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The role of Runx2 in the development of the tendon-bone attachment unit
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The role of Runx2 in the development of the tendon-bone attachment unit
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Content
The Role of Runx2 in the Development of the Tendon-bone Attachment Unit
By
Siyan Wang
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
[Biochemistry and Molecular Medicine]
August 2020
Copyright 2020 Siyan Wang
ii
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my mentor, Dr. Amy E. Merrill, for giving
me the wonderful opportunity to join her lovely laboratory to achieve my passion for scientific
research. Thanks to her guidance, constant encouragement, and selfless support in research and
writing that allows me to finish this research project and my thesis. I would also like to thank my
other thesis committee members, Dr. Francesca Mariani, and Dr. Jian Xu, for their insightful
guiding comments and suggestions.
Besides, I would like to thank all my lab members, Yi Sui, Ryan R. Roberts, Lauren
Bobzin, Audrey Nickle, Creighton T. Tuzon, and Diana Rigueur for their patience help for me to
overcome all the difficulties in my research. I appreciate their kindness and joy in creating a
harmonious atmosphere of the team that giving me the sense of support and optimism.
I am proud to become a part of USC, a part of Keck School of Medicine and grateful to all
the professors who have taught me that enriched my knowledge in my study, as well as all the
faculty who have offered me help. I would also thank all my friends for their accompany.
Lastly, I would like to express my gratitude to my parents for their unconditional love and
support for the past two years. And thank my grandma for all the good memories.
iii
TABLE OF CONTENTS
Acknowledgements ....................................................................................................................... ii
List of Tables ................................................................................................................................ v
List of Figures ............................................................................................................................... vi
Abbreviations ................................................................................................................................ vii
Abstract ......................................................................................................................................... viii
Chapter 1: Introduction ................................................................................................................ 1
1.1 Overview of the musculoskeletal system .......................................................................... 1
1.1.1 Skeletal development in the limb ............................................................................. 2
1.1.2 Muscle and tendon development in the limb ........................................................... 4
1.1.3 Development and modularity of the tendon-bone attachment unit and the
bone eminence ......................................................................................................... 5
1.1.4 Clinical significance of the enthesis ........................................................................ 8
1.2 Molecular mechanisms regulate the development of the tendon-bone attachment unit..... 9
1.2.1 TGF- β signaling pathway ....................................................................................... 9
1.2.2 Scx/BMP4 pathway ................................................................................................. 9
1.2.3 FGF signaling pathway ............................................................................................ 10
1.2.4 HH signaling pathway .............................................................................................. 10
1.2.5 Runx2 ....................................................................................................................... 11
1.3 Objectives and hypothesis ................................................................................................. 11
Chapter 2: Material and Methods ................................................................................................ 13
2.1 Mice .................................................................................................................................. 13
2.2 Genotyping ........................................................................................................................ 13
2.2.1 DNA extraction ........................................................................................................ 13
2.2.2 PCR .......................................................................................................................... 14
2.3 Skeletal preparations ......................................................................................................... 14
2.4 HBQ staining .................................................................................................................... 15
2.5 Fluorescent in situ hybridization ....................................................................................... 15
2.6 Immunofluorescence ......................................................................................................... 16
Chapter 3: Results ........................................................................................................................ 18
3.1 Runx2 is necessary for normal deltoid tuberosity formation ............................................ 18
3.2 Runx2 haploinsufficiency alters histogenesis of the deltoid tuberosity ............................ 20
3.3 Runx2 regulates development of Scx
+
/Sox9
+
progenitors ................................................ 22
iv
Chapter 4: Discussion .................................................................................................................. 29
4.1 Runx2 regulates development of tendon-bone attachment unit in the limb ..................... 29
4.2 Altered musculoskeletal system integration dictates DT formation and causes
bone bending ..................................................................................................................... 35
4.3 The possible explanation of variant DT formation defect in Runx2
+/-
mutants ................ 36
4.4 Potential signaling mechanism affected during tendon-bone attachment unit
development in Runx2
+/-
embryos .................................................................................... 37
4.5 Future directions ............................................................................................................... 38
References ..................................................................................................................................... 36
Supplementary .............................................................................................................................. 41
v
LIST OF TABLES
Table 2.1 PCR genotyping primers ............................................................................................... 14
Table 2.2 Primary antibodies for immunofluorescence ................................................................ 17
Table 2.3 Secondary antibodies for immunofluorescence ............................................................ 17
vi
LIST OF FIGURES
Figure 1 Limb bud morphology and development ....................................................................... 3
Figure 2 Endochondral bone formation ........................................................................................ 4
Figure 3 Bone eminence formation ............................................................................................... 7
Figure 4 Runx2
+/-
forelimbs exhibit hypoplastic or missing deltoid tuberosity, as well as
delayed bone mineralization .......................................................................................... 19
Figure 5 Haploinsufficiency of Runx2 leads to delayed chondrocyte hypertrophy and
mineralization of deltoid tuberosity and disrupts tendon insertion ................................ 21
Figure 6 Reduction of Scx
+
/Sox9
+
cells at E13.5 in Runx2
+/-
mutant mouse embryos ................ 23
Figure 7 Runx2
+
/Scx
+
cells in the tendon-bone attachment site are disorganized in Runx2
+/-
mouse embryos ............................................................................................................... 25
Figure 8 Scx
+
/Sox9
+
cells remain in the perichondrium at the tendon-bone attachment site
in Runx2
+/-
mutant embryos at E14 ................................................................................ 27
Figure 9 The RNA expression domain of Runx2 and Scx overlap in the tendon-bone
attachment unit at E14 ................................................................................................... 28
Figure 10 Schematic summarizing the working hypothesis on the role of Runx2 during
tendon-bone attachment unit development ................................................................... 30
Figure S1 Cartilage condensation on the humerus at E13.5 ......................................................... 41
vii
ABSTRACT
The enthesis, a graded connective tissue between bone and tendon, delivers the
force of muscle contraction to bone. The enthesis is prone to injury and has poor
capacity for repair. To advance strategies for enthesis repair it is important to
understand how the unique connective tissue is formed during embryonic
development. The goal of my project is to study the molecular regulators
controlling development of the tendon-bone attachment unit progenitor cells in the
limb. One of the candidate genes I proposed to be involved is Runx2, a
transcription factor of the RUNX family, which is essential for bone formation.
Haploinsufficiency of Runx2 leads to Cleidocranial dysplasia (CCD) in
humans. The CCD mouse model fails to develop the deltoid tuberosity, suggesting
that Runx2 is necessary for proper development tendon-bone attachment unit
progenitor cells in the limb. Previous studies have shown that the tendon-bone
attachment unit is formed modularly by a pool of Scx
+
/Sox9
+
progenitor cells that
forms the tendon’s terminus and the bone eminence into which it inserts. In this
study, I found that Runx2 is expressed by Scx
+
/Sox9
+
progenitor cells and
necessary for their differentiation into the bone eminence. These results extend our
understanding of tendon-bone attachment unit development by identifying Runx2
as a molecular regulator.
viii
ABBREVIATIONS
Deltoid tuberosity (DT)
Tendon-bone attachment unit (T-B AU)
Scleraxis (Scx)
SRY-Box transcription factor 9 (Sox9)
Runt‐related protein 2 (Runx2)
Bone morphogenetic proteins (BMP)
Transforming growth factor-β (TGF-β)
Fibroblast growth factor (FGF)
Indian hedgehog (IHH)
Sonic hedgehog (SHH)
Hedgehog (HH)
GLI family zinc finger 1 (Gli1)
1
CHAPTER 1: INTRODUCTION
1.1 Overview of musculoskeletal system
The musculoskeletal system is an organ system with a sophisticated assembly of
muscular and skeletal systems, incorporating muscles, bones, tendons, ligaments, cartilage and
other connective tissue that stabilize and connect the bones (Standring et al., 2016). The major
function of the musculoskeletal system is to provide the body with support and stability, and
allow different body movements to happen. Besides, it can also protect vital structures, provide
the body shape, store minerals and also participate in hematopoietic process (Kahn et al., 2008).
To generates body movements, muscle contraction first generates momentum, and the presence
of tendons inserted into the bones ensures the transmission of force, together, the bones serve as
levers and the traction control of the muscle produce body movements (Genin et al., 2009; Liu et
al. 2012; Zelzer et al., 2014).
Even though the physical properties of tendon and bone are vastly different, there is a
specialized graded tissue located in between call the enthesis, that not only ensures the effective
transmission of force that facilitates joint motion but also dissipates the pressure accumulated by
the contracting muscle at the tendon insertion site on the bone to maintain the integrity of the
attachment during body movements. Gradient cell types that form the mature enthesis can be
classified as 4 zones, from bone to tendon they are: bone, fibrocartilage, mineralized
fibrocartilage, and tendon (Lu et al., 2013; Benjamin et al., 2006; Benjamin et al., 2001;
Angeline et al., 2012). Although the development of each component of the musculoskeletal
system has been well-studied, the integration of tendon and bone still remains largely unknown.
Before understanding the development of the assembly of the musculoskeletal system, it
is helpful to have a basic understanding of the development of each musculoskeletal tissue.
2
1.1.1 Skeletal development in the limb
The limb skeleton is formed from cartilaginous templates followed by endochondral
ossification. The formation of the forelimb bud is initiated by the aggregation and condensation
of the undifferentiated mesenchymal cells from lateral plate mesenchyme at E9.0 and become
distinct at E10.5. These mesenchymal cells form the primordia of the limb skeleton then are
specified as chondroprogenitor, differentiate into chondrocytes and generate primary cartilage
skeleton (Akiyama et al., 2005). Sox9, a specific transcription factor required for chondrogenesis
during the differentiation of mesenchymal cells to chondrocytes and an earliest known marker of
chondrogenic progenitors and chondrocytes, is detectable in the limb bud since E10.0 (Akiyama
et al., 2002; Akiyama et al., 2008). The specification of the limb segments: stylopod, zeugopod,
and autopod are observed in a proximal to distal manner (Martin et al., 1990; Wanek et al.,
1989). A thin layer of undifferentiated mesenchymal cells makes up the perichondrium
surrounding the nascent cartilage. At E12.5, the differentiation of the chondroprogenitors is
completed; a cartilaginous template is also formed. As the chondrocytes proliferate, they secrete
Type II Collagen marking the differentiation of the chondrocytes. The growth of the cartilage
element proceeds, a hypertrophic zone is then established at the center, where chondrocytes stop
proliferating, enlarge and undergo hypertrophy from E13.5. Hypertrophic chondrocyte
differentiation is critical for bone formation, it can direct the mineralization of surrounding
matrix, attract blood vessels, and direct perichondrium cells surrounding the hypertrophic zone to
differentiate into osteoblasts, followed by perichondrium to periosteum transformation, bone
collar formation, and calcification. As the primary ossification center is established, the
cartilaginous template will be gradually be replaced by mineralized bone. Runx2, a transcription
3
factor as an initial marker for chondrocyte differentiation and osteogenesis, starts robust
expression in the limb at E12.5 (Takeda et el., 2001; Otto et al., 1997; Akiyama et al., 2005). As
the bone further enlarges, a secondary ossification center appears in the epiphyses. The
proliferating chondrocytes located between the primary and secondary ossification center
composes the growth plate and determines the future length and shape of the bone (Kronenberg,
2003).
Figure 1. Limb bud morphology and development.
Representative forelimb (FL) and hindlimb (HL) bud morphology are showed from E9.5 to
E13.5. Light blue indicates mesenchymal condensations, and cartilage is marked by thick black
lines (figure panel adapted from Taher et al., 2011).
4
Figure 2. Endochondral bone formation.
a, mesenchymal cells condensation. b, cells of condensations become chondrocytes (c). c,
hypertrophic zone establishment (h). d, perichondral cells adjacent to hypertrophic chondrocytes
become osteoblasts, forming the bone collar (bc). Mineralization and blood vessel invasion of
the hypertrophic chondrocytes. e, primary spongiosa (ps) formation. f, proliferation of the
chondrocytes lengthening the bone. g, the secondary ossification center (soc) forms, the growth
plate forms orderly columns of proliferating chondrocytes (col), hematopoietic marrow (hm)
expands in marrow space (figure panel adapted from Kronenberg, 2003).
1.1.2 Muscle and tendon development in the limb
During embryonic development, limb muscle derives from Pax3 and Pax7 co-expressing
somitic progenitors that arise and migrate from the dermomyotome by E10.5 (Biressi et al.,
2007; Murphy and Kardon, 2011). These progenitor cells in the limb will finally give rise to two
types of cells: muscle or endothelial. When destined to the muscle fate, cells will undergo a
myogenesis process, starting as myoblasts. The basic muscle pattern is established by E10.5-12.5
in the mouse. At E13.5, muscles are individuated and distinct. Full patterning of muscle in the
limb is observed by the end of the embryonic myogenesis phase at E14.5. Since then, muscle
5
development continues until P21; fetal and neonatal myogenesis are also critical for muscle
growth and maturation (Mathew et al., 2011; Huang, 2017).
Tendons, which interconnect muscle and bone, arise from a the same mesodermal
compartment in the limb as cartilage, and also differentiate in a proximal to distal direction.
Scleraxis (Scx), a basis helix-loop-helix transcription factor, is a distinct early marker of tendon
and ligament progenitors. Scx expression is first detected at E10.5 in mouse limb bud in the sub-
ectodermal limb bud mesenchyme (Schweitzer et al., 2001; Huang et al., 2015), regulating
tendon/ ligament differentiation and maturation. Both transient and persistent expression of Scx
are important for musculoskeletal integration (Yoshimoto et al., 2017). Tendon formation starts
at E12.5, as tendon progenitors begin to align between the differentiating cartilage and muscle
tissues (Schweitzer et al., 2010). At E13.5, condensation of loosely organized tendon progenitors
occurs and gives rise to distinct functional tendons (Murchison et al., 2007), and tendon
differentiation markers, such as Tnmd, Col1a1 and Mkx, start to express (Havis et al., 2014;
Lejard et al., 2011; Ito et al., 2010). Most limb tendons are formed at E14.5 (Huang et al., 2013).
1.1.3 Development and modularity of the tendon-bone attachment unit and the bone
eminence
The tendon-bone attachment tissue, consisting of the distal end of the tendon, the
mineralized side of the bone, and the transitional zone in between, is referred to as the tendon-
bone attachment unit during embryonic development, the mature form is referred to as the
enthesis. Despite the functionally important of the tendon-bone attachment, the establishment
and regulatory mechanisms of the attachment unit haven't been studied very thoroughly. Studies
are usually initiated by looking at the development of the bone eminence at the tendon insertion
site. The bone eminence is the protrusion on the bone that exhibits various morphologies, and
6
which functions as the anchoring point of the tendon or ligament thus integrating of the
musculoskeletal system. For example, the deltoid tuberosity is a bone eminence on the humerus
attached by the deltoid muscle tendon (Figure 3A, A'). In mice, the development of bone
eminence is based on a modular model: a distinct pool of progenitor cells located externally to
the established primary cartilage rudiment gives rise to a secondary cartilage that is added on to
the cartilage template and that subsequently differentiates into the bone eminence during
development (Figure 3C). The specification of the bone eminence progenitor occurs later than
the primary cartilage. For example, when the chondrocytes in the primary cartilage humerus are
expressing the differentiation marker Col2a1 at E12.5, the chondrocytes in presumptive DT
remain undifferentiated (Blitz et al., 2013). The rudimentary cartilage of the humerus is formed
at E12.5, while the DT appears at E14.5 (Figure 3B). At the molecular level, primary cartilage
progenitors and bone eminence progenitors also show different gene expression patterns.
Primary cartilage progenitors express only Sox9, while bone eminence progenitors are Scx and
Sox9 double positive (Sugimoto et al., 2013; Blitz et al., 2013). The tendon-bone attachment unit
progenitors are also shown to be co-expressing Scx and Sox9 (Blitz et al., 2013; ). Cells at the
tendon attachment site are derived from a Sox9
+
lineage (Akiyama et al., 2005), while
chondrocytes of the bone eminence originate from a Scx
+
lineage (Sugimoto et al., 2013). It is
still unclear how a double-positive progenitor pool gives rise to two distinct populations that
undergo differentiation into two different cell types.
7
Figure 3. Bone eminence formation.
Anatomical sketches of forelimb bones showing the outlines of major eminences on each bone
(A), and muscles that are inserted into bone eminences (A’). (B) Skeletal preparations from
E12.5 and E14.5 wild-type embryos demonstrate morphological changes in the cartilage
template of forelimb during development. Arrows indicate eminences: deltoid tuberosity (black
arrow) and great tuberosity (green arrow) of the humerus and the olecranon of the ulna (red
arrow). C, modular model of deltoid tuberosity development: the eminence is derived from a
distinct pool of progenitor cells located outside the primary cartilaginous elements. Orange
circles indicate differentiated cartilage cells; green ovals indicate eminence progenitor cells
(figure panel adapted from Blitz et al., 2013).
C
8
During development, it has been observed that the Scx
+
/Sox9
+
progenitor population
reduces in size, and then two expression domains finally become completely segregated to Scx
+
tenocytes and Sox9
+
chondrocytes at E13.5 detected by ISH (Blitz et al., 2013). Thereafter, the
T-B AU further develops into a more complex, graded tissue postnatally and the T-B AU is
replaced by cells with a Gli1 lineage during enthesis maturation (Benjamin et al., 2006;
Felsenthal et al., 2018). Differentiation, maturation and maintenance occurs postnatally. The
mineralization of the mature attachment is similar to the growth plate (Zelzer et al., 2015).
Muscle loading is also critical for maturation of the developing enthesis.
1.1.4 Clinical significance of the enthesis
Although the enthesis is plays an important role in the musculoskeletal system, it is also
the place where overuse damage or mechanical injury commonly happens and can cause
significant pain and disability, such as the rotator cuff tear on the shoulder (Benjamine et al.,
2009; Lu et al., 2013; Thomopoulos et al., 2002). However, the mature enthesis is not
regenerated during tendon-bone healing (Galatz et al., 2015) and surgical intervention is often
need to re-join the tendon using sutures. The ideal result of therapeutic treatment for enthesis
injury would be to restore its natural tendon-bone insertion which is critical for its proper
function. Even though surgical treatment like using stronger sutures to re-attach the tendon to the
bone could be applied, it could still be followed by a high rate of substantial failure which means
incomplete healing, persistent anatomic defects, and chances to recurrence (Angeline et al.,
2012). Besides, little knowledge is known about the native healing process of the enthesis as well
as its embryonic development, so in order to determine potential therapeutic treatment of the
9
tendon-bone injury, the molecular mechanisms of tendon-bone attachment development should
be investigated.
1.2 Molecular mechanisms regulate the development of the tendon-bone
attachment unit
Knowing that the T-B AU and the containing bone eminence are derived from
Scx
+
/Sox9
+
progenitors, the investigation of the molecules and signaling pathways involved
during tendon-bone attachment unit development is usually initiated by analyzing mutants
lacking a bone eminence. The molecular mechanisms underlying the formation of the gradient
tissue of the enthesis still remain largely unknown.
1.2.1 TGF- β signaling pathway
TGFβ signaling is a key regulator of tendon-bone attachment unit formation, regulating
the specification of Scx
+
/Sox9
+
progenitors. TGFβ signaling regulates chondrogenesis of the
cartilaginous side of the T-B AU, as well as tendon formation (Zelzer et al., 2014; Pryce et al.,
2009). At E12.5, Tgfbr2 expression is detected in Scx
+
/Sox9
+
T-B AU progenitors. Limb
mesenchyme specific ablation of Tgfbr2 lead to absence of a bone eminence, which is attributed
to the loss of mechanical load of muscle contraction or tendon formation (Blitz et al., 2009; Seo
and Serra, 2007), mutant embryos also exhibit lack of Sox9
+
cells and dramatically reduced Scx
expression (Blitz et al., 2013), indicating TGFβ signaling is necessary for specification of
tendon-bone attachment unit progenitors.
10
1.2.2 SCX/BMP4 pathway
Scx (Scleraxis), a bHLH transcription factor marking tendon progenitors and all
tendinous tissue, is essential for tendon differentiation and proper integration of the
musculoskeletal system (Yoshimoto et al., 2017; Murchison et al., 2007). Scx is also important
for DT initiation, since Scx
-/-
embryos result in lack of the DT at E14.5 (Blitz et al., 2009).
Bmp4 is thought to be acting downstream to Scx, co-expressed with Scx at the
attachment site in the tendon cells at E13.5, and Bmp4 expression that activates BMP signaling
is Scx dependent. Blocking Bmp4 expression in limb mesenchyme failed to generate DT and
progenitor cells remain undifferentiated (Blitz et al., 2009). Implying that SCX/BMP4 pathway
is regulating bone eminence and T-B AU development.
1.2.3 FGF signaling pathway
FGF signaling is important for tendon development, inducing the initial induction of Scx-
expressing tendon progenitor cells in the early limb bud (Schweitzer et al., 2010). FGF signaling
is also implicated in skeletal morphogenesis. Moreover, FGF signaling specifies the secondary
cartilage in the avian jaw, Fgfr2 regulates early development of Scx
+
/Sox9
+
cells and loss of
Fgfr2 induces biased differentiation of these cells into Sox9
+
chondrocytes, determining the
progenitor cell fate at the tendon-bone interface in mouse mandible (Roberts et al., 2019).
1.2.4 HH signaling pathway
After the onset of T-B AU formation during embryonic development, Gli1
+
lineage cells
at the T-B AU are induced by SHH signaling pathway will eventually replace Sox9
+
progenitors
during postnatal development to form a mature enthesis, and IHH signaling from mineralized
11
fibrocartilage and bone is required for the maintenance of Gli1 expression in the enthesis
postnatally (Felsenthal et al., 2018).
1.2.5 Runx2
Runx2 (Runt-related protein of transcription factor 2) also known as Cbfa1 (Core-binding
factor subunit alpha-1) is a member of RUNX family of transcription factors. Runx2 plays
multiple important roles during skeletal development. Runx2 is an initial marker of the
osteogenic lineage, activating the differentiation of multipotent mesenchymal cells into
osteoblasts, and is also required for the proliferation of osteoblast progenitors (Ducy et al., 1997;
Otto et al., 1997; Komori et al., 2002; Kawane et al., 2018). Runx2 deficient mice exhibit
normalized maturation of chondrocytes and completely lack of vascular invasion into cartilage,
showing Runx2 is an essential regulatory factor for chondrocyte maturation and vascular
invasion during endochondral ossification (Inada et al., 1999; Kim et al., 1999; Hinoi et al.,
2006).
Heterozygous loss-of-function mutations in RUNX2 leads to Cleidocranial dysplasia
(CCD), an autosomal-dominant congenital disorder, characterized by hypoplastic clavicles,
patent fontanelles, wide pubic symphysis (Mundlos et al., 1997; Hall et al., 2001; Kaissi et al.,
2013) and other defects in skeletal patterning and growth. Mice generated with a heterozygous
mutation in Runx2 that recapitulates CCD, exhibit missing deltoid tuberosities of the humerus in
newborns (Otto et al., 1997), indicating the involvement of Runx2 in development of the tendon-
bone attachment unit at the deltoid tuberosity. For this project, I am only focusing on the
development of this specific T-B AU on DT utilizing this CCD mouse model.
12
1.3 Objectives and hypothesis
The modular model of the formation of the tendon-bone attachment unit development
involves the specification and differentiation of Scx
+
/Sox9
+
progenitor cells, but the molecular
regulations of this process remain largely unknown. To study the molecules that participate in T-
B AU formation, one usually starts by analyzing mutants that have failed to generate a bone
eminence. Studies showing the missing deltoid tuberosity in Runx2
+/-
mice is a hint of Runx2
involvement. The overall goal of this project is to study the role of Runx2 in development of the
tendon-bone attachment unit. Therefore, the aim is to determine: 1) how does haploinsufficiency
of Runx2 alter histogenesis of the deltoid tuberosity, and 2) whether Runx2 marks T-B AU
progenitor cells within the tendon-bone attachment unit during developing deltoid tuberosity. My
working hypothesis is that Runx2 is co-expressed with Scx and Sox9 that together form a triple
positive T-B AU progenitor population which would further develop into different
subpopulations and give rise to the gradient tissue in the T-B AU.
13
CHAPTER 2: MATERIALS AND METHODS
2.1 Mice
The Runx2 knockout mouse line has been described previously (Otto et al., 1997). The
Scx-GFP transgenic line used to mark Scx
+
cells, as well as tendon and ligament, has been
previously described (Pryce et al., 2007). Runx2 heterozygous mutant mice were mated with Scx-
GFP mice to create Scx-GFP; Runx2
+/
embryos. The plug date was defined as E0.5 for all timed
matings. Postnatal samples were staged according to the date of birth. To harvest embryonic
samples, timed pregnant females were euthanized by carbon dioxide (CO2) overdose followed by
cervical dislocation. For embryonic collections, the gravid uterus was dissected out and rinsed in
cold PBS. Embryos were surgically separated from the amniotic sac by removing umbilical cord
and the placenta. Perinatal and embryonic samples were euthanized by decapitation and then
skinned and eviscerated. Samples were rinsed with cold PBS three times to remove the blood.
Tail samples were collected for DNA isolations and genotyping. All experimental protocols
involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) of
the University of Southern California.
2.2 Genotyping
2.2.1 DNA extraction
To isolate DNA from tail tips of embryonic samples, the tissue was incubated in 100µl
tail lysis buffer ( 10 mM Tris (pH=8.0); 100 mM NaCl; 10 mM EDTA (pH=8.0); 0.5% SDS;
distilled H2O.) with Proteinase K (Thermo Fisher Scientific, #25530-015, 10 mg/ml in UltraPure
DNase/RNase-Free Distilled Water). for 1.5 hours at 52 °C. Next, 10µl 3M NaCH3COOH and
200µl 100% ethanol was added to each tail digest, mixed, incubated at -80°C for 30 minutes, and
14
centrifuged at 12,000 rpm at 4 °C for 15 minutes. The supernatant was decanted and 600 µl 70%
ethanol added. Samples were centrifuge as above again and the supernatant decanted.
Remaining DNA pellets were dried and resuspended in 30µl UltraPure DNase/RNase-Free
Distilled Water (Thermo Fisher Scientific).
To isolate DNA from postnatal samples, tail tips were digested by Mouse Quick
Genotyping DNA Preparation Kit according to the manufacturer’s instructions (Bioland, #GT11-
01).
2.2.2 PCR
PCR was performed using 2x Taq PCR Premix (Bioland) according to the manufacturer’s
instructions. Following primers were used:
Allele Primer sequence (5’-3’) Product size
Runx2 KO
Runx2 MUT F: AAGATGGATTGCACGCAGGTTCTC
~ 1 kb
Runx2 MUT R: CACGGAGCACAGGAAGTTGGG
Runx2 wildtype
Runx2 WT F: CCGGCCACTTCGCTAACTTGTG
~ 500 bp
Runx2 WT R: ACCTTGAAGGCCACGGGCAGG
Scx-GFP
GFP F: GCACGACTTCTTCAAGTCCGCCATGCC ~ 280 bp
GFP R: GCGGATCTTGAAGTTCACCTTGATGCC
Table 2.1 PCR genotyping primers.
2.3 Skeletal preparations
Samples were skinned, eviscerated, and fixed in 95% ethanol for 3-5 days. To stain for or
cartilage, samples were placing in Alcian Blue stain [0.15 mg/ml Alcian Blue 8GX (Sigma A
3157) in 80% ethanol and 20% glacial acetic acid] overnight and de-stained in 95% ethanol for
one day at room temperature. Samples were then cleared by incubating in 0.25%-1% KOH [w/v]
for 6-24 hours. To stain bone, sample were then incubated in Alizarin Red stain [0.02mg/ml
Alizarin Red S (Sigma A 5533) in 0.25%-1% KOH] overnight at room temperature. Stained
15
specimens were then gradually equilibrated to 75% glycerol for storage and imaging. These
experiments were performed on at least three biological replicates, which we defined as three
littermate pairs (control and mutant) derived from three different litters.
2.4 HBQ staining
For histological staining, whole embryos were fixed in 4% PFA at 4ºC for 30-45 minutes
and rinsed with PBS an equal amount of time. E18.5 and P2 samples were then decalcified with
BBC Biochemical Rapid Cal Immuno (Thermo Fisher Scientific) overnight at 4 ºC. Samples
younger than E18.5 did not require decalcification. Forelimbs were dissected, dehydrated in an
ethanol series to 100%, equilibrated in Citrosolv, and embedded in paraffin. Paraffin embedded
tissues were sectioned in the longitudinal plane of the humerus at a thickness of 8 µm using
microtome (LM2235, Leica). Connective tissues were discriminated using Hall-Brunt quadruple
stain (HBQ) (Hall,1986). These experiments were performed on at least two biological
replicates, which we defined as three littermate pairs (control and mutant) derived from three
different litters.
2.5 Fluorescent in situ hybridization
RNA transcripts for Scx, Sox9, and Runx2 were detected using RNAscope Fluorescent
Multiplex Assay (ACD) as per the manufacturer’s instructions at E14. Paraffin sections were
baked on a hot plate for 1 hour at 65°C, deparaffinized using xylene, dehydrated in 100%
ethanol, and allowed to air dry completely. Endogenous peroxidase activity was quenched using
the hydrogen peroxide provided within the kit and then slides were washed in deionized water.
Antigen retrieval was performed using the provided Target Retrieval Reagent for 15 minutes in
16
an Oster steamer heated to 99°C, followed by dehydration in 100% ethanol and complete air
drying. The slides were treated with ACD Protease Plus in a humidified slide chamber for 20
min at 40°C. Dual hybridization of the Scx (ACD, 439981-C2) and Sox9 (ACD, 401051-C2)
probes or Scx and Runx2 (ACD, 414021-C3) probes was performed in a 40°C humidified slide
chamber for 2 hours. Slides were then stored in 5× SSC solution overnight at room temperature.
Amplification steps were performed as prescribed by manufacturer and signal development for
channels two and three were carried out using TSA Plus fluorophores Cyanine 3 (PerkinElmer,
NEL744E001KT) and Cyanine 5 (PerkinElmer, NEL705A001KT) diluted 1:750 in the ACD-
provided TSA buffer. Slides were counterstained using Vectashield mounting medium with
DAPI and imaged using confocal microscopy. All steps, other than those recommended by the
manufacturer to complete in a humidified slide chamber at 40°C, were carried out using plastic
five-slide capacity mailers at room temperature. Slides were images using the Leica TCS SP5/8
confocal system confocal microscope. These experiments were performed on one biological
replicate.
2.6 Immunofluorescence
Embryonic samples were fixed in 4% paraformaldehyde (PFA) for 30 minutes and rinsed
with PBS for equal time. Forelimbs were dissected, equilibrated in sterilized 10% sucrose/PBS
for 2 hours and then into sterilized 30% sucrose/PBS overnight at 4°C. Forelimbs were
suspended in optimal cutting temperature (O.C.T.) compound (Electron Microscopy Sciences)
for 1 hour at 4°C, then embedded in O.C.T-filled cryomold using powered dry ice. The
embedded tissues were sectioned in the longitudinal plane of the humerus at a thickness of 8 µm.
17
Frozen sections were washed with PBST (1× PBS with 0.1% Triton X-100) and blocked
with 10% donkey serum (Sigma-Aldrich) for 1 hour at room temperature. All sections were
incubated with primary antibodies (see Table 2.2) overnight at 4 °C. Sections were washed with
PBST and incubated with Alexa Fluor secondary antibodies (see Table 2.3) at 1:500/PBST for 1
hour at room temperature, washed with PBST and mounted with Vectashield containing DAPI
(Vector Labs). Slides were imaged with a Keyence microscope (BZX710). These experiments
were performed on at least three biological replicates, which was defined as three littermate pairs
(control and mutant) derived from three different litters. See Table S1 for antibody vendor
information and dilutions.
Antigen Species Source Catalog number Dilution
Runx2 rabbit CST 12556 1:1500
Sox9 rabbit Novus Biologicals ab5450 1:1000
GFP goat Abcam NBP1-85551 1:1000
Table 2.2 Primary antibodies for immunofluorescence.
Source Catalog number Description Dilution
Thermo
Fisher
Scientific
A-11055
Donkey anti-goat IgG Alexa Fluor 488
conjugate
1:500
A10042
Donkey anti-rabbit IgG Alexa Fluor 568
conjugate
Table 2.3 Secondary antibodies for immunofluorescence.
18
CHAPTER 3: RESULTS
3.1 Runx2 is necessary for normal deltoid tuberosity formation
The deltoid tuberosity is the tendon-bone attachment site for the deltoid muscles on the
humerus. It was previously shown that newborn Runx2
+/-
mutant mice are missing the deltoid
tuberosity (Otto et al., 1997), which suggests that Runx2 is necessary for the development of this
tendon-bone attachment unit in the limb. To more completely investigate the role of Runx2 in the
development of the tendon-bone attachment unit, I first examined the morphogenesis of the
deltoid tuberosity during critical stages of its development. To examine the development of the
deltoid tuberosity, I performed whole-mount skeletal preparations in Runx2
+/-
mutant embryos
along with their littermate controls. At E13.5, just prior to formation of the deltoid tuberosity
(Blitz et al., 2013), no significant difference in the primary cartilage condensation of the humerus
between the Runx2
+/-
mutant and controls (Figure S1). At E14.5, when the cartilage template of
the deltoid tuberosity is first detected, the Runx2
+/-
mutant deltoid tuberosity appeared greatly
reduced or missing. The establishment of the mineralized area in the humerus was also delayed
in the mutant (Figure 4A-A”). Since variations in the Runx2
+/-
mutant embryos were observed
consistent within an animal, I classified hypoplastic deltoid tuberosities as mild and missing
deltoid tuberosity as severe (Figure 4A’,A”). Besides, mutants showed smaller size of the
forelimbs than wildtypes starting at E14.5. As the deltoid tuberosity grows and ossifies between
E16.5-P2, Runx2
+/-
mutants that exhibited the mild phenotype had delayed mineralization of the
deltoid tuberosity (Figure 4B-D”). At E18.5 and P2, mild and severe mutant animals retained a
small cartilage remnant at the site where the deltoid tuberosity should have formed (Figure
4D’,D”). Mutants also showed varying extent of bending of the humerus (Figure 4C’, C”, D’).
19
Together this data shows that Runx2 is necessary for proper formation of the early cartilage
condensation that gives rise to the deltoid tuberosity.
Figure 4. Runx2
+/-
forelimbs exhibit hypoplastic or missing deltoid tuberosity, as well as
delayed bone mineralization.
Skeletal preparations of E14.5, E16.5, E18.5, and P2 forelimbs from control (A-D) and Runx2
+/-
littermates that are characterized as mildly affected (A’-D’) and severely affected (A”-D”).
Cartilage is stained blue by Alcian Blue and ossified bone is stained red by Alizarin Red. Arrows
demarcate the deltoid tuberosity. Affected deltoid tuberosity is indicated by asterisk. dt, deltoid
tuberosity. Scale bars: 200 µm in A-B”; 500 µm in C-D”.
E14.5
E16.5
Control Runx2
+/-
mild severe
E18.5
P2
dt
A A’ A”
B B’ B”
C C’ C”
D D’ D”
*
*
*
*
*
*
* *
20
3.2 Runx2 haploinsufficiency alters histogenesis of the deltoid tuberosity
To elucidate how haploinsufficiency of Runx2 alters the histogenesis of the deltoid
tuberosity, I analyzed longitudinal paraffin sections of the forelimb from E13.5- P2 followed by
HBQ staining (Figure 5). At E13, no differences were noted between the wildtype and
Runx2
+/-
embryos in the mesenchyme that prefigures the cartilage template of the deltoid
tuberosity or in the cartilage template of the humerus (Figure 5A-A”). At E14.5, the cartilage
template of the DT had emerged in the wildtype forelimb but was hypoplastic or missing in the
Runx2
+/-
forelimb (Figure 5B-B”). At E16.5, hypertrophic and pre-hypertrophic chondrocytes
were observed in the wildtype DT, while this process is delayed in Runx2
+/-
DT (Figure 5C’,C”,
asterisk). Also, it was noted at this stage that in the Runx2
+/-
forelimb, the perichondrium/
periosteum was thicker at the tendon attachment (Figure 5C-C”). At E18.5, the wildtype DT
began to ossify, but bone mineralization was delayed in mutants (Figure 5D-D’). It was also
apparent in Runx2
+/-
forelimbs at this stage that tendon attachment was incomplete and that the
perichondrium/periosteum remained thickened. By P2, the DT is mostly ossified in the control
(Figure 5E). Yet, in mildly affected Runx2
+/-
forelimbs where the DT was hypoplastic,
mineralization was delayed, and the cartilaginous template remained (Figure 5E’). In severely
affected Runx2
+/-
forelimbs where the DT was missing, the tendon was inserted directly into a
thickened perichondrium/periosteum (Figure 5E”). These results demonstrate that the process of
endochondral bone formation, including chondrocyte hypertrophy and mineralization, within the
deltoid tuberosity is delayed or inhibited in the DT of the Runx2
+/-
forelimbs. Additionally, this
shows that histogenesis of the normal tendon-bone insertion in the Runx2
+/-
forelimb is
disrupted.
21
Figure 5. Haploinsufficiency of Runx2 leads to delayed chondrocyte hypertrophy and
mineralization of deltoid tuberosity and disrupts tendon insertion.
Histological longitudinal sections of the humerus at E13.5, E14.5, E16.5, E18.5, and P2 from
wildtype (A-E), mildly affected mutants (A’-E’) and severely affected mutants (A”-E”).
Cartilage is stained blue, ossified bone is stained red, and muscle is stained pink. Arrows
demarcate the deltoid tuberosity, affected deltoid tuberosity is indicated by an asterisk,
arrowheads indicate thickened perichondrium. Scale bars: 100 µm.
Runx2
+/-
Control
mild severe
E13.5
E16.5
E18.5
P2
E14.5
A
B
C
D
E
A’
B’ B”
C’
D’
C”
D”
E’ E”
*
*
* *
*
22
3.3 Runx2 regulates the development of Scx
+
/Sox9
+
progenitors
Recent studies have identified Scx
+
/Sox9
+
progenitors, which arise between the primary bone
template and the developing tendon, as the progenitors of the deltoid tuberosity and the tendon-
bone attachment unit (Blitz et al., 2013). Based on my findings that haploinsufficiency of Runx2
affects the histogenesis of the deltoid tuberosity and tendon insertion, next I investigated the
extent to which Runx2 marks Scx
+
/Sox9
+
progenitor cells within the developing tendon-bone
attachment unit and deltoid tuberosity. To do so, I examined the expression of Runx2, Sox9, and
the Scx-GFP reporter in the forelimb of wildtype and Runx2
+/-
forelimbs. Scx-GFP transgenic
reporter mice (Pryce et al., 2007) that mark tenocytes within tendon and the tendon-bone
attachment unit were crossed with Runx2
+/-
mice to generate Scx-GFP; Runx2
+/-
embryos. At
E13.5, when Scx
+
/Sox9
+
progenitor cells are abundant in the forming deltoid tuberosity (Eyal et
al., 2019), I conducted immunofluorescence for anti-GFP and anti-Sox9 on longitudinal sections
of forelimbs. In wildtype forelimbs, Scx
+
/Sox9
+
progenitor cells were detected as expected in the
developing deltoid tuberosity at the tendon insertion site (Figure 6A-D, arrowhead). In Runx2
+/-
forelimbs, the spatial distribution of Scx
+
cell was abnormal with very few Scx
+
cells adjacent to
the bone. In addition, the number of Scx
+
/Sox9
+
progenitors was greatly reduced in mutants
(Figure 6E-H, asterisk). Together this data suggests that Runx2 is necessary for normal
development of Scx
+
/Sox9
+
progenitors that form the DT and tendon-bone attachment unit.
23
Figure 6. Reduction of Scx
+
/Sox9
+
cells at E13.5 in Runx2
+/-
mutant mouse embryos.
Longitudinal sections through the forelimb at E13.5 were immunofluorescently stained for
endogenous Scx-GFP with anti-GFP and anti-Sox9. In the wildtype DT, Scx-GFP (green) and
Sox9 (red) identified Scx
+
/Sox9
+
progenitor (yellow, arrowhead). In Runx2
+/-
mutant embryos,
Scx
+
/Sox9
+
cells are reduced (asterisk) and few Scx
+
cells neighbored the bone. Individual
fluorescent channels with DAPI are shown. Scale bars: 200 µm.
Control Scx-GFP;Runx2
+/-
anti-GFP DAPI Sox9 DAPI anti-GFP Sox9 Merge
A
B
C
D
E
F
G
H
*
*
24
Studies have shown that Runx2 is expressed in the limb bud from E10.5 and has prominent
expression in the humerus from E12.5 (Kim et al., 1998). However the extent to which Runx2 is
expressed in Scx
+
/Sox9
+
progenitor cells, the developing DT, or the developing tendon-bone
attachment unit has yet to be been reported. To understand if the aberrant patterning of
Scx
+
/Sox9
+
progenitors in Runx2 mutants was caused by an autonomous role for Runx2, I also
performed immunofluorescence staining with antibodies against GFP and Runx2 at E13.5. In
wildtype forelimbs, Runx2 expression was observed in the pre-hypertrophic chondrocytes,
hypertrophic chondrocytes, and adjacent perichondrium (Figure 7B). Runx2 expression in the
perichondrium was co-localized with Scx-GFP in the wildtype forelimb in the perichondrium
and the tip of the forming tendon at the tendon insertion site (Figure 7C, D, arrowheads). In the
Runx2 mutants, Scx
+
cells of the tendon were more loosely dispersed and displaced from the
developing bone (Figure 6E, Figure 7E). While reduced Scx
+
/Runx2
+
cells were detected in the
mutant samples, they were disorganized and displaced from the bone at the developing tendon-
bone attachment site. The co-localization of Runx2 and Scx-GFP was only observed in the
perichondrium at the tendon insertion site (Figure 7G, H, arrowheads), but not in the distal end
of the tendon (Figure 7G, H, asterisk). Together these results suggest that Scx
+
/Runx2
+
are an
important progenitor population that gives rise to the tendon-bone attachment unit at the deltoid
tuberosity.
25
Figure 7. Runx2
+
/Scx
+
cells in the tendon-bone attachment site are disorganized in Runx2
+/-
mouse embryos.
Longitudinal sections through forelimb at E13.5 were immunofluorescently stained for
endogenous Scx-GFP with anti-GFP (green) and anti-Runx2 (red). Scx
+
/Runx2
+
cells were
observed in the developing tendon-bone attachment unit and DT of both wildtype and Runx2
+/-
forelimbs (arrowheads). Scx
+
/Runx2
+
cells were reduced in the distal end of forming tendon at
the tendon attachment site in mutants (asterisks). Individual fluorescent channels with DAPI are
shown. Scale bars: 200 µm.
Control Scx-GFP;Runx2
+/-
anti-GFP DAPI Runx2 DAPI anti-GFP Runx2 Merge
A
B
C
D
E
F
G
H
*
*
26
To determine how Scx
+
/Sox9
+
progenitor cell differentiation is affected in the
Runx2
+/-
mutant forelimb, I performed double fluorescent in situ hybridizations for Scx and Sox9
on paraffin sections of E14 forelimbs. I found that expression of Scx and Sox9 was mostly
mutually exclusive in wildtype forelimbs (Figure 8C,D, asterisk) at this stage, as described
previously (Blitz et al., 2013). Scx was expressed in the forming tendon and Sox9 was expressed
in the cartilage beneath the perichondrium at the attachment site (Figure 8A, B), a significant
reduced overlapping region of Scx and Sox9 expression domain were identified in the
perichondrium of the mutants (Figure 8C,D, asterisk). In Runx2
+/-
mutant forelimbs, the
expression of Sox9 and Scx was expanded and overlapped in the perichondrium. Thus,
undifferentiated Scx
+
/Sox9
+
cells remain in the perichondrium at the tendon-bone attachment
site (Figure 8E-H, arrowhead). Next I examined the development of Scx
+
/Runx2
+
in the
Runx2
+/-
mutant forelimb by performed double fluorescent in situ hybridizations for Scx and
Runx2. In wildtype forelimbs, Runx2 was expressed in the cartilage, perichondrium, and
neighboring tendon at the tendon-bone attachment site (Figure 9A, B). In addition, Runx2
expression overlapped with Scx expression in the perichondrium and the forming tendon (Figure
9C, D, arrowhead). In the Runx2 mutant forelimb, Scx
+
/Runx2
+
cells were found in the
perichondrium and the forming tendon as seen in the wildtype (Figure 9G, H, arrowhead), but
the overlapping region is reduced in the tendinous side of the attachment site (Figure 9G, H,
asterisk). Irregular and dispersed distribution of the Scx expression domain was also observed in
the mutant. Together these findings suggest Runx2 regulates the differentiation of Scx
+
/Sox9
+
progenitor cells into chondrocytes and tenocytes located at each sides of the tendon-bone
attachment unit. Loss of a single copy of Runx2 delays this process and leads to progenitors
27
remaining in the perichondrium. This data also suggests that Runx2 regulates development of a
novel Scx
+
/Runx2
+
cells in the tendon-bone attachment unit.
Figure 8. Scx
+
/Sox9
+
cells
remain in the perichondrium at
the tendon-bone attachment
site in Runx2
+/-
mutant
embryos at E14.
Double fluorescent in situ
hybridization for Scx (green) and
Sox9 (red) in the deltoid
tuberosity longitudinal paraffin
sections at E14.
The Runx2
+/-
mutant deltoid
tuberosity shows cells co-
expressing Scx and Sox9 in the
perichondrium region
(arrowhead), which is absent in
the control (asterisk). Individual
fluorescent channels with DAPI
are shown. Dashed lines
demarcate both boundaries of the
perichondrium at tendon-bone
attachment site. Scale bars: 25
µm. n=1 littermate pair.
*
Control Runx2
+/-
*
Scx DAPI Sox9DAPI Scx Sox9 Merge
A
B
C
D
E
F
G
H
28
Figure 9. The RNA expression
domain of Runx2 and Scx
overlap in the tendon-bone
attachment unit at E14.
Double fluorescent RNA in situ
hybridization for Scx (green) and
Runx2 (red) in the deltoid
tuberosity, longitudinal paraffin
sections at E14.
Runx2 and Scx expression
domains overlap in both mutant
and control (arrowhead). Runx2
expression is reduced at the
tendon-bone insertion in
Runx2
+/-
mutant deltoid
tuberosity (asterisk). Individual
fluorescent channels with DAPI
are shown. Dashed lines
demarcate both boundaries of
the perichondrium at the tendon-
bone attachment site. Scale bars:
25 µm. n=1 littermate pair.
Control Runx2
+/-
ScxDAPI Runx2DAPI ScxRunx2 Merge
A
B
C
D
E
F
G
H
*
*
*
29
CHAPTER 4: DISCUSSION
4.1 Runx2 regulates development of tendon-bone attachment in the limb
This study shows that Runx2 regulates proper development of tendon-bone attachment at the
deltoid tuberosity. First, I identified that Runx2 is essential for deltoid tuberosity formation
during mouse limb development. Haploinsufficiency of Runx2 leads to hypoplasia or absence of
the deltoid tuberosity, which is coincident with delayed hypertrophic and mineralization of the
chondrocytes histologically during endochondral ossification of the bone eminence. These
results suggest that Runx2 is necessary for correct spatial formation of the developing DT, as
well as the patterning of Scx
+
/Sox9
+
progenitors that form the tendon-bone attachment unit of the
DT. I showed that in wildtype limb at E13.5, Runx2 is expressed in the
perichondrium/periosteum of the DT, as well as in the Scx
+
/Sox9
+
that form the tendon-bone
attachment unit. In the Runx2
+/-
limb at this stage, I discovered the pattern of Scx
+
cells are
dispersed and that the number of Scx
+
/Sox9
+
progenitor cells is reduced, followed by a delay in
their differentiation into Scx
+
tenocytes and Sox9
+
chondrocytes. These findings indicate an early
role for Runx2 in the establishment of Scx
+
/Sox9
+
cells, as well as their differentiation in the DT.
Interestingly, I found that the reduction in Scx
+
/Sox9
+
cells in the Runx2
+/-
limb was correlated
with an increase in perichondrial/periosteal thickness. Since Runx2 is a marker for periosteal
cells, this suggests the possibility that 1) Runx2
+
cells in the perichondrium/periosteum are the
origin of tendon-bone attachment unit progenitors and 2) the tendon-bone connection unit cells
are derived from a Runx2
+
/Scx
+
/Sox9
+
progenitor cell pool that undergo differentiation into
Runx2
+
/Scx
+
osteo-fibrogenic progenitors and Runx2
+
/Sox9
+
osteo-chondral progenitors that
eventually form the gradient tissue characteristic of the tendon-bone attachment unit (Figure 10).
30
Figure 10. Schematic summarizing the working hypothesis on the role of Runx2 during
tendon-bone attachment unit development.
(A) Cell lineage of the tendon-bone attachment unit. Runx2
+
cells from the perichondrium/
periosteum at the tendon-bone attachment site give rise to Runx2
+
/Scx
+
/Sox9
+
progenitors.
During tendon-bone attachment unit development, Runx2 expression promotes this triple
positive progenitor cells pool differentiate into 2 subgroups: Sox9
+
/Runx2
+
osteochondral
progenitor that give rise to the bone eminence in the proximal side of the attachment, and
Scx
+
/Runx2
+
osteofibrogenic progenitors that give rise to the mineralized fibrocartilage and
fibrocartilage. (B) Schematic assembly of deltoid muscle and deltoid tuberosity. Cell model in
wildtype (C) and Runx2
+/-
mutants (D) tendon-bone attachment unit on the DT. Reduction of
Runx2 expression delays the generation of Runx2
+
/Scx
+
/Sox9
+
progenitors and its differentiation
temporally, causing abnormal tendon-bone integration. c, clavicle; dm, deltoid muscle; dt,
deltoid tuberosity; h humerus.
A
B
C D
31
4.2 Altered musculoskeletal system integration dictates DT formation and
causes bone bending
Studies have demonstrated that mechanical load created by contracting muscles upon
formation of proper musculoskeletal assembly is critical for normal bone eminence formation. In
immobilized mice, the absence of muscle contraction during development leads to lack of DT
formation (Rot-Nikcevic et al., 2005; Nowlan et al., 2009). In humans, contracture of the deltoid
muscle causes prominence or hypertrophy of the DT (Ogawa et al., 1999; Bhattacharyya, 1966).
This indicates the contribution of muscle contraction of bone eminence formation.
During fetal development, DT and other bone eminences are formed through a two-phase
process. The initiation phase, which establishes the forming bone eminence, is tendon-sufficient
but muscle-independent. Once established, bone eminence growth is subsequently regulated by
muscle contraction during the growth phase, allowing for dynamic coordination at the tendon-
bone attachment (Blitz et al., 2009). The reduction or absence of DT over time, as well as the
disrupted patterning of Scx
+
cells observed in Runx2
+/-
mutants, imply the tendon insertion failed
at the initiation phase and that insufficient anchoring leads to deficient muscular input during the
growth phase into that affects the formation of tendon-bone attachment unit in response causing
defects of DT formation.
In addition, studies have shown that imbalanced mechanical load of an abnormally patterned
tendon-bone attachment unit can cause long bone bending. Human patients with
haploinsufficiency of SOX9 causes Campomelic Dysplasia (Lazjuk et al., 1987), as well as
FGFR2 mutations in Bent Bone Dysplasia syndrome (Merrill et al., 2012) exhibit bowing and
shortening of the long bone. Since Sox9 is necessary for the formation of Scx
+
/Sox9
+
progenitor
cells and since FGF signaling regulates tendon-bone attachment in the limb and development of
32
Scx
+
/Sox9
+
progenitors in the jaw (Salva et al., 2019; Roberts et al., 2019), abnormalities of
tendon-bone attachment unit development are linked to bowed long bones. In this work,
shortened humerus are observed in all mutants, and humerus bending is also occasionally
observed (Figure 4C’, C”, D’). This suggests the possibility that the defects in the tendon-bone
attachment unit progenitors due to the haploinsufficiency of Runx2 caused humerus bending.
4.3 The possible explanation of variant DT formation defect in Runx2
+/-
mutants
In this study, I found that the DT defect in Runx2
+/-
was variable and could be classified as
either hypoplastic or absent. To explain the cause of this variation, I first checked whether this
variation could be correlated with sidedness (right vs. left) at each embryonic stages by
collecting whole-mount skeletal preparation stained embryos, and found no significant
relationship.
Considering the ideas discussed above that sufficient tendon formation with subsequent
proper muscle contraction ensures normal formation of the bone eminence, this variation may be
explained by stochastic differences in tendon insertion among Runx2
+/-
mutants. For example,
Runx2
+/-
mutants that form a hypoplastic DT may have some tendon insertion and some
mechanical input, while those that are missing the DT have little to no tendon insertion and no
mechanical input.
Another possible explanation is differences in Runx2 expression level in Runx2
+/-
mutants
due to allele-specific expression, where one parental allele could be preferentially expressed.
Varying levels of Runx2 expression could impact the size of the Runx2
+
/Scx
+
/Sox9
+
progenitor
pool, and ultimately the size of the DT.
33
Finally, one possibility needs to be ruled out is that there might be a difference based on sex.
To address this, I would like to collect a certain amount of male and female littermates at the
same stage with the wildtype and Runx2
+/-
mutants and study the variation of DT formation by
whole-mount skeletal preparation.
4.4 Potential signaling mechanism affected during tendon-bone attachment
unit development in Runx2
+/-
embryos
Although this work reveals a new insight into Runx2 as a new molecule that directs the
patterning and development of the tendon-bone attachment unit, the signaling mechanism
underlying this process remains unexamined. The known molecular pathways involved in the
fetal development of tendon-bone attachment unit are TGFβ signaling, BMP signaling, and FGF
signaling. Abnormality in any one of the three signaling pathway could result in loss of bone
eminence or tendon insertion defects, suggesting that the reduced or absent DT observed in this
study may be associated with impairment of these three signaling pathways.
TGFβ signaling is a key regulator of tendon-bone attachment unit progenitor specification,
necessary for tendon induction and organization in tendon-bone attachment unit. Embryos with
ablated TGFβ signaling in limb mesenchyme exhibited lack of Sox9
+
cells at the attachment site
(Blitz et al., 2013), which is consistent with the phenotype of Runx2
+/-
mutants in this work
(Figure 6F, G; asterisk). Moreover, dispersed patterning of Scx
+
cells and reduction of
Scx
+
/Sox9
+
progenitor cells are also observed in the Runx2 mutant, may indicate the
involvement of TGFβ signaling.
In SCX/BMP4 pathway, Bmp4 expression induced by Scx regulates differentiation of
Scx
+
/Sox9
+
progenitors and mediates bone eminence formation (Blitz et al., 2009). Forelimbs
34
from mutant embryos with Bmp4 ablated from limb mesenchyme displayed remaining
undifferentiated Sox9
+
progenitor cells at the attachment site from E12.5 (Blitz et al., 2013). In
this study, E14 Runx2
+/-
mutant embryos exhibited loosely patterned Scx
+
cells with delayed exit
of Scx
+
/Sox9
+
progenitors in the perichondrium, suggesting that the delayed differentiation of
progenitors may be associated with SCX/BMP4 pathway signaling.
During embryogenesis, FGF signaling induces Scx expression to regulate tenocytes
differentiation in the limb (Edom-Vovard et al., 2002; Pryce et al., 2009). FGF signaling is also
known to regulate the differentiation of Scx
+
/Sox9
+
progenitors in the jaw, loss of Fgfr2 induces
biased differentiation of progenitors into Sox9
+
cells (Roberts et el., 2019). Implying the
possibility that abnormal patterning of Scx expression in cells on the tendinous side of the
attachment site and the remaining Scx
+
/Sox9
+
progenitors observed in mutants in this study could
be associated with FGF signaling.
4.5 Future directions
Due to the technical limitations, expression of Sox9 and Runx2 could not be co-stained using
immunofluorescence, leaving the relationship between them unexplained in this study.
Therefore, my first direction is to address the question that whether the expression of Runx2
overlaps with Sox9 in tendon-bone attachment unit progenitors. I would perform FISH by using
Sox9 and Runx2 probes . Lineage tracing of Runx2
+
cells at various developmental time points
could also be applied to understand the tissue origin of Runx2
+
cells, to detect Sox9 and Scx
expression within Runx2
+
cells, and to determine whether Runx2
+
cells are the source of the
tendon-bone attachment unit.
35
This study also needs to be added with the quantification of the data, such as measuring the
length of forelimbs when smaller forelimbs were observed in Runx2
+/-
mutants by skeletal
preparations, measuring the thickness of the perichondrium in HBQ sections, as well as counting
the number of co-expressing cells detected by IF and FISH, to enhance the credibility of the
findings of study.
The future direction of this study should also focus on understanding the molecular
mechanism of how the haploinsufficiency of Runx2 results in the tendon-bone attachment unit
defects during development. To study this, I would assess the expression of different signaling
molecules, such as Fgfr2, pSmad2/3 to detect Tgfβ activity, pSmad1,5,8 to detect BMP signaling
activity, and Gli1 to detect HH activity, to examine whether any of these signaling pathways is
altered in Runx2
+/-
mutant embryos.
To further solidify the finding by HBQ staining that haploinsufficiency of Runx2 leads to
delayed chondrocyte hypertrophy and mineralization of deltoid tuberosity, other types of staining
could be applied to the tendon-bone attachment unit, such as Von Kossa staining to illustrate the
mineralization; EdU staining to detect changes in proliferation; TUNEL staining to detect
apoptosis; staining Col1a1, Col2a1, and Osteocalcin for proliferating chondrocytes,
differentiating chondrocytes, and bone formation; staining Ihh for pre-hypertrophic
chondrocytes; Col10a1 for hypertrophic chondrocytes.
Finally, I’m curious about whether the anatomic defects in the deltoid tuberosity tendon-bone
attachment unit during fetal development in Runx2
+/-
embryos could lead to any functional
defect, altering the strength of the forelimbs or behavior, in their adulthood. So to test this, I
would conduct strength tests of the forelimbs and perform a gait study in the future.
36
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41
SUPPLEMENTARY
Figure S1. Cartilage condensation on the humerus at E13.5.
Wholemount skeletal preparations at E13.5 from control and Runx2
+/-
littermates. Black arrows
indicate the humerus, showing no significant difference in cartilage condensation at E13.5. h,
humerus. Scale bars: 500 µm.
E13.5
Control Runx2
+/-
h
Abstract (if available)
Abstract
The enthesis, a graded connective tissue between bone and tendon, delivers the force of muscle contraction to bone. The enthesis is prone to injury and has poor capacity for repair. To advance strategies for enthesis repair it is important to understand how the unique connective tissue is formed during embryonic development. The goal of my project is to study the molecular regulators controlling development of the tendon-bone attachment unit progenitor cells in the limb. One of the candidate genes I proposed to be involved is Runx2, a transcription factor of the RUNX family, which is essential for bone formation. Haploinsufficiency of Runx2 leads to Cleidocranial dysplasia (CCD) in humans. The CCD mouse model fails to develop the deltoid tuberosity, suggesting that Runx2 is necessary for proper development tendon-bone attachment unit progenitor cells in the limb. Previous studies have shown that the tendon-bone attachment unit is formed modularly by a pool of Scx⁺/Sox9⁺ progenitor cells that forms the tendon’s terminus and the bone eminence into which it inserts. In this study, I found that Runx2 is expressed by Scx⁺/Sox9⁺ progenitor cells and necessary for their differentiation into the bone eminence. These results extend our understanding of tendon-bone attachment unit development by identifying Runx2 as a molecular regulator.
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Wang, Siyan
(author)
Core Title
The role of Runx2 in the development of the tendon-bone attachment unit
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Medicine
Publication Date
01/23/2021
Defense Date
05/27/2020
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deltoid tuberosity,enthesis,limb development,OAI-PMH Harvest,RUNX2,tendon-bone attachment unit
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Merrill, Amy (
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deltoid tuberosity
enthesis
limb development
RUNX2
tendon-bone attachment unit