, it is possible that Hh
signaling has a similar role within the Sox9 expressing population during osteophyte formation as
it does during rib regeneration. Inhibiting Hh signaling within the Sox9+ population could prevent
cartilage formation and therefore inhibit osteophyte formation.
4.2 Materials and methods
Mice and animal housing
Mice housing and breeding were done as previously described in section 3.2.
Femur fracture
Closed, mid-diaphyseal femur fractures were performed on Sox9-
CreERT2;tdTomato;Smo
fl/fl
mice and Sox9-CreERT2;tdTomato mice by Venus Vakhshori from
56

the Lieberman lab. Mice were injected with Tamoxifen for 3 consecutive days starting 7 days prior
to injury. Mice were weighed to determine how much post-operative pain medicine
(buprenorphine SR (ZooPharm)) should be administered at a dose of 0.5µL/gram. Isoflourane
was then administered. The fur from the right limb was removed and the leg was cleaned 3 times
with an iodine-based solution and 70% ethanol. The mice were then placed on their backs with
the right knee flexed. A scalpel was used to make a 1.5 cm incision over the knee joint and the
patella was displaced exposing the end of the femur. A 26-gauge needle was inserted into the
medullary canal of the femur and an X-ray was used to ensure the needle was placed properly.
The patella was positioned over the knee and the incision was closed using absorbable sutures.
The fracture was created using a modified Bonnerans and Einhorn’s fracture apparatus
(Bonnarens and Einhorn, 1984) (Marturano et al., 2008). The mice were 3 months of age when
the Tamoxifen injections started and were sacrificed at 10- and 28-days post fracture. Both males
and females were used.
DMM
DMM surgeries were performed on Sox9-CreERT2;tdTomato;Smo
fl/fl
mice and Sox9-
CreERT2;tdTomato mice by Gage Crump. Mice were injected with Tamoxifen for 3 consecutive
days starting 7 days prior to injury. Isoflourane was administered. The fur from the right limb was
removed and the leg was cleaned 3 times with an iodine-based solution and 70% ethanol. The
mice were placed on their backs with the right knee flexed. A scalpel was used to make a 1 cm
incision over the knee and then the knee joint capsule was dissected with forceps. The hind paw
was held while the patella and patellar ligament were dislocated with forceps. Sterile saline was
dripped on the surface of the articular cartilage throughout the process. The medial meniscus,
which is located between the medial condyle of the femur and the medial plateau of the tibia, was
identified. The medial meniscus was then cut using a surgical blade, being careful to not injure
the articular cartilage or other ligaments. The knee joint capsule and skin were closed with 7-0
sutures. Mice were sacrificed at 14 days post-surgery. Males were between 8-10 weeks old when
tamoxifen injections were started.
Histological analysis, immunofluorescence, and ISH
Done as previously described in 2.2 and 3.2
4.3 Results
Femur Fractures
Femur fractures were performed by Venus Vakhshori from the Lieberman lab on Sox9-
CreERT2;tdTomato;Smo
fl/fl
mice and Sox9-CreERT2;tdTomato mice under the Pre tamoxifen
induction schedule. In the absence of an injury, there are Sox9+ progenitors in the periosteum of
57

the femur (Figure 4.1A). Upon femur fracture, a large callus composed of cartilage and bone
forms (N=2). Lineage tracing using the tdTomato reporter showed that the callus is filled with
Sox9-expressing progenitors, which can contribute to cartilage and bone at 10 dpf in both control
and Smo KO calluses (Figure 4.1B,C). Similar to the rib, the Sox9-expressing cells can participate
in femur fracture repair regardless of Smo expression and only contribute to a fraction of total
repair cells (N=4).
Femur fractures were analyzed at 10 days post fracture (dpf), when there is a large callus
composed of both bone and cartilage, and at 28 dpf, when the fracture is fully bridged by bone.
Comparison between the control and Smo KO showed no significant differences at either time
point (Figure 4.2A, B and 4.4A, B). At 10 dpf there is no difference in the amount of cartilage
formation, based on Safranin-O staining, between the control and KO (Figure 4.2A, B) (Control
N=4, Smo KO N=5). If the repair callus was composed of hybrid skeletal cells that express high
levels of both chondrocyte and osteoblast associated genes was determined next. In both the
control and Smo KO, the callus is filled with cells co-expressing both Col2a1 and Col1a1. There
are hybrid skeletal cells with chondrocyte morphology and osteoblast morphology in both the
control and KO calluses (Figure 4.3A, B). At 28 dpf there is no difference noticeable between the
control and Smo KO (Figure 4.4A, B) (Control N=3, Smo KO N=3). The fractures were all still able
to repair, as seen by complete bridging with trabecular bone. These results indicate that Hh
signaling does not have the same crucial role in the Sox9 expressing periosteal subpopulation
during femur fracture repair as it does during rib regeneration. Smo is not required by the Sox9
expressing progenitors for the induction of a cartilage repair callus during femur fracture repair.
DMM
DMM surgeries were performed by Gage Crump on Sox9-CreERT2;tdTomato mice to
determine the contribution of Sox9 expressing cells in osteophyte formation. Prior to DMM
surgery, Sox9+ cells could be seen in the articular cartilage, growth plate cartilage, and in some
of the periosteal cells (Figure 4.5A, B). Tamoxifen was administered to Sox9-CreERT2;tdTomato
mice using the Pre induction schedule, to only include the cells that expressed Sox9 prior to DMM
in the lineage trace of the subsequent osteophyte formation. Mice were examined at 14 days post-
surgery for osteophyte formation by H&E staining of paraffin sections (Figure 4.6A). At this time
osteophyte formation could be seen on the medial side of the tibia and appeared to be composed
of mostly cartilage, as seen by Safranin-O staining (Supplemental Figure 4.1), with a little bone
formation in only some of the osteophytes (Figure 4.6A). Lineage tracing showed that some
tdTomato+ cells could be seen throughout the osteophyte in regions of both bone and cartilage
(Figure 4.6C) (Control N=4, Smo KO N=5). Although these results show that Sox9+ cells give rise
58

to osteophytes, the osteophytes were not composed solely of Sox9+ cells. Additionally, the
contribution of Sox9+ progenitors to the osteophyte varied between animals.
Double RNA-ISH for Col1a1, the osteoblast-associated gene, and Col2a1, the
chondrocyte-associated gene, were performed on sections of the osteophytes to determine if it
was composed of osteoblasts, chondrocytes, or hybrid cells. While some of cells in the osteophyte
expressed only the osteoblast or only the chondrocyte associated gene, some cells were hybrid,
expressing both Col1a1 and Col2a1 (Figure 4.6C). These hybrid skeletal cells have been
observed in both femur fracture repair and large-scale rib repair. Double RNA-ISH for Col1a1 and
Col2a1 performed in combination with the tdTomato antibody showed that the lineage-traced
Sox9-expressing cells were able to become hybrid cells in the osteophyte (Figure 4.6B) (N=4).
During large-scale bone repair, Hh signaling is required for the generation of a large
cartilage callus filled with hybrid skeletal cells. Since osteophytes also develop through a cartilage
intermediate with hybrid cells Sox9-CreERT2;tdTomato;Smo
fl/fl
mice were used to determine if Hh
signaling has a similar role during their formation. The Pre tamoxifen induction schedule was used
on these mice and osteophyte formation was examined at 14 days post-surgery. Despite the KO
of Smo, osteophyte formation could still be seen histologically, although their size appeared to be
smaller when compared to the control animals (Figure 4.7A and 4.6A) (N=5). Safranin-O staining
confirmed that the osteophytes were composed of cartilage and again showed that the osteophyte
was decreased in size in the Smo KO when compared to controls (Supplemental Figure 4.1).
Double RNA-ISH for Col1a1 and Col2a1 showed that some cells within the osteophyte were
hybrid, while others were expressing for only Col1a1 or Col2a1 (Figure 4.7B, C). Lineage tracing
experiments showed that Smo knockout cells could still contribute to the osteophyte and, in
combination with the double RNA-ISH, showed that these cells were still able to become hybrid
(Figure 4.7B). Overall the osteophytes in control and Smo KO mice appeared to be very similar,
with only a slight decrease in size in the Smo KOs.
4.4 Discussion
The lack of a phenotype in the Smo KO mice during femur fracture repair was surprising
(Figure 4.2, 4.3 and 4.4). Although I predicted that the femur would be able to fully repair through
intramembranous ossification, I did not predict that the cartilage callus would still be able to form.
Even though the Sox9 expressing progenitors could not respond to Hh signaling, none of the cells
involved in the fracture callus appear to be inhibited from differentiating into cartilage or hybrid
skeletal cells, in contrast to what is seen during rib regeneration. It is possible that Hh signaling
has a role in femur fracture repair similar to the role is has during bone development, but due to
the small contribution of Sox9 expressing cells, no phenotype could be observed. It is also
59

possible that there are different repair mechanisms involved fracture repair and large-scale
injuries. Although the overall steps appear to be the same between fracture and large-scale repair,
it is possible that the mechanisms are different. The signaling pathways, as well as the role of
specific progenitors, could vary between the different circumstances of repair. Different repair
mechanisms are used during wound healing of the skin and skin regeneration. Although some
signals and mechanisms are shared, the end results are different (reviewed in (Takeo et al.,
2015)). A similar phenomenon could occur between fracture repair and large-scale regeneration.
Another possibility is that the progenitors marked by Sox9-expression in the femur and rib
periosteum are different from one another. Although these periosteal progenitors are marked by
the expression of Sox9, it is possible that they are very different from one another. The other
genes expressed by the two cell populations as well as their chromatin structures could be
different. Between all the bones in the body, many of the developmental processes are different,
with varying surrounding tissues and signals. For example, a previous study indicated that Hox
genes are involved in proper differentiation in bone repair (Rux et al., 2016). Hox genes are
regionally restricted so bones throughout the body will not have the same Hox genes activated.
Analyzing the cells that compose the periosteum of various bones in-depth and how they respond
to injury will help determine if this is the case. Techniques such as single cell RNA-seq can help
determine if the cells in the periosteum differ between the bones throughout the body.
Similar to the rib resection, hybrid osteochondral cells participated in both the femur
fracture repair callus and osteophyte formation. This supports the proposal that periosteal
progenitors maintain their osteoblast gene expression, such as Col1a1 (Kawanami et al., 2009),
while differentiating into chondrocytes in response to injury. It also supports the proposal that
repair does not simply recapitulate development.
The lineage tracing of the Sox9 expressing cells showed that cells from this lineage can
give rise to osteophytes. Although Sox9 expression marks a broad population of cells including
the periosteum, growth plate cartilage, and articular cartilage, based on additional lineage tracing
experiments, the periosteum is most likely the source. Another Cre line (Prg4-CreER), that marks
cartilage, has been shown to not contribute to osteophyte formation (unpublished data, G.
Crump). Taken together, it would appear that the Sox9-expressing periosteal population are a
source of cells that give rise to osteophytes. Sox9 expression does not marks all of the cells in
the periosteum and it only marks some of the cells in the osteophyte, indicating that there are
other progenitor populations that contribute to osteophyte formation.
The Sox9+ progenitor cells do not appear to have a coordinating role in osteophyte
formation since osteophytes could still form in Smo KO mice. Although it appears that Hh signaling
60

is not required in Sox9-expressing progenitors to form osteophytes, it may still have a role in their
development and maturation. Overall the Smo KO mice appeared to develop smaller osteophytes
when compared to controls, but the difference was not quantified. It is possible that Hh signaling
may induce proliferation of the cells in the osteophyte and the decrease in size is due to a
decrease in the proliferation of the Sox9 expressing cells. Since the Sox9 expressing population
makes up only a fraction of the cells in an osteophyte, knocking out Smo in only this population
may explain why the osteophytes appear to be smaller in the KOs. Although bone repair and
osteophyte formation share developmental steps the signaling pathways and cellular responses
could be different. A recent study comparing femur fracture calluses and osteophytes actually
indicated that the two have different formation pathways (Hsia et al., 2018). How different cellular
responses lead to similar outcomes, such as new bone formation, will be important to determine
in the future.

61

4.5 Figures

Figure 4.1 Sox9-CreERT2 marks cells that participate in femur fracture repair
(A)Tamoxifen induction schedule for the femur fractures. Tamoxifen was injected for 3 consecutive
days starting 7 days prior to surgery. A tdTomato antibody was used to detect cells from the Sox9
expressing lineage. In an uninjured mouse, there are some Sox9 expressing cells in the periosteum of
the femur. (B) 10 days after a fracture, these cells can be seen in the fracture callus. Some tdTomato
expressing cells have osteoblast morphology, while others have chondrocyte morphology. (C) Similar
results were seen in Smo knock out calluses at 10 days post fracture.
62

Figure 4.2 Callus formation is not affected by the Smo knock out
Near adjacent H&E (top) and Safranin-O stained sections (bottom) show that there is no significant
difference in the repair callus of control (A, left) and Smo KO calluses (B, right) at 10 days post
fracture. Both sets of calluses are large and filled with both bone and cartilage. (A) Shows a control
callus while (B) shows a Smo knock out callus.
63

Figure 4.3 Hybrid cells are not affected by the Smo knock out
Hybrid osteochondral cells, expressing both bone and cartilage associated genes, are found in the
fracture callus of both (A) control and (B) Smo knock out calluses. Osteoblast like cells lining the
newly formed trabecular bone express the osteoblast-associated gene Col1a1 and the chondrocyte
associated-gene Col2a1. Similarly, cells with chondrocyte morphology express both genes. The top
set of enlarged boxes show a region of chondrocyte like cells, while the bottom set show an example
of osteoblast like cells. The color channels have been separated to show that many of the same cells
express both genes.
Figure 4.4 Smo knock out does not affect the ability for a femur fracture to fully repair
H&E staining of 28 days post fracture calluses. (A) In control mice, bone has bridged the fracture and
is being remodeled. (B) The Smo knock out callus shows similar results to the control, indicating that
Hh signaling is not required in the Sox9 expressing periosteal cells for full repair to occur.
64

Figure 4.5 Sox9+ progenitors are found in the periosteum near the knee joint
Sox9creERT2;tdTomato mice were injected with Tamoxifen for 3 days followed by a 5 day chase. (A)
An antibody against the tdTomato protein shows that there are some Sox9 expressing progenitors in
the periosteum near the knee joint on the tibia. (B) In addition, growth plate cartilage and articular
chondrocytes are Sox9-expressing in both the tibia and femur. A is an enlarged image of the white
box in B.
65

Figure 4.6 Characterization of osteophyte
Sox9creERT2;tdTomato mice were injected with Tamoxifen for 3 days starting 7 days prior to the
DMM surgery. The animals were analyzed at 14 days post surgery for osteophyte formation. (A) H&E
staining shows that an osteophyte has formed on the tibia. The enlarged view of the yellow box
shows that there is a mix of both immature and hypertrophic chondrocytes present. (B) A double ISH
of Col2a1 (green) and Col1a1 (red) was combined with an IHC for the tdTomato protein (purple). (C)
The channels have been separated to more clearly determine which cells are positive. These results
show that some of the Sox9+ cells lineage trace into the osteophyte. Additionally, many of the cells in
the osteophyte are hybrid skeletal cells being positive for both Col2a1 and Col1a1.
66

Figure 4.7 Smo is not required in Sox9+ progenitors for osteophyte formation
Sox9creERT2;tdTomato;Smo
c/c
mice were also used for the DMM surgery with the same
Tamoxifen induction schedule as previously mentioned. (A) Smo KO mice were also examined at
14 days post surgery. The enlarged yellow box shows that there are some mature, hypertrophic
chondrocytes, although there are fewer then in the control mice. (B) A double ISH of Col2a1
(green) and Col1a1 (red) was combined with an IHC for the tdTomato protein (white). (C) The
channels have been separated to more clearly determine which cells are positive. These results
show that, even in the absence of Smo, some of the Sox9+ cells still lineage trace into the
osteophyte. Additionally, some of the cells in the osteophyte are also positive for both Col2a1 and
Col1a1.
67

Supplemental Figures

Supplemental Figure 4.1 Safranin-O staining of osteophytes
Safranin-O staining confirms the presence of cartilage in the forming osteophyte at 14 days post
surgery. The image on the right is a control mouse, while the left shows a Pre Smo KO mouse.
While both show cartilage formation, there is much more in the control when compared to the KO.
68

Chapter 5 Conclusion
Large scale rib regeneration occurs through a cartilage intermediate, with a repair callus
that is composed of chondrocytes, osteoblasts, and hybrid osteochondral cells, which express
high levels of both chondrocyte and osteoblast associated genes. These hybrid cells were also
observed in the callus of femur fractures and during the formation of osteophytes. Hybrid
osteochondral cells are not observed at the growth plate and are rare during development,
indicating that they have be selectively utilized during repair responses.
In an uninjured rib, there are some Sox9 expressing cells in the periosteum. These cells
are able to participate in forming the repair callus of a large scale rib resection. Although they
compose only 20% of the cells within the repair callus, these results indicate that they have an
important role coordinating the differentiation of the all the progenitors of the repair callus. When
only the Sox9+ population loses the ability to respond to Hh signaling, there is a callus wide failure
to differentiate into cartilage, bone, and hybrid cells causing a complete failure to regenerate the
resected rib bone. Although it is unknown exactly why this occurs, it is possible that in response
to Hh signaling, the periosteal Sox9+ population produces a yet to be determined signal that
induces the differentiation of the remaining callus cells.
Sox9-expressing cells can be found in the periosteum of other bones throughout the body,
such as the femur and tibia. Although these cells are able to participate in femur fracture repair
and the formation of osteophytes, they do not appear to have the same coordinating role that they
do during rib regeneration. When the Sox9-expressing cells are unable to respond to Hh signaling,
the femur fracture is still able to repair with no significant reduction in cartilage or bone formation.
Similarly, osteophytes are still able to form when the Sox9-expressing cells are KO for Smo.
Although there are many similarities between rib regeneration, femur fracture repair, and the
formation of osteophytes, the role that Hh signaling plays within the Sox9+ population does not
appear to be the same in each of these contexts. It is clear that further experiments must be done
to understand the differences between these repair mechanisms and bone formation processes.
The differences could lie in the progenitor cells themselves or in the signaling involved in each
process.
This is the first example of a progenitor population having more than just a participatory
role during bone repair. The Sox9-expressing population was shown to be required for the
induction of the cartilage callus and complete repair. Furthermore, these results have indicated
that a cartilage callus is required for large-scale bone regeneration. Additionally, this work has
partially elucidated the unique role of Hh signaling during large-scale bone regeneration. Although
69

further investigation is required, it has the potential to lead to improved therapies for bone
regeneration.

FUNDING
Funding was from the National Institutes of Health [T32 HD060549 to S.T.K, R21
DE023899 and R35 DE027550 to J.G.C.; R21 AR064462 and R01 AR069700 to F.V.M.]; the
James H. Zumberge Research and Innovation Fund to F.V.M; and a University of Southern
California Regenerative Medicine Initiative Award to F.V.M. and J.G.C.

70

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Abstract (if available)

Abstract While many bone fractures can repair on their own, non-unions and critical sized defects are still major clinical problems. Between 5-10% of fractures become non-unions, which are fractures that will not fully healing without additional medical involvement, such as surgery. Large-scale bone injuries from trauma or bone resections due to cancer are examples of critical sized defects. Current treatment options for bone repair are very limited, with the current gold standard being autologous bone grafting, which involves some risks. To improve clinical treatment options the process of bone repair needs to be better understood. The goal of this project was to determine the role of Hedgehog signaling in large-scale bone repair

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Asset Metadata

Creator Kuwahara, Stephanie T. (author)

Core Title The role of Hedgehog signaling in Sox9 expressing progenitors during large scale bone repair

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Development, Stem Cells and Regenerative Medicine

Publication Date 06/24/2019

Defense Date 04/25/2019

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

Tag bone repair,cartilage,Hedgehog,OAI-PMH Harvest,skeletal,Sox9-expressing progenitors

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Merrill, Amy (committee chair), Crump, Gage (committee member), Evseenko, Denis (committee member), Frenkel, Baruch (committee member), Mariani, Francesca (committee member)

Creator Email skuwahar@usc.edu,stkuwahara@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-178499

Unique identifier UC11660438

Identifier etd-KuwaharaSt-7510.pdf (filename),usctheses-c89-178499 (legacy record id)

Legacy Identifier etd-KuwaharaSt-7510.pdf

Dmrecord 178499

Document Type Dissertation

Format application/pdf (imt)

Rights Kuwahara, Stephanie T.

Type texts

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

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

Repository Name University of Southern California Digital Library

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