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Fgfr2 regulates cell fate at the interface between tendon and bone
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Content
Fgfr2 regulates cell fate at the interface between tendon and bone
by Ryan Richardo Roberts
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
Genetics, Molecular and Cellular Biology
December 2017
Copyright 2017 Ryan Richardo Roberts
I | P a g e
TABLE OF CONTENTS
Acknowledgements III
Funding Sources III
Abstract IV
Chapter 1: Introduction 1
FGF Signaling 1
Congenital Disorders with FGFR2 mutations 2
Development of the Jaw 4
Tendon Entheses 5
Types of Tendon Entheses 7
Development of Tendon Enthesis 9
Chapter 2: Modeling craniofacial and skeletal congenital birth
defects to advance therapies 12
Abstract 13
Introduction 14
Craniosynostosis 15
Craniofacial Dysmorphologies 17
Dental Abnormalities 19
Skeletal Dysplasias 20
Bone Mineral Density 23
Future Directions 26
Acknowledgments 27
Figure 1 27
Chapter 3: Fgfr2 regulates the cell fate at the interface between
tendon and bone 29
Abstract 30
Introduction 31
Experimental Procedures 34
II | P a g e
Results 37
Discussion 45
Conclusions 50
Figure 2 52
Figure 3 53
Figure 4 54
Figure 5 55
Figure 6 56
Figure 7 57
Figure 8 58
Figure 9 59
Figure 10 60
Figure 11 61
Figure 12 62
Supplement Table 1 63
Supplement Table 2 63
Supplement Figure 1 63
Supplement Figure 2 64
Chapter 4: Discussion and Future Directions 66
References 70
III | P a g e
ACKNOWLEDGEMENTS
I would like to thank my faculty mentor, Amy E. Merrill Ph.D. for all her support and
guidance throughout my Ph.D. training. Her belief in me and the project has been
instrumental to my success. I would like to thank all my labmates, past (Cynthia
Neben and Brian Idoni) and present (Joanna Salva, Lauren Bobzin, Taylor Stucky,
Diana Rigueur and Creighton Tuzon) for all their feedback over the years. I would
like to thank my dissertation committee, Cheng-Ming Chuong Ph.D. and Takahiro
Ohyama Ph.D. for their guidance on my project. I would like to thank the staff at
CCMB and the PIBBS office for all their logistical support. Last, but not least, I
would like to thank my family, friends and all the other mentors I have had
throughout my life. It is their support, belief and guidance that is been the
foundation for my success.
FUNDING SOURCES
James H. Zumberge USC Faculty Research and Innovation Fund to A. M.
Basil O’Connor Starter Scholar Research Award #5-FY12-166 , March of Dimes
to A. M.
NIH/NIDCR Training Grant #T90DE021982 to R. R.
NIH #1R01DE025222 to A.E.M.
NIH #1R01DE025222 diversity supplement
IV | P a g e
ABSTRACT
Fibroblast Growth Factor (FGF) signaling plays a critical role in skeletal
development, as mutations in Fibroblast Growth Factor Receptor 2 (FGFR2)
manifest with at least 9 distinct skeletal birth defects. Loss-of-function mutations
in FGFR2 cause Lacrimoauriculodentodigtal (LADD) syndrome, which is a
disorder characterized by posterior shortening of the jaw. We hypothesized that
the posterior jaw shortening in LADD indicates a role for FGFR2 in the
development of the jaw processes. To test this hypothesis, we employed a
conditional knockout mouse in which Fgfr2 is ablated within the neural crest-
derived skeletal precursors of the jaw. We found that Fgfr2
flx/flx
; Wnt1-Cre mice
have jaw abnormalities at the tendon/ligament-to-bone attachment sites, also
know as entheses, on the condyle, angular process, and ramus. Histological and
molecular markers indicate that at these regions, endochondral-like bone has
replaced the developing enthesis. The progenitor cells of the enthesis co-express
Scx and Sox9 have been shown to differentiate into chondrocytes and tenocytes.
I found that Fgfr2 regulates differentiation of Scx
+
/Sox9
+
progenitor cells of the
developing enthesis by regulating expression of Notch2, Delta1 and Jagged1.
Notch signaling pathway plays a crucial role in establishing cell fate. Lineage
tracing analysis in addition to a tendon specific knockout of Fgfr2 suggest that the
change in cell fate is autonomous to the enthesis progenitor cells and not caused
by the ablation of Fgfr2 in the underlying bone. Altogether, this suggests a role for
Fgfr2 in cell fate determination of the enthesis progenitor cells in the jaw.
1 | P a g e
Chapter 1
INTRODUCTION
FGF Signaling
Fibroblast growth factors (FGF) are critical regulators of multiple biological
processes including embryonic development, wound healing, and cancer
progression. The 22 known FGFs in humans share high sequence homology
along the 140 amino acid core region, which is critical for binding heparin and
extracellular heparan-like glycosaminoglycans. All members but FGF1 and FGF2
have traditional secretory peptide domains that are processed through the ER
secretory pathway (Powers et al., 2000). FGF1 and FGF2 both have nuclear
localization domains, with the latter being further shuttled in the nucleolar
subdomain where it has been shown to regulate ribosomal biogenesis (Neben et
al., 2014; Powers et al., 2000). FGF2 is translated into four isoforms 18kDa,
22.5kDa. 23.1kDa and 24.2kDa, with only the 18kDa being secreted from the cell
and the larger isoforms participating in the nuclear role (Powers et al., 2000). FGF
signaling regulates many different cellular processes in a wide array of cell types
through Fibroblast Growth Factor Receptors (FGFR) 1-4, that are part of the Ig
superfamily of tyrosine kinase receptors (Powers et al., 2000). The receptors share
about 64% sequence homology at the amino acid level and share a base structure
of three extracellular Ig domains with an acidic region between the first and second
Ig domains, a transmembrane region and a cytosolic tyrosine kinase domain
(Powers et al., 2000). Fgfr2 has been shown to regulate the balance between
cellular differentiation and cellular proliferation (Neben et al., 2014; Powers et al.,
2 | P a g e
2000; Su et al., 2014). Being a single pass membrane tyrosine kinase, Fgfr2
transmits extracellular growth factor signaling by dimerizing with another Fgfr2
protein. This causes the intercellular domain of Fgfr2 to auto-phosphorylate
relaying the external signal at the membrane to the PLC , AKT, MAPK and STAT
signaling cascades affecting gene expression (Powers et al., 2000). Alternative
splicing of Fgfr2 produces isoforms, IIIb or IIIc, which are expressed in tissues
specific manner and have different ligand specificity. Isoform IIIb is expressed in
epithelial tissues and binds Fgf1, -3, -7, -10, and -22. Isoform IIIc is expressed in
the mesenchyme where it binds Fgf1, -2, -4, -5, -6, -8, -9 and 16-19. Spatial and
temporal expression of the ligands and receptor modulate a paracrine like
communication between the epithelial and mesenchyme (Powers et al., 2000; Su
et al., 2014).
Congenital Disorders with FGFR2 mutations
Mutations in FGFR2 causes 9 known autosomal dominant rare human genetic
disorders with the causes ranging from increased ligand affinity, change in ligand
specificity, increase in tyrosine kinase activity, receptor mis-localization or
complete loss of function. The common phenotypes of the 8 gain-of-function
mutations with a spectrum of severity are craniosynostosis, facial dysmorphism
and appendicular skeletal abnormalities (Merrill et al., 2012; Neben et al., 2014;
Robin N. H., 2011; Rohmann et al., 2006). This suggests that FGFR2 plays a
significant role in the development of the skeleton. Lacrimo-auriculo-dento-digital
(LADD) syndrome also known as Levy-Hollister syndrome is caused by the loss of
3 | P a g e
function mutations in FGFR2 (Rohmann et al., 2006). Though it does share the
facial dysmorphism with the gain of function mutations, it does not share the
craniosynotosis and the severe syndactyly phenotypes (Rohmann et al., 2006).
Like its name suggests, LADD syndrome is characterized by cup shaped low set
ears, hearing loss, hypoplastic salivary and lacrimal glands, hypodontia, enamel
hypoplasia and microretrognathia (Rohmann et al., 2006). Abnormal genitalia and
respiratory and renal dysfunction as also been reported. LADD is cause by both
sporadic and hereditary missense (A628T and A648T) or nonsense
(R649S A650) mutations that are focused in exon 16 of FGFR2 tyrosine kinase
domain (Rohmann et al., 2006). The mutations C106F in FGF10 and D513N in
FGFR3 also cause LADD syndrome, which highlight the redundant roles of the
FGF receptors (Rohmann et al., 2006).
Development of the Jaw
The branchial arches form in pairs on both sides of the midline foregut and is
anatomically comprised of an outer ectoderm covering a mesoderm core and lined
on the inner surface by endodermal tissue. The mesodermal core is derived from
the paraxial mesoderm, lateral plate mesoderm and the cranial neural crest cell
(CNCC). The paraxial and lateral plate mesoderms form the musculature and the
majority of the soft connective tissue in the face, while the CNCC form the bone,
cartilage and ligaments. The vertebrate face, which is derived from a common
plan, forms from the primordial buds that surround the stomodeum or primitive
mouth. The jaw or mandibular bone is formed from the CNCC that are primarily
4 | P a g e
derived from the neural fold at the anterior rhombencephalon or hindbrain region.
These CNCC migrate through the first branchial arch into the mandibular
prominence where the cells proliferate during the fourth week of gestation in
humans. Concurrently, the mandibular prominence separates from the maxillary
prominence. At week five, the opposing pairs of mandibular arches converge
along the midline bringing together the two halves of the jaw. From this point
onward, the mandible will continue to grow and change in proportion to the rest of
the face until birth.
The mandible is divided into the ventral body and dorsal ramus with the posterior
angle separating the two. At the anterior end of the ramus lie the coronoid,
condylar, and angular processes, which are attachment sites for muscles critical
for mastication. The buccinators, mentalis, quad lagilinfer, triangularis, and
platysma muscles insert into the outer surface of the mandibular body. On the
outer surface of the ramus inserts the masseter, which extends from the angle to
just below the coronoid process. The temporalis muscle is inserted into the
coronoid process and wraps around to the lingual side. Attaching to the lingual
side of the ramus just below the condyle is the pterygoideus externus muscle, while
the pterygoideus internus attaches to angle. There are also two ligaments
sphenomandibular and stylomandibular, which extends from the sphenoid bone to
the lingual surface of the ramus or from the styloid process to the dorsal base of
the ramus, respectively. The digastricus and mylo-hyoideus muscles insert into
the lingual side of the body.
5 | P a g e
The condyle forms the temporomandibular joint (TMJ) with the glenoid fossa of the
temporal bone, which is the hinged synovial joint located ventrally to each ear that
is responsible for the opening and closing of the jaw. Like other synovial joints, it
is surrounded by an articular capsule that is lined on the inner surface with the
synovial membrane, which produces the synovial fluid. One distinguishing feature
that only occurs in a few synovial joint is the articular disc, a fibrous, avascular
outgrowth of the capsule that forms medial-laterally through the center of the joint.
This causes the TMJ to have two synovial cavities: one with the head of the
condyle and the other with the glenoid fossa. TMJ disorders (TMD) are muscle-
skeletal disorders that affect the masticatory muscles and the TMJ. They affect
between 5-12% of the population with higher rates in younger people and women
being the most affected. These disorders can be caused by injury, malformation
of the joint or musculature, osteoarthritis and rheumatoid arthritis with the most
common symptom being chronic pain (Broussard, 2005; Furquim et al., 2015;
Israel et al., 1998). TMDs are considered functional pain disorders similar to
irritable bowel syndrome and chronic fatigue syndrome (Furquim et al., 2015).
Tendon Entheses
Tendon, which is a fibrous connective tissue, facilitates body movement by
delivering high tensile forces from muscle to bone. Collagen Type I is about 80%
of the dry weight of a tendon, which is comprised of a hierarchical structure of
functional units. The hierarchical structure of a tendon which is subdivided into
6 | P a g e
fasicicle, fibril, subfibrils and microfibrils is essential for its mechanical function
(Alexander, 2011; Benjamin et al., 2008). The fibril are crimped in their resting state
which allows the tendon to stretch by about 10% of its length with a tensile strength
of 80 MN/m
2
(Alexander, 2011). Fibroblast-like cells called tenocytes are interlaced
between the collagen fibers are responsible for producing the extracellular matrix
components. Type I is also the primary collagen found in bone which gets most of
its mineral density from impure calcium phosphate. The rigidity gain from the
calcium phosphate only allow for a stretch of about 2 to 3% with a tensile strength
of 180 MN/m
2
(Alexander, 2011)
. Since tendon and bone have vastly different
mechanical properties, they are joined through a transitional connective tissue
known as an enthesis (Alexander, 2011; Benjamin and McGonagle, 2009; Liu et
al., 2011; Liu et al., 2012). The enthesis can be defined as the site of insertion for
the joint capsule, ligament or tendon into the bone and serves as an interface
between soft and hard tissues. Entheses allow for the transmission of force from
the contracting muscle to the bone, which makes movement possible (Benjamin
and McGonagle, 2009; Liu et al., 2011). The interplay between muscle movement
and the enthesis is further highlighted by the requirement of muscle loading and
contraction for proper enthesis development, which happens post birth (Schwartz
et al., 2015; Thomopoulos et al., 2007; Zelzer et al., 2014). The enthesis is prone
to rheumatic diseases as well as common orthopaedic overuse injuries
(Apostolakos et al., 2014). The mature enthesis has a poor healing capacity
following damage; forming a fibrotic scar that makes the enthesis stiff and
mechanically unstable; significantly increasing the likelihood of reoccurring injury.
7 | P a g e
Post injury of the enthesis, there is a release of cytokines and growth factors like
Insulin Growth Factor 1 (IGF1), Platelet Derived Growth Factor (PDGF) and
Transforming Growth factor (TGF ) that causes inflammation and the
recruitment of macrophages (Apostolakos et al., 2014). During this initial phase
the platelet cells release Fibril and Fibronectin with contribute to scar formation.
The formation of the scar replaces the normal graded structure of the fibrocartilage
enthesis (Apostolakos et al., 2014). Current clinical strategies to repair critical
entheseal injuries consist of suturing the tendon back onto the bone, which never
regains normal functionality. To better supplement these current clinical
strategies, researchers are pioneering new scaffolding and mesenchymal stem
cell techniques, which show promising results (Apostolakos et al., 2014; Liu et al.).
To advance clinical strategies for enthesis regeneration and repair we must gain a
better understanding of the molecular mechanisms that regulate enthesis
morphogenesis during development.
Types of Tendon Entheses
Tendons attach to bone through fibrous or fibrocartilaginous entheses
(Apostolakos et al., 2014; Benjamin et al., 2006). Fibrous entheses typically insert
into the metaphysis or diaphysis of long bones and are characterized by a dense
fibrous connective tissue that contain short penetrating mineralized collagen fibers.
The fibers insert directly into the bone or periosteum distributing stress across
large surface areas (Apostolakos et al., 2014). Fibrous entheses are not prone to
overuse injuries due to a slight insertion angle change during use (Apostolakos et
8 | P a g e
al., 2014). The low propensity for injury makes fibrous enthesis the least studied
of the two enthesis types. Fibrocartilage entheses, on the other hand, insert into
the epiphesis or apopthesis and are prone to overuse injuries due to large insertion
angle changes during use, with a prime example being a rotator cuff tear
(Apostolakos et al., 2014). A fibrocartilage enthesis is characterized by tendon-
bone insertions containing a transitional tissue, graded from tendon to
fibrocartilage to mineralized fibrocartilage to bone that is adapted to withstand
complex stress concentrations (Apostolakos et al., 2014). Each zone of the
fibrocartilage can be defined by it cellular and collagen components. The tendon
layer consist primarily of fibroblast cells that produce both type I and type III
collagens. The uncalcified fibrocartilage is made up of fibrochondrocytes that
produce collagens I-III as well as the proteoglycan aggrecan. This layer serves to
dissipate the strain from the bending collagen fibers of the tendon (Apostolakos et
al., 2014). The tidemark which as a flat straight surface marks the boundary
between soft and hard tissue that separates the fibrocartilage and mineralized
fibrocartilage zones (Apostolakos et al., 2014). The mineralized or calcified
fibrocartilage layer is also composed of fibrochondrocytes and expresses
predominantly type II collagen but additionally expresses collagen types I and X.
Like the fibrocartilage zone, the mineralized fibrocartilage is avascular which is
thought to be important to minimize the spread of infection from the highly
vascularized bone to the tendon (Apostolakos et al., 2014). Unlike the tidemark,
the interface between the mineralized fibrocartilage zone and the bone is irregular
in shape with the two surfaces highly interdigitated (Apostolakos et al., 2014). This
9 | P a g e
interlocking is thought to provide structural integrity to the enthesis and is essential
for its function. The bony end of the fibrocartilage enthesis known as the eminence
displays all the typical hallmarks of bone consisting of osteoblast, osteocytes and
osteoclasts with a Type 1 collagen matrix (Apostolakos et al., 2014). The
eminences are the physical bumps and ridges on the bone that are sites of tendon
insertions.
Formation of the Enthesis
Fibrocartilage entheses are derived from a distinct pool of Scx
+
/Sox9
+
progenitors
that are established secondarily to the primary cartilage anlagen of the bone.
During enthesis morphogenesis, these bipotent progenitor cells differentiate into
either Scx
+
tenocytes or Sox9
+
chondrocytes to a connective tissue (Blitz et al.,
2013; Blitz et al., 2009; Sugimoto et al., 2013). In the appendicular skeleton,
Scx
+
/Sox9
+
progenitor cells are specified by TGFβ signaling. Conditional knockout
of Tgfβr2 in limb mesenchyme leads to dramatic reductions in Scx
+
/Sox9
+
cells and
loss of enthuses (Blitz et al., 2013). Later, BMP signaling induces differentiation of
Scx
+
/Sox9
+
progenitor cells into Sox9
+
chondrocytes. Conditional knockout of
Bmp4 in limb mesenchyme blocks differentiation of Scx
+
/Sox9
+
progenitors cells
in to chondrocytes (Blitz et al., 2013; Blitz et al., 2009). During enthesis maturation,
hedgehog signaling is required for mineralization. Conditional knockout of
Smoothened significantly limits the production of mineralized fibrocartilage
(Dyment et al., 2015). While we are beginning to understand the molecular cues
10 | P a g e
that influence enthesis development, those signals that determine the initial pattern
of differentiation across the Scx
+
/Sox9
+
progenitor field remain limited.
The mechanism that spatially resolves bipotency of Scx
+
/Sox9
+
progenitor cells
has been difficult to solve because the upstream regulators that promote Scx
expression and commitment to the tenocyte cell lineage are not well understood.
Several lines of evidence suggest that FGF signaling has tendon-inducing
activities during development. FGFs emanating from the myotome are both
necessary and sufficient to induce syndetome, a compartment of the somites that
contains tendon progenitor cells for the axial skeleton in mouse and chick (Brent
et al., 2005; Brent et al., 2003; Brent and Tabin, 2004; Tozer and Duprez, 2005).
FGF is a potent inducer of tendon markers such as Scx and loss of FGF signaling
reduces or blocks Scx expression in axial and limb tendons during chick
development (Brent et al., 2003; Brent and Tabin, 2004; Edom-Vovard et al., 2002;
Havis et al., 2016). However, in contrast to the chick limb, it appears that FGF
signaling inhibits Scx expression and tenocyte cell fate in mouse limb explants and
mouse mesenchymal stem cells (Havis et al., 2016; Havis et al., 2014).
Additionally, Fgf signaling inhibits Scx expression in mouse chondrocyte-like TC6
Cells (Kawa-uchi et al., 1998). Since tendon and/or enthesis defects have yet to
be reported in mouse models that harbor mutations in FGF ligands or receptors, it
remains unclear if FGF signaling has an inhibitory effect on tenocyte cell fate in
mouse limb mesoderm during development in vivo.
11 | P a g e
Here we demonstrate that FGF signaling regulates development of the graded
connection between tendon and bone in the mammalian jaw. Masticatory muscles
open and close the jaw by transmitting contractile forces across tendon entheses
anchored into the bone of the mandible. The mandible is a compound structure,
developing from an intramembranous bone whose proximal processes terminate
in a cap that crowns a wedge of secondary cartilage (Anthwal et al., 2008; Atchley
and Hall, 1991). Secondary cartilages of the condyle and angular processes form
in close association with the tendons for the masticatory muscles, facilitate
mandibular growth, and enable articulation of the mandible at the
temporomandibular joint (TMJ). We show that the secondary cartilages of the
condyle and angular processes contain Scx
+
/Sox9
+
progenitor cells and that
conditional inactivation of Fibroblast growth factor receptor 2 (Fgfr2) alters the
spatial pattern of their differentiation, subsequently disrupting formation of graded
entheses. We find that Fgfr2 signaling promotes formation of Scx+ tenocytes in
Scx
+
/Sox9
+
progenitor cells by spatially regulating Notch/Jagged expression
across the developing enthesis. Correspondingly, conditional loss of Jagged1 in
neural crest-derived mesenchyme disrupts cell fate in Scx
+
/Sox9
+
progenitor cells
and enthesis development.
12 | P a g e
Chapter 2
Modeling craniofacial and skeletal congenital birth defects to advance
therapies
Cynthia L. Neben
1
, Ryan R. Roberts
2
, Katrina M. Dipple
3
, Amy E. Merrill
2
, and
Ophir D. Klein
1,4*
1
Department of Orofacial Sciences and Program in Craniofacial Biology,
University of California, San Francisco, San Francisco, California, USA
2
Center for Craniofacial Molecular Biology, Ostrow School of Dentistry and
Department of Biochemistry and Molecular Biology, Keck School of Medicine,
University of Southern California, Los Angeles, California, USA
3
Departments of Pediatrics and Human Genetics, David Geffen School of
Medicine and InterDepartmental Program Biomedical Engineering, Henry
Samulei School of Engineering and Applied Sciences, University of California,
Los Angeles, California, USA
4
Department of Pediatrics and Institute for Human Genetics, University of
California, San Francisco, San Francisco, California, USA
*To whom correspondence should be addressed: University of California, San
Francisco, 513 Parnassus Ave, HSE1509, San Francisco, CA 94143, USA. Tel:
+1 4154764719; Fax: +1 4154769513; Email: ophir.klein@usc.edu
This work was previously published in Human Molecular Genetics, Volume
25, Issue R2, 1 October 2016, Pages R86–R93
13 | P a g e
Abstract
Craniofacial development is an intricate process of patterning, morphogenesis,
and growth that involves many tissues within the developing embryo. Genetic
misregulation of these processes leads to craniofacial malformations, which
comprise over one-third of all congenital birth defects. Significant advances have
been made in the clinical management of craniofacial disorders, but currently very
few treatments specifically target the underlying molecular causes. Here, we
review recent studies in which modeling of craniofacial disorders in primary patient
cells, patient-derived induced pluripotent stem cells (iPSCs), and mice has
enhanced our understanding of the etiology and pathophysiology of these
disorders while also advancing therapeutic avenues for their prevention.
14 | P a g e
Introduction
The craniofacial complex is one of the most intricate and sophisticated parts of the
human body. Its patterning and morphogenesis involve a dynamic interplay
between the ectoderm, mesoderm, and endoderm, and a critical role is played by
neural crest cells, which give rise to the majority of skeletal and connective tissues
in the craniofacial region. These interactions are established and maintained by
numerous genes, including those encoding a variety of transcription factors,
growth factors and receptors (Minoux and Rijli, 2010; Thesleff, 2006). Disruption
of gene expression or function results in devastating craniofacial anomalies, which
have a collective incidence rate of 1 in 600 births (2004). Much of our current
understanding of the etiology and pathophysiology of craniofacial disorders has
been uncovered through the use of model systems. Mice are considered by many
to be the gold standard for disease modeling, as they are anatomically and
physiologically comparative to humans and can be genetically manipulated to
mimic human phenotypes (Rosenthal and Brown, 2007). Primary patient cells and
patient-derived iPSCs have proven to be a valuable complement to the mouse
model system by either highlighting species-specific differences or further
validating the observations already made in mice (Tiscornia et al., 2011). The
modeling of craniofacial disorders has not only informed genetic risk assessment
and patient prognosis but also identified potential targets for pharmaceutical
intervention. In this review, we highlight recent efforts that have provided new
information to advance the treatment of five general classes of human genetic
disorders, with particular emphasis on the craniofacial region. In addition, we
15 | P a g e
discuss how this information furthers our understanding of the molecular
mechanisms regulating normal craniofacial development.
Craniosynostosis
The cranial sutures are fibrous joints that form between the five principal flat bones
of the skull vault during embryogenesis. From the early fetal period through the
first years of life, the cranial sutures are primary sites of bone growth and allow the
skull vault to expand with the growing brain. Formation and maintenance of the
suture, which include the osteogenic fronts of the jointed bones and their
interposed mesenchyme, is critical to its function as a growth center. Dysfunction
in genes that regulate organization, proliferation, and/or differentiation within the
suture can lead to its premature fusion, a relatively common birth defect known as
craniosynostosis (Ishii et al., 2015). Serious clinical problems associated with
craniosynostosis include craniofacial deformities, increased intracranial pressure,
and impaired brain development leading to learning difficulties or developmental
delay. Treatment plans involve surgery that removes and reshapes large areas of
the calvaria; however, for many patients suture re-fusion necessitates repeated
surgeries (Foster et al., 2008). Thus, there is a clinical need to develop less
invasive, more effective therapies for craniosynostosis. Recent studies that apply
our current knowledge of the molecular players in normal and abnormal suture
development have advanced the potential for these new therapies.
16 | P a g e
The pathogenesis of syndromic craniosynostosis is commonly associated with
gain-of-function mutations in Fibroblast Growth Factor receptors (FGFR) 1-3. FGF
signaling promotes proliferation and differentiation in osteogenic cells, most
notably in the cranial sutures (Iseki et al., 1999; Rice et al., 2000). Apert syndrome
is usually caused by dominant mutations in FGFR2 that increase ligand-dependent
activation and subsequently enhance osteoblast differentiation (Yang et al., 2008).
New findings show that expression or nanogel-mediated delivery of a soluble form
of FGFR2 harboring the Apert mutation S252W blocks enhanced FGFR2 signaling
and inhibits craniosynostosis in a mouse model for Apert syndrome (Morita et al.,
2014; Yokota et al., 2014).
The Bone Morphogenetic Protein (BMP) pathway plays a critical role in
development of the skull vault. Increased BMP signaling is associated with
craniosynostosis (Gong et al., 1999; Komatsu et al., 2013; Warren et al., 2003),
and antagonists of the pathway are being tested as a possible treatment to prevent
post-operative re-fusion. In a recent study, delivery of the BMP antagonist
GREMLIN1 via hydrogel that rapidly polymerized upon injection prevented bone
re-growth in a mouse model for re-synostosis (Hermann et al., 2014).
Hypophosphatasia, a metabolic disorder with craniosynostosis, is caused by loss-
of-function mutations in ALPPL, the gene encoding Tissue-nonspecific Alkaline
Phosphatase (TNAP). TNAP is an osteoblast surface protein that induces
17 | P a g e
hydroxyapatite crystal growth by increasing inorganic phosphate (Hessle et al.,
2002; Murshed et al., 2005). A recent report shows that craniosynostosis in Alppl
knockout mice is rescued by subcutaneous injection of a mineral-targeted form of
recombinant TNAP (Liu et al., 2015).
Craniofacial Dysmorphologies
Dysmorphic craniofacial features can often be quantified as anthropometric
measurements outside the normal variance, and these can be isolated or occur in
a syndrome. Current treatments are directed towards addressing the specific
features on a patient-by-patient basis. While craniofacial dysmorphologies can
have phenotypic overlap, the underlying mechanism of disease is quite disparate.
Due to their genetic heterogeneity, an exciting frontier in the treatment of
craniofacial dysmorphologies is the possibility of targeted therapeutics such as
genome editing (Goodwin et al., 2013; Niihori et al., 2006; Tekin et al., 2009;
Vincent et al., 2016).
Brachio-ocular-facial (BOF) syndrome is associated with missense mutations in
the TFAP2A gene encoding AP-2, a transcription factor with early roles in neural
crest cell specification and survival (Knight et al., 2005; Milunsky et al., 2008;
Milunsky et al., 2011). Generation of the first fully penetrant cleft lip and palate
mouse model caused by mutations in Tfap2a revealed that one cause of clefting
can be subtle changes in FGF pathway gene expression in the facial prominences.
18 | P a g e
Manipulation of Fgf8 gene dosage partially rescued the phenotype, suggesting that
FGF signaling and/or downstream effectors may be possible targets of
pharmacological intervention in BOF syndrome and nonsyndromic cases of
clefting associated with TFAP2A mutations (Green et al., 2015).
Heterozygous mutations in BRAF are found in 50-75% of patients with cardio-
facio-cutaneous (CFC) syndrome (Niihori et al., 2006). BRAF is a serine threonine
kinase that regulates the RAS-MAPK signaling pathway, and therefore CFC
syndrome is classified as a RASopathy (Jindal et al., 2015). Braf
Q241R/+
mice exhibit
embryonic/neonatal lethality with liver necrosis, edema, and craniofacial
abnormalities, effectively mimicking the phenotypes of human patients.
Interestingly, co-treatment with MEK inhibitors and histone demethylase inhibitors
rescued the pathophysiology (Inoue et al., 2014). This finding has implications not
only for the potential therapies of CFC syndrome but for other RASopathies as
well. It will be important to examine the epigenetic contributions to heart and
skeletal defects in these disorders as well as the treatment potential of combined
inhibition of HRAS signaling and histone demethylases.
Treacher Collins syndrome (TCS) is an autosomal dominant disorder which
presents with hypoplasia of the facial bones, cleft palate, and low set, malformed
ears (Trainor et al., 2009). In a mouse model of TCS, haploinsufficiency of the
Tcof1 gene encoding the nucleolar phosphoprotein treacle reduces ribosome
19 | P a g e
biogenesis, causing deficient proliferation and extensive apoptosis of
neuroepithelial cells via a nucleolar stress-induced, p53 pathway (Jones et al.,
2008; Rubbi and Milner, 2003). The recent discovery that treacle also functions in
DNA damage response/repair to limit oxidative stress-induced neuroepithelial cell
death identifies a novel underlying contributor to the pathogenesis of TCS.
Excitingly, in utero treatment with antioxidants prevents DNA damage and
minimizes cell death in the neuroepithelium to substantially ameliorate the
craniofacial anomalies in Tcof1
+/-
embryos (Sakai et al., 2016). While previous work
has shown that genetic and pharmacological inhibition of p53 can suppress the
neuroepithelial apoptosis in Tcof
+/-
embryos, maternal antioxidant dietary
supplementation may be a safer potential therapeutic for patients with TCS, given
the risk of tumorigenesis associated with p53 manipulation (Jones et al., 2008;
Sakai et al., 2016).
Dental Anomalies
Developmental dental anomalies are defined as marked deviations from the
normal color, contour, size, number, and degree of formation of teeth. These
malformations can occur either as part of a syndrome or as an isolated finding
(Klein et al., 2013). In Costello syndrome (CS), a RASopathy associated with
craniofacial, cardiac, musculoskeletal, and neurodevelopmental abnormalities,
characteristic dental phenotypes include class III malocclusion, enamel
hypomineralization, and soft tissue hyperplasia (Goodwin et al., 2014a). Nearly all
individuals with CS have a heterozygous mutation in HRAS that results in
20 | P a g e
constitutive activation of Ras signaling (Aoki et al., 2005; Estep et al., 2006). A CS
mouse model expressing HRas
G12V
phenocopies many aspects of the syndrome
and was used to understand the cellular mechanism underlying the
hypomineralization of the enamel (Goodwin et al., 2014b). In this model, enamel-
forming ameloblasts lacked polarity and the ameloblast progenitor cells were
hyperproliferative. Furthermore, inhibition of MAPK led to complete rescue of the
dental phenotype, whereas modulation of either MAPK or PI3K signaling corrected
the defect in progenitor cell proliferation in CS mice (Goodwin et al., 2014b). This
work defined for the first time distinct roles of Ras signaling in tooth development
and provided additional evidence for the use of Ras inhibitors in treating CS and
other RASopathies.
Hereditary conditions involving nonsyndromic enamel conditions are referred to as
amelogenesis imperfectas (AIs). The X-linked form of hypoplastic AI is associated
with missense mutations in Amelogenin, an extracellular matrix protein secreted
by ameloblasts (Kim et al., 1994; Lagerstrom et al., 1991). The murine Y62H
Amelogenin mutation similarly results in eruption of malformed tooth enamel with
severely compromised mechanical properties (Barron et al., 2010). Recent work
has demonstrated that this specific mutation disrupts proper intracellular trafficking
of amelogenin and induces ER stress-related apoptosis in ameloblasts, classifying
AI as a protein conformational disease for the first time (Brookes et al., 2014).
Treatment with 4-phenylbutyrate, which can act to relieve conformational
abnormalities of the protein, rescued the enamel phenotype in affected female
21 | P a g e
mice by promoting cell survival over apoptosis, offering a potential therapeutic
option for patients with this form of AI (Brookes et al., 2014; Iannitti and Palmieri,
2011).
Skeletal Dysplasias
Skeletal dysplasias represent one of the largest classes of birth defects, with over
450 recognizable conditions (Warman et al., 2011). The craniofacial defects in
these disorders result from the combinatorial interactions of transcription factors,
growth factors and receptors responsible for the intricate genetic patterning and
morphogenesis of craniofacial structures (Neben and Merrill, 2015). With the
advent of next-generation DNA sequencing, clinical phenotypes can be linked to
key cellular processes of skeletal development, including proliferation,
differentiation, and apoptosis. Dominant missense mutations in FGFR3 that
reduce chondrocyte proliferation are associated with achondroplasia (ACH) and
thanatophoric dysplasia (TD), the most common genetic forms of dwarfism
(Henderson et al., 2000; Rousseau et al., 1994; Shiang et al., 1994). Craniofacial
findings include macrocephaly, frontal bossing, and midface hypoplasia in ACH,
and macrocrania, cloverleaf skull, and frontal bossing in TD. The severity of these
chondrodysplasias is linked with the degree of constitutively activated FGFR3
signaling through MAPK or STAT1, and as such, therapeutic strategies have
focused on decreasing excessive downstream signaling (Krejci et al., 2008;
Laederich and Horton, 2010). Recent work in patient-specific induced pluripotent
stem cells (iPSCs) has identified statins as a potential drug to treat FGFR3-
22 | P a g e
mediated chondrodysplasias. Treatment with statins rescued cartilage formation
in chondrogenically differentiated TD1 and ACH iPSCs and led to significant
recovery of bone growth in an ACH mouse model (Yamashita et al., 2014). While
the precise mechanism of action remains to be determined, the success of statin
treatment highlights a previously unappreciated role for anabolic activity during
chondrogenesis (Baker et al., 2012; Simopoulou et al., 2010; Yudoh and
Karasawa, 2010).
Maintaining the proper balance between proliferation and differentiation is also
critical for bone formation. Examination of the pathophysiology of Bent Bone
Dysplasia Syndrome (BBDS) revealed an unexpected nuclear route for FGF
signaling to regulate osteoprogenitor cell proliferation and differentiation via
ribosome biogenesis (Neben et al., 2014). BBDS is a dominant disorder
characterized by bent long bones in the lower extremities and craniofacial
abnormalities including poorly mineralized calvaria, craniosynostosis, midface
hypoplasia, micrognathia, low-set ears, and prenatal teeth. BBDS results from
mutations in the transmembrane domain of FGFR2 that redistribute the receptor
from the plasma membrane to the nucleolus, where it activates ribosomal DNA
transcription by halting RUNX2-mediated repression (Merrill et al., 2012; Neben et
al., 2014). Inhibition of ribosomal RNA synthesis by small molecules has been
shown to be effective in preclinical cancer models in mice and may be a potential
therapeutic strategy to specifically target the pro-proliferative role of FGFR2 in the
23 | P a g e
nucleolus of BBDS patients and likely other FGFR2 gain-of-function disorders
(Bywater et al., 2012; Devlin et al., 2016; Drygin et al., 2011; Haddach et al., 2012).
Cherubism is a condition caused by excessive osteoclast activity in the mandible
and maxilla, which drives progressive proliferation of fibrous tissues and leads to
severe facial deformities. Spontaneous regression of bone lesions is usually
observed at puberty, and surgical intervention is only considered when functional
or aesthetic concerns arise (Papadaki et al., 2012; Reichenberger et al., 2012).
Recently, two independent studies presented promising pharmacological
therapeutic approaches to inhibit or delay the progression of cherubic lesions. Most
patients with cherubism have gain-of-function mutations in the gene encoding
SH3BP2, an adapter protein involved in the immune response. Sh3bp2 knock-in
mice develop massive infiltration of macrophages into skeletal elements, including
the jaw, which can be rescued by genetic inhibition of TNF-α expression (Ueki et
al., 2007). Consistent with the role of TNF-α, treatment with the anti-TNF-α inhibitor
etanercept significantly reduced facial swelling and bone loss in neonatal mice.
Furthermore, this phenotypic rescue was not recapitulated in adult mice,
emphasizing the importance of early diagnosis and treatment of cheribusm
(Yoshitaka et al., 2014). An effective therapy for patients with actively growing and
established inflammatory lesions may be bone marrow (BM) transplants.
Transplantation of wild type BM cells to Sh3pb2 knockin mice rescued the systemic
inflammation and bone loss in adult cherubism that could not be ameliorated by
etanercept treatment (Yoshitaka et al., 2015). Treatment with tacrolimus, an
24 | P a g e
immunosuppressor that has been shown to inhibit activation of the
calcineurin/NFATc pathway and osteoclastogenesis (Amaral et al., 2010; Duarte
et al., 2011; Lietman et al., 2008), led to significant clinical improvement in a 4-
year old boy with an aggressive form of cherubism; specifically noted was
stabilization of jaw size and intraosseous osteogenesis (Kadlub et al., 2015).
Future studies are needed to determine the precise mechanism of action of
tacrolimus and whether combined treatment with anti-inflammatories may further
ameliorate the pathophysiology of cherubism.
Bone Mineral Density
Bone mineral density (BMD) is determined by relative rates of bone deposition and
resorption, which are carried out by osteoblasts and osteoclasts, respectively.
Mutations in the genes controlling osteoblast and osteoclast function cause
congenital disorders with abnormal BMD. While these conditions present with
generalized skeletal abnormalities, the craniofacial findings have important clinical
complications. In osteopenic and osteoporotic disorders, where bone resorption
exceeds deposition, calvaria are undermineralized, malformed, and fractured. In
osteopetrotic disorders, where bone deposition outpaces resorption, there is focal
or widespread thickening of the calvaria, skull base, and facial bones. Recent
studies have supported the use of biologics to restore the balance between bone
anabolism and catabolism in congenital BMD disorders.
25 | P a g e
Genetic studies of congenital BMD disorders have demonstrated that the
Wnt/LRP5 pathway increases bone density by promoting osteoblast production
and function. Loss-of-function mutations in the Wnt co-receptor LRP5 cause the
low bone mass disorder osteoporosis-pseudoglioma syndrome (OPPG), while
LRP5 gain-of-function mutations cause the high bone mass disorders Van Buchem
disease, osteosclerosis, and osteopetrosis (Ai et al., 2005; Boyden et al., 2002;
Gong et al., 2001; Little et al., 2002; Van Wesenbeeck et al., 2003). LRP5
mutations in the high bone mass disorders increase the co-receptor activity by
disrupting binding of the inhibitor sclerostin, which is inactivated in the high bone
density disorder sclerosteosis (Balemans et al., 2007; Balemans et al., 2001;
Brunkow et al., 2001; Semenov et al., 2005). These studies laid the groundwork
for development of an inhibitory antibody against sclerostin that is now in phase 3
clinical trials for the treatment of postmenopausal osteoporosis (McClung et al.,
2014). New evidence supports repurposing anti-sclerostin to treat the very
syndromes that advanced its discovery: depletion of sclerostin, either genetically
or through the use of anti-sclerostin, increases the BMD of mouse models for
OPGG (Chang et al., 2014; Kedlaya et al., 2013). These findings also provide the
rationale for use of a recombinant Wnt/LRP5 inhibitor or inhibitory antibody against
LRP5 to block bone overgrowth in the osteopetrotic disorders.
There is strong evidence to suggest that anti-sclerostin will increase BMD in other
skeletal fragility syndromes, such as osteogenesis imperfecta (OI) and hereditary
hypophosphatemic rickets, despite differences in the molecular pathologies. While
26 | P a g e
OI is largely caused by deficiencies in type I collagen production, modification, or
secretion, mouse models for OI gain a significant increase in bone mass and
strength when Wnt/LRP5 signaling is increased, through either expression of LRP5
gain-of-function mutation or treatment with anti-sclerostin (Grafe et al., 2015;
Jacobsen et al., 2014; Roschger et al., 2014; Sinder et al., 2013; Sinder et al.,
2014). Additionally, anti-sclerostin significantly improves osteomalacia in DMP1
knockout mice, a model for hereditary hypophosphatemic rickets (Grafe et al.,
2015).
Mouse models with reduced BMD have enabled identification of promising new
targets for protein-based therapies. Defective type I collagen biosynthesis in OI
increases the bioavailability of TGFβ, leading to excessive TGFβ signaling (Grafe
et al., 2014). Promotion of osteoclast bone resorption by TGFβ signaling provides
a rationale for the use of inhibitory antibodies against TGFβ. Indeed, anti-TGFβ
improves bone mass in mouse models for OI (Grafe et al., 2014). Knockout of
Nell1, which codes for a secreted bone-inducing factor, leads to age-related
osteoporosis (Desai et al., 2006). Correspondingly, delivery of recombinant NELL1
was shown to increase bone formation via the Wnt pathway in both small and large
animal models of osteoporosis (James et al., 2015).
Future Directions
Studies that model congenital disorders in primary patient cells, iPSCs, and mice
have advanced therapeutic opportunities for craniofacial disorders (Figure 1). New
27 | P a g e
technologies such as CRISPR/Cas9 that increase the speed, efficiency, and
simplicity in genome editing will allow for rapid generation of cells lines and animal
models that carry human disease-causing mutations (Cong et al., 2013; Harrison
et al., 2014; Jinek et al., 2013; Mali et al., 2013). Specifically, genome editing
techniques offer a way to model Mendelian disorders in large animals, whose
disease states may more closely resemble humans than the mouse models.
Strategies for CRISPR/Cas9-modification in monkey, pig, and goat embryos have
recently been reported. Genome editing will also aid in the study of congenital
disorders associated with allelic or locus heterogeneity, which can complicate the
diagnosis and treatment of these conditions (Lu et al., 2014; Warman et al., 2011).
Introducing patient-specific mutations will help to identify genotype-phenotype
correlations and subtle differences in the mechanistic effects of specific mutation
(Hai et al., 2014; Ni et al., 2014; Niu et al., 2014). One of the most exciting clinical
applications of genome editing is the possibility of correcting disease-causing
genes. Indeed, the therapeutic potential of CRISPR/Cas9 is currently being
investigated in patient-derived iPSCs, organoid cultures, and mouse models (Li et
al., 2015; Osborn et al., 2015; Schwank et al., 2013; Wu et al., 2013; Xie et al.,
2014; Yin et al., 2014). These studies raise high hopes for improving the clinical
diagnosis, treatment, and outcome of patients with craniofacial and skeletal
malformations.
28 | P a g e
Acknowledgements
This work was supported by the National Institutes of Health [R01DE025222 to
A.E.M, U01-DE024440 and R01-DE024988 to O.D.K]; and March of Dimes [#6-
FY15-233 to A.E.M.].
Conflict of Interest Statement. All authors state that they have no conflicts of
interest.
Figure 1. Pipeline that turns emerging insights into potential therapeutics for
craniofacial disorders. Human genetic studies identify critical genes linked to
craniofacial disease. Mechanistic studies, using primary patient cells, patient-
specific iPS cells, and/or animal models, probe the disease gene’s role in
craniofacial biology. Once the biological function of the gene is discovered,
therapeutic targets can be identified. Having an in-depth view of the target’s
biology aids in selecting therapeutic modalities, such as biologics, small molecules,
stems cells, and possibly gene editing.
29 | P a g e
Chapter 3
Fgfr2 regulates the cell fate at the interface between tendon and bone
Ryan R. Roberts
1,2
, Lauren Bobzin
1,2
, Camilla Teng
2
, Deepanwita Pal
3
, Creighton
Tuzon
1,2
, Ronen Schweitzer
3
, Amy E. Merrill
1,2,*
1
Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, University
of Southern California, Los Angeles, CA, 90033
2
Department of Biochemistry and Molecular Biology, Keck School of Medicine,
University of Southern California, Los Angeles, CA, 90033
3
Research Division, Shriners Hospital for Children, Portland, OR 97239, USA
*
Correspondence: amerrill@usc.edu
30 | P a g e
ABSTRACT
Tendon is joined to bone through a transitional connective tissue known as the
enthesis. The enthesis arises from a specialized population progenitor cells that
co-express Scleraxis (Scx) and Sox9, transcriptional regulators of tenocyte and
chondrocyte differentiation, respectively. Scx
+
/Sox9
+
progenitors differentiate into
either Scx
+
tenocytes or Sox9
+
chondrocytes; however, the mechanism that
spatially resolves their bipotency to produce the morphologically graded enthesis
is not understood. We demonstrate that FGF signaling patterns differentiation in
fields of Scx
+
/Sox9
+
progenitors within the mammalian lower jaw. Conditional
inactivation of Fgfr2 in mandibular entheses induces abnormal distribution of the
Scx
+
and Sox9
+
lineages, ectopic bone formation, and incomplete tendon insertion.
Changes in the pattern of Scx
+
/Sox9
+
cell differentiation is preceded by disruptions
in Notch-Jagged expression and conditional deletion of Jagged2 leads to abnormal
development of mandibular entheses. These results suggest that Fgfr2 establishes
a spatial gradient of cell fate via Notch-Jagged during enthesis morphogenesis.
31 | P a g e
INTRODUCTION
Tendons facilitate body movement by delivering high tensile forces from muscle to
bone. Since tendon and bone have vastly different mechanical properties, they are
joined through a transitional connective tissue known as an enthesis that is graded
from tendinous to osseous. The graded morphology of the enthesis is important
for efficient load transfer and minimizing stress concentrations across the hard-soft
tissue interface (Apostolakos et al., 2014). Due to its function, the enthesis is prone
to rheumatic disease as well as common orthopaedic injuries (Apostolakos et al.,
2014). The mature enthesis has poor healing capacity following damage and, by
failing to replicate the normal developmental program, forms a mechanically
unstable attachment that lacks its formerly graded structure (Apostolakos et al.,
2014). To advance clinical strategies for enthesis regeneration and repair we must
gain a better understanding of the molecular mechanisms that regulate enthesis
morphogenesis during development.
Tendons attach to bone through fibrous or fibrocartilaginous enthesis (Benjamin et
al., 2006). Fibrous enthesis contain short tendon fibers that insert directly into the
periosteum and distribute stress across large surface areas. Fibrocartilage
entheses, on the other hand, are characterized by tendon-bone insertions
containing a transitional tissue that is adapted to withstand complex stress
concentrations. Fibrocartilage entheses are derived from a distinct pool of
Scx
+
/Sox9
+
progenitors that are established secondarily to the primary cartilage
anlagen of the bone. During enthesis morphogenesis, these bipotent progenitor
32 | P a g e
cells differentiate into either Scx
+
tenocytes or Sox9
+
chondrocytes to produce a
connective tissue that is graded from tendon to fibrocartilage to mineralized
fibrocartilage to bone (Blitz et al., 2013; Blitz et al., 2009; Sugimoto et al., 2013).
In the appendicular skeleton, Scx/Sox9
+
progenitor cells are specified by TGFβ
signaling. Conditional knockout of Tgfβr 2 in limb mesenchyme leads to dramatic
reductions in Scx
+
/Sox9
+
cells and loss of entheses (Blitz et al., 2013). Later, BMP
signaling induces differentiation of Scx/Sox9
+
progenitor cells into Sox9
+
chondrocytes. Conditional knockout of Bmp4 in limb mesenchyme blocks
differentiation of Scx/Sox9
+
progenitors cells in to chondrocytes (Blitz et al., 2013;
Blitz et al., 2009). During enthesis maturation, hedgehog signaling is required for
mineralization. Conditional knockout of Smoothened significantly limits the
production of mineralized fibrocartilage (Dyment et al., 2015). While we are
beginning to understand the molecular cues that influence enthesis development,
those signals that determine the initial pattern of differentiation across the
Scx
+
/Sox9
+
progenitor field remain limited.
The mechanism that spatially resolves bipotency of Scx/Sox9
+
progenitor cells has
been difficult to solve because the upstream regulators that promote Scx
expression and commitment to the tenocyte cell lineage are not well understood.
Several lines of evidence suggest that Fibroblast growth factor (FGF) signaling has
tendon-inducing activities during development. FGFs emanating from the
myotome are both necessary and sufficient to induce syndetome, a compartment
of the somites that contains tendon progenitor cells for the axial skeleton in mouse
33 | P a g e
and chick (Brent et al., 2005; Brent et al., 2003; Brent and Tabin, 2004; Tozer and
Duprez, 2005). FGF is a potent inducer of tendon markers such as Scx and loss
of FGF signaling reduces or blocks Scx expression in axial and limb tendons during
chick development (Brent et al., 2003; Brent and Tabin, 2004; Edom-Vovard et al.,
2002; Havis et al., 2016). However, in contrast to the chick limb, it appears that
FGF signaling inhibits Scx expression and tenocyte cell fate in mouse limb
explants and mouse mesenchymal stem cells (Havis et al., 2016; Havis et al.,
2014). Since tendon and/or enthesis defects have yet to be reported in mouse
models that harbor mutations in FGF ligands or receptors, it remains unclear if
FGF signaling has an inhibitory effect on tenocyte cell fate in mouse limb
mesoderm during development in vivo.
Here we demonstrate that FGF signaling regulates development of the graded
connection between tendon and bone in the mammalian jaw. Masticatory muscles
open and close the jaw by transmitting contractile forces across tendon entheses
anchored into the bone of the mandible. The mandible is a compound structure,
developing from an intramembranous bone whose proximal processes terminate
in a cap that crowns a wedge of secondary cartilage (Anthwal et al., 2008; Atchley
and Hall, 1991). Secondary cartilages of the condyle and angular processes form
in close association with the tendons for the masticatory muscles, facilitate
mandibular growth, and enable articulation of the mandible at the
temporomandibular joint (TMJ). We show that the secondary cartilages of the
condyle and angular processes contain Scx/Sox9
+
progenitor cells and that
34 | P a g e
conditional inactivation of Fibroblast growth factor receptor 2 (Fgfr2) alters the
spatial pattern of their differentiation, subsequently disrupting formation of graded
entheses. We find that Fgfr2 signaling promotes formation of Scx+ tenocytes in
Scx/Sox9
+
progenitor cells by spatially regulating Notch/Jagged expression across
the developing enthesis. Correspondingly, conditional loss of Jagged1 in neural
crest-derived mesenchyme disrupts cell fate in Scx/Sox9
+
progenitor cells and
enthesis development.
EXPERIMENTAL PROCEDURES
Mice
To conditionally knockout Fgfr2 in neural crest cells, the Fgfr2
flx/flx
(JAX Stock No.
007569) mice were crossed with the Wnt1-Cre2 driver Cre2 (JAX Stock No.
022137). The Fgfr2
flx/flx
and Wnt1-Cre2 lines have been previously described
(Lewis et al., 2013; Yu et al., 2003). To conditionally knockout Fgfr2 in enthesis
progenitor cells, Fgfr2
flx/flx
mice were crossed with the Scx-Cre/GFP driver (Blitz
et al., 2009). Scx-GFP was used as a reporter for enthesis progenitors, as well as
tendon and ligament (Pryce et al., 2007). To conditionally knockout Jagged 1 and
Notch 2 in neural crest cells, the Wnt1-Cre2 driver was crossed with Jagged1
flx/flx
(JAX Stock No. 010618) and Notch2
flx/flx
(JAX Stock No. 010525) mice,
respectively. Jagged1
flx/flx
and Notch2
flx/flx
lines have been previously described
(Kiernan et al., 2006). The Ai9 allele (JAX Stock No. 007909) was used as a
lineage marker for those tissues targeted by Wnt1-Cre2 and Scx-Cre/GFP
(Madisen et al., 2010). Scx
-/-
mice have been previously described (Murchison et
35 | P a g e
al., 2007). Embryonic samples were collected form timed pregnant females.
Postnatal samples were staged according to the date of birth.
Immunofluorescent analysis
Embryos and postnatal samples were fixed in 4% paraformaldehyde for 15
minutes to 3 hours, depending on the tissue size. Postnatal tissues were then
decalcified with 10% EDTA, pH 7.4 for 1-3 weeks at 4
o
C. Embryonic and postnatal
tissues were equilibrated 30% sucrose/PBS at 4
o
C, embedded in O.C.T.
compound (EMS), and sectioned in the coronal plane at 8 μM. Frozen sections
washed with PBST (0.1% Triton X-100) and blocked with 10% serum for 1 hour at
room temperature. When mouse primary antibodies were used, the sections were
pre-incubated with Anti-fab fragment for 1 hour at room temperature (Sup. Table
1). All sections were incubated with primary antibodies overnight at 4
o
C. See Sup.
Table 1 for antibody venter information and dilutions. Sections were then washed
with PBST and incubated with Alexa Fluor secondary antibody at a 1:500/PBST
for 1 hour at room temperature, washed with PBST, and mounted with Vectashield
containing DAPI (VWR). Slides were imaged on the Leica TCS SP5/8 confocal
system.
Skeletal preparation
Samples were skinned, eviscerated, and fixed in 95% ethanol for 3-5 days. Fixed
samples were incubated in Alcian Blue stain (0.15 mg/ml Alcian Blue 8GX in 80%
ethanol and 20% glacial acetic acid) overnight and destained in 95% ethanol for 2
36 | P a g e
days. Samples were then cleared with 0.5-1% KOH [w/v] for 1-5 days, depending
on size, and incubated in Alizarin red solution (0.02 mg/ml Alizarin red S, Sigma-
Aldrich in 0.5-1% KOH) for an additional 1-5 days. Stained specimens were
equilibrated in glycerol for imaging.
Histology
For histological staining the tissues were fixed in 95% ethanol, decalcified with
BBC Biochemical Rapid Cal Immuno (Fisher Scientific) overnight at room
temperature, embedded in paraffin, and sectioned in the coronal plane at 10 M.
Connective tissues were discriminated using Hall-Brunt quadruple stain (HBQ)
(Hall, 1986).
Micro-computed tomography (μCT)
All μCT scans were performed by the USC Molecular Imaging Center using
a μCT50 (Scanco Medical). Samples were rotated 360° and X-ray settings were
standardized to 90 kV and 155 µA, with an exposure time of 0.5 seconds per frame
to yield a nominal resolution of 20 μM. A 0.5-mm-thick aluminum filter was
employed to minimize beam-hardening artifacts. Morphometric analysis was
performed using the Amira 6.2 or Avizo 7.1 software packages. Equally threshold
isosurface renderings were measured using the 3D measuring tool. All jaw
measurement landmarks were derived from (Guerreiro et al., 2013).
37 | P a g e
RNA Isolation and Gene Expression Analysis
RNA was isolated from tissues using Qiagen RNeasy Mini Prep Kit according to
the manufacturer’s instructions. The RNA concentration was measured, and 50 ng
of total RNA was used for amplification using KAPA SYBR FAST one-step
quantitative real-time PCR reagents per the manufacturer’s instructions using
primers listed in Sup. Table 2. Expression analysis was monitored on a BioRad
CFX96 Real Time System based on relative gene expression using CT
calculation.
RESULTS
Scx is essential for proper formation of the jaw processes and tendon
insertion
The graded connection units are formed from a pool of Scx/Sox9
+
progenitor cells
that differentiate from the condensing mesenchyme (Blitz et al., 2013; Blitz et al.,
2009; Sugimoto et al., 2013). Scx, which delineates the fate of tendon cells upon
maturation of the connection unit, is thought to be essential for its maintenance
(Blitz et al., 2013; Blitz et al., 2009; Sugimoto et al., 2013). To determine the role of
Scx within the angular and condylar processes of the mouse jaw the Scx
-/-
mice
were used. As early as 30 days post birth the Scx
-/-
mice exhibited ectopic bone
on the angular and condylar processes by CT (Fig 1A-B). Further analysis of the
skeletal preparation of the P30 mice showed a persistence of cartilage matrix on
the condyle (Fig 1C-D). The ectopic bone becomes more pronounced over the
period of 1.5 years, with a bony protrusion extending from the condyle and a large
38 | P a g e
ridge presenting on the ramus (Fig 1E-F). To better understand the mechanism
for ectopic bone formation, skeletal preparation of P7 mice were analyzed. The
Scx
-/-
showed enhanced cartilage matrix staining indicative of premature
differentiation of the normally undifferentiated progenitor cell pool at the posterior
end of the processes (Fig 1G-H).
The Scx null mice are deficient in the differentiation of the force-transducing and
intermuscular tendons in both the limbs and tail (Murchison et al., 2007). There
was less of a defect in the muscle attaching tendons in the Scx
-/-
mice, which was
the expected phenotype in the soft tissue surrounding the jaw processes. In the
jaw region, there were disruptions in muscle attachment at the interface where the
disc merges with the pterygoid and masseter muscles on both sides of the condyle
in P5 mice (Fig 1I-J). Around the angular process at P5, there was a loss of almost
all of the intermuscular tendons similar to what was previously described in the
trunk regions of Scx
-/-
mice (Murchison et al., 2007), in addition to an overall
dysmorphic process. There was also a disruption in the muscle attaching tendons
at the regions where we see ectopic bone at P30 (Fig 1K-L). This data suggest
that loss of Scx leads to ectopic bone formation at the expense of proper insertion
of the muscle.
The condyle and angular process are bona fide connection units
Since Scx seems essential for proper muscle attachment on the angular and
condylar processes, we wanted to determine if the processes have the molecular
39 | P a g e
hallmarks of a typical connection unit. Immunofluorescence for Scx and Sox9 were
performed on coronal cross-sections of control E16.5 angular and condyle
processes. Both processes were Scx
+
/Sox9
+
indicating that the processes are
bona fide connection units (Fig 2A-B). Additionally, Fgfr2 is expressed throughout
the entire process and its ligand Fgf2 around the periphery indicating a role for
FGF signaling in their development (Fig 2C-D).
Wnt1 derived Neural crest ablation of Fgfr2 leads to ectopic bone at the
entheseal sites of tendon insertion in the craniofacial complex.
Knowing that Fgfr2 and Fgf2 are expressed at the right place and time during the
development of the jaw processes, we wanted to determine the role of Fgf
signaling in the connection unit. To address this, Wnt1-cre mice were crossed with
Fgfr2
fl/fl
mice to generate Wnt1-cre; Fgfr2
fl/fl
mice to determine the role of Fgfr2 in
the developing craniofacial complex. Mutant mice and Wild-type controls were
analyzed by CT at P30. These conditional knock our (cKO) mice exhibited
ectopic bone on the zygomatic arch, ramus of jaw, and on the angular and condular
processes (Fig 3A-B, 3J-K). The ectopic bone seen on the jaw processes (Fig 3J-
K) is morphologically similar to the phenotype of the Scx-null (Fig 1). Overlay of
Scx-GFP expression from the tendon specific reporter mouse line on the Control
mouse, illustrates that defects occur at tendon insertion entheseal sites (Fig 3C).
There is a dislocation of the TMJ due to an upward rotation of the articular surface
of the condyle cause by ectopic bone at the base of the process (Fig 3D-E).
Morphological analysis showed that Wnt1-cre; Fgfr2
flx/flx
mice have shorter lengths
40 | P a g e
and heights of their jaws (Fig 3F, I), which may be partly due to the mutant mice
being significantly smaller by weight at P30 (Fig 3M). There is no difference in
weight embryonically (Fig 3M), suggesting this size difference is caused perhaps
by a mastication defects. Despite this, ratios of condyle length to the overall length
of the jaw show that the condyles are significantly shorter in the mutants (Fig 3L),
which exhibit retro-micrognathia similar to the LADD syndrome patients.
Premature loss of the growth plates leads to retro-micrognathia
A skeletal preparation stage series of the Wnt1-cre; Fgfr2
flx/flx
mice was done to
better understand the progression of the ectopic bone phenotype. There was really
no noticeable bone defects embryonically as late as E18.5 (Fig 4A-B). Bone
defects were visible at P2, with both the angular and condylar processes having
noticeable smaller growth plates, indicating a possible reduction of the overall
progenitor pool (Fig 4C-D). By P5 there was visible ectopic bone and a greater
loss of the growth plates at the base of each process with the angular process
being more severely affected (Fig 4E-F). There is a complete loss of the angular
growth plate and partial loss of the growth plate at the base of the condyle a P10.
Both processes are noticeably truncated at this point (Fig 4G-H). This data
suggests that the ectopic bone phenotype occurs postnatally, and is due to the
progenitor cells in the growth plate prematurely differentiating into bone.
Additionally, the premature loss of the growth plate causes the retro-micrognathia
phenotype.
41 | P a g e
There is a novel interaction between Fgfr2 and Scx
In the Scx-/- mice there were defects in the three types of tendon attachments (Fig
1). Though the force-transducing and intermuscular tendons around the
processes are cranial neural crest derived, we did not expect a severe defect of
these tissues in the Fgfr2-cKO. The intermuscular tendons were the least affected
in the Wnt1-cre; Fgfr2
flx/flx
mice with the masseteric cutaneous tendons still being
visible (Fig 5D-F). On the lingual side of the condyle there was a disruption in the
attachment of the superior and inferior pterygoid muscle (Fig 5C-D). At the
interface between the disc and the masseter muscle there was a severe reduction
of the tendon attachment on the zygomatic arch (Fig 5C-D). On the angular
process, ectopic bone is visible at all regions of muscle or intermuscular tendon
insertion (Fig 5E-F). There is a tearing away of muscle from the surface of the
process, suggesting improper muscle attachment (Fig 5E-F). Loss of Fgfr2 in the
craniofacial complex exhibits a phenotype similar to the Scx
-/-
, characterized by
ectopic bone on the processes in regions of muscle and tendon attachment. This
suggests that Fgfr2 and Scx are acting through a similar mechanism and may be
part of the same pathway. Fgfr2 and Scx have been shown to interact within the
chick model (Brent and Tabin, 2004) but not in the mouse, so this would suggest
a novel interaction.
42 | P a g e
The boundaries between the layers of the connection unit are disruption in
the Fgfr2-cKO
To further address the molecular interaction between Fgfr2 and Scx, we evaluated
the expression of Scx-GFP in the Wnt1-Cre; Fgfr2
flx/flx
mice. There was a global
reduction of Scx-GFP in the craniofacial complex of the mutant mice (Fig 6A-B).
Additionally, there is a reduction of the Scx-GFP expressing cells that intercalate
into the processes at the site of tendon insertion (Fig 6C-D). This region is void of
Scx-GFP expressing cells suggesting a loss of tendon anchoring within the
processes. The reduced anchoring is thought to be responsible for the tearing of
the tissue seen in Figure 4. This data also suggest a loss of either the mineralized
fibrocartilage or the fibrocartilage domain seen in a typical connection unit.
To determine whether the mineralized fibrocartilage or fibrocartilage layer is lost in
the Fgfr2-cKO, specific markers for each layer were analyzed by
immunofluorescence. The layers of the connection unit express specific collagens
with Col II located in the mineralized fibrocartilage layer, Col III in the fibrocartilage
layer and Col I in both layers including the tendon (Apostolakos et al., 2014). Col
I was globally downregulated (Fig 7A-B) in the entire angular process (Fig 7A-B).
There is a substantial expansion of Col II expression domain with the expression
level being slightly low at the insertion site (Fig 7C-D). Col III expression is also
expanded throughout the process but unlike Col II the overall expression appear
to be elevated at the insertion site (Sup. Fig 1). This suggests that there is mixing
between the fibrocartilage and mineralize fibrocartilage layers, which hints to a loss
43 | P a g e
of boundaries in the mutant. Incidentally, Col III is also a marker for tissue damage,
which could partly account for its significant increase in expression.
The Scx/Sox9+ cells are established in the Wnt1-Cre; Fgfr2
flx/flx
mice
The phenotype seen in the Fgfr2-cKO is either due to defects in the initial
establishment or the later maintenance of the Scx
+
/Sox9
+
cells. Immuno-
fluorescent staining at E16.5 showed that the mutant mice do establish the initial
Scx
+
/Sox9
+
cells, which indicates that the phenotype is cause by later maintenance
of the progenitor cell population (Fig 8A-B). The mutant mouse does produce a
truncated non-functioning form of Fgfr2 that is detectable by the Santa Cruz Bek
antibody, which indicates an expansion of the Fgfr2 expression domain to tendon
like tissue around the processes (Fig 8A-B). Fgf2, which is normally expressed in
the surrounding tissue and around the periphery of the process, sees its
expression expanded, infiltrating into the process (Fig 8C-D).
Since Fgfr2 did not affect the establishment of the bipotent progenitor population,
we then wanted to determine if the defect was autonomous to the Scx expressing
cells. To answer this question, we generated a Scx-Cre; Fgfr2
flx/flx
mouse line and
we observed a similar but milder defect on both the condyle and angular process
when compared to the Wnt1-Cre; Fgfr2
flx/flx
mice (Sup Fig 2A-H). This suggests
that Fgfr2’s role in the processes is autonomous to the Scx expression cells. The
milder phenotype also hints to the role of Fgfr2 at time points before and after Scx
expression.
44 | P a g e
Notch signaling is altered in the jaw entheses of Wnt1-Cre; Fgfr2
flx/flx
mice
The mixing or loss of the fibrocartilage and mineralize fibrocartilage layer suggest
that there is a loss of cell fate boundaries in the Fgfr2-cKO mouse. This led us to
look at the Notch signaling pathway, which is a key regulator of cell boundary
specification (Appel et al., 2001; Kiernan et al., 2006; Matsuda and Chitnis, 2010).
Notch signaling regulates cell fate boundaries through either lateral induction or
lateral inhibition (Appel et al., 2001). Lateral induction refers to a preferred cell
that expresses a Jagged ligand inducing the surrounding secondary Notch
receptor expressing cells to adapt a similar cell fate (Appel et al., 2001). In contrast,
lateral inhibition refers to a preferred cell that expresses a Delta ligand, inhibiting
the surround Notch receptor expressing cell from adopting the same cell fate
(Appel et al., 2001). In the Zebrafish ear, fgf signaling promotes support cell
formation by indirectly inducing the expression of deltaD ligand activates Notch
signaling in the surrounding cells, inhibiting hair cell fate (Appel et al., 2001;
Kiernan et al., 2006; Matsuda and Chitnis, 2010). Examples like this made the
Notch signaling pathway a prime candidate for regulating the boundaries between
the layers of the enthesis. Notch2 which is normally expressed throughout the
angular process at E16.5 is downregulated in the Wnt1-Cre; Fgfr2
flx/flx
mouse.
Additionally, the down-regulation of Notch2 was confirmed by qPCR. Conversely,
Jagged1, which is also expressed throughout the process, sees its localization
relegated to the outside edge of the process where the insertion site should later
form. At E16.5 Delta1 in the Control is expressed at the outer edge of the process
45 | P a g e
at the presumptive insertion site, which sees its expression become downregulated
and irregular in the Wnt1-Cre; Fgfr2
flx/flx
mouse.
As a proof of concept, Wnt1-Cre; Jagged1
flx/flx
mice were generated to determine
if these mice have a similar phenotype to the Wnt1-Cre; Fgfr2
flx/flx
mice. At E16.5
the Jagged1-cKO mice had a vastly reduced Scx expression in and around the
angular process. There was also a reduction in Sox9 expression within the
process. This resulted in an eroded articular surface of the condyle and a reduction
in size of the angular process. Additionally, there was ectopic mineralization on
the surface of the condyle.
DISCUSSION
The condyle and the angle of the jaw are essential for the functions of speech and
mastication. In addition to the condyle forming the Temporomandibular hinge joint
with the temporal bone; both the angle and condyle are sites of muscle and
ligament attachments. There is a decent body of work using gene ablation studies
that have identified genes that are important in the formation of the condyle and
angular process in mice but very little is known about the establishment of the
entheses in the craniofacial complex. Entheses have traditionally been studied in
the long bones of the limbs. In order to better facilitate the repair of the enthesis
post injury, it is paramount to have a better understanding of factors that regulate
the specification and maintenance of the enthesis. This study serves to recognize
46 | P a g e
the jaw processes as bona fide entheses and establishes the role of Fgfr2 in
maintenance of these entheses through the Notch signaling pathway.
The most well characterized entheseal site is the deltoid tuberosity (DT) of the
humerus, which is absent in the Scx
-/-
mice (Murchison et al., 2007). Since these
mice have deficiencies in the force transducing tendons and force is required for
enthesis maintenance, it possible to infer that absence of the DT is due to
inadequate forces being exerted on the processes (Benjamin et al., 2006;
Murchison et al., 2007; Thomopoulos et al., 2007). Interestingly, like other
entheses, the DT is formed from a progenitor pool of Scx
+
/Sox9
+
cells (Blitz et al.,
2013; Blitz et al., 2009). Upon maturation, it is the parsing out of these cells into
either Scx
+
tendon or Sox9
+
chondrocyte that is thought to be responsible for
formation of the layers of the enthesis (Blitz et al., 2013; Blitz et al., 2009). Taken
together, it is evident that the role of Scx in the enthesis is more complicated than
what was initially surmised, with it possibly being important in both establishment
and maintenance of the enthesis. With Scx expression localized at the muscle
bone interface of the condyle (Purcell et al., 2012), this led us to ask about the role
of Scx in the craniofacial complex focusing on the jaw processes. Unlike the DT,
both the condyle and the angular process are present in the Scx
-/-
, though there
was evidence of ectopic bone, delayed maturation, structural defects and
deficiencies in muscle insertion. This suggests that Scx in the craniofacial complex
plays a more important role in the maintenance verses in the specification of the
47 | P a g e
jaw processes. This brought into question to whether the condyle and angular
processes are actual entheses.
Tgf 2 have been shown to be essential to form the DT and jaw processes, with
loss of Tgf 2 leading to both their absences (Blitz et al., 2009; Oka et al., 2008;
Oka et al., 2007; Zhao et al., 2008). Additionally, both the DT process are formed
for secondary cartilage condensate from the primary bone anlage, which is one of
the hallmarks of an enthesis (Blitz et al., 2009), with the second hallmark being the
formation from a Scx
+
/Sox9
+
progenitor pool. Like the DT, the Jaw processes are
formed from a pool of Scx
+
/Sox9
+
cells, which suggest that the condyle and angular
processes are entheses. Additionally, Like in the previous study by Purcell et al.,
2012, we observed that that Fgfr2 is expressed in jaw processes. Fgfr2 was
expressed in conjunction with its ligand Fgf2, which was expressed in the
surrounding muscle and tendons. Fgf2 expression is expressed in the same tissue
as Sprty1 and Sprty2, which are Fgf signaling inhibitors (Purcell et al., 2012). This
suggests that Fgf2 coming from the muscle and tendon, while Sprty1/2 inhibits the
spread of Fgf signaling to the surrounding tissue stimulates Fgfr2. Both Scx and
Fgfr2 are spatial-temporally expressed during the establishment of the jaw
processes.
We wanted to better understand the role of Fgf signaling in entheseal development
and determine if there is a link to Scx in the craniofacial complex. It is evident from
the Fgfr2 skeletal defects that Fgfr2 plays an important role in skeletogenesis.
48 | P a g e
Fgfr2 is expressed in the condensing mesenchyme and is a marker for the
precondrogenic condensate (Su et al., 2014). As development progresses it is
expressed in the perichondrium and periosteum. In chicks, fgfr2 is shown to
promote the expression of scx in the differentiating somites (Brent and Tabin,
2004). When we ablate Fgfr2 in the craniofacial complex, there was ectopic bone
defects at the sites of Scx expression, including the jaw processes. This causes
a premature loss of the growth plate and a reduction in the length of the jaw
processes leading to retromicrognathia. Additionally, there was a dislocation of
the TMJ joint, which is thought to affect mastication causing the delay in growth of
the mice. Interestingly, both the soft and hard tissue defects seen in the Fgfr2
mutant, were very similar to the defects seen in the Scx
-/-
mice, which led to
evaluate the role of Fgfr2 on Scx expression. There is a global downregulation of
Scx expression in the affected tissue when Fgfr2 is ablated, which is more evident
at the site of tendon insertion. This does suggest that Fgfr2 regulate Scx
expression in the facial region though this does not indicate whether the interaction
is direct. One potential explanation is the embryonic origin of the mesenchymal
tissue with bones and entheses of the face deriving forming from the cranial neural
crest. Additionally, there is aberrant expression of collagen markers that are
normally specific to each zone suggesting the loss of the boundaries of the
entheseal layers.
We then wanted to determine if the mixing of the boundaries between entheseal
layers is due to a defect in the establishment of the Scx/Sox9
+
progenitor cells.
49 | P a g e
The double positive progenitor cells are present in the mutant suggesting that the
defect is not due to the establishment of the progenitor cell pool. Though we do
see an expansion of the Fgf2 expression domain, which could be due to the mutant
receptor not serving as a sink on the process boundary to the ligand coming from
the surrounding tissue. This coupled with the altered collagen matrix of the
processes could allow for the structure to be more permeable to the ligand.
Additionally, with the surrounding tissue not getting the normal feedback from the
jaw processes may cause an increase in Fgf2 production.
Through lateral inhibition or lateral induction, Notch signaling has been shown to
be a key regulator of establishing tissue boundaries and regulating cell fate. This
is done through a three-way feedback loop where a high expressing Delta ligand
cell promotes Notch receptor and Jagged1 ligand expression while inhibiting Delta
in a neighboring cell. The Notch-Jagged expressing cell is now able to further
promote Notch-Jagged expression in an adjacent cell. FGF signaling regulates
delta in both the zebrafish ear to induce support cells and in the chick spinal cord
promoting neuronal differentiation through lateral inhibition. Interestingly, Jagged
inhibits cartilage cell fate in vascular smooth muscle cells of mice. Taken together,
this led us to ask if Notch signaling has been affected in the jaw processes of the
Fgfr2 mutant mice. We observed a decrease in Delta1 expression at the boundary
between the process and tendon, in addition to a reduction in Notch2 and a change
in localization of Jagged1 from within the process to along the outside edge in the
Fgfr2-cKO. The down-regulation of Delta1 around the outer edge of the process
50 | P a g e
may serve to promote homogeneity at the tendon insertion site, resulting in the
ectopic bone observed at the later stages. Delta probably promotes the boundary
between soft non-mineralized and mineralized cartilage of the enthesis and in its
premature absence leads to a mixing of the layers. The attenuation of Notch2 and
Jagged1 expression within the process is due to the loss of Delta, which is no
longer able to promote their expression. This leads to a loss of lateral induction
within the process, which then releases Jagged1’s inhibitory hold on Sox9
expression. This increases Sox9 expression along the presumptive insertion site,
shifting the balance in the Scx
+
/Sox9
+
progenitor cells promoting an early cartilage
fate. Conversely, Notch2 may also regulates Scx expression similar to Notch’s role
in up-regulating another bHLH transcription factor Atoh1. The loss of Notch2 would
cause a down regulation of Scx expression. All Together, this would account for
the premature loss of the growth plate and the void region of Scx expression in the
P2 insertion site. To further understand the role of Jagged in the establishment of
the connection unit, we used a Wnt1-Cre; Jagged1
flx/flx
mouse line, where we
observed a complete loss of Scx expression. This led to a smaller process and
signs of ectopic bone similar to the Fgfr2-cKO. This emphasized the role of Notch-
Jagged signaling in promoting the balance between Scx and Sox9 in the
development of the jaw processes.
Lastly, we wanted to elucidate whether the phenotype observed in the Wnt1-Cre;
Fgfr2
flx/flx
mice was autonomous to the Scx expressing cells. We generated a Scx-
Cre; Fgfr2
flx/flx
mice that had a similar but milder phenotype to the Wnt1-Cre; Fgfr2
51 | P a g e
flx/flx
mice (Sup. Fig 2A-H). The milder phenotype may be attributed to Scx
expression being downstream of Fgfr2, with the majority of Fgfr2’s role temporally
before Scx is expressed. Additionally, this data does suggest that the phenotype
is autonomous to the Scx expressing cells.
CONCLUSION
Fgfr2 is important in maintaining the Scx
+
/Sox9
+
progenitor pool of the enthesis.
This is facilitated by Fgf2 being expressed in the tendinous tissue adjacent to the
site of the developing enthesis. This then activates Fgfr2 in the presumptive
enthesis, which then promotes the expression of Notch2. Notch2 then interacts
with the Delta1 on the tendinous cell simultaneously reinforcing the expression of
Delta1 in the tendinous cell and Notch2 in the newly forming enthesis through
lateral inhibition. The now high Notch2 expressing cell of the enthesis promotes
the expression of Jagged1, which interacts Notch2 on an adjacent enthesis cell.
This promotes the expression of Notch2 and Jagged1 in the adjacent cell through
lateral induction. This then serve to downregulate the expression of Sox9 and
promoting the expression of Scx maintaining the balance of both the cartilage and
tendon markers in the enthesis cells (Fig 12).
52 | P a g e
Figure 2: CT comparison of the dorsal end of the jaw in (A) Control and (B) Scx
-
/-
mice at P30. Skeletal preparation comparison of dorsal end of the jaw in (C, E,
G) Control and (D, F, H) Scx
-/-
at P30, 1.5 years and P7. HBQ analysis of coronal
sections through the (I, J) condyle and (K, L) angular processes in (I, K) Control
and (J, L) Scx
-/-
at P5.
53 | P a g e
Figure 3: CT analysis of the face from P30 (A, C) Control and (B) Wnt1-Cre; Fgfr2
flx/flx
mice. (C) Control mice with an overlay of Scx-GFP. CT analysis of the TMJ
from P30 (D) Control and (E) Wnt1-Cre; Fgfr2
flx/flx
mice. (F, I, L) Average length
(mm) of the jaw: (length-I) mandibular symphysis to condyle articular surface;
(length-II) mandibular symphysis to angular process; (height) Angular process to
base of condyle articular surface and (condyle) condyle base to condyle articular
54 | P a g e
surface. CT of jaw processes in Control and Wnt1-Cre; Fgfr2
flx/flx
mice. (M)
Average fold difference in weight of (black bar) Control and Wnt1-Cre; Fgfr2
flx/flx
mice at E18.5 and P30.
Figure 4: Skeletal Preparation of (A, C, E, G) Control and (B, D, F, H) Wnt1-Cre;
Fgfr2
flx/flx
mice at E18.5, P2, P5 and P10.
55 | P a g e
Figure 5: Alcian Blue Staining of P5 (A) Control and (B) Wnt1-Cre; Fgfr2
flx/flx
mice.
Modified HBQ staining of coronally sectioned P5 (C, D) condyle and (E, F) angular
processes in (C, E) Control and (D, F) Wnt1-Cre; Fgfr2
flx/flx
mice: SPT, superior
belly of the pterygiod); IPT, inferior belly of the pterygoid); MPT, medial belly of
the pteryoid, SL, Superior lamina (elastin fiber that attached the disc to the
temporal bone); IL, Inferior lamina (collagen fibers that attach the disc to the
condyle); TL, tendinous lamina of the medial pterygoid; and Mas, Masseter.
56 | P a g e
Figure 6: Whole mount overlay of Scx-GFP reporter expression in P2 (A) Control
and (B) Wnt1-Cre; Fgfr2
flx/flx
mice. Scx-GFP reporter expression counterstained
with DAPI in (C, C’) Control and (D, D’) Wnt1-Cre; Fgfr2
flx/flx
mice. C’ and D’
zoomed in images of respective squared outline region.
57 | P a g e
Figure 7: Dual immuno-fluorescence of coronal sections from the angular process
in (A, C) Control and (B, D) Wnt1-Cre; Fgfr2
flx/flx
P2 mice: (A, B) Col I-purple, (C,
D) Col II-red and (A-D) Scx-GFP-green.
58 | P a g e
Figure 8: Dual immune-fluorescence of coronally sectioned E16.5 angular
processes from (A) Control and (B) Wnt1-Cre; Fgfr2
flx/flx
mice: Scx-purple and
Sox9-green. Dual immune-fluorescence of coronally sectioned E16.5 angular
processes from (C) Control and (D) Wnt1-Cre; Fgfr2
flx/flx
mice: Fgfr2-green and
59 | P a g e
Fgf2-red. Fgfr2
flx/flx
allele produces truncated protein product, acts as a functional
null.
Figure 9: Dual immuno-fluorescence of coronal sections from the angular process
in (A) Control and (B) Wnt1-Cre; Fgfr2
flx/flx
E16.5 mice: (A, B) Jagged1-green and
Fgfr2-green. (C) Quantitative PCR of Notch1 and Notch2 expression in Control
and Wnt1-Cre; Fgfr2
flx/flx
mice from pooled condyle and angular processes
samples.
60 | P a g e
Figure 10: Dual immuno-fluorescence of coronal sections from the angular process
in (A, C) Control and (B, D) Wnt1-Cre; Fgfr2
flx/flx
E16.5 mice: (A, B) Jagged1-green
and Delta1-red (C, D) Jagged1-purple and Fgfr2-green.
61 | P a g e
Figure 11: Dual immuno-fluorescence of coronal sections from the angular process
in (A) Control and (B) Wnt1-Cre; Jagged1
flx/flx
E16.5 mice: (A, B) Sox9-green and
Scx-red. Skeletal preparation of (C) Control and (D) Wnt1-Cre; jagged1
flx/flx
P17
mice.
62 | P a g e
Figure 12: Model of Fgf and Notch signaling in the enthesis.
63 | P a g e
Supplement Table 1: Immunofluorescence Antibodies
Antibody Species Source Catalog # Dilution
Bek (C-17) rabbit Santa Cruz Biotechnology, Inc sc-122 1:200
Collagen I rabbit Abcam ab34710 1:500
Delta1 rabbit Abcam ab10554 1:100
Collagen II mouse Developmental Hybridoma Bank CIIC1 1:100
FGF-2 (3) mouse Santa Cruz Biotechnology, Inc sc-135905 1:500
Jagged1 (C-
20)
goat Santa Cruz Biotechnology, Inc sc-6011 1:200
Notch2 (25-
255)
rabbit Santa Cruz Biotechnology, Inc sc-5545 1:100
Scleraxis (D-
14)
goat Santa Cruz Biotechnology, Inc sc-87425 1:50
Sox9 rabbit Novus Biologicals
NBP1-
85551
1:1000
Supplement Table 2: Mouse qPCR Primers
Gene Primer Sequence Tm Product Chr
Notch1 F GCTGACTGCATGGATGTCAATG 60.2 146 2
R CCCTGGTAGATGAAGTCAGAGATG 60 2
Notch2 F GATGGTGCATACTGTGATGTGC 60.0 82 3
R GGCACAAGTGTTCAACAGGTAC 60.0 3
64 | P a g e
Supplement Figure 1: Dual immuno-fluorescence of coronal sections from the
angular process in (A) Control and (B) Wnt1-Cre; Fgfr2
flx/flx
P2 mice: Col III-red
and Scx-GFP-green.
Supplement Figure 2: Skeletal Preparation of (A, C, E) Control and (B, D, F) Scx-
Cre; Fgfr2
flx/flx
mice at P3, P5 and P30. mCT analysis at P30 in (G) Control and
(H) Scx-Cre; Fgfr2
flx/flx
mice
65 | P a g e
Chapter 4
Discussion
Throughout my Ph.D. research in the Merrill Laboratory, I have had three main
projects. The first looked at the role of MicroRNA in Jaw development, using the
chick as a model. The chick presumptive jaw was dissected out a different stages
of development and process for microarray analysis. The microarray yielded
promising targets but this project ended mainly because of its slow progression.
The other two projects where derived from morphological observations of Wnt1-
Cre; Fgfr2
flx/flx
mouse. The initial observations were centered on the bones of the
face and cranium. A few of these observations were a foramen of the frontal
suture, asymmetry of the face, ectopic bone on the face and jaw, shortening of the
jaw processes, premature fusion and delayed ossification of the hyoid bone,
shortening of the tympanic ring bone, and bone defects on the middle ear ossicles.
The second project, focused on understanding the defects on the middle ear
bones. Preliminary data suggested that the mice had ectopic bone in the synovial
joint spaces connecting the ossicles and ectopic bone at the insertion sites of the
tensor tympani and stapedius muscles. These defects led to hearing loss in these
mice. This project is ongoing and was taken over by a post-doctoral fellow in the
Merrill Lab.
The third project pursued the understanding of the defects observed on the jaw
and its processes, which became my primary dissertation project. This project
established the role of Fgfr2 in the maintenance of the interface between the
66 | P a g e
tendon and bone, known as the enthesis in the craniofacial complex. This is
facilitated by the Fgfr2 expressing cells of the enthesis receiving a signal from the
inserting tendon in the form of Fgf2. The enthesis cells react by promoting the
expression of Notch2 and Jagged1. Notch2 then interacts with the Delta1 ligand
on the inserting tendon cell and through lateral inhibition, inhibit a cartilage fate in
the tendon cells. The Notch2 expression in the enthesis through lateral induction,
promotes the expression of Notch2 and Jagged1 in a neighboring enthesis cell.
This serves to regulate the expression of Scx and Sox9 in the enthesis cell
maintaining the balance between the two transcription factors in the progenitor
cells.
This project serve to better understand the defects seen the Fgfr2 loss of function
mutation disorder, LADD syndrome. We now know that the retro-micrognathia
seen in the patients is likely due to a premature loss of `the progenitor cells of the
jaw processes. This data will not only help to understand the bone related defects
seen in LADD syndrome but the hypoplastic glandular defects as well. The majority
of LADD syndrome patients exhibit both hypoplastic salivary and lacrimal gland,
which affects their ability to produce saliva and tears, respectively. This affects
their ability to lubricate their eyes and to swallow food (Rohmann et al., 2006).
Notch receptors and ligands are expressed in the acinar cells of the salivary gland
and serves to regulate cell growth and differentiation. Inhibiting Notch signaling in
the salivary gland cells inhibits their ability to differentiate (Dang et al., 2009). Since
my data suggest that Fgfr2 promotes Notch signaling; perhaps the loss of Fgfr2 in
67 | P a g e
LADD syndrome leads to the loss of Notch signaling affecting the maturation of the
salivary gland.
The enthesis does not repair well post injury and often leads to enthesopathy,
characterized by chronic pain and inflammation. Other causes of enthesopathies
are osteoarthritis, rheumatoid arthritis and ankylosing spondylitis (Karten et al.,
1962). Of specific interests is ankylosing spondylitis, with patients having fusion
of their vertebrates caused by ectopic bone growth at the enthuses (Tseng et al.,
2016). Though the ankylosing spondylitis and LADD syndromes bone phenotypes
manifest at different locales on the bone, both seem to share common etiologies,
suggest a molecular link between the disorders. It is possible that my work could
help to elucidate the molecular mechanism behind ankylosing spondylitis and other
enthesopathies.
The current strategy to repair the enthesis post injury, involves the suturing or
stapling of the tendon onto the bone. The enthesis then heals through a three
stage process; inflammation, repair and remodeling which leads to a fibrovascular
scar (Apostolakos et al., 2014; Zelzer et al., 2014). This scar does not recapitulate
the graded structure of the normal enthesis and thus is mechanically weaker and
prone to re-injury. It is thought that to better facilitate repair a more complete
understanding of the development of the enthesis is needed. It is known that
development of the enthesis requires the spatial-temporal interplay between
various growth factors and mechanical force. One promising clinical strategy is to
68 | P a g e
use a scaffold infused with growth factors to induce the formation of the layers of
the enthesis during repair (Apostolakos et al., 2014; Liu et al.). Understanding the
role that Fgfr2 plays in the maintenance of the enthesis could be crucial for this
kind of strategy.
Overall my project will help to advance our scientific knowledge of Fgfr2 signaling
in bone and enthesis development. Clinically, this work could help in devising new
strategies to treat enthesopathies and entheseal injuries. Additionally, this project
will help to shed light on the molecular mechanism behind LADD syndrome, which
could help to provide relief to the patient symptoms.
Future Direction
1. The data from the Wnt1-cre; Fgfr2
flx/flx
mutant suggest that there is a mixing
of the layers of the enthesis when you lose Fgfr2. It is still possible to delve
deeper to understand specifically how the layers are affected. A brief follow-
up to this project could examine the progression of how the mixing occurs
and determine how this contributes to the ectopic bone phenotype. Different
histological staining such as Von Kossa, which would mark the boundary
between mineralized and unmineralized fibrocartilage and Safranin O,
which would mark both fibrocartilage layers, together would provide new
insight on the defects that are observed. Additionally, immunofluorescence
staining for Gli1 and IHH, which are expressed differentially throughout the
layers of the enthesis (Dyment et al., 2015; Schwartz et al., 2015), would
69 | P a g e
also shed new light on the mixing phenotype. This combined with the
previous collagen staining would really provide a more detailed
understanding of the enthesis defect.
2. The maintenance of an enthesis requires mechanical input from muscle
movement (Benjamin et al., 2006; Hall, 1986; Kahn et al., 2013). Very little
is known about the molecular mechanism that responds to the mechanical
input to allow for enthesis maintenance. Since the source of Fgf2 to the
enthesis is the surrounding muscle and tendon, it is possible that one of the
roles of FGF signaling is to respond to the mechanical input. The phenotype
that we observe in the Wnt1-Cre; Fgfr2
flx/flx
mice maybe due to the enthesis
being unable to respond to the muscle stimuli. A future direction could focus
on the role of Fgfr2 in responding to mechanical forces in the jaw.
3. From my preliminary observations, there is a change in pigmentation and a
disheveled appearance of the fur on the Scx-Cre; Fgfr2
flx/flx
mice. Fgfr2 has
been shown to negatively regulate Shh signaling in the hair follicle
(Mukhopadhyay et al., 2013) and the role of Scx has never been described
in this tissue. A future direction could aim to define the role of Scx in the
hair follicle and further elucidate the relationship between Scx and Fgfr2.
70 | P a g e
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Abstract (if available)
Abstract
Fibroblast Growth Factor (FGF) signaling plays a critical role in skeletal development, as mutations in Fibroblast Growth Factor Receptor 2 (FGFR2) manifest with at least 9 distinct skeletal birth defects. Loss-of-function mutations in FGFR2 cause Lacrimoauriculodentodigtal (LADD) syndrome, which is a disorder characterized by posterior shortening of the jaw. We hypothesized that the posterior jaw shortening in LADD indicates a role for FGFR2 in the development of the jaw processes. To test this hypothesis, we employed a conditional knockout mouse in which Fgfr2 is ablated within the neural crest-derived skeletal precursors of the jaw. We found that Fgfr2ᶠˡˣ/ᶠˡˣ
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Asset Metadata
Creator
Roberts, Ryan Richardo
(author)
Core Title
Fgfr2 regulates cell fate at the interface between tendon and bone
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
09/29/2019
Defense Date
09/29/2017
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
bone,craniofacial,Development,enthesis,FGFR2,jaw,LADD syndrome,mandible,OAI-PMH Harvest
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Chuong, Cheng-Ming (
committee chair
)
Creator Email
jamdownian.rr@gmail.com,rrrobert@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c40-439899
Unique identifier
UC11265507
Identifier
etd-RobertsRya-5802.pdf (filename),usctheses-c40-439899 (legacy record id)
Legacy Identifier
etd-RobertsRya-5802.pdf
Dmrecord
439899
Document Type
Dissertation
Rights
Roberts, Ryan Richardo
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
Tags
craniofacial
enthesis
FGFR2
jaw
LADD syndrome