Expression Analysis of CTHRC1 in the Murine Embryo during Mid-facial Development

Transcription

1 Expression Analysis of CTHRC1 in the Murine Embryo during Mid-facial Development by Dr. Laurene Dao-Pei Yen A thesis submitted in conformity with the requirements for the degree of Masters of Science in Dentistry (Orthodontics) Department of Orthodontics University of Toronto Copyright by Laurene Dao-Pei Yen, 2014

2 Expression Analysis of CTHRC1 in the Murine Embryo during Midfacial Development Abstract Dr. Laurene Dao-Pei Yen Masters of Science in Dentistry (Orthodontics) Department of Orthodontics University of Toronto 2014 Collagen Triple Helix Repeat Containing 1 (CTHRC1) has been shown to regulate collagen expression, cell migration and interact with the Wnt/planar cell polarity pathway. Objective: Determine CTHRC1 expression in the midface of mouse embryos during craniofacial development. Methods: qpcr and immunohistochemistry were used to determine the expression of Cthrc1 at embryonic (E) days of development E8.5-E13.5. Results: CTHRC1 expression was in the notochord and neural tube at E8.5, mesenchyme of the midface at E9.5- E10.5 and areas of cartilage formation at E11.5-E13.5. Conclusions: The expression of CTHRC1 in the developing craniofacial region suggests a role of CTHRC1 in migration of cranial neural crest cells and in chondrocyte proliferation and differentiation. We speculate the functions of CTHRC1 during craniofacial development are via its interactions with the Wnt/PCP pathway, shown previously to play a significant role during craniofacial development. Support: Donald G. Woodside Fund and NSERC. ii

3 Acknowledgments I would like to thank my supervisor, Dr. Siew-Ging Gong for your guidance and support throughout my thesis. Thank you for the constant encouragement, especially during the writing of my thesis. I would also like to express my sincere thanks to the members of my committee, Dr. Bernhard Ganss and Dr. Bryan Tompson for their scientific guidance and advice throughout my MSc degree. I would like to thank the Donald G. Woodside Fund for funding part of my project. A special thanks to Mr. James Holcroft, for his help with the quantitative PCR portion of my thesis, and Feryal Sarraf, for her technical expertise in the use of the microscopes and digital cameras in the histology lab. Thank you to my husband, Dr. Alexander Unterberger for his constant support and encouragement throughout my degree. I could not have done it without you! iii

4 Table of Contents Abstract ii Acknowledgments iii List of Figures viii Chapter 1: Introduction Development and morphogenesis of the vertebrate face Role of neural crest cells in craniofacial development Formation and migration of cranial neural crest cells Induction and specification of CNCCs and NCCs Delamination Migration NCC positional identity NCC differentiation and proliferation Facial prominences and CNCC derivatives of the face Facial development and facial prominences Craniofacial developmental anomalies Proteins and genes involved in formation of craniofacial structures Fibroblast growth factors and bone morphogenetic proteins Sonic hedgehog Wnt pathways Wnt pathways and craniofacial development 17 iv

5 Wnt5A Collagen Triple Helix Repeat Containing 1(Cthrc1) Background CTHRC1 structure Expression pattern of Cthrc Possible functions of Cthrc Cthrc1 role in collagen deposition Role of Cthrc1 in osteogenesis, osteoclastic bone formation and bone remodeling Role in cell motility and tissue repair Genes and signalling pathways involved with CTHRC Cthrc1 activation of Wnt/PCP pathway Cell-specific action of Cthrc Rationale for study Hypothesis/Aims 31 Chapter 2: Materials and Methods Embryos RT-qPCR RNA isolation Reverse transcription of RNA samples Quantitative PCR 33 v

6 2.3 Histological processing and Paraffin Embedding Immunohistochemistry Documentation and analysis of Cthrc1 expression 35 Chapter 3: Results Quantitative expression of Cthrc1 mrna transcripts during midface development Expression of CTHRC1 protein CTHRC1 peptide competition assay Spatial expression of CTHRC1 in the midface at different developmental stages CTHRC1 expression in the mandible and developing tooth bud 43 Chapter 4: Discussion Summary of Cthrc1 expression during mouse embryo midface development Summary of qpcr mrna levels of Cthrc1 mrna during E8-5-E Summary of immunolocalization experiment results of CTHRC1 protein from E8.5- E Involvement of Cthrc1 in midface development Role of Cthrc1 in cell migration Role of Cthrc1 in epithelial/mesenchyme interactions Role of Cthrc1 in regulation of collagen formation and deposition Role of Cthrc1 in bone formation Summary of roles of Cthrc1 in midface development 55 vi

7 4.3 Future Directions for Cthrc1 studies Midface development in Cthrc1 knockout models Cthrc1 expression in mouse models specifically targeting effects on midface development Colocalization studies of CTHRC1 with other proteins with a known function in midface development Conclusions 58 References 59 Copyright Acknowledgements 68 vii

8 List of Figures Figure 1: Migration and skeletal fates of the CNCCs 4 Figure 2: A schematic diagram of the pharyngeal arches 7 Figure 3: The canonical Wnt signaling pathway 15 Figure 4: The non-canonical Wnt signaling pathway 17 Figure 5: Summary of WNT5A functions during cartilage development and disease.20 Figure 6: The structure of the CTHRC1 protein 21 Figure 7: A model of selective activation of the Wnt/PCP pathway by CTHRC1 30 Figure 8: Temporal expression profile of Cthrc1 mrna across E8.5-E Figure 9: Peptide competition assay on consecutive coronal sections through the anterior part of the midface in an E13.5 embryo 37 Figure 10: CTHRC1 peptide expression in E Figure 11: CTHRC1 protein expression in E10.5 embryos 39 Figure 12: CTHRC1 protein expression in E Figure 13: CTHRC1 protein expression at E Figure 14: CTHRC1 protein expression at E Figure 15: CTHRC1 protein expression in tooth bud formation 44 viii

9 1 Chapter 1 Introduction Development of the craniofacial region is a complex process. It is regulated by different genes, proteins and signalling pathways. In this chapter, the development and morphogenesis of the vertebrate face and the involvement of neural crest cells will be discussed. These topics will be followed by the sequence of formation of the facial structures and a discussion of craniofacial developmental anomalies. 1.1 Development and morphogenesis of the vertebrate face Role of neural crest cells in craniofacial development Development and morphogenesis of the vertebrate face is a complex and highly orchestrated process. A major population of cells that contribute to the craniofacial region is the neural crest cells (NCCs) that form along the entire length of the developing embryo. NCCs are transient migratory cells that develop at the border between the neural plate (NP) and the epidermis. NCCs delaminate from their site of origin along the dorsal neural tube and migrate ventrally along different pathways. NCCs contribute to many different cell types and tissues in the body, such as the enteric nervous system, melanocytes, connective tissues, and myofibroblasts which line blood vessels, neurons and glia of the peripheral nervous system, pigment cells, endocrine cells, cardiac structures, smooth muscle cells and tendons (1). NCCs in the rostral region of the developing embryo are known as cranial neural crest cells (CNCCs). In contrast to trunk NCCs, CNCCs can give rise to osteoblasts and chondrocytes (2) and are the major contributor to structures of the facial skeleton, cartilages and bones of the jaws, middle ear and neck, cranial nerves, smooth muscles, dermis and facial connective tissues (3). During the migration of the CNCCs and after their arrival, their interactions with the surrounding ectoderm, endoderm and neuroectoderm are integral for normal development of the face (4-6). This multi-step process is regulated both temporally and spatially and is made up of the coordinated action of numerous genes and signalling pathways (7). Due to this high level of regulation, craniofacial development is susceptible to perturbations, resulting in facial dysmorphologies. Many birth defects associated with craniofacial malformations can be attributed to the defects in the generation, proliferation, migration and differentiation of CNCCs

10 2 (8). Understanding the complex and sequential mechanisms of craniofacial development and how they relate to the formation of facial structures is essential to the orthodontic specialty Formation and migration of cranial neural crest cells The development of the NCCs and CNCCs can be divided into a number of stages: a) Induction b) Delamination c) Migration d) Differentiation Induction and Specification of CNCCs and NCCs Induction of NCCs is a multistep process resulting in a molecular cascade of events involved in establishing their migratory and multipotent characteristics. The origin of NCCs can be traced to the border of the NP in a region of ectoderm situated between the NP and the non-neural ectoderm (NNE). Immediately beneath the ectoderm there is a layer of mesoderm, and together with the NP and NNE, these tissues are collectively believed to contribute to the induction of the NC. Induction of NCCs has been shown as early as during gastrulation during embryonic development (9). Induction involves a complex set of extracellular signals which transform the fate of cells along the medio-lateral and anterior-posterior axes of the embryo (10). The signals that position the NCCs along the axes are released from the NP, the epidermis and the lateral mesoderm. For complete induction of NCCs, there must be a transformation of the naive ectoderm into the neural crest. A precise combination of extracellular signalling molecules operate to activate the expression of transcription factors that define the neural crest territory and control subsequent neural crest development (11). Molecules such as wingless-type MMTV integration site (WNTs), Notch, bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are secreted from the adjacent epidermis and underlying mesoderm and separate the NNE (epidermis) from the NP during neural induction (12-14). The induction of the expression of regulatory transcription factors (Msx1/2, Pax3/7, Zic1, Dlx3/5, Hairy2, Id3, Ap2) specify the NP border that subsequently trigger the expression of NC specifiers, a second set of transcription factors (Snail2, FoxD3, Sox9/10, Twist, cmyc, and Ap2). NC specifiers are proposed to

11 3 ultimately control neural crest behavior, from epithelial-mesenchymal transition (EMT) and delamination to migration and differentiation (15). One theory proposes that NCCs are specified during gastrulation (9) whereas the second, more classical, theory postulates that NCCs are specified during the time of neural tube closure. After specification, contact-mediated signalling between tissues in the dorsal neural tube results in EMT in neural ectoderm cells at the neuroectoderm and NNE border (1) Delamination After induction and specification at the neuroepithelium, NCCs undergo EMT and leave their site of origin through a delamination process. Delamination is the splitting of the NCCs from their surrounding tissues whereas EMT is a series of events at the molecular level which orchestrates a change from an epithelial to mesenchymal phenotype (11, 16). EMT is a multistep process in which the NCCs lose their apico-basal polarity and disassemble intercellular adhesion complexes required for epithelial formation (16), allowing the NCCs to separate from the neuroepithelium and ectoderm (11). Contact-mediated signaling between tissues in the dorsal neural tube stimulates cells at the neural/non-neural border to undergo EMT(17). This results in a highly invasive phenotype characteristic of NCCs, behaviour which is also shared with metastatic cells (18). In the cranial region, CNCCs delaminate all at once, whereas the trunk NCCs delaminate progressively, leaving the neuroepithelium one by one after neural tube closure (19) Migration The CNCCs follow migratory pathways that are conserved among vertebrate species. CNCC migration is directed along well-defined routes that end in the ventral part of the brain and branchial arches (20). Interactions between the CNCCs and their local environment are critical in CNCC directional and collective migration (21). A number of cellular mechanisms have been shown to operate during the directed migration of the NCCs. NCCs become polarized in the direction of their migration with a tail at the back of the cell and filopodia and lamellipodia at the side towards their migration. A mechanism known as contact inhibition of locomotion (CIL) exists when two migrating NCCs make contact and they retract their protrusions and change direction (22, 23). The non-canonical WNT planar cell polarity (PCP) signalling pathway

12 4 (section 1.3.3) and cell-cell contact are crucial in controlling the polarity of migrating NCCs (8). Even though the NCCs experience CIL when they contact each other, they also tend to migrate in large groups, more so than what would be predicted given the CIL phenomenon. This is a result of a phenomenon called co-attraction, which allows the collective migration of NCCs despite low cell adhesion and dispersion due to CIL (14). NCCs have also been shown to move around barriers introduced into the migration path and can re-target their direction (24). The migration of CNCCs proceeds along a well-defined route, ending in the ventral part of the brain and the pharyngeal arches (PA) (25). NCCs from the diencephalon and anterior mesencephalon migrate into the frontonasal process (FNP) and NCCs from the posterior mesencephalon and hindbrain migrate to the PAs (8). CNCCs in the hindbrain region are compartmentalized in 7 distinct regions called rhombomeres (r). At first, the CNCCs migrating to the PAs migrate as a continuous wave but then they split into three distinct segregated streams (8). CNCCs from the posterior mesencephalon, r1 and r2 fill the first PA (PA1) and NCCs from r4 fill pharyngeal arch 2 (PA2) (Figure 1 ) (3, ). In the post-otic hindbrain, NCCs from r6-r8 colonize PA3-6 (21). The subpopulations of NCCs that migrate to and target the PAs migrate in stereotypical streams, maintaining a spatial segregation. This segregation has an important impact on craniofacial patterning and the early anteroposterior patterning of NCCs by establishing the segmental pattern in which the pharyngeal region of the vertebrate head is formed (26, 27).

13 5 Figure 1: Migration and skeletal fates of the CNCCs. A) Embryo showing the colonization of the head and pharyngeal arches by fore-, mid- and hindbrain NCCs. There are 7 segments in the hindbrain known as rhombomeres (r) from which crest cells emigrate in three major paths to the pharyngeal or branchial arches (BA), coded in blue, yellow and green. B) A skull drawing showing comparative contributions of the NCC populations to cranial skeletal elements of the human. The major bones are coded to match the origin of the contributing migratory neural crest streams. Reproduced from Gong, (3, 25). To allow proper positioning in the PA and the proper assembly of structures, the subpopulations of NCCs are guided by a complex set of cues to which they respond to locally during their migration (28). From the dorsal neural tube into the craniofacial region the NCCs follow wellmarked paths by communication with the surrounding neural, facial and pharyngeal epithelia and cephalic mesoderm (17). Examples of signalling molecules that guide the NCCs include: FGF-2 and FGF-8, which function in a chemotactic manner (29); Ephrins, which are ligands for Eph receptors (30); and semaphorin proteins (31) NCC positional identity The positional identity of NCCs is regulated by the homeodomain (HD) transcription factors of the homeobox gene family (Hox) (32, 33). The HD expression is induced and maintained in NCCs in later developmental stages via signals from the surrounding local environment (8). Anteroposterior NCC positional identity is thought to be acquired pre-migration (34, 35); however, it is not permanent and there is some degree of plasticity. For example, intrinsic molecular programs of the NCCs can be switched to new programs if exposed to ectopic environmental cues (34, 36, 37). The involvement of the Hox genes was first shown in a mouse model where targeted inactivation of Hox2a resulted in homeotic transformation of PA2 elements into PA1-like skeletal elements (38). Previous experiments by Santagati et al. (28) and Pasqualetti et al. (39) in Xenopus demonstrated that the skeletal pattern of mandibular and hyoid crest is not irreversibly committed before migration of NCC. However, positional information has to be maintained throughout the post-migratory states in order to provide information about size, shape and orientation of PA2 skeletal elements.

14 6 Dorso-ventral axis patterning information is regulated by the NCCs through the Dlx homeobox code. The involvement of Dlx genes has been investigated mainly through loss of function mutations in the mouse (40). Namely, the partitioning of PA1 is achieved with the two Dlx combinations; Dlx1/2 for the maxilla and Dlx 1/2/5/6 for the mandibular process. Mutations of Dlx1 or Dlx2 primarily affect the maxilla and mutations of Dlx 5 affect the mandibular process (41-43). It is clear the diversity of NCC-derived skeletal elements among vertebrates arises from modification of the levels, timing or spatial expression of HD factors. These factors play a crucial role in spatial identity and modifying the expression of pattern genes can result in aberrant development of structures (8) NCC differentiation and proliferation Once the NCCs have arrived in the facial prominences, they begin to proliferate and form the facial structures. Many factors regulate the proliferation of NCCs, such as the Sonic hedgehog (SHH) protein and WNT ligands. Disruption of these factors can affect the process of craniofacial morphogenesis and cause aberrant proliferation and morphogenesis of NCCs (4). There are two theories as to how NCCs determine which structures to make once they migrate to their final destination. The first theory is that the NCCs have an intrinsic program that is activated once they leave the neural tube. This intrinsic program contains information for molecular patterning for the formation of facial structures. The second theory is that the NCCs acquire facial patterning information from the surrounding tissue at their destination (44). The dominant theory is that of the latter theory, that the NCCs retain multipotency into the later stages of embryonic development (1) Facial prominences and CNCC derivatives of the face During the migration towards their final destinations ventrally, extensive interactions occur between the CNCCs and the surrounding mesoderm and overlying ectoderm and endoderm. The CNCCs in each facial prominence have distinctive molecular signatures and interact with different epithelia during development (1). By the end of fourth week of human embryonic development, most of the CNCCs have reached their final destinations. Within the pharyngeal arch is a center mass of mesodermal cells,

15 7 surrounded by CNCCs and externally covered by ectoderm and internally bordered by endoderm (Figure 2) (45). Continued interactions between surface ectoderm, CNCCs, mesoderm and pharyngeal endoderm are needed for the proper development of the facial structures (46, 47). For example, coordinated interactions between CNCCs and the mesoderm cells result in the differentiation of myoblast and skeletal precursors to form the musculature and skeleton of the facial region (48, 49). Signals that emanate from the CNCCs instruct and inform mesodermal cells to differentiate into myoblast precursors and how to organize themselves around the developing skeletal elements Facial development and facial prominences The major portion of the face in the human is formed starting between the fourth and eighth weeks of development. The face is derived from buds of tissue called facial prominences that undergo symmetrical and asymmetrical growth. Appropriate fusion events are required to result in mature facial structures (50). These facial primordia appear in the fourth week of development around the primordial stomodeum through inductive influences of organizing centers located in the prosencephalon and rhombencephalon (51). Figure 2: A schematic diagram of the pharyngeal arches. (A) The arches are indicated by different colors. The first arch is divided into maxillary (1a, orange) and mandibular (1b, yellow)

16 8 component. The second arch (2, green); third arch (3, purple); fourth arch (4, pink) and the sixth arch (6, blue). B. A coronal cut through the human embryo showing the tissue contribution to each arch. The arches (1,2,3,4) are composed of a core of neural crest (nc, yellow) and mesoderm (mes, green) surrounded by both surface ectoderm (se, pink) and pharyngeal endoderm (pe, purple). Reproduced from Cordero et al (1). At about the 4 th to 5 th week range (E9.5 in mice) of human development, CNCCs are migrating and arriving at the facial prominences (52). A total of seven prominences in the face will eventually be present ventral to the forebrain the FNP, two lateral nasal processes (LNP), two maxillary processes and two mandibular processes. The FNP surrounds the ventrolateral part of the forebrain which give rise to the optic vesicles of the eyes and also will give rise to the forehead, middle of the nose, upper lip, philtrum and primary palate (53). The lateral region of the FNP, also known as the median nasal process (MNP), will fuse with the LNPs and the maxillary processes to create the alae and columnellae of the nose. Initially, around the 5 th week of embryonic development, a pair of placodes form ventral to the developing forebrain. Further growth and proliferation of the placodes lead to a deepening, resulting in the formation of nasal pits which are surrounded by the lateral nasal process (LNP) and medial nasal processes (MNP) (51). These nasal placodes are bilateral oval thickenings of the surface ectoderm and develop on the inferolateral parts of the FNP. Mesenchyme in the margins of the placodes proliferates to produce horseshoe shaped elevations known as the MNP and LNP (Figure 2). The maxillary and mandibular processes are derivatives of the first pharyngeal arch and give rise to the upper and lower jaws, respectively. The maxillary processes enlarge and grow medially towards each other due to proliferation of the mesenchyme within the processes. This causes the MNPs to move towards the median plane and each other. The LNPs are separated from the maxillary processes by the cleft of the nasolacrimal groove. Merging of the LNP with the maxillary process starts around the 6 th week of development. Between the 7 th and 10 th weeks of development the MNPs merge with each other and with the maxillary and lateral nasal processes resulting in a continuity of the upper jaw, lip and separation of the nasal pits from the stomadeum. When the MNPs merge they form the intermaxillary segment which is made up of the philtrum of the upper lip, the premaxillary part of the maxilla and the primary palate (55). The lateral parts of the upper lip, most of the maxilla and secondary palate arises from the maxillary processes (51). The secondary palate begins to form in the early 6 th week from the

17 9 mesenchymal projections of the maxillary processes. The palatal shelves are outgrowths of NCC-derived mesenchyme covered by a layer of oral and surface epithelia on their outer surface. Within the palatal shelves is NCC derived mesenchyme. For the palate to form, the maxillary processes expand and undergo rotation to a horizontal position. The palatal shelves must have outgrowth in order to fuse. As the jaws grow, the tongue becomes relatively smaller and moves inferiorly, allowing the lateral palatine processes to elongate and ascend to a horizontal position. These palatal processes fuse in the median plane as well as with the nasal septum and primary palate (55). The maxillary processes merge with the mandibular prominences and the mesenchyme from the second pair of PA invades the primitive lips and cheeks and will later differentiate into the facial muscles of expression which are supplied by the facial nerve, a nerve of the second PA. The mesenchyme in the first PA differentiates into the muscles of mastication which are innervated by the trigeminal nerve from the first PA. At the 9 th -12 th week of development the nasal septum develops in a downward growth fashion from the fused MNPs and begins fusing with the palatine processes. Bone will gradually develop in the primary palate and extends from the maxillae and palatine bones into the lateral palatine processes to form the hard palate. In the midfacial region, NCCs condense within the single median frontonasal process to form the pre-cartilaginous nasal capsule, which undergoes chondrogenesis to form the nasal septum. The nasal septum has been proposed to play the role of pacemaker or growth center for the subsequent growth of the face and the skull. The nasal septum contributes to the overall changes in morphology observed during the development of the facial skeleton (56). To date, the fundamental mechanisms involved in the initiation, growth, boundary setting and differentiation of the cartilaginous and skeletal structures of the frontonasal region are not well characterized. Tooth formation also occurs around this stage. At 37 days of embryonic development in humans, a thickened band of epithelium forms in the upper and lower jaws around the mouth. The primary epithelial bands give rise to the dental lamina and the vestibular lamina. Localized thickening of the epithelium, also known as placodes, form within the epithelial bands (57). Beneath the epithelium lies an embryonic connective tissue called ectomesenchyme into which NCCs have migrated. Epithelial outgrowths descend into the ectomesenchyme at the sites of

18 10 deciduous teeth and ectomesenchymal cells proliferate and accumulate around these outgrowths. Within the first twelve days of development in mice, the epithelium possesses the potential to induce tooth formation but after 12 days of development this potential is assumed by the ectomesenchyme such that the ectomesenchyme can elicit tooth formation form different epithelia. Experiments have shown that first arch ectomesenchyme combined with foot epithelium can induce enamel organ formation from the plantar epitheliulm (58). If the epithelial enamel organ is recombined with skin mesenchyme, the organ loses its dental characteristics and assumes those of the epidermis. These experiments indicate that odontogenesis is initiated by factors from the epithelium initially and then later the ectomesenchyme. 1.2 Craniofacial developmental anomalies Due to the highly regulated nature of growth and fusion of the facial processes, craniofacial development is extremely susceptible to genetic and environmental perturbation that results in craniofacial malformations. There are many opportunities for congenital facial anomalies to occur. Anomalies result mainly from maldevelopment of the neural crest tissue which give rise to the connective tissue and skeletal primordia of the face (51). Facial dysmorphologies are a component of 75% of birth defects (7). Additionally, perturbances in the critical and highly regulated roles of CNCC specification, delamination, migration and differentiation during craniofacial development is best illustrated when abnormalities occur in craniofacial birth defects such as Treacher Collins syndrome, cleft lip and palate, 22q11.2 microdeletion syndrome and CHARGE syndromes. One of the most common craniofacial malformations is clefting of the lip and palate and clefting of the secondary palate alone. Cleft of the primary lip and palate has an incidence of 1/700 and clefting of the secondary palate alone has an incidence of 1/1500. Environmental factors such as a large tongue or ankylosis can cause clefting of the palate because the tongue will not descend, preventing the palatal shelves from fusing (1). Genetic factors, such as mutations in transcription factors like TBX 22 (59) and the gene Wntb9, play a role in secondary palatal clefting (60). In mammals, Wnt signalling is critical for the proliferation of CNCCs within the maxillary processes (61). Transforming growth factor (TGF-α and TGF-β) and epithelial growth factor receptor (EGFR) play a role in the fusion process of the epithelial cells on the palatal processes of the secondary palate. Egfr-/- mice have midline defects with an elongated primary palate,

19 11 clefting of the palate and micrognathia (62). Any disruption in the timing or extent of growth of the facial prominences can result in facial malformations. Treacher Collins syndrome is an example of abnormal regulation and proliferation of CNCCs. It is characterized by downward slanting eyes, micrognathia, conductive hearing loss, underdeveloped zygoma, drooping part of the lateral lower eyelids, and malformed or absent ears. The syndrome is caused by a mutation in TCOF1, which encodes the Treacle protein. A decrease in the Treacle protein results in a significant reduction in the number CNCCs, due to apoptosis and a decrease in migration and proliferation (63), and results in severe craniofacial hyperplasia and dysplasia. In summary, early development of the craniofacial region starts with the highly regulated process of CNCC development and their eventual contribution to the major structures of the craniofacial region. During the formation of the CNCCs, interactions between the surrounding surface ectoderm, neuroectoderm and endoderm are crucial for normal development of the face (1) and under strict molecular regulation. Many genes have been characterized to be significant players in the regulation of CNCC development and morphogenesis. 1.3 Proteins and genes involved in formation of craniofacial structures A number of genes, proteins and specific signalling pathways have been shown to be involved at different stages of craniofacial formation. During early craniofacial development, epithelialmesenchymal interactions are especially crucial in determining the types of tissues and extent of outgrowth of the different structures. Patterning and differentiation events later in development are also regulated by a series of molecules with specific functions. A review of the major signalling pathways and specific molecules with known roles in regulating some of the major events in craniofacial development will be discussed. For example, members of the BMP family, Wnt/β-catenin pathways and FGFs are necessary for NCC generation and survival (64-66). In the final part of this section, a description of Cthrc1, the focus of the current study, will be conducted. Early on, the prosencephalic organizing center, located at the rostral end of the notochord beneath the forebrain, induces the visual, inner ear apparatuses and the upper third of the face.

20 12 The rhombencephalic center induces the middle and lower thirds of the face (52). The forebrain establishes many signalling centres in the ectoderm that covers the frontonasal zone. Interactions between CNCCs and epithelia from the forebrain and facial ectoderm are required for proper development of the FNP (67). The forebrain provides SHH signalling which imprints on the forebrain ectoderm, controlling differential cell proliferation, and transforming the FNP into the nasal region. The nasal placodes provide morphogenetic information to the mesenchyme of the lateral nasal prominence and act as a signalling center for the lateral nasal skeleton, inducing cartilage and bone (68). Many genes involved in signal transductions, transcription regulation, binding and catalytic activities have been identified in the developing first PA. Examples are Msx-1 and 2, Pax8, Bmp4, 5 and 7, Fgfr11 and Tgf-β. Reciprocal epithelial and mesenchymal signalling events in the first PA induce transcription factors that function to differentiate oral versus aboral and rostrocaudal surfaces and patterning of skeletal elements (68). An example of the signalling between the endoderm, mesoderm and epithelium is that of TBX1, a T-box transcription factor. Tbx1 is expressed in the endoderm, mesoderm and epithelium of the palatal shelves and FNP (69). Decreased Tbx1 expression in the endoderm in PA development and epithelia of the palatal shelves results in secondary NCC abnormalities (70). Tbx1 knockout mice show defects in the PAs and pouches (70). The secreted peptide, Endothelin I, signals from the facial epithelia and mesoderm to the intervening CNCCs that form the PA1 skeleton. Mice with a homozygous mutation in EdnI have malformed bones of the jaw and throat and defective musculature (71). Recent genetic studies in humans have indicated that mutations in the Endothelin pathway may play a role in Waardenburg-Shah syndrome and Hirschprung disease which are caused by defects in NCC behaviour. Epithelial-mesenchymal interactions have been shown to play a key role in tooth development. Examples of genes that are expressed in the NCC-derived ectomesenchyme which initiate tooth formation are Lim-homeobox genes, Lhx-6 and Lhx-7. It has been shown that Fgf8 signalling from the epithelium induces the expression of the Lim-homeobox genes in the mesenchyme. Fgf8 from the epithelium also induces Pax-9 expression in the mesenchyme (57).

21 Fibroblast growth factors and bone morphogenetic proteins FGFs and BMPs have many roles during craniofacial and pharyngeal skeletal morphogenesis. The roles of FGF include serving as survival factors for CNCCs (72, 73), directing CNCCs to adopt the ectomesenchymal fate (74), acting as chemoattractants to promote lateral migration of endodermal cells for segmentation of the pharyngeal endoderm into pouches and the correct patterning of the CNCC-derived skeletal elements (26). For example, a Fgf8 conditional inactivation in the ectoderm of PA1 in a mouse model results in NCC apoptosis and absence of most PA1 skeletal elements (73). FGF signalling is also involved in NCC spatial identity and the establishment of anteroposterior and dorsoventral polarity of PAs (75, 76). BMPs are osteogenic agents that induce differentiation of mesenchymal cells toward an osteoblastic lineage and stimulate differentiation and functions of osteoblasts during bone modeling (77, 78). BMPs are critical in regulating bone formation (79) and play an integral role during craniofacial and pharyngeal skeletal morphogenesis, such as patterning of the maxilla and mandible (8), mesenchyme proliferation (80) and the pathogenesis of craniofacial synostosis (81). At early stages of development, FGF8 and BMP4 pattern the maxilla-mandibular region and define it from the premandibular domain (82) before the CNCCs arrive. The Fgf8 expression domain is induced by SHH signalling from the endoderm and delimited by Bmp4 which is expressed on both sides of adjacent Fgf-8 expressing ectoderm (82-84). BMP4 and FGF8 control the location of incoming NCCs by activating the patterning genes Dlx1, Barx1 and Msx1. These epithelial-mesenchymal interactions are needed for specifying the identity of the pre-mandibular and maxilla-mandibular regions. Fgf8 also has a role in anteroposterior and dorsoventral PA patterning and left-right symmetry of the craniofacial skeleton (85) Sonic hedgehog SHH signalling has many roles in craniofacial development. Disruption of SHH signalling in chick, mouse and zebrafish models results in severe head skeleton abnormalities due to defects in CNCC survival, proliferation and patterning (86, 87). If SHH is absent in the foregut endoderm, development of Meckel s cartilage is prevented as well as some associated PA1 structures, due

22 14 to NCC apoptosis (88). SHH also has a late-stage role in NCCs to promote differentiation into cartilage and establishing skeletal polarity in the mediolateral axis of the embryo (89, 90). SHH signalling from facial ectoderm is involved in the specification of NCC spatial identity (91). Excess SHH results in supernumerary Meckel s cartilage. SHH also has a late-stage role in NCCs to promote differentiation into cartilage and establishing skeletal polarity in the mediolateral axis of the embryo (89, 90). A signalling center called the frontonasal ectodermal zone (FEZ) exists in the ectoderm overlying the FNP. It regulates the growth and dorsoventral polarity of the upper beak in birds (91). Mice have two FEZ in the left and right MNP (92). When the FEZ is grafted ectopically it can reprogram the fate of underlying NCCs (91), showing an epithelial-mediated patterning instruction. FGF8 and SHH are both expressed in the FEZ facial epithelia and they promote cartilage outgrowth by inducing BMP4 expression the underlying NCCs (92). It has been proposed that the persistence of FGF8 expression in the facial ectoderm of the duck, but not the chick, is what contributes to the distinct morphology of the duck beak by inducing it to produce more cartilage (93). SHH is also involved in epithelial-mesenchymal interactions during tooth initiation. SHH is expressed in the dental ectoderm of mice at E11. Shh knockout mice have little development of facial processes. Addition of SHH soaked beads to oral ectoderm can induce local epithelial proliferation to produce invaginations like those of tooth bud formation, implicating Shh in tooth initiation (57) Wnt pathways In vertebrates, Wnt proteins have roles in regulating body axis specification, patterning of germ layers and tissues, cell adhesion, cell migration, cell proliferation, cell differentiation, morphogenetic movements during embryogenesis and organogenesis, and growth and metastasis of cancers/tumours (94). WNTs are secreted glycoproteins that interact with Frizzled (Fz) proteins on the cell surface. Fz proteins are a family of seven transmembrane G protein-coupled receptor proteins and they mediate cell signalling from the cell surface to cytoplasm (95). The Wnt signalling pathway can be divided into two subsets: the β-catenin-dependent canonical pathway and the β-catenin-independent non-canonical pathways.

23 15 In the β-catenin-dependent canonical pathway, Wnt proteins combine and interact with an Fz/low density lipoprotein receptor (LRP) complex on the surface of target cells (Fig. 3). This interaction leads to stabilization of β-catenin proteins (96) and subsequent signalling cascades. In the resting state β-catenin in the cytoplasm is phosphorylated. During activation, the accumulated β-catenin translocates to the nucleus and interacts with transcriptional factors to activate transcription of target genes (97). This canonical Wnt pathway is involved in cell fate, regulation of body axis, patterning of neuroectoderm and amplification of neural progenitors (94). Figure 3: The canonical Wnt signalling pathway. In resting cells, β-catenin is assembled in a multiprotein complex with CK1α, GSK3β, APC and Axin and is primed for phosphorylation by GSK3β. Phosphorylated β-catenin interacts with β-trcp and is degraded by ubiquitation (A). In

24 16 the activated state, Wnt proteins act on the Fz/LRP complex on the surfaces of the target cells. Upon Wnt-Fz signaling, Wnt-Fz and LRP coordinate Dvl activation, which results in recruitment of axin to the plasma membrane. Activated Dvl dissociates the multiprotein complex which leads to the inactivation of GSK3β, which can no longer phosphorylate β-catenin. Excess free cytoplasmic β-catenin translocates to the nucleus and binds to TCF/LEF transcription factors, causing transcriptional activation of target genes (B). Reproduced from Miao et al., 2013 (95). The β-catenin-independent non-canonical Wnt signalling pathways has two categories: the Wnt/Ca 2+ pathway and the PCP pathway (Figure 4) (95). These pathways are involved in cell proliferation, cell adhesion and cell differentiation, among other biological roles (98). The Wnt/Ca 2+ pathway leads to the release of intracellular calcium through G-proteins and also involves activation of phospholipase C and protein kinase C (PKC) (99, 100). The non-canonical Wnt-PCP pathway regulates cytoskeleton organization via actin polymerization through activation of small GTPases (Figure 4). It also regulates the establishment of polarity within the plane of the epithelium and allows cells to obtain directional information during embryogenesis (101). Fzd activates JNK and directs asymmetrical cytoskeletal organization and coordinated polarization of cells within the plane of epithelial sheets (102, 103). The Wnt-PCP pathway also controls convergent extension movements during gastrulation and is involved in the directional migration of cells in the developing palatal shelves (104).

25 17 Figure 4: The non-canonical Wnt signalling pathway. The non-canonical Wnt signalling pathway includes the Wnt/Ca 2+ signalling pathway and the planar cell polarity Wnt pathway. In Wnt/Ca 2+, certain Wnt-Fz interactions activate the cytoplasmic protein Dvl, which increases the level of cytosolic Ca 2+. Subsequent activation of Ca 2+ -sensitive enzymes CamKII, PKC and others induces the target gene expression and corresponding biological effects. In the Wnt/PCP pathway, activated Dvl activates the small GTPases Rho and Rac, which leads to the activation of Jun kinase (JNK) and rho kinase (ROK). Subsequently, transcription factors, such as the AP1 family, are activated and target gene expression is induced. Reproduced from Miao et al., 2013 (95) Wnt pathways and craniofacial development The Wnt/β-catenin signalling pathway components are highly conserved and are crucial for the patterning and morphogenesis of many developmental processes, including face, limb, skeleton, central nervous system, skin and ectodermal appendages like teeth and hair (105, 106). In

26 18 craniofacial development, Wnt/β-catenin is crucial for NCC induction and migration and for proper fusion of facial prominences (66, 107, 108). Studies in the chick and mouse have shown that Wnt signalling in specific regions of the facial prominences correlate with outgrowth and fusion of processes (61, 109). In particular, mouse studies have demonstrated that the LNPs were the primary region of Wnt-β-catenin signalling (61). Mice with a loss-of-function (LOF) or gain-of-function (GOF) mutation for β-catenin resulted in aberrant Wnt signalling in the ectoderm and embryos showed facial dysmorphologies at the crucial stage of fusion and growth of prominences of mid-face development. Between E9.5 and E12.5 (7), LOF mutants of β-catenin had a hypoplastic facial morphology, e.g., underdeveloped mandibular processes, and a narrow FNP which resembled a beak at birth. GOF mutants had a lack of controlled directional growth in their facial prominences, resulting in increased size of the maxillary and mandibular processes and improper fusion of the processes. GOF mutants also had ectopic cartilage throughout the head region resulting in malformations of the head. Both the GOF and LOF mutants had alterations in the shape of their face, which would affect the underlying cranial skeleton. The proper growth of facial prominences is needed for cartilage formation as the mesenchymal cells beneath the ectoderm require proliferation signals to establish a critical number of progenitor cells (110). The progenitor cells respond to localized and intrinsic patterning signals from the ectoderm to form cartilage of the correct shape and size (110). The strictly canonical WNTs (2B, 7A, 9B and 16) and WNT4 and WNT6 (canonical and non-canonical Wnt ligands) are expressed in the epithelia while WNT5A, 5B and 11 are limited to mesenchyme (109). These data demonstrate that ectodermal Wnt/β-catenin signalling plays a crucial role in the formation and patterning of the craniofacial skeleton Wnt5A Mutations in the human Wnt pathway genes have been shown to have an effect on the craniofacial and limb skeleton (111). For example, a recessive missense mutation of WNT5A or WNT5A receptor ROR2 results in micrognathia, clefting and rhizomelic limb shortening seen in Robinow syndrome.

27 19 Wnt5a was exclusively expressed in the mesenchyme in the facial region of developing chicken embryos (109). Of the WNTs found in the chicken embryo face (WNT5A, WNT5B and WNT11), 5A was the most abundant and was found in all facial prominences. At stage 21 of chicken embryo development there are strong WNT5A signals throughout the mesenchyme of the maxillary and mandibular prominences, lateral nasal processesand lateral edges of the frontonasal mass, with the highest expression in Meckel s cartilage. In the non-canonical pathway, WNT5A binds to Fz receptors or can bind to the Ror2 receptor (112). When WNT5A binds these receptors the JNK/ PCP pathway or calcium signalling pathways are activated, leading to changes in actin cytoskeleton, cell polarity and cell movement. In development, WNT5A is expressed in cartilage blastema, which may promote chondrogenesis. However, in excess it induced rapid loss of the cartilage matrix due to induction of metalloproteinase and aggrecanase enzymes. WNT5A regulates matrix stability but not the initial steps of chondrogenesis. It keeps canonical signalling low in cartilage blastema and in this manner, promotes chondrogenesis and cartilage differentiation (113). See Figure 5 for the roles of WNT5A in development. Some other Wnt ligands of significance to craniofacial development are WNT3A and WNT9B. A deletion of Wnt3a causes death upon birth and these embryos have mandibular defects. Wnt9b is involved in lip fusion and its targeted deletion results in cleft lip in animal models. A deletion of Wnt5a leads to truncation of the upper and lower jaws (113) (114).

28 20 Figure 5: Summary of WNT5A functions during cartilage development and disease. A) During normal development, there is synthesis of WNT5A in the cartilage blastema and by the newly differentiated chondrocytes. The secreted WNT5A acts back on the same cells to inhibit canonical activity. The net result of reducing canonical activity is to promote chondrocyte differentiation and matrix secretion. Reproduced from Hosseini-Farahabadi et al. (113). 1.4 Collagen Triple Helix Repeat Containing 1 (Cthrc1) Background Collagen Triple Helix Repeat Containing 1 (Cthrc1) was originally discovered in a screen for novel sequences induced in a rat arterial injury model, being expressed in adventitial cells of remodelling arteries and dermal fibroblasts during skin wound healing (115). Cthrc1 was also identified in a microarray study of the midface region of the mouse embryo at E10.5 (Gong unpublished data, 2006). Cthrc1 mrna was upregulated in the MNP compared to the LNP, suggesting a possible critical role in the development of the midface. It is for this reason as well as information that will be discussed in the following section that the CTHRC1 gene and protein was chosen as a gene of interest for a role in development of the midface.

29 CTHRC1 Structure CTHRC1 protein is a 30 kda secreted N-glycoprotein containing a short collagen motif with an NH 2 -terminal peptide for extracellular secretion, a short collagen triple helix repeat of 36 amino acids and a COOH terminal globular domain (Figure 6). The helix repeat comprises 12 Gly-X-Y repeats, a domain believed to be responsible for trimerization of the protein and protection from cleavage (116). The normal active form of CTHRC1 is an N-glycosylated trimer anchored on the cell surface (94). Figure 6: The structure of the CTHRC1 protein. Adapted from Lindner (117). CTHRC1 is exclusive to vertebrates and is highly conserved throughout evolution, showing little homology to other currently known proteins (115). Its short collagen-like motif is similar to the collagen domains present in the C1q/tumour necrosis factor-α related proteins, (118). CTHRC1 is thought to belong to the adiponectin/complement factor family (119) that all contain the conserved collagen domain with the 12 GLY-x-y repeats and a globular domain in the C- terminal half. Adiponectin is a protein hormone that modulates a number of metabolic processes, including glucose regulation and fatty acid oxidation (120). Similar to adiponectin, CTHRC1 exists in monomeric, dimeric and trimeric forms (55). Under reducing conditions in Western blot analysis, CTHRC1 exists as a 28 kda protein (119). The biological activity of CTHRC1 is restricted to the highly conserved 200 amino acids at the C-terminal region (94) which contains an N-glycosylation site that stabilizes the CTHRC1 protein by decreasing its turnover rate. Upregulation of CTHRC1 is seen to coincide with its increased glycosylation rate in certain cells

30 22 such as human oral squamous cell carcinoma (121). N-glycosylation also promotes tethering of CTHRC1 to the cell membrane, which promotes actin polymerization and cell polarity (94) Expression Pattern of Cthrc1 During development, Cthrc1 mrna expression has been identified in visceral endoderm, developing kidney and the heart of mouse embryos, with specific abundance in the cartilage primordia, growth plate cartilage (excluding the hypertrophic zone), bone matrix and periosteum (122). Prior to neural crest migration (E8.5 in mice), Cthrc1 mrna was seen in the notochord and the floor plate of the anterior ventral neural tube. By E9.5 transcripts were seen in somites, branchial arches, otic placode and the hindbrain-midbrain junction with the somatic expression becoming more pronounced by E10.5. Expression of the gene was observed in developing bone formed via endochondral and intramembranous ossification, such as skull bones, ribs, vertebrae and cartilage primordia (122). Cthrc1 transcripts have been shown to be expressed prominently in chondrocytes during E14.5 of mouse development (119). Although chondrocytes from condensing mesenchyme contained high levels of CTHRC1 protein and mrna, hypertrophic chondrocytes no longer expressed the mrna. At much later time points (E18.5) developing incisors show CTHRC1 in the dentin line and at the interface of epithelial ameloblasts and underlying mesenchyme. Postnatally, the expression of Cthrc1 in adult tissues has been shown at low levels and restricted to basal expression in bone, brain and mature bone-resorbing osteoclasts. However, Cthrc1 expression surges in pathological states such as arterial injury, skin wounds or cancer (115, 122, 123). During development, Cthrc1 transcripts are prominently expressed in chondrocytes (119). In mouse pups, high levels of CTHRC1 protein expression were seen in chondrocytes of the resting and proliferating zone of the growth plate (122). Conversely, non-proliferating chondrocytes in adult articular cartilage and fibrous cartilage of the meniscus did not express CTHRC Possible Functions of Cthrc1 Several studies have suggested that Cthrc1 plays important roles in collagen regulation, postnatal bone remodeling, and cell migration. The following section will review the literature on the

31 23 involvement of Cthrc1 in these processes and discuss the possible role of Cthrc1 in the processes in development Cthrc1 Role in collagen deposition The localization of CTHRC1 to tissues rich in collagen, such as cartilage (collagen type II), bone matrix (collagen type I) and skin (collagen type I and III), and to active sites of collagenous matrix deposition suggests a role for Cthrc1 in modulating collagen matrix synthesis. The role of Cthrc1 in collagen deposition has mainly been seen in studies in vascular remodelling after injury. Cthrc1 was first discovered in a screen for differentially expressed sequences in ballooninjured versus normal arteries where it was expressed by fibroblasts of the remodeling adventitia and by smooth muscle cells of the neointima and was shown to decrease or inhibit collagen matrix deposition (115). In vitro overexpression of CTHRC1 caused a dramatic reduction in collagen type I mrna, procollagen protein levels and collagen deposition (115, 124). Transgenic mice that constitutively overexpress Cthrc1 under a CMV promoter have brittle bones, caused by a reduction in collagenous bone matrix (124). The blood vessels of Cthrc1 transgenic mice had widespread cartilaginous metaplasia of the tunica media which suggests that Cthrc1 causes the shifting of type I collagen expression profile typical of bone to a collagen type II pattern typically found in cartilage. This shift to the subtype of collagen found in cartilage would further support findings showing chondrocytes of the growth plate of developing bones with abundant expression of Cthrc1 mrna (122). Vascular remodeling after injury is controlled by TGF-β signalling that mediates negative aspects of vessel repair such as neointimal lesion formation, smooth muscle cell proliferation, increased collagen deposition and lumen narrowing (125). Cthrc1 expression patterns overlap significantly with those of TGF-β family members in calcified tissues and cartilaginous matrix, as well as developing bone and cartilage (122, 124), periosteum, osteocytes (126), developing skull bones, ribs, vertebrae, cartilage primordia, hyptertrophic and proliferative zones of growth plate chondrocytes and all zones of endochondral ossification (122). CTHRC1 interacts with the TGF-β signalling pathway to modulate collagen regulation (59). CTHRC1 can be regulated by TGF-β and conversely, may also regulate TGF-β responsiveness and affect TGF β target genes (124). For example, Cthrc1 mrna levels are increased in response to TGF-β but CTHRC1 protein is also a cell type-specific inhibitor of TGF-β which impacts collagen type I and III

32 24 deposition (61), neointimal formation and differentiation of smooth muscle cells (122, 124). In transgenic mice that have upregulated Cthrc1 gene expression, TGF β signalling is reduced in smooth muscle cells (124). CTHRC1 s signalling interactions with TGF-β have been shown to be via activation of Smad 2/3 complexes (127). The promoter region of Cthrc1 contains a putative Smad binding site (128). It has been suggested that the activation of Cthrc1 transcription could be regulated by TGF-β signalling through Smad proteins (123). Different studies have shown that CTHRC1 regulates extracellular collagen deposition by inhibiting phosphorylation of Smad2/3 activation via inhibition of TGF-β signalling. Phosphorylated Smad 2/3 increases expression of CTHRC1 that in turn inhibits the deposition of extracellular collagen controlled by Smad 2/3 phosphorylation (123). Smooth muscle cells that overexpressed CTHRC1 protein had reduced levels of phospho- Smad 2/3 (122). Cthrc1 transcript induction by TGF-β, the CTHRC1 inhibition of TGF-β sensitive reporters and the reduction of phospho-smad 2/3 levels in vivo provide evidence that these two proteins are working as antagonists to maintain balance in extracellular matrix components. The mechanism by which CTHRC1 disrupts TGF-β signalling, leading to a reduction in collagen is not clear. Since CTHRC1 is a secreted protein, it is likely to function as a ligand in a signalling pathway with downstream effects on collagen promoter activity (124) Role of Cthrc1 in osteogenesis, osteoclastic bone formation and bone remodelling The skeletal system fulfills mechanical, supportive, and protective roles in animals (129). Its formation is controlled by tightly regulated programs of cell proliferation, differentiation, survival, and organization (130). The initiation of the skeletal system is from mesenchymal condensations, in which skeletal precursor cells, also known as osteochondral progenitors, give rise to either chondrocytes to form the cartilage or osteoblasts to form the bone. Bone is a mineralized connective tissue consisting by weight of 28% type 1 collagen and 5% noncollagenous structural matrix proteins, synthesized by osteoblasts. These constituents accumulate as the uncalficied matrix, osteoid, that acts as a scaffold for the deposition of apatite crystals of bone to make up the remaining 67% of bone. This mineral is in the form of small plates which lodge in the holes and pores of collagen fibrils (57). Some osteoblasts become trapped in the bone matrix and are then referred to as osteocytes. The osteoclast is a

33 25 multinucleated cell which resides against the bone surface where they can bind and secrete enzymes to demineralize the bone matrix and degrade the organic matrix. The difference in the initial fate choices of osteochondral progenitors to either chondrocytes or osteoblasts determines whether ossification is endochondral or intramembranous (57). Intramembranous ossification is the direct laying down of bone into the primitive connective tissue (mesenchyme). During intramembranous ossification, bone develops directly within the soft connective tissue whereby osteoblasts differentiate from the mesenchyme and begin to produce bone matrix (57). In contrast, the initiation of endochondral ossification requires the presence of cartilage as a precursor (131). During endochondral ossification, condensation of mesenchymal cells gives rise to cartilage cells. This cartilage will eventually be replaced by bone. Cartilage consists of collagen of which the main type is collagen type II, in contrast to bone which is primarily collagen type I. Perichondrium forms around the periphery of the cartilage which gives rise to a cartilage model that will eventually be replaced by bone through the action of osteoblasts. Prechondrogenic mesenchymal condensations in the developing mouse face are seen as early as E8.5 and 9.5 and cartilage primordia forms at E (132, 133). Overall, collagen deposition is critical in both endochondral and intramembranous bone formation as the collagen matrix acts as a scaffold in endochondral bone formation and is a component of the bone material in intramembranous bone formation and makes up the components of cartilage. Bone mass is regulated by a process of continual remodelling, which is based on the action between osteoblastic bone formation and bone resorption, and is coordinated by mediation of many signalling pathways such as parathyroid hormone, TGF-β and BMPs (134). Bone turnover rates of 30%-100% in childhood are common and slow down in adulthood (57). CTHRC1 has been shown to be involved in postnatal bone formation mainly through effects on osteoblasts. CTHRC1 is expressed in bone tissue in vivo, and is a gene identified as a downstream target of bone morphogenetic protein-2 (BMP-2) in osteochondroprogenitor-like cells (119). Micro-computed tomography and bone histomorphometry analyses showed that Cthrc1-null mice have low bone mass due to decreased osteoblastic formation and conversely, transgenic mice overexpressing Cthrc1 under an osteoblastic promoter displayed high bone mass due to an increase in osteoblastic bone formation (135). Cthrc1 osteoblast-specific

34 26 overexpressing and null mice also had increased and decreased levels of osteoblast specific genes, ALP, Col1a1 and Osteocalcin, respectively, with no change in osteoclast numbers. In vitro, CTHRC1 stimulated differentiation and mineralization of osteoprogenitor cells with a particular acceleration of osteoblast proliferation (135). CTHRC1 has been shown to be induced by BMP2 and is not required for skeletal development, though it is required for bone maintenance homeostasis (135, 136). In addition to TGF-β, CTHRC1 has also been shown to be regulated by BMP-4 (115, 128), suggesting that CTHRC1 may act as one of the downstream targets for BMP-Smad signalling and that its expression in growth plate cartilage and bone matrix controls collagen deposition though regulation of Smad2/3 and TGF-β signaling (122, 124). In addition to transgenic and knockout studies, in vitro studies have further supported a role of CTHRC1 in bone formation through its effects on osteoblasts via osteoclasts. Osteoclasts placed on a substrate containing hydroxyapatite had upregulated expression of Cthrc1 mrna compared to the dentin slices, suggesting the CTHRC1 protein production is closely linked with osteoclast attachment to calcified tissue (119). The same result was observed when osteoclasts were placed in an environment with high extracellular calcium and phosphate. Cthrc1 expression increased in a high-turnover state (RANKL injections in vivo) and decreased in conditions associated with suppressed bone turnover (aging and after alendronate treatment) (119). BrdU incorporation assays showed that CTHRC1 stimulates osteoblast proliferation in vitro and in vivo. Colonyforming unit (CFU) assays in bone marrow cells showed CTHRC1 stimulates differentiation of osteoprogenitor cells and osteoblasts (136). Experiments using ST2 cells, a stromal line derived from mouse bone marrow showed that CTHRC1 targets stromal cells to stimulate osteogenesis by binding to a putative cell surface receptor on ST2 cells to stimulate osteoblastic differentiation as well as recruitment in order to promote bone formation (119). The impact of CTHRC1 on bone mass was examined in mice with ovariectomies. Ovariectomised mice are estrogen deficient and develop bone loss secondary to bone resorption. (135). Wild-type mice showed a 47% trabecular bone loss after ovariectomy while transgenic mice overexpressing Cthrc1 showed only 38% trabecular bone loss, suggesting a stimulatory effect of CTHRC1 on bone formation.

35 27 More recently it has been shown that CTHRC1 is secreted by resorbing active osteoclasts and acts as a coupling agent between bone resorption and formation. In vitro experiments showed that Cthrc1 expression stimulated osteogenic differentiation of osteoblasts, measurable by increased alkaline phosphatase and osteocalcin and had bone formation stimulating activity comparable to that of BMP-2 and FGF-1(119). Mice containing a systemic or osteoclast specific knockout of Cthrc1 were born appearing grossly normal but with absence of Cthrc1 mrna. There was no difference in mrna levels between the two types of knockout mice, indicating that mature osteoclasts are the major source of CTHRC1 in vivo. Both types of knockout mice had low bone mass and abnormalities similar to osteoporosis. Histomorphometirc analysis showed decreased osteoid surface and bone formation rate compared to control mice. These results concluded that CTHRC1 protein is produced and secreted by mature osteoclasts and acts on osteoblastic cells to stimulate osteoblastic differentiation and recruitment which promotes bone formation and maintains bone mass and trabecular structure through regulation of bone formation. From these studies the common theme is that Cthrc1 plays a role in postnatal bone formation due to regulation of collagen deposition as well as having an effect on osteoblasts. Taken with results from Cthrc1 transgenic and knock out mouse experiments, evidence of a role in collagen deposition and in vitro studies on osteoblast proliferation, CTHRC1 seems to have an anabolic effect on bone formation though collagen matrix deposition and regulation of osteoclasts and osteoblasts Role in cell motility and tissue repair CTHRC1 is also shown to be involved in cell migration (137). Inhibition of Cthrc1 in vitro decreases cell migration, while overexpression increases cell migration (115, 128). CTHRC1 enhances migration of smooth muscle cells and fibroblasts in a scratch wound assay, enabling CTHRC1 overexpressing cells to migrate into the wound area significantly faster than control cells (115, 122). Enhanced expression of CTHRC1 is involved in vascular remodelling; in rat fibroblasts it promotes cell migration and inhibits collagen I synthesis in these cells (124). Under normal conditions in blood vessels, CTHRC1 resides in the cytoplasm of smooth muscle cells in an unprocessed form, despite the presence of a signal peptide. Under conditions of vascular injury, CTHRC1 is released from the smooth muscle cells and cleavage of the N-terminal

36 28 propeptide results in a molecule with an increased ability to inhibit collagen matrix deposition (138). The ability of CTHRC1 to reduce collagen matrix deposition correlates with increased cell migration.(139). This limitation of collagen matrix deposition has been suggested as one mechanism by which CTHRC1 promotes cell migration (128). CTHRC1 expression is also linked to cellular proliferation, such as Schwann cell proliferation (140) and enhanced proliferation of osteoblasts (135). CTHRC1 has the same positive effect on migration in tumour cell lines and has been found in increased levels in highly metastatic tumours, which suggests it plays a role in cancer progression by promoting cell migration (128). Cthrc1 transcripts and protein are increased in malignant melanoma, cancers of the GI tract, lung, breast, liver and pancreatic cancers when compared to normal tissues (141) and are highly active in degrading extracellular matrix proteins in several of these malignant tumours (128, 142, 143). A significant increase of CTHRC1 has been observed in patients with metastatic bone cancer compared to non-metastatic tumours (121). A germline mutation in Cthrc1 was identified in patients with Barrett s esophagus and esophageal adenocarcinoma (143). Cthrc1 was among 48 genes that are significantly associated with risk of recurrence of patients with stage II/III colon cancer (144). CTHRC1 expression has been used as a cancer prognostic indicator. Lack of CTHRC1 has been demonstrated in non-invasive stages of melanoma in contrast to enhanced expression in primary invasive melanomas and metastatic melanomas (142). Inhibition of Cthrc1 using short interfering RNA showed decreased invasion of melanoma cell lines and patients with bone metastases had a significant increase in CTHRC1 stromal expression compared to patients without. In conclusion, the current literature supports a role for Cthrc1 in cell migration, collagen deposition and bone formation Genes and signalling pathways involved with CTHRC Cthrc1 Activation of Wnt/PCP pathway Cthrc1 is also suggested to interact with components of the Wnt/PCP pathway in the noncanonical Wnt pathway by forming a CTHRC1-Wnt-FZD/Ror2 complex to selectively activate the Wnt/PCP pathway (94). The normal active form of CTHRC1 is an N-glycosylated trimer

37 29 anchored on the cell surface. CTHRC1 is a positive regulator of the non-canonical Wnt signalling and simultaneously inhibits the canonical branch (94). Results from Yamamoto et al. (94) suggest that CTHRC1 is a Wnt cofactor protein that selectively activates the Wnt/PCP pathway by stabilizing an extracellular ligand-receptor interaction. In vitro, CTHRC1 has been shown to interact with multiple extracellular components of Wnt signalling including both canonical and non-canonical Wnt proteins, Fzd proteins and the Wnt/PCP co-receptor Ror2 but not with the canonical Wnt co-receptor LRP6 or the PC component Vangl2 (94, 139) (Figure 7). The CTHRC1 protein is thought to positively regulate the Wnt/PCP pathway by promoting interaction between the Wnt ligand and the Fz receptor complex (94). In conditions where appropriate receptors are available, Wnt proteins may activate the canonical or non-canonical pathways but when CTHRC1 is present the interaction of Wnt proteins with the Fzd/Ror2 complex is enhanced and the selective activation of the Wnt/PCP pathway in this environment suppresses the canonical pathway (145). Through the Wnt/PCP pathway, CTHRC1 may regulate cell motility in embryogenesis and cancer cell migration and invasiveness in adult tissues (128). It has also been speculated that the Wnt/PCP pathway activation by CTHRC1 contributes to the promotion of cell motility because the PCP pathway regulates actin polymerization through GTPase signalling, which alters cellular morphology and increase cellular motility (94). This Wnt/PCP pathway plays an important role in controlling cell polarity and movement and has been implicated in chondrocyte maturation and cartilage formation during ontogenesis (146). The Wnt/PCP pathway regulates chondrocyte maturation and cartilage formation as genetic alterations in the pathway are associated with chondroplasia, dysregulation of collagen deposition and changes in cartilage morphology (94, 139, 147), thus further supporting a role for Cthrc1 in collagen deposition and cartilage formation (139).

38 30 Figure 7: A model of selective activation of the Wnt/PCP pathway by CTHRC1. CTHRC1 is cell-surface anchored and enhances the interaction of Wnt proteins, Fzd proteins and Ror2 complex to activate the Wnt/PCP pathway (94). The interaction of Wnt proteins with Fzd/Ror2 is selectively enhanced in the presence of Cthrc1. Adapted from Yamamoto et al.(94) Cell-specific action of Cthrc1 Cthrc1 s cell-specific behaviour is well characterized, with different actions or effects depending on the tissue it is expressed in. CTHRC1 inhibits TGF-β signalling in smooth muscle cells but not in endothelial cells (124). There are differing results on the effect of CTHRC1 on Col1a1 expression upregulation of CTHRC1 in osteoblasts results in increased expression of Col1a1 whereas in PAC1 cells, a smooth muscle cell line, upregulated CTHRC1 produced reduced mrna levels of Col1a1 and collagen deposition (115, 124). High levels of CTHRC1 were also associated with reduced collagen deposition in smooth muscle cells but increased collagen deposition in osteoblasts (123, 135). These results suggest CTHRC1 function may differ between cell types and that it may work through its own signalling pathway that is functional in a celltype specific manner (135). In summary, many roles of CTHRC1 are supported by various studies in the literature. Transgenic mouse studies have suggested CTHRC1 has a possible role in bone formation, specifically in osteoblast regulation and collagen matrix deposition. In disease states, CTHRC1 is associated with increased cell motility and migration and alterations in collagen matrix deposition. These roles in cell migration, collagen matrix deposition and regulation of bone formation have been demonstrated in vitro as well as in postnatal animals. Cell migration and motility, bone formation and regulation of collagen expression are all important steps in

39 31 embryogenesis and therefore the involvement of CTHRC1 in these processes during development is highly probable. The role of CTHRC1 in cell migration, collagen matrix deposition and bone formation makes it a gene of interest to study in craniofacial formation as these processes are crucial in midface development. 1.5 Rationale for Study Many craniofacial abnormalities can be attributed to defects in the generation, proliferation, migration and differentiation of cranial NCCs and epithelial-mesenchymal induction interactions. Any perturbation in these processes can result in craniofacial malformations and developmental anomalies of the facial structures. Although existing studies describe the involvement of a number of well-known genes in midfacial development, there still remain a great number of candidate genes whose role in development remains unexplored. Given the evidence that the CTHRC1 protein and mrna play roles in cell migration, collagen deposition, and bone formation, we speculate that it plays such roles in the early and later events during the development of the midface. 1.6 Hypothesis/Aims The hypothesis is that CTHRC1 protein has an expression pattern in the developing mouse embryo midface indicative of a role in craniofacial development of the mouse with respect to cell migration, bone formation and collagen deposition. The aims of this study are to perform a temporal and spatial analysis of CTHRC1 expression in at crucial time points of the developing midface in mouse embryos.

40 32 Chapter 2 Materials and Methods 2.1 Embryos Timed pregnant wild-type CD-1 mice were obtained from the Toronto Centre for Phenogenomics. Embryos were removed from the uteri of mice at embryonic (E) stages E8.5, E9.5, E10.5, E11.5, E12.5 and E13.5. Embryos were isolated from the uteri with the aid of a Nikon SMZ800 dissecting microscope, at a temperature of 4 C. Whole embryos were dissected for time points E8.5 and E9.5 and the head was dissected from time points E11.5, E12.5 and E13.5. Approximately five embryos were used for each time point of immunohistochemical staining with the CTHRC1 antibody. Another group of eight embryos at stages E8.5, E9.5, E10.5, E11.5 and E13.5 were used for reverse transcriptase quantitative polymerase chain reactions (RT-qPCR). The whole embryo was collected for stage E8.5. The head was dissected from the body of the embryos of stage E9.5, E10.5, E11.5 and E13.5. Tissues were stored at -80 C. 2.2 RT-qPCR To obtain a temporal profile of the expression of the Cthrc1 gene mrna, RT-qPCR was performed on RNA samples from embryos at stages E8.5, E9.5, E10.5, E11.5 and E RNA isolation Tissues of embryos harvested at specific embryonic stages, as described above, were homogenized in TRI Reagent (Sigma Aldrich) for 5 minutes. Chloroform was added to samples, which were shaken for 15 seconds. Tissue samples were centrifuged at 12,000g for 15 minutes at 4 C. The aqueous phase was removed and mixed with isopropanol, incubated for 10 minutes at room temperature and then centrifuged at 12,000g for 8 minutes at 4 C. The supernatant was removed to isolate the RNA pellet, which was washed with 75% ethyl alcohol (ETOH) and centrifuged at 7500 g for 5 minutes at 4 C. The ETOH supernatant was removed and the RNA pellet was resuspended in distilled H 2 0. Samples were run on a 2% agarose gel and stained with

41 33 ethidium bromide to determine the integrity of the RNA; specifically, to determine the presence of the small (2 kb) and large (5 kb) ribosomal RNA Reverse transcription of RNA samples Reverse transcription of the RNA from each time point sample was performed using the Maxima First Strand cdna Synthesis Kit for RT-qPCR (#K1641; Thermo Fischer Scientific). A reaction mix was made with the sample template RNA, H 2 0, Maxima Enzyme Mix and 5X reaction mix. Reaction samples were incubated for one cycle of 10 minutes 25 C, 30 minutes 72 C, 5 minutes 85 C and then stored at 4 C Quantitative PCR Primers for Cthrc-1 were designed using Ensembl software on the Cthrc1 gene ( ). Primers were selected that skipped the intron between exons 1 and 2 (3,323 bases). The primers spanned basepairs of the Cthrc1 gene, producing an amplified fragment of 218 basepairs. The primer sequences were: Forward primer: 5 -T G C T G C T G C T A C A G T T G T C C-3 Reverse primer: 5- T C C C T T T T C C C C T T T G A A T C-3. All reactions were performed in a 96-well plate using the itaq Universal SYBR Green Supermix (Biorad). GAPDH was used as the reference gene. PCR reactions of cdnas from tissue samples at each time point sample were run in duplicate at two different dilutions (1:5 and 1:10), for quality control. PCR conditions were as follows: 40 cycles of 94 C, 58 C and 72 C to allow for denaturing of the cdna, binding of the primer and extension of the primer, respectively. cdna quantities were determined using the CFX96 Touch Real-Time PCR Detection System and CFX manager software 3.0 (Biorad) and plotted on a graph using the formula: 2 CT(target) / 2 CT(reference). CT target was the change in cycle threshold value for the tissues at each time point compared to E8.5 and CT reference was the change in cycle threshold values of GAPDH at the corresponding time points compared to E8.5. The values from this ratio were plotted using Excel software (Microsoft Office 2007).

42 Histological Processing and Paraffin Embedding Embryos were transferred to Bouin s fixative (Polysciences, Inc.) to incubate for 24 hours, followed by storage in 70% ETOH at room temperature (20 C). Prior to paraffin embedding the embryos were stored in 80% ETOH for 1 hour, 100% ETOH for 1 hour, followed by incubation in methyl benzoate overnight. Afterwards, embryos were transferred to toluene for 2 hours. All embryos were processed for paraffin sectioning according to Gong (2001)(148). Sections were mounted on Superfrost slides (Fisherbrand). Embryos were oriented such that they were cut in a coronal, transverse or sagittal plane. The choice of orientation for different time points depended on the need to capture the pattern of CTHRC1 expression in specific midfacial structures at different stages of development in the midface. Therefore, embryos at E8.5 and E9.5 were oriented only in sagittal or coronal planes and E were oriented in the coronal or transverse planes. 2.4 Immunohistochemistry Immunohistochemical reactions using a rabbit polyclonal antibody to Cthrc1 (ab85739; Abcam) were conducted on tissue sections at different embryonic stages according to Gong (2001)(148). To ensure specificity of the α-cthrc1 antibody, a peptide competition assay was performed to rule out false positive results. Transverse sections of embryos at E13.5 were selected based on a distinct pattern of expression. The Cthrc1 peptide (ab101727; Abcam) was incubated with the primary antibody at 5x the primary antibody concentration used for the immunohistochemistry at 4 C for 24 hours prior to the staining. The staining was then carried out as described in Gong (2001)(148), with additional slides receiving the peptide-blocked primary antibody instead of the primary antibody alone. Subsequent steps for the staining of these slides were as described in Gong (2001)(148). The signal detection was compared in the sections stained with the peptideblocked primary antibody and the primary antibody alone. The sections on the slides were deparaffinised and rehydrated with washes of 100% ETOH, 95% ETOH and 70% ETOH. Tissue sections were washed for 30 minutes in phosphate buffered saline (PBS). Sections were treated with 0.3% H for 20 minutes to quench endogenous peroxidase activity and washed for 10 minutes with PBS. Antigen retrieval was conducted by treating sections with 0.01 NaCitrate buffer (ph 6.0) for 10 minutes at 90 C, followed by washes

43 35 for 10 minutes in PBS. Tissues were then incubated in a blocking solution of 1.5X normal goat serum (Zymed), 0.01% Saponin and 0.1% Bovine Serum Albumin (Sigma) to prevent nonspecific binding of the secondary antibody. The tissue sections were subsequently incubated with the primary antibody at a 1:100 dilution in PBS overnight at 4 C. The next day, tissue sections were washed with PBS for 30 minutes and incubated with a polyclonal goat-anti-rabbit biotinylated IgG (R&D Systems) at a dilution of 3:100 for 40 minutes at room temperature. The tissues were washed for 30 minutes with PBS and then incubated with Vectastain Elite ABC kit (Vector Laboratories) for 1 hour. Lastly, sections were washed in PBS for 30 minutes and the antibody signal was detected using a DAB Peroxidase Substrate Kit (Vector Labs). The sections were then rehydrated and coverslips were placed using Permount mounting media (Fischer Scientific). Negative controls were performed concurrently, without the presence of the primary antibody. 2.5 Documentation and analysis of Cthrc1 expression Images of immunohistochemically stained histological sections were taken using Spot Advanced software and the Spot Diagnostic Camera attached to an Olympus BX51 camera at 10x and 4x magnification. Signal localization was determined by images taken with the digital camera and observations were recorded for each developmental stage. Location as well as spatial distribution of the signal was recorded.

44 36 Chapter 3 Results 3.1 Quantitative expression of Cthrc1 mrna transcripts during midface development Analysis of Cthrc1 expression in the midface tissues of embryos at E8.5, E9.5, E10.5, E11.5 and E13.5 developmental stages by quantitative polymerase chain reaction (qpcr) revealed a specific temporal pattern. There was an increase of Cthrc1 mrna from time points E8.5 to E9.5. At time point E10.5 there was a decrease in mrna levels from the E9.5 time point. At E11.5 there was an increase of the Cthrc1 transcripts back to a level similar to what was seen at E9.5. At the last time point of E13.5 the highest level of Cthrc1 transcript increase was seen, as compared to E8.5 (Figure 8). Figure 8: Temporal expression profile of Cthrc1 mrna across E8.5-E13.5. RNAs were extracted from tissues of embryos from each time point, reverse transcribed to produce the corresponding cdna and amplified by qpcr. Cthrc1 mrna levels were compared to levels at time point E8.5. Overall, the results from the qpcr revealed that expression of Cthrc1 mrna peaked at E9.5 relative to E8.5, followed by a decrease in expression at E10.5 and then a steady increase after

45 37 E11.5. The results confirmed the presence of Cthrc1 mrna in the midface during these time points of embryonic development. 3.2 Expression of CTHRC1 protein The spatial expression of CTHRC1 protein was assayed at six different time points of embryonic development: E8.5, E9.5, E10.5, E11.5, E12.5 and E13.5. Tissue sections of the sagittal, transverse and coronal planes through the developing craniofacial region were used for the expression analysis. A distinct spatial pattern of expression of CTHRC1 was clearly observed at each time point (Figures 10-15) CTHRC1 peptide competition assay at E13.5 to confirm specificity of primary CTHRC1 antibody A peptide competition assay of the CTHRC1 antibody revealed the specificity of the antibody for its antigen. Compared to the distinct expression of CTHRC1 in the nasal septum and nasal capsule (Figure 9C; see below for more details of expression), no staining was observed in the areas of CTHRC1 expression on a consecutive tissue section of the nasal capsule and septum where the antibody was blocked with the CTHRC1 peptide (Figure 9A). The negative control (absence of primary antibody) also showed no staining (Figure 9B), similar to that of the blocked primary antibody. These results indicate that the primary antibody, raised against the CTHRC1 peptide, was specific to the protein with little cross-reactivity or non-specific binding. Figure 9: Peptide competition assay on consecutive coronal sections through the anterior part of the midface in an E13.5 embryo. A) Peptide block at 5X antibody concentration showing no signal. B) Negative control staining with goat anti-rabbit secondary antibody showing minimal

46 38 background signal. C) CTHRC1 primary antibody (1:100) and secondary goat anti-rabbit antibody showing a distinct pattern of CTHRC1 expression in the nasal septum (asterisks) and nasal capsule (arrow) Spatial expression analysis of CTHRC1 in the midface at different developmental stages At the earliest observed time point of E8.5, CTHRC1 expression was limited to the midline of the embryo, most strikingly in the notochord and neural tube (Figure 10). In the midline, expression was observed in the notochord, extending from the rostral end and along the length of the dorsal side of the embryo (asterisks, Figure 10A and C). In addition, CTHRC1 expression was also seen in the neuroectoderm in the area of the ventral brain vesicle and at the rostral end of the embryo (arrows, Figure 10B and D). Expression of the protein was observed in a small area of the oral ectoderm (open arrowhead, Figure 10D). Figure 10: CTHRC1 protein expression in E8.5 of coronal (A, B and D) and sagittal (C) sections. A) Asterisk shows CTHRC1 localized to the notochord (nc) and arrow shows CTHRC1 expression in neuroectoderm (ne). B) Arrow shows CTHRC1 expression in neuroectoderm (ne). C) Sagittal section showing CTHRC1 localized (asterisks) to notochord (nc) along rostral-caudal length of the embryo. D) CTHRC1 expression in the neuroectoderm (arrow) and oral ectoderm (open arrowhead).

47 39 By E9.5 and E10.5, CTHRC1 protein was exclusively expressed in the mesenchyme (Figure 11). Expression was present in the facial prominences, specifically the mesenchyme of the frontonasal and medial nasal processes and the maxillary and mandibular processes (Figure 11 A and B). In the more anterior or ventral region of the medial portion of the midface, e.g., the developing frontonasal region, CTHRC1 was widely expressed throughout the whole area, including the medial-most or central portion of the FNP (Figure 11B and C). Another consistent pattern of CTHRC1 expression in the craniofacial region was the mesenchymal expression of CTHRC1 adjacent to the ectoderm and neuroectoderm. CTHRC1 mesenchymal expression in the frontonasal and medial nasal processes was adjacent to the ectoderm of the respective facial processes (Figure 11A and C). Also, in certain areas, CTHRC1 protein was strongly expressed in mesenchymal tissues immediately adjacent to the neuroectoderm of the telencephalic brain vesicles (arrowhead, Figure 11D). Figure 11: CTHRC1 protein expression in E10.5 embryos, in coronal (A, B and D) and sagittal (C) sections. A) CTHRC1 protein expression in mesenchyme of the frontonasal process (FNP)

48 40 (arrows). B) CTHRC1 expression in the frontonasal process (FNP, arrow), maxillary process (Mx, tailed arrow) and mandibular process (Md, open arrow). C) Expression in FNP adjacent to ectoderm (arrow) and in ventricle of heart (H). D) Distinct expression in mesenchyme next to neuroectoderm (MeN, arrowhead) and mesenchyme in Md process (open arrow). Figures A, B and D are sections from three E10.5 representative embryos, with A and B sections being more ventral and D located more dorsally. By stage E11.5 of embryonic craniofacial development, expression of the CTHRC1 protein was observed in a distinctive pattern in areas of cartilage formation and deposition. As cellular condensations are initiated in different parts of the developing craniofacial region, CTHRC1 expression was clearly expressed in many of these structures. The maxilla at this stage presents with a nasal septal cartilage analgen, a rod like structure that is flanked with two wing-like cartilaginous processes in the developing nasal capsule. In the maxilla and forming snout of the mouse, distinct expression was observed to localize at the forming nasal septum and nasal capsule (Figure 12 A and B). nc ns Figure 12: CTHRC1 protein expression in E11.5 through transverse sections of the maxilla nasal septum and nasal capsule. A) CTHRC1 expressed in areas of the nasal capsule (nc) and nasal septum (ns) (arrows). B) CTHRC1 in nasal capsule (nc) and nasal septum (ns). A, B = 4x and 10X magnifications, respectively.

49 41 CTHRC1 was still strongly expressed in the nasal septum and other areas of cartilage formation of the midface at the later time points of E12.5 and E13.5. With further development of the nasal cartilaginous areas, the staining of CTHRC1 appeared as an almost complete ring-like pattern with a central rod staining, within which contains the bilateral nasal cavities (Figures 13A, 13B and 14). Figure 13: CTHRC1 protein expression at E12.5 in coronal sections of the nasal capsule and a transverse section of the mandible. A) A coronal section of the snout showing CTHRC1 expression in the nasal capsule (nc). B) A coronal section showing CTHRC1 expression in the

50 42 nasal septum (ns). C) A transverse section showing CTHRC1 expression in Meckel s cartilage (mc). At E13.5, the same pattern of expression of the CTHRC1 protein was seen in the maxilla and mandible, but the signal became more distinct and covered a larger surface area as the regions of cartilage formation and deposition increased with further development of the midface. Expression was also seen in other areas of cartilage formation in the mouse embryo head, such as in the developing bones of the calvarium (Figure 14A). c mc nc M x Figure 14: CTHRC1 protein expression at E13.5 in sections of head (A), the maxilla (C and D) and midface including mandible (D). A) CTHRC1 expression in Meckel s cartilage (arrow, mc) and bones of skull (arrow, c). B) More dorsal transverse section of midface and mandible showing CTHRC1 expression in nasal septum (ns) and nasal capsule bones (nc). C) More anterior section of CTHRC1 expression in nasal septum (ns) and nasal capsule bones (nc). D) Arrows showing Cthrc1 expression in nasal capsule (arrow, nc) and bones of maxilla (arrow, Mx).

51 CTHRC1 expression in the mandible and developing tooth bud In the developing mandible of embryos at stages E9.5, E10.5 and E11.5, the pattern of CTHRC1 expression was consistent with the pattern seen at E10.5 in the maxillary process and frontal nasal process. CTHRC1 expression was localized to the mesenchyme and was always adjacent to areas of the ectoderm. For instance, the protein was expressed in embryos at stages E9.5 and 10.5 in areas of outgrowth, with a higher expression in the anterior and ventral portion of the facial processes (Figure 11 A and C) and the oral half of the mandibular process (open arrow, Figure 11B). As cartilaginous structures are laid down in the mandible, the expression of CTHRC1 was clearly evident. One structure is Meckel s cartilage, a bilateral cartilaginous bar across the mandibular process. A strong CTHRC1 signal was localized in the area corresponding to Meckel s cartilage (Figure 13C and 14A). The distinct pattern of CTHRC1 expression in Meckel s cartilage was only observed at later time points, specifically E12.5 (Figure 13C) and E13.5 (Figure 14A). CTHRC1 expression was also observed at time point E12.5 lateral to the Meckel s cartilage in an area that most likely represents the membranous ossification of the body of the mandible (arrow, Figure 13C). CTHRC1 also appeared to be expressed in areas of tooth formation. Initially, its expression was generally throughout the mesenchyme with a stronger signal near the epithelial thickenings on the margins of the stomodeum, which corresponds to the formation of the dental placode (Figure 11B, 11D and 15A). At E11.5, the formation of what is equivalent to the cap stage of tooth development has started in the mandible. CTHRC1 expression at this time was seen in the mesenchyme directly below areas of epithelia corresponding to the enamel organ (Figure 15B and C). This distinct pattern of mesenchymal expression in the area corresponding to the dental papilla continued until the last observed time of E13.5 (Figure 15D).

52 44 Figure 15: CTHRC1 protein expression in tooth bud formation. A) E10.5: Mandible, arrows showing CTHRC1 expression in mesenchyme of the mandibular process (Md) near the ectoderm. B) E11.5: Tongue (t) and Mandibular process (Md), arrow indicating CTHRC1 expression in mesenchyme near tooth buds. C). E11.5: Arrows pointing to CTHRC1 expression in mesenchyme near tooth bud. D) E13.5: Arrows showing expression in mesenchyme adjacent to developing tooth.