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The role of the nasal pit and Fibroblast Growth Factor signaling in avian craniofacial development Szabo Rogers, Heather Lynn 2007

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The role of the nasal pit and Fibroblast Growth Factor signaling in avian craniofacial development by H E A T H E R L Y N N S Z A B O R O G E R S B.Sc. Specialization in Cel l Biology, University of Alberta, 2000 A. THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Dental Science) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A July 2007 © Heather Lynn Szabo Rogers, 2007 Abstract We characterized the role of FGFs (Fibroblast growth factors) during upper lip fusion in the chicken embryo model. We first undertook an expression study of members of the Sprouty family, intracellular inhibitors of FGF signaling, in the upper face to assess where active FGF signaling is occurring. We found that Sprouty 1, 2, and 4 are expressed in nested, overlapping domains within the fusing upper lip in the frontonasal mass and maxillary prominence. Furthermore the areas that expressed Sprouty family members are adjacent to areas that also express Fg/8. Next we tested the requirement for FGF signals in the developing lip by implanting beads soaked in an FGF receptor antagonist, SU5402. We hypothesized that loss of FGF signaling would induce a cleft lip through reduced proliferation and increased cell death. We were surprised to find that mesenchyme in the zone of fusion was FGF-independent. In contrast, clefts were induced when beads were placed distant to the zone of fusion, near the top of the nasal slit. We then showed that cranially implanted SU5402 beads decreased active FGF signaling along the lateral edge of the frontonasal mass. Furthermore we showed that the cranially implanted SU5402 beads resulted in reduced proliferation of the cranial region of the frontonasal mass, and increased cell death along the lateral edge of the frontonasal mass. In contrast, beads in the fusion zone had limited effects on gene expression, cell proliferation and cell death. We therefore identified an FGF-dependent growth centre in the cranial frontonasal mass. We proposed a new theory of facial fusion, whereby the FGF-dependent cranial growth centre displaces the globular process towards the maxillary prominence. This led us to hypothesize that the FGF-rich nasal pit epithelia which is directly adjacent to the frontonasal mass growth center influences morphogenesis of the upper beak. We found that the ectopic nasal pits had the capacity to form ectopic cartilage and bone in competent facial mesenchyme. Although we could not attach specific identities to the induced skeletal elements, these experiments show for the first time that the nasal pit epithelium is capable of providing skeletogenic patterning information. ii Table of Contents: Abstract ii Table of Contents: iii List of Tables vi List of Figures vii List of Abbreviations viii Acknowledgements ix Dedication.'. x Co-Authorship Statement xi 1 Chapter 1: G E N E R A L INTRODUCTION 1 1. l O V E R V I E W O F C R A N I O F A C I A L D E V E L O P M E N T l 1.1.1 Neural crest cell origins of facial skeletogenic mesenchyme 1 1.1.2 Placodal Development 2 1.1.3 Tissue interactions in facial prominence morphogenesis 3 1.1.4 The mechanics of lip fusion 6 1.1.5 Typical cleft lip with or with out cleft palate in humans ... . .7 1.1.6 Chicken Embryos as a model organism for upper lip development 9 1.2 F I B R O B L A S T G R O W T H F A C T O R S I G N A L I N G W I T H I N T H E C O N T E X T O F F A C I A L D E V E L O P M E N T ..9 1.2.1 FGF ligands and receptors 9 1.2.2 Intracellular F G F signaling ; 11 1.2.3 Knockout Mouse models of FGF signaling in the face 1 2 1.3 R A T I O N A L F O R T H E T H E S I S : 13 1.3.1 Hypothesis I, Chapter 2 : FGF signaling is required for lip fusion to take place 13 1.3.2 Hypothesis II, Chapter 3: Tissue interactions between the nasal pit and facial mesenchyme are required for craniofacial skeletal patterning 14 1.4 R E F E R E N C E S : 2 3 iii 2 CHAPTER 2: FGF signaling from the nasal pit regulates a potent growth centre that controls development of the upper lip 31 2.1 I N T R O D U C T I O N 32 2.1.1 Embryological origin of cleft lip with or with out cleft palate: 32 2.1.2 Chicken as a model for C L / P 32 2.1.3 Signaling molecules in the developing upper lip 33 2.1.4 Knockout models for clefting 34 2.2 M E T H O D S : 36 2.2.1 Embryological Methods: 36 2.2.2 Cell proliferation and death analysis: 37 2.2.3 Whole mount in situ hybridization and immunohistochemistry : 38 2.2.4 High Density facial micromass culture: 39 2.3 R E S U L T S : 41 2.3.1 Sprouty 1, 2, and 4 are expressed in partially overlapping domains in the frontonasal mass41 2.3.2 Loss of FGF signaling in the F N M induces a cleft beak phenotype: 42 2.3.3 Skeletal effects underlying the cleft beak phenotype 43 2.3.4 Loss of F G F signaling in the F N M inhibited the differentiation of osteogenic and chondrogenic precursors: 45 2.3.5 SU5402 directly affects downstream FGF signaling within the frontonasal mass 45 2.3.6 FGF signaling controls levels of cell proliferation and cell death in the frontonasal mass ...47 2.3.7 Position specific effects on expression of signals required for facial fusion 49 2.3.8 Dlx5, an olfactory marker, is decreased in cranial treatment 52 2.4 DISCUSSION: 53 2.4.1 F G F signals are required in the frontonasal mass contribution to the upper lip 53 2.4.2 Role of the nasal pit in the context of upper lip development 53 2.4.3 Frontonasal mass and maxillary derivatives are affected in the cleft embryos: 54 2.4.4 Chicken as a model for C L / P : 57 2.4.5 Model for facial fusion: 58 2.5 R E F E R E N C E S : 82 iv 3 Chapter 3: Tissue interactions between the nasal pit and facial mesenchyme suggest new roles for the nasal pit in craniofacial skeletal patterning 89 3.1 I N T R O D U C T I O N : 90 3.1.1 Development of the cranial placodes: 90 3.1.2 Tissue interactions in the developing upper face 92 3.2 M E T H O D S : 94 3.2.1 Embryological Manipulations: 94 3.2.2 Grafting Experiments 95 3.3 R E S U L T S : 98 3.3.1 Exogenous FGF8 provides incomplete patterning information 98 3.3.2 Extirpation of the nasal pit at stage 20 leads to mild patterning changes in the upper face ..99 3.3.3 Nasal pits can organize ectopic structures of ectopic bone and cartilage 101 3.4 DISCUSSION: 106 3.4.1 Autonomous differentiation of the nasal pit epithelium was supported in an ectopic location 106 3.4.2 The nasal pit secretes a combination of permissive and instructive signals 107 3.4.3 Stage 26 host mesenchyme is the most restricted in its response to nasal pit epithelium ...107 3.4.4 Older nasal pit epithelia can induce a larger variety of structures 107 3.4.5 FGFs in the nasal pit are not the only organizing molecule in the nasal pit 108 3.4.6 In situ development: lessons from extirpation 109 3.5 R E F E R E N C E S : 124 4 Chapter 4 - Discussion 129 4.1 T H E C H I C K E N E M B R Y O IS A N A P P R O P R I A T E M O D E L F O R C L E F T LIP 129 4.2 T H E C L E F T R E S U L T S F R O M R E D U C E D FGF S I G N A L I N G IN T H E U P P E R F A C E 129 4.3 T H E U P P E R F A C E IS S C U L P T E D B Y T H E C O O R D I N A T E D A C T I O N S O F S P E C I F I C R E G I O N S O F E P I T H E L I A 131 4.4 M O R P H O G E N E S I S IN T H E F A C E IS D R I V E N B Y D I S T I N C T P R O L I F E R A T I O N P A T T E R N S 132 4.5 T F I R E E FGF I N D E P E N D E N T R E G I O N S IN T H E F A C E 134 4.6 F U T U R E DIRECTIONS 136 4.7 R E F E R E N C E S : 139 v List of Tables Table 2-1: Dose response to SU5402 implanted into the lateral frontonasal mass 78 Table 2-2: Cranial bead position induces a cleft beak 79 Table 2-3: Quantification of increased apoptosis in the frontonasal mass 80 Table 2-4: Gene expression decreases along the cranial-caudal axis in response to the cranial bead position 81 Table 3-1: Skeletal structures induced by supernumerary grafted nasal pits into maxillary mesenchyme 111 List of Figures Figure 1.1: Scanning electron micrographs 15 Figure 1.2: Fate map of the chicken embryo face 16 Figure 1.3: Schematic of lip fusion and typical cleft lip 18 Figure 1.4: Schematic of FGF signaling 19 Figure 1.5: Expression of FGF ligands and receptors 21 Figure 2.1 Sprouty 1, 2 and 4 are expressed in overlapping domains in the mesenchyme 59 Figure 2.2 Loss of FGF signaling in the frontonasal mass induced a cleft beak 62 Figure 2.3: Reduced staining in micromass cultures treated with SU5402 64 Figure 2.4 The expression of FGF signaling inhibitors is reduced in the frontonasal mass 65 Figure 2.5: SU5402 reduced the levels of phosphorylated M A P K 67 Figure 2.6: Cell Proliferation is reduced in the cranially treated frontonasal mass 68 Figure 2.7: Cell death is increased in the cranially treated frontonasal mass 70 Figure 2.8: FgfS and Shh expression are not affected in cranially treated embryos, while Bmp4 is downregulated for a short time 72 Figure 2.9: The expression of a non-canonical Wnt, Wnt5a, is decreased in response to SU5402 treatment 73 Figure 2.10: Msxl and Msx2 expression fails to be maintained in response to SU5402 treatment. 74 Figure 2.11: Dlx5 is decreased in the olfactory epithelial domain 76 Figure 2.12: Model for FGF signaling in the fusing facial prominences: 77 Figure3.1: Methods of grafting 112 Figure 3.2: FGF8b induced ectopic bone and cartilage 114 Figure 3.3: Nasal pit extirpation results in morphological changes in the nasal capsule 115 Figure 3.4: Ectopic pieces of bone and cartilage are in induced by nasal pits grafted to Stage 20 hosts 117 Figure 3.5: Organized outgrowth of bone and cartilage is induced by nasal pits into Stage 15 hosts 118 Figure 3.6: The grafts were made of epithelium 120 Figure 3.7: The nasal pit grafts undergo normal morphogenesis and differentiate into nasal structures 122 Figure 4.1: FGF dependent and independent regions control outgrowth and patterning of the face 137 v i i List of Abbreviations B M P - bone morphogenetic protein CL/P - Cleft lip with or with out cleft palate CPO - cleft palate only FGF - fibroblast growth factor fnm frontonasal mass gP globular process; lnp lateral nasal prominence mdp mandibular prominence mnp medial nasal prominences mxp maxillary prominence np nasal pit v i i i Acknowledgements I wish to acknowledge the support by the Joseph Tonzetich Fellowship. With this generous support I was able to provide some answers to the question that is inevitably posed by every child with a cleft: "Why?". First and foremost, I wish to thank my supervisor, Dr. Joy Richman, for challenging and providing me with a facility to grow and develop into a scientist. I wish to thank my supervisory committee: Dr. Virginia Diewert, Dr. Jane Roskams, and Dr. Cal Roskelley. Each of you shaped my research. I wish to thank Kathy Fu, Marcela Buchtova, and Julia Boughner for the never-ending enthusiasm, welcoming smiles, and helpful advice. ix Dedication To Matt, my rock. x Co-Authorship Statement Chapter 2 was submitted in a shorter revised format to Development. Dr. Joy Richman and I contributed to the concept of the manuscript. I performed all of the experiments except for the Pystl wholemount in situ hybridizations. The analysis was performed mainly by myself in consultation with Dr. Joy Richman. The manuscript was written by myself, and revised by Dr. Joy Richman. Chapter 3 is in preparation for publication. The concepts evolved from the work presented in chapter 2. Dr. Joy Richman and I collaborated in the design of grafting experiments, which we performed together. The analysis was performed in consultation with Dr. Joy Richman. The manuscript in preparation was written by myself and revised by Dr. Joy Richman. xi Chapter 1: GENERAL INTRODUCTION 1.1 Overview of Craniofacial development 1.1.1 Neural crest cell origins of facial skeletogenic mesenchyme Craniofacial development begins very early in development, when a population of ectodermal cells the cranial neural crest, undergo an epithelial to mesenchymal transformation, to become ectomesenchymal cells (Le Lievre and Le Douarin, 1975). The ectomesechymal cells migrate from the dorsal edge of the neural tube into the presumptive facial region and fill epithelially encapsulated facial prominences surrounding the primitive oral opening (Le Lievre, 1978; Le Lievre and Le Douarin, 1975). Following interactions with the surface epithelium of the facial prominences (Hall, 1981), the ectomesenchymal cells differentiate into the bones and cartilages of head and neck (Le Lievre and Le Douarin, 1975). Sequential signals are required to instruct the facial prominences to form specific skeletal elements beginning with the formation and migration of the cranial neural crest. It is apparent from early stages that trunk and cranial neural crest cells are not equivalent. Hox-expressing neural crest cells, the neural crest contribution to the components of the second branchial arch and more caudal structures do not contribute to the bones and cartilages of the head (Couly et al., 2002; Couly et al., 1998). When transplanted more cranially, hindbrain neural crest cells migrated into the face but these cells did not contribute to the facial skeleton. Instead, they formed cellular aggregates surrounding the nasal pits (Couly et al., 2002). Complete extirpation of the neural folds followed by replacement with the diencephalic crest, showed that approximately one-third of the diencephalon substituted for the whole diencephalic crest population and allowed normal facial development (Couly et al., 2002). The authors conclude that the diencephalic neural crest population is not prepatterned before neural crest migration, and must interact with another tissue for skeletal patterning (Couly et al., 2002). Extirpation of the foregut endoderm prevented facial development, while the implantation of an supernumerary foregut endoderm induced duplications of the facial skeleton (Couly et al., 2002). The direction of the duplicated cartilages depend on the orientation of the foregut endoderm (Couly et al., 2002). The excised region of foregut endoderm expressed sonic hedge hog (Shh), and mandibular development can also be rescued by the application of Shh soaked beads after extirpation (Brito et al., 2006). Shh-soaked beads reduced the amount of cell death observed as the result of foregut endoderm removal, and rescues the expression of Shh in the mandibular 1 prominence (Brito et al., 2006). Thus the foregut endoderm provided instructive signals to the migratory neural crest cells. The neural crest cell-derived mesenchyme determines jaw pattern. The clearest evidence comes from interspecific exchanges of neural folds between quail and duck embryos. The donor neural crest cells give rise to the ectomesenchyme and the beak shape is consistent with the donor rather than the host. Therefore, quail neural folds grafted into duck hosts lead to the development of a duck with a quail face and vice versa (Schneider and Helms, 2003). In a similar experiment, a more detailed analysis of the retroarticular process, a component of the mandible which differs in shape and size between the quail and duck, showed that the pattern of the R A P was consistent with the origin of the donor neural crest (Tucker and Lumsden, 2004). Donor cells were reacting to patterning signals provided by host endoderm and first arch ectoderm by attaining mandibular identity, while final shape and size of the retroarticular process was determined by the donor, and presumed to be prepatterned before the graft was harvested (Tucker and Lumsden, 2004). 1.1.2 Placodal Development A second vital contributor to facial development are the ectodermal placodes. At the point of neural crest induction, the facial ectodermal placodes (olfactory, otic and lens placode) are being induced in the surface ectoderm apposed to the anterior neural ridge, in a region called the pre-placodal region. The facial placodes gives rise to distinct cell types, ranging from neurons, to non-neurogenic lens cells. The placodal precursors share expression of Eya, Dach and Six genes which wil l delineate where the first thickenings placode wil l form (Bailey et al., 2006; Schlosser, 2005). The current hypothesis suggests that the pre-placodal region is specified in a default state of becoming a lens placode, which is then induced to form other placodes through environmental interactions and differential expression of target genes (Bailey et al., 2006). The olfactory placode is induced in part by the presence of FGF8 from the anterior neural ridge at early stages and then at later stages, the presence of frontonasal mass neural crest cells which elucidates the olfactory program from the default lens state (Bailey et al., 2006). Within the developing face, the olfactory placode is positioned cranial to the stomodeal ectoderm, and makes up the lateral edge borders of the frontonasal mass. Very quickly after it can be recognized externally in the developing chicken embryo, the placode inVaginates into the craniofacial mesenchyme, and within 24 hours, the olfactory neurons and gonadotrophin releasing neurons have migrated towards the telencephalon (Drapkin and Silverman, 1999). 2 Once the placode has been established, the developing olfactory epithelium undergoes development in a four step process: stem cell population, neuronal precursors, immediate neuronal precursors and finally differentiated neuronal cell types (Kawauchi et al., 2005). Each step is defined by the expression of a specific characteristic gene: Sox2 for stem cells; Mashl for neuronal precursors; neurogeninl for immediate neuronal precursors and then olfactory receptor neurons for differentiated neurons (Kawauchi et al., 2005). The olfactory epithelium is unique in that it is one of the only tissues in the body that is able to replenish itself throughout the lifetime of the animal. The function of Fg/S in the developing olfactory epithelia was dissected using a Foxgl-Cre excision of Fgf8 in the facial and neurectoderm (Kawauchi et al., 2005). These embryos develop hypoplastic olfactory epithelia, and have reduced FGF signaling in the medial nasal prominences and reduced numbers of olfactory epithelial stem cell precursors (Kawauchi et al., 2005). The conditional null embryos appear to have shortened snouts but the underlying skeletal abnormalities were not analysed (Kawauchi et al., 2005). The link between the olfactory placode and skeletal development of the face were studied many years ago in the amphibian. Work in Amblystoma, showed that the extirpation of the nasal pit increased the size of the premaxillary and maxillary bones (Burr, 1916), while the grafting of nasal pits to the flank of Triton induced ectopic limbs (Balinsky, 1933). Based on these results, it would be interesting to see whether the nasal pit provides instructive signals to the face in amniotes. 1.1.3 Tissue interactions in facial prominence morphogenesis After the neural crest has migrated into the face, and the cranial placodes are visible, then further development of the pharyngula embryo occurs through the zootypic and phylotypic stages. The pharyngula embryo is one that has a recognizable vertebrate bauplan. This stage of development begins with three facial prominences, the mandible, lateral nasal prominence and the frontonasal mass. The maxillary prominence appears shortly after this as cells migrate from the proximal first pharyngeal arch (mandibular arch) (Lee et al., 2004). As the maxillary region is being set up, the interactions of several signaling molecules are patterning the facial mesenchyme, and few have been described. The maxillary region can be transformed into a frontonasal mass by the application of beads soaked in retinoic acid and Noggin (Lee et al., 2001). The mandible has nested expression of all members of the Dlx family, Dlxl-6, with Dlx5/6 being expressed in the most restricted distal domain of the first branchial arch. Knockouts of Dlx5/6 have a homeotic transformation of the first branchial arch into the 3 maxillary prominence, including the development of vibrissae and rugae on the lower jaw and loss of the nasal capsule (Depew et al., 2002). In pharyngula staged avian embryos, the oral opening is surrounded cranially by the frontonasal mass, laterally by maxillary prominence, and caudally by the mandibular prominence. The lateral frontonasal mass is surrounded by the nasal pits, which also form the medial edges of the lateral nasal prominence (Fig. 1.1). The neural crest-derived ectomesenchymal cells populating each prominence migrate from distinct areas of the closing neural tube. The frontonasal mass is filled with neural crest cells derived from diencephalon and cranial mesenchephalon (Couly et al., 1996; Kontges and Lumsden, 1996; Lumsden et al., 1991). The maxillary prominence mesenchymal cells are derived from the midbrain (Lumsden et al., 1991). The mandibular prominence ectomesenchymal cells originated from the rostral midbrain and rhombomere neural crest (Couly et al., 1996; Kontges and Lumsden, 1996). For the most part the early shape of the pharyngula-stage embryo face is conserved in amniotes (Fig. 1.1). The one difference between the facial form of early chicken embryos and mouse and human embryos is nomenclature of facial prominences. The square part of the upper face between the olfactory slits in chicken embryos is named as one prominence: the frontonasal mass, which has mesenchymal thickenings at the lateral-caudal edge of the frontonasal mass, named the globular processes. In mouse and human embryos, the region between the olfactory slits is divided into the medial nasal prominences and medial furrow. The lateral edge of the chicken frontonasal mass is functionally equivalent to the medial nasal prominences of the mouse and human embryo. Once the ectomesenchymal cells reach the facial prominences, then another series of epithelial to mesenchymal interactions occur. The frontonasal mass or mandibular prominence requires ectoderm to grow out into the prenasal or Meckel's cartilage at stage 20 and 24 (Richman and Tickle, 1989; Wedden, 1987). Facial mesenchyme requires epithelial signals for osteogenesis to occur either when grafted to the limb or chorioallantoic membrane (Hall, 1980). The form of the facial bones is resident in the mesenchyme of facial prominences, so the grafts differentiate into bones and cartilage from the region of the face that the mesenchyme is derived from (Richman and Tickle, 1989). At early pharyngula stages, facial ectoderm is required for outgrowth of the facial mesenchyme, but the specific regions and molecular mechanisms underlying this growth is just starting to be explored. At stage 20, the dorsal and ventral edge of the medial frontonasal mass has already been demarcated, as shown by the injection of an avian retrovirus encoding alkaline 4 phosphatase in distinct dorsal/ventral regions (Hu et al., 2003). At stage 20, the expression of FgfS, extends to the edge of the region that is marked by the dorsal frontonasal mass, while transcripts of Shh are found in the region marked by the ventral injection. These regions were named the frontonasal mass ectodermal zone (Hu et al., 2003). The frontonasal ectodermal zone provides permissive signals to duplicate the region of the face that is is transplanted to (Hu et al., 2003). It duplicated the frontonasal mass and mandible of a stage 25 host embryo (Hu et al., 2003). The same region of stage 25 ectoderm does not have the ability to duplicate structures. Thus frontonasal mass mesenchyme is competent to respond to signals in the epithelium until at least stage 25, while the instructive ability of the epithelium is lost by stage 25 (Hu et al., 2003). These experiments suggest that the epithelium becomes restricted in its ability to induce ectopic structures at younger stages compared to the competence of responding mesenchyme to form ectopic structures. The relative competence of facial mesenchyme to respond to exogenous epithelial signals has not been directly tested. Similarly to the endoderm grafts (Couly et al., 2002), the direction of outgrowth of the frontonasal mass is determined by the dorsal-ventral polarity of the frontonasal ectodermal zone graft (Hu et al., 2003). The facial mesenchyme does not require the epithelium to independently grow out in grafts by stage 28 in chicken embryos (MacDonald et al., 2004), and E l 3 in mouse embryos (Hall, 1980). Chondrogenesis has initiated at approximately day E6, or stage 29, followed by intramembranous ossification on E8 or stage 34, and finally all of the bones have begun to ossify by the endpoint of our experiments on E l 3, stage 38 (Hamburger and Hamilton, 1951). The maxillary prominence gives rise to the maxillary bone, palatine bone and the jugal bone (Lee et al., 2004) (Fig. 1.2A,B); the lateral nasal prominence gives rise to the nasal chonchae (MacDonald et al., 2004). The osteogenic derivatives of the lateral nasal prominence have not been determined but likely include the nasal, lacrimal and prefrontal bones (Fig. 1.2A,B). The frontonasal mass gives rise to the prenasal cartilage and the premaxillary bone, although, the experiments correlating the frontonasal mass derivatives did not stain for bone morphology (Matovinovic and Richman, 1997; Richman and Tickle, 1989; Wedden, 1987). The mandibular prominence gives rise to Meckel's cartilage (Richman and Tickle, 1989), and the bones of the lower jaw (Fig. 1.2A,B). 5 1.1.4 The mechanics oflip fusion The upper lip forms in chicken embryos at stage 28 when three prominences: the cranial maxillary prominence, globular process of the frontonasal mass and the lateral nasal prominence fuse together to form a continuous smooth edged upper beak. In chicken embryos the equivalent to the upper lip is the tomium or lateral edge of the beak (Fig. 1.3A-D). Upper lip development occurs in four steps: outgrowth, contact, fusion and mesenchymal confluence. Outgrowth of the prominences occurs by mesenchymal proliferation. The medial region of the frontonasal mass is an area of low proliferation at the time of lip fusion where as at the lateral edges of the frontonasal mass, has higher proliferation (Peterka and Jelinek, 1983; MacDonald et al., 2004; McGonnell et al., 1998; Wu et al., 2004). Once the prominences have reached appropriate size, epithelial contact occurs between the frontonasal mass and maxillary prominence creating a bilayered epithelial seam. The fate of the epithelial seam within the zone of fusion disappears due to epithelial to mesenchymal transformation (Sun et al., 2000) or apoptosis (Ashique et al., 2002). The evidence of epithelial to mesenchymal transformation comes from studies that specifically labeled the epithelium of the medial nasal and maxillary prominences, and followed fusion in tissue culture. Following lip fusion, labeled cells were observed in the mesenchymal bridge of the fused upper lip, which suggested that some cells within the bilayered epithelial seam underwent epithelial to mesenchymal transformation (Sun et al., 2000). This paper also suggested that apoptosis is required to slough off the periderm, the most superficial layer of the ectoderm in the seam (Sun et al., 2000). The application of Noggin soaked beads in the lateral frontonasal mass decreased the level of proliferation within the mesenchyme, without affecting mesenchymal cell death (Ashique et al., 2002). Near the beads, the epithelium is thickened and has increased cell survival, suggesting that normal fusion of the upper lip requires apoptosis of the epithelial seam (Ashique et al., 2002). The next step of primary palate development involves the movement of mesenchymal cells through the area of the seam to form a strong mesenchymal bridge across the zone of fusion (Wang et al., 1995). Finally the grooves between the facial prominences are filled out through cell proliferation of the mesenchyme (merging). Thus cell proliferation, apoptosis, epithelial-mesenchymal transformation and cell movement all play a part in the mechanics ofl ip fusion. 6 1.1.5 Typical cleft lip with or with out cleft palate in humans Failure of any of the steps of upper lip development (outgrowth, contact, fusion and mesenchymal confluence) results in a clinically significant event: cleft lip with or without cleft palate (CL/P) (Fig. 1.3E-G). This is the most common congenital anomaly present in the human population. Depending upon ethnicity, there are between 1 and 3 humans are affected with cleft lip with or with out cleft palate in every 1000 live births (Cox, 2004; Jiang et al., 2006). Non-syndromic cleft lip with or with out cleft palate is also called typical cleft lip and is a multigenic, multifactorial condition. Cleft lip is most often unilateral and can range in severity from a notch in the upper lip to severe with opening through the upper lip into the nostril (Fig. 1.3F). Cleft lip in humans occurs between 6 to 10 weeks of gestation. Clefts of the secondary palate (CP) occur in the midline separating the palatal shelves, and can occur as a secondary consequence of cleft lip. Isolated CP is a temporally distinct malformation and occurs between weeks 10 and 12 of gestation (Fig. 1.3G). The secondary palate develops from palatal shelves that are outgrowths of the maxillary prominences, they elevate, grow towards the midline and fuse. Development of the secondary palate and upper lip have similarities in the growth of the fusing edges together, contact of the bilayered epithelial seam, and seam degradation. C L / P is also increased as a result of environmental factors, including the presence of a polymorphism in a cytochrome P450 gene: CYP1 A l , and maternal cigarette smoking which greatly increases the rate of cleft lip (Shi et al., 2007) and maternal use of anti-convulsants (Holmes et al., 2001). Although cleft lip can be associated with syndromes, typical cleft lip is not a single gene disorder. Human genetic studies compared the sequences of several genes in individuals with orofacial clefting amd a family history of clefting to a non-cleft population (Vieira et al., 2005). This analysis identified several polymorphisms in transcription factors {MSX1, MSX2, and TBX10) and one intracellular modulator of the FGF (Fibroblast growth factor) signaling pathway (SPRY2) (Vieira et al., 2005). Msxl is expressed near the fusing upper lip in chickens (Ashique et al., 2002) and is also associated with human syndromic forms of orofacial clefting and tooth agenesis (van den Boogaard et al., 2000). Furthermore, an analysis of these cleft populations focused solely on FGF ligands and FGF receptors and identified missense mutations in the FGFs and FGFRs that are expressed in the facial prominences (Riley et al., 2007). These studies provide a foundation to initiate investigations to test i f alterations in the FGF signaling or pathways that regulate Msxl/2 expression are causative in C L / P , however determining which missense mutation is causative is complicated by crosstalk between many pathways. 7 There are mouse strains that mimic human non-syndromic clefting. Work from the Juriloff lab has shown that the A strains of mice are more susceptible to clefts than other strains. Genetic studies in the A/WySn strain of mouse suggested that there are at least two loci that determine the rate of nonsydromic clefting in this strain (Juriloff et al., 2001b; Juriloff and Mah, 1995). The clfl loci is found on mouse chromosome 11, and encompasses a region that encodes members of the Wnt family and the Dlx family (Juriloff and Harris, 1999; Juriloff and Mah, 1995). The clfl gene is localized on mouse chromosome 13 and is epistatic to the clfl loci (Juriloff et al., 2001a). The clfl loci includes the canonical Wnt (Wingless-type M M T V integration site family) gene, Wnt9b. Long range PCR of the Wnt9b locus revealed the presence of an IAP (intra-cisternal A particle) 6.6 kb from the Wnt9b 3' U T R (Juriloff et al., 2005). The transposon is present in mouse strains that are known to have the clfl loci of A/WySn, and together with the high degree of similarity between the long-terminal repeats, suggested that this transposon is a relatively new insertion (Juriloff et al., 2005). IAP transposons are regulated by methylation and can modify the gene expression over a long range (Whitelaw and Martin, 2001). To determine whether the IAP transposon 3' of the Wnt9b gene was causing a loss of function, a breeding experiment was carried out with the Wnt9b conditional nulls. The phenotype of the Wnt9b null embryos included agenesis of the kidneys and incompletely penetrant cleft lip with or without cleft palate (Carroll et al., 2005). To create a Wnt9b7c//7/c//2 genotype, Wnt9b+ /" heterozygotes were crossed to A/WySn female (Juriloff et al., 2006). The Wnt9b7c//7 compound mutants had an increased rate of cleft lip compared to the normal rate expected for the A/WySn strain. Furthermore, analysis of these embryos showed that 93% of the cleft lip embryos were mutant in Wnt9b" and had the mutant, A/WySn derived clfl and clfl loci (Juriloff et al., 2006). The Wnt9b" with the A/WySn clfl and clfl loci was also correlated with increased prevalence of the more severe bilateral cleft lip phenotype (Juriloff et al., 2006). This genetic analysis confirmed that loss of Wnt9b can not complement the clefting liability in the clfl loci, leading to the conclusion that decreased levels of Wnt9b are causative for non-syndromic C L / P in A/WySn mice (Juriloff et al., 2006). Thus far no data linking Wnt9b to human clefting has been reported. The Wnt9b story is impressive but also serves to reinforce the idea that understanding the molecular causes of cleft lip with or with out cleft palate is bound to be cumbersome because of multiple interacting genetic factors. We can use the chicken embryo model system to specifically alter levels of multiple signaling molecules to characterize their role in upper lip development. 8 1.1.6 Chicken Embryos as a model organism for upper lip development. My research question is centered on the molecular signals that take place during fusion of the upper lip, and 1 am using the chicken as a model for cleft lip for several reasons. First, the chicken embryo is accessible during lip fusion. Second, longitudinal studies on effects of manipulations to the face can be studied in the whole embryo and within the context of the face itself. Third, we have means of increasing or blocking signaling directly in the chicken fusion zone by implanting beads soaked in agonists or antagonists of the pathways of interest (Ashique et al., 2002). Fourth, we can directly assess epithelial-mesenchymal interactions in the context of facial development by extirpating facial tissues (Wang et al., 2001) or recombining and grafting fragments of the face to host embryos (MacDonald et al., 2004). Fifth, what we learn from chickens wil l be applicable to mammals because the steps of fusion and signaling pathways are conserved. However, chicken embryos are not an appropriate model system for secondary palate development, since they have a naturally cleft secondary palate. 1.2 Fibroblast Growth Factor signaling within the context of facial development 1.2.1 F G F ligands and receptors Fibroblast growth factor (FGF) signaling modulates many developmental processes including development of the upper and lower jaws (Macatee et al., 2003; Trumpp et al., 1999), the olfactory placode (Bailey et al., 2006), the olfactory epithelium (Kawauchi et al., 2005), and the limb (Tickle, 2003). FGF signaling occurs through the binding of an FGF ligand to an FGF receptor, and activation of an intracellular phosphorylation cascade (Fig. 1.4A). FGF ligands share a common 120 amino acid core which interacts with the FGFR, most have an amino terminal signal peptide for secretion and all share a high affinity for heparin sulfate proteoglycans (reviewed in (Bottcher and Niehrs, 2005; Ornitz and Itoh, 2001)). The human FGF family comprises 22 ligands divided into seven subfamilies on the basis of homology (Itoh and Ornitz, 2004). There are at least 20 FGF ligands encoded in the chicken genome (our own in silico analysis of the chicken genome), although the expression of only FgfS, 10 and 75 and 19 have been reported for chicken embryos (Fig. l ' .5A,B). In the upper face, a continuous band of Fgf8 expressing epithelia is found between the nasal pits, the commissural plate and the optic vesicle in E9.5 and El0.5 mouse (Bachler and Neubuser, 2001; Crossley and Martin, 1995; Karabagli et al., 2002) and stage 15 -21 chicken (Hu et al., 2003; Song et al., 9 2004) embryos. At stage 20, Fgfl8, a member of the Fgf8 synexpression group is expressed in the nasal pit of chicken embryos (Ohuchi et al., 2000), while the third member of the group, Fgfl7 is expressed in the epithelia lining the medial wall of the nasal pit in El0.5 mouse embryos (Bachler and Neubuser, 2001). The expression of Fg/S, Fgfl7 and Fgfl8 overlaps in the area surrounding the nasal pits at later stages (Fig. 1.5A,B) (Bachler and Neubuser, 2001; Kawauchi et al., 2005; Ohuchi et al., 2000). Another ligand FgflO, is expressed in the epithelia near the nasal pits (Bachler and Neubuser, 2001; Havens et al., 2006), and the mesenchyme of the maxillary prominence (Havens et al., 2006). The FGFRs are single spanning transmembrane proteins that have an extracellular domain, a juxtamembrane domain and an intracellular tyrosine kinase domain. The extracellular domain binds to the FGF ligands and contains three immunoglobin-like repeats. Each F G F R is alternatively spliced to give at least seven different isoforms that differ in the number of Ig-like repeats or others that lack a transmembrane domain. The biological significance of the different isoforms is unknown (Bottcher and Niehrs, 2005). FGFR], 2, 3 are expressed in most of the tissues of the face, with FGFR1 being expressed ubiquitously (Fig. 1.5A,B). Ligand specificity is determined by the membrane adjacent, third Ig-like repeat. The 111b or IIIc Ig-like repeat is encoded by one common exon and then either the 'b ' exon or ' c ' exon which is post-transcriptionally controlled by differential splicing to encode the Ig loop (Johnson et al., 1991). The Illb splice or IIIc splice form can be activated by either acidic or basic FGF in Xenopus oocytes (Johnson et al., 1990). The 111c isoform is preferentially expressed in mesenchyme whereas the Illb isoform is found in epithelium (Orrurtreger et al., 1993). Ligand specificity is determined by the availability of heparin sulfate proteoglycans and the differential splicing of the receptor. The ligand binding specificities of the receptors were characterized in an in vitro BaF3 cell line expressing each receptor splice variant followed by the application of purified ligands followed by quantifying the mitogenic response (Ornitz et al., 1996; Zhang et al., 2006). Through this method, Fgf8, the FGF ligand with the clearest role in facial development, binds most specifically to the FGFR3 IIIc splice variant of the receptor. Extracellular FGF ligand binding requires the presence of a heparin sulfate proteoglycan (Fig. 1.4A). The monomeric ternary crystal structure of F G F - F G F R binding has been solved (Schlessinger et al., 2000). For signaling to occur, a ligand bound F G F R must recruit a second ligand bound receptor to initiate downstream signaling (Spivak-Kroizman et al., 1994). Ligand binding induces a conformational change in the juxtamembrane position that activates the intrinsic tyrosine kinase domain. The FGFR tyrosine kinase domain can phosphorylate members 10 of the Ras /MAPK, phosphatidyl inositol 3-kinase, or phospholipase C gamma kinase signaling cascades (Bottcher and Niehrs, 2005) (Fig. 1.4A). The medial nasal prominences and the pharyngeal arches have high levels of phosporylated (activated) E R K (Corson et al., 2003). The application of an FGFR-antagonist, SU5402, to whole embryo culture reduced the abundance of phospho-ERK in the medial nasal promiences (Corson et al., 2003). Thus, one of the pathways that FGFR-derived signals can mediate in the medial nasal prominences is the Ras /MAPK pathway. Sprouty proteins are intracellular inhibitors of FGF signaling (Hacohen et al., 1998). Comparing the regions of phosphorylated E R K (Corson et al., 2003) to the areas of Sprouty 1, 2 transcripts (Minowada et al., 1999) in the medial nasal prominences suggests that FGF signaling in the medial nasal prominences works through the activation of M A P K and is decreased by Sprouty 1, 2. In addition, these experiments correlate the presence of Sprouty transcripts with an active FGF signaling state in the medial nasal prominences of mouse embryos. 1.2.2 Intracellular F G F signaling Sprouty was first identified in Drosophila, by loss of function mutations that increased the amount of branching in the trachea which resulted from overactive FGF signaling (Hacohen et al., 1998). Tracheal branching requires breathless (a Drosophila FGF ligand) and loss of function of Sprouty increased the expression of downstream FGF effectors (Hacohen et al., 1998). Three human Sprouty {hSpryl, 2, 4 (Hacohen et al., 1998; Leeksma et al., 2002)), four mouse (mSpryl-4; (Minowada et al., 1999)), and four chicken {cSpryl-4 (Minowada et al., 1999) have been identified. Sprouty proteins share homology in the cysteine-rich (22 in Drosophila; 19 in human) carboxy terminus domain-(Hacohen et al., 1998). A conserved tyrosine, tyrosine 53 in XSprouty2, is phosphorylated in response to FGF stimulation, but not EGF stimulation (Hanafusa et al., 2002). Phosphorylated Sprouty2 is recognized by the SH2 (Src Homology domain 2) of Grb2 (Hanafusa et al., 2002) (Fig. 1.4C). A phosphorylated octapeptide including Tyr53 prevents the recruitment of Grb2 to SOS, and downstream phosphorylation cascades (Hanafusa et al., 2002). Binding of Grb2 to Sprouty2 prevents Grb2 from binding the SOS (son of sevenless) Raf GEF (guanine exchange factor) protein, and therefore Ras /MAPK activation (Hanafusa et al., 2002). Without Sprouty the Grb2/SOS interaction activates Ras/Raf leading to the activation of M A P K (mitogen activated protein kinase). Activation of M A P K facilitates transcription of target genes and response to the FGF signal. The activation of M A P K can be decreased by Pystl/Mkp3/Dusp6, a dual specificity phosphatase that binds to the noncatalytic domain of E R K 2 and provides post-transcriptional control of E R K 2 by dephosphorylating the 11 activated threonine and tyrosine residues (Camps et al., 1998). Pystl is expressed in the medial nasal prominences at the time of facial fusion (Dickinson et al., 2002). Direct transcriptional targets of FGF signaling are Etsl and Pea3 (Firnberg and Neubuser, 2002) and Spry2 (Liu et al., 2003) (Fig. 1.4C). At the time of facial fusion, Etsl and Pea3 are heavily expressed in the lateral nasal prominence and not the frontonasal mass (Firnberg and Neubuser, 2002). FGF8 can also induce Sprouty2 in chicken embryo lateral plate mesoderm, and overexpression of Sprouty2 prevents the differentiation of chondrogenic precursors in the limb, resulting in failure of endochondral bone formation and a chondrodysplasia phenotype (Minowada et al., 1999). Alternatively, overexpression using electroporation with a mammalian expression vector encoding Pystl, results in limb truncation and the exacerbation of the normal areas of cell death (Eblaghie et al., 2003), in response FGF signaling not PI3K activation (Smith et al., 2006). Within two hours of bead FGF4 or FGF8 bead implantation, Pystl expression is induced in the neurectoderm and limb bud, the ectopic activation of Pystl can be prevented with co-implantation of beads soaked in SU5402 or a M A P K inhibitor (Eblaghie et al., 2003). Thus Spry2, and Pystl are indicators of active FGF signaling. 1.2.3 Knockout Mouse models of FGF signaling in the face Full knockouts of Fgf8 are embryonic lethal at gastrulation (Meyers et al., 1998). A series of hypomorphic (Abu-Issa et al., 2002; Frank et al., 2002) and conditional knockouts have been constructed using AP2-Cre (Macatee et al., 2003), FoxGl-Cre (Kawauchi et al., 2005) and Nestin-Cre (Trumpp et al., 1999). Loss of FGF8 in the facial ectoderm results in truncations of the upper and lower jaw as well brain dysmorphologies. FGF ligands require heparan sulfate proteoglycans to bind their cognate receptors (Schlessinger et al., 2000) and modulating the amount of heparan sulfate wil l also affect growth factor signaling including FGF signaling. Knockout mice lacking an enzyme that transfers heparan sulfate to proteoglycans, Ndstl (GlcNAc N-deacetylase/N-sulfotransferase 1) develop severely reduced maxillary, mandibular and frontonasal processes and as a consequence are missing most of the upper and lower jaw bones (Grobe et al., 2005). At later stages of development these embryos also have hypoplastic nasal epithelium (Pallerla et al., 2007). The facial phenotype of members of the FGF signaling pathways suggest that FGF signaling is required for facial development. 12 1.3 Rational for the Thesis: The work presented in thesis will focus on the role of FGF signaling in the developing upper lip, and then characterize the tissue interactions between the nasal pit epithelia and the facial mesenchyme. 1.3.1 Hypothesis I, Chapter 2: F G F signaling is required for lip fusion to take place. Thus far, work in upper lip development has focused on naturally occurring mutations in mouse (Juriloff et al., 2006) or knockout mouse models with a cleft lip (Liu et al., 2005), or interactions of the signaling molecules in facial development, (Ashique et al., 2002; Hu and Helms, 1999; Song et al., 2004). Increased Noggin in the upper face resulting in increased Fgf8 expression (Ashique et al., 2002), while the Cpp chicken mutant line has increased Fg/8 expression which prevents the outgrowth of the frontonasal mass (Ashique et al., 2002; MacDonald et al., 2004), however no one has directly tested the role of FGF signaling in upper lip development. Human molecular genetic studies suggest that mutations in Sprouty2, several FGF ligands and receptors can be attributed to the pathogenesis of C L / P (Riley et al., 2007; Vieira et al., 2005). We endeavor to characterize the role of FGF signaling in the developing of the upper lip. Aim I: To map expression of FGFs and other markers of active F G F signaling in the developing face. Aim II: To antagonize F G F signaling in different positions in the embryonic face in order to determine where F G F activity was most essential. Aim III: To determine the consequences of blocking F G F signaling on skeletal morphogenesis in the whole skull and at the cellular level. Aim IV: To determine whether defects were caused by changes in cell proliferation or apoptosis. Aim V: To determine if F G F signaling had been decreased in the frontonasal mass using the expression of markers of F G F activity. Aim VI: To determine if other signaling pathways and transcription factors were dependent on endogenous F G F signals. 13 1.3.2 Hypothesis II, Chapter 3: Tissue interactions between the nasal pit and facial mesenchyme are required for craniofacial skeletal patterning. We hypothesized from the work presented in chapter 2 that the nasal pit epithelia contributes to the pattern of the upper face. The removal of the nasal pit epithelia from the Amblystoma embryo resulted in the overgrowth of the premaxillary and maxillary bone concomitant with loss of the nasal cartilages (Burr, 1916). The grafting of ectopic nasal pits to a larval Xenopus to second host embryo induced localized hyperplasia near the graft (Stout and Graziadei, 1980). The addition of a nasal pit to the flank of Triton taenatius induced an ectopic limb (Balinsky, 1933). Conditional knockouts of Fgf8, in the facial ectoderm, resulted in hypoplastic nasal epithelia and truncations of the snout (Kawauchi et al., 2005). Removal of the nasal pit ectoderm in chicken embryos, results in the loss of olfactory nerve (Lutz et al., 1994), and nasal conchae (Wang et al., 2001). However, no one has tested the skeletogenic inductive effects of the nasal pit epithelia in facial mesenchyme. Aim I: To determine if the bones surrounding the nasal capsule are affected by nasal pit removal. Aim II: To determine if FGF8b protein can induce ectopic skeletal elements in competent facial mesenchyme. Aim III: To determine whether nasal pits can induce ectopic nasal capsules. Aim IV: To determine if host mesenchyme becomes restricted during organogenesis. Aim V: To determine if the nasal pits can autonomously develop in an ectopic location 14 Figure 1.1: Scanning electron micrographs rhxp.. y B " st. 28 Inpj — 0 - . St. 30 \ By E10.75 ( ^ - i Figure 1.1: Scanning electron micrographs of facial prominence stage embryos. The face shape is similar between chicken embryos (A-C) , and mouse embryos (D-F). Original S E M images provided by V . M . Diewert (UBC). (A, D) Distinct facial prominences are present in the pre-fusion stages. The facial prominences need to grow together to meet within the zone of fusion. (B, E) Approximately a day later, the facial prominences have increased in size and are fusing. (B) In the chicken upper lip, the globular process (light blue) is fusing with the maxillary prominence (green). (C, F) The upper face is fused and the upper lip is intact. Key: Scale bars: A , B , C-F, 1mm; C, 2mm. Arrows, Nasal pit; arrowhead, zone of fusion; light blue, globular process; yellow, frontonasal mass (fnm) (A-C), medial nasal prominences (mnp) (D-F); blue, lateral nasal prominence (lnp) (A-F); Green, maxillary prominence (mxp) (A-F). 15 Figure 1.2: Fate map of the chicken embryo face Figure 1.2 Fate map of the chicken face. The bone and cartilage derivatives derived from each facial prominence. (A) The ectomesenchymal cells were labeled in the stage 24 face (Lee et al., 2004) or individual prominences were from the stage 24 face were grafted to the limb bud (Richman and Tickle, 1989; Wedden, 1987), and apply to the stage 26 face presented here. These fate mapping experiments were followed longitudinally until bone and cartilage differentiation. (B) The frontonasal mass derivatives: premaxillary bone and the prenasal cartilage are coloured yellow (Richman and Tickle, 1989), the lateral nasal prominences derivatives nasal bone, and lacrimal bone (Song et al., 2004) and nasal conchae (MacDonald et al., 2004) are coloured blue. The maxillary derivatives include the palatine bone, the maxillary bone and the jugal and are coloured grey (Barlow and Francis West, 1997; Lee et al., 2004), while the mandibular derivatives are pink (Richman and Tickle, 1989) including the quadrate (Wilson and Tucker, 2004), the distal quadratojugal is derived from the maxillary prominence while the proximal region is likely derived from the mandibular arch (Lee et al., 2004; Wilson and Tucker, 2004). The second branchial arch derivatives are coloured purple, and the origins of the white areas have not been determined. 16 Key: an, angular; ba2, second branchial arch; c, columnella; de, dentary; e, eye; fnm, frontonasal mass; gp, globular process; ios, intraorbital septum; j , jugal; 1, lacrimal; lnp, lateral nasal prominence; pf, prefrontal; mc, Meckel's cartilage; mdp, mandibular prominence; mxb, maxillary bone; mxp, maxillary prominence; nb, nasal bone; nc, nasal conchae; p, palatine; pmx, premaxillary bone; pnc, prenasal cartilage; pt, pterygoid; q, quadrate; qj, quadratojugal; rap, retroarticular process; sa, surangular; sp, splenary; v, vomer. 17 Figure 1.3: Schematic of l ip fusion and typical cleft lip A Stage 26 i i f\ Stage 28 | \ Stage 30 ns 111. .If ns nc gp V H stomodeal g opening C D 1 Typical cleft lip Isolated cleft palate Figure 1.3: Schematic of lip fusion in chicken and typical cleft lip (A-D) Lip fusion in chicken embryos. Lip fusion occurs in four steps, the globular process of the frontonasal mass and the maxillary prominence grow towards each other at stage 26 (A, B). (C) The prominences meet at stage 28, and form a bilayered seam. (D) By stage 30, the epithelial seam has disappeared and a full mesenchymal bridge is present resulting in a normal upper lip. (E-G) Representation of human orofacial clefts in the palatal view. (E) Normal upper jaw morphology (F) Typical cleft lip can extend from the nostril through the upper lip, and alveolar ridge, extending back to the incisive foramen. Although the severity of the cleft lip can range from a small lip pit to the most severe form presented here. Often, cleft palate occurs secondarily to most severe cases of cleft lip. (G) In cases of isolated cleft palate the lip is intact, and a medial cleft is present which connects the oral and nasal cavity. Key: ar, alveolar ridge; ena, external nasal aperture; fnm, frontonasal mass; gp, globular process; if, incisive foramen; lnp. lateral nasal prominence; Is, labium superioris; mdp, mandibular prominence; mxp, maxillary prominence; n, nose; np, nasal passage; ns, nasal slit; sp, secondary palate. 18 Figure 1.4: Schematic of F G F signaling Figure 1.4: Schematic of F G F signaling (A) Secreted Fgf8 binds to mesenchymally expressed FGFR3 IIIc, with the help of heparin sulfate proteoglycan, in the D i l i Ig-loop of the receptor. Ligand binding induces a conformational change which activates the kinase domain of the receptor, and transphosphorylates its dimerization partner. (B) Activated FGF receptor can also signal through phospholipase C y or phosphatidyl inosital 3-kinase pathway. In the face, the expression of Fg/8 is adjacent to the mesenchymal phosphorylated E R K (Corson et al., 2003). (C) The phosphate group is recognized by the SH2 domain on FRS2 which is membrane associated and myristoylated. FRS2 is an adaptor protein that recruits Grb2 to the activated receptor. Sprouty2 can inhibit the recruitment of SOS to binding Grb2 and preventing downstream signaling. Otherwise, SOS binds Grb2 and activates Ras/Raf a kinase that phosphorylates and activates M A P K . Pystl will dephosphorylate M A P K and can prevent downstream activation of gene transcription. Phosphorylated M A P K translocates into the nucleus to activate transcription factors, and transcribe FGF responsive genes such as Pea3 and Etsl. SU5402 binds near the A T P loading site within the kinase domain and prevents receptor autophosphorylation. 20 igure 1.5; Expression of FGF ligands and receptors A) Chicken Key: FgfS, 17, 18 subfamily B FGFR1 •FGFR2 MFGFR3 Stage 20 Stage 24 Figure 1.5: Expression of FgfS subfamily and FGFR1, 2, 3 in the face of chicken and mouse embryos prior to lip fusion (A) Chicken embryos, stage 20 and 24; (B) Mouse embryos E10.5 and E l 1.5. The expression of FgfS, 17, and 18 are schematized on the right half of the upper face embryos, while the FGFRs are on the left half of the upper face. The expression of FGFR1 is found through out the face, while FGFR2, 3 are more restricted. Later, after fusion, FGFR2 is only present in the medial frontonasal mass (Wilke et al., 1997). 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The complete mammalian FGF family. Journal of Biological Chemistry 281, 15694-700. 30 CHAPTER 2: FGF signaling from the nasal pit regulates a potent growth centre that controls development of the upper lip Heather L . Szabo Rogers and Joy M . Richman Department of Oral Health Sciences, Life Sciences Institute, University of British Columbia, Vancouver B C , Canada ' A version of this chapter has been submitted for publication. Szabo Rogers H L and Richman J M FGF signaling from the nasal pit regulates a novel growth centre in the avian frontonasal mass that controls lip fusion. Development. 31 2.1 INTRODUCTION 2.1.1 Embryological origin of cleft lip with or with out cleft palate: Facial development starts at the time of neurulation, the facial bones (the bones of the jaw and upper face) are formed from migratory neural crest cells that migrate from the dorsal edge of the closing neural tube to fill the discrete facial epithelially encapsulated prominences (Le Lievre and Le Douarin, 1975). Once in the face, the neural crest cells proliferate and attain prominence specific form, then the individual prominences that make up the upper face must fuse together to make a continuous upper lip. In humans, the upper lip is formed by fusion of three facial prominences: the medial nasal prominence, the lateral nasal prominence and the maxillary prominence (Jiang et al., 2006). These facial prominences must first reach the correct size so that they can meet within the zone of fusion. Following the approximation of the prominences, epithelial contact occurs, followed shortly by disappearance of the bilayered epithelial seam and mesenchymal confluence (Jiang et al., 2006). Slight alterations in any of these processes will create a clinically significant malformation, cleft lip with or without cleft palate (CL/P) (Cox, 2004). Failure of fusion wil l result in a space between the maxillary and premaxillary bones. C L / P can either be syndromic or non-syndromic in origin. Non-syndromic C L / P is the most common congenital anomaly present in approximately 1/800 births, and its genetic origin is enigmatic. The genetic causes of non-syndromic CL/P have been difficult to identify because its development is also influenced by environmental and genetic factors. Population screens have identified missense mutations in FGFR1, FGFR2 and FGFR3, FGF8, FGF 10 and SPRY2 in populations from Iowa and Philippines (Riley et al., 2007; Vieira et al., 2005). Often the cleft occurs on the left side of the upper lip while leaving the midline intact. The bones underlying the soft tissue cleft are present, but there is a reduction in size of the premaxillary and maxillary bones with concomitant loss of teeth in the cleft area (Cox, 2004; Jiang et al., 2006). In humans, C L / P is an anatomically, and temporally distinct malformation from isolated cleft palate (CPO). CPO results from the failure of fusion of the medial palatal shelves in the roof of the mouth leaving the upper lip unaffected and intact. Often, CP can occur as a secondary effect of C L . 2.1.2 Chicken as a model for CL/P We have employed the chicken embryo model system to characterize some of the molecular mechanisms underlying non-syndromic CL/P . We chose the chicken embryo to model C L because we can specifically target the fusing upper lip just prior to fusion. Similarly 32 to humans, the upper lip in chicken embryos develops within the zone of fusion between the maxillary prominence and frontonasal mass. Lip fusion is initiated at stage 28, when the lateral caudal corners (or globular processes) of the F N M approximate with the maxillary prominence and fuse. At stages just prior to fusion, the chicken embryo face is made up in a square conformation, it undergoes cell proliferation and attains a conformation similar to the medial nasalprocesses in mouse and human just prior to lip fusion (Tamarin et al., 1984; Wi l l and Meller, 1981; Yee and Abbott, 1978). Similar to human C L / P , if fusion fails in the chicken embryo face, a cleft tomium (tomium- chicken lip) phenotype will develop resulting in bony defects and a space occurring where the premaxillary bone (derived from the frontonasal mass), and the maxillary bone (derived from the maxillary prominence) articulate to provide structural support to the upper lip (Lee et al., 2004; Peterka and Jelinek, 1983). Chicken embryos are inappropriate models for CP because the secondary palate of chickens is naturally patent. 2.1.3 Signaling molecules in the developing upper lip The mouse and human FGF (fibroblast growth factor) family consists of at least 22 ligands, (Ornitz and Itoh, 2001). The FGF ligands can bind to and signal through 4 receptor tyrosine kinases (Ornitz and Itoh, 2001). The FGF signaling cascade is initiated by two ligands binding to heparin sulfate proteoglycans, which in turn induces the dimerization and a conformational change of the FGFR and facilitates an intracellular signaling cascade (Bottcher and Niehrs, 2005; Thisse and Thisse, 2005). The four FGFRs are differentially spliced and expressed in either the mesenchyme or epithelia. Epithelial to mesenchymal signaling is mediated by the expression domain of the 'c ' receptor splice type (Orrurtreger et al., 1993). The 'c ' receptor splice type is expressed in the mesenchyme and binds with highest affinity to epithelially expressed ligands (Ornitz et al., 1996; Zhang et al., 2006). In chicken embryos, Fgfrl is expressed in the mesenchyme of lateral edges of the F N M and the olfactory epithelium, while Fgfr2, and Fgfr3 are restricted to more medial regions of the F N M (Hebert et al., 2003; Matovinovic and Richman, 1997; Wilke et al., 1997). In the maxillary prominence, Fgfrl is expressed ubiquitously in the mesenchyme, while Fgfr3 is expressed in the mesenchyme of the distal maxillary prominence (Wilke et al., 1997). Just prior to lip fusion, in mouse and chicken embryos at least three FGF ligands (FgfB, FgflO, FgflT) are expressed within the zone of fusion (Bachler and Neubuser, 2001; Havens et al., 2006; Karabagli et al., 2002). Sprouty (Spry) proteins are intracellular inhibitors of activated FGFR signaling. Spry expression can be used as a reporter of active FGF signaling because Spry expression can be induced by exogenous FGF 33 ligands within 25 minutes of application, and are directly regulated by FGF signals (Chambers et al., 2000; Liu et al., 2003; Minowada et a l , 1999). The maxillary prominence and frontonasal mass express unique combinations of epithelial signaling molecules. Within the zone of fusion, the maxillary prominence epithelial component expresses Shh, Bmp4, Wnt9b, while FgfS and Noggin are not expressed, and the frontonasal mass epithelial component expresses Noggin, Bmp4, and Wnt9b, but not FgfS, or Shh (Ashique et al., 2002; Hu and Helms, 1999; Lan et al., 2006; Song et al., 2004). Msxl/2 are expressed in neural crest derived facial mesenchyme and are indicators of both FGF and B M P signaling (Ashique et a l , 2002). The reduction of either B M P or S H H signaling induced cleft lip in chicken embryos (Ashique et a l , 2002; Hu and Helms, 1999). 2.1.4 Knockout models for clefting Knockouts of a non-canonical Wnt, Wnt5a, resulted in severe truncations of the upper face including loss of the premaxillary and maxillary bones (Yamaguchi et a l , 1999). Nestin-cre driven excision of Fgf8 in the ectoderm of the facial prominences resulted in mandible and upper face agenesis (Trumpp et a l , 1999). Hypomorphic FgfS knockouts resulted in significant defects in craniofacial structures including absence of many of the upper face bones, and confirmed a role for FGF signaling in DiGeorge syndrome (Abu-lssa et a l , 2002; Frank et a l , 2002). A very small percentage of humans with DiGeorge syndrome also have C L / P (Oskarsdottir et a l , 2005). An ectodermal knockout of FgfS driven by an ubiquitously expressed epithelial driver: AP2ct-cre driver has severe facial anomalies, including loss of most of the upper face and mandible (Macatee et a l , 2003). A finer ectodermal tissue specific knockout of Fgf8 was performed using a F o x G l driven cre-line to excise Fgf8 specifically within the facial and olfactory epithelium, these embryos develop misshapen nasal cavities, and the face is foreshortened, with loss of bones in both the frontonasal and mandibular regions (Kawauchi et a l , 2005). The severe nasal phenotype results from the loss of a stem cell niche in the olfactory epithelium (Kawauchi et a l , 2005). Here, we focused our study on the role of asymmetric FGF signaling within the zone of fusion. To tease apart the roles of FGF signaling in upper lip development we altered levels of FGF signaling in the face in a very specific temporal and spatial manner by using the FGF pan antagonist SU5402 (Mohammadi et a l , 1997). SU5402 binds to highly conserved residues in FGFR1-3, (but not within the platelet derived growth factor receptor or the epidermal growth factor receptor) and prevents downstream FGF receptor signaling (Mohammadi et a l , 1997). 34 To address the role of FGF signaling just prior to the time of fusion in chicken embryos (Hamburger and Hamilton, stage 26), we implanted beads soaked in SU5402 into the developing upper beak in chicken embryos. Surprisingly, we found that cranial FGF signaling in the frontonasal mass controls the level of cell death and proliferation in the entire frontonasal mass, and is a powerful morphogenetic center for upper lip development. 35 2.2 METHODS: 2.2.1 Embryological Methods: Embryos: Fertile white leghorn chicken eggs were obtained from the Poultry Unit at the University of Alberta. The eggs were incubated at 38 °C in a humidified incubator until stage 20, or 26. The eggs were windowed the day before the experiment to synchronize the stage of the embryos. A l l embryo work was performed under the approval of the U B C Animal Care Committee. Beads: Stock and working concentrations of SU5402 ( S U G E N , U S A and E M D Biosciences, U K ) were dissolved in D M S O (dimethyl sulfoxide) and kept in a dark environment, at -20°C. The formate form of A G 1X2 beads (Biorad) were dried and then soaked in 5 ul of SU5402 and 0.01% Fast Green. Fast green improved bead visualization. After soaking for an hour, 150 um beads were selected and separated from the rest of the beads and were kept in the dark until implantation. Surgery: Stage 20, and 26 embryos were prepared for surgery by tearing the membranes surrounding the head and applying a small drop of undiluted neutral red (0.333% in water, Fisher Science N-129). For stage 26 embryos, the F N M was visualized by using the eye as fulcrum with blunt forceps to turn the head; for stage 20 embryos the lateral edge of the F N M was easily visualized in situ, so turning the head was not needed. Bead implantation was performed by first making a shallow cut in the lateral F N M followed by inserting the bead. Careful documentation of the original bead location was made with the ocular reticule. The embryos were collected at 3, 6, 9, 12 and 16 hours, and 10 days following surgery. Skeletal preparation and skull analysis: To study bone and cartilage morphology, stage 39 embryos were collected, fixed in 100% ethanol, permeabilized and defatted with acetone and then stained with alcian blue and alizarin red to stain cartilage and bone respectively (Plant et al. 2000). Each process of the bones that coalesce to support the upper beak (maxilla, premaxilla and palatine) was compared to the normal, contralateral side. Our scoring methodology used three categories -normal, reduced or absent. The reduced category included bony processes that 36 were greater than 50% shorter in length. We compared the effect of bead position on skeletal morphology in the three different treatment groups by Chi square analysis (p<0.05). 2.2.2 Cell proliferation and death analysis: BrdU analysis: Two hours before collection, approximately 50 ul of 102 M BrdU (bromodeoxyuridine; dissolved in PBS (phosphate buffered saline)) was injected into the ventricle or atrium of the heart. Embryos were collected 12 hours after bead implantation and fixed overnight in 4% P F A and embedded in paraffin. Seven u.m sections were placed on TESPA-(3-aminopropyltriethoxysilane, Sigma) coated slides (FISHER plus). The sections were pre-treated with 5 p-g/ml proteinase K (37°C; 10 min), exonucleases (300 U/ml Exonuclease III (Amersham); 15 U/ml Sau3Al (NEB) and then incubated for 30 minutes at 37°C with neat primary antibody (Amersham, G E Healthcare). The secondary Alexa Fluor 488 (Molecular Probes, Invitrogen) or FITC (Jackson Labs) labeled goat anti-mouse antibody was diluted 1:50 and incubated at room temperature for 30 minutes. The secondary antibody was rinsed off with PBS and the slides were mounted using Prolong Gold Antifade with DAP1 to stain nuclei (Molecular Probes, Invitrogen). We analysed sections that were at least 14 u.m apart, so that different cells would be represented in each of our counts. Photographs of each section were made into a composite image and pseudocoloured using the green and blue channels in Adobe Photoshop 7.0. We used the WCIF Image J plugins: ITCN (Image-based Tool for Counting Nuclei) for BrdU positive cells, and nucleus counter for DAPI stained nuclei. To determine i f there were differences in cell proliferation related to bead position we divided the lateral frontonasal mass into three regions to distinguish cells from the top of the nasal slit from those in the globular process. The superior region lined up with the top edge of the nasal slit, the inferior region included the corner or globular process of the frontonasal mass. Each area was approximately 200 u,m wide by 100 u.m in height and contained approximately 800 cells. We calculated the proliferation index by dividing the number of BrdU positive cells by the DAPI positive (total cell number) for each region. We compared each region counted, bead position and compared SU5402 treatment to D M S O using multifactorial A N O V A ( M A N O V A ) with Fisher Least Significant Difference post hoc testing (p<0.05; Statistica). 37 Nile Blue Assay for wholemount cell death analysis: Bead implantation was performed without neutral red staining and embryos were collected 3, 6, and 9 hours after bead implantation followed immediately by staining with 0.3% nile blue sulphate in PBS for 35 minutes at room temperature. The entire embryo was washed in PBS at room temperature for an additional 40 minutes and photographed immediately. To ensure that the staining was even, we confirmed the presence of normal areas of cell death were stained equally on both sides of the body, including patches within the somites, near the posterior necrotic zone of the limb, the midline of the mandibular arch, and the groove between the lateral nasal prominence and maxillary prominence. The embryos were placed in a glass chamber, and photographed under a coverslip on a Ziess Axioskop. Images were captured with a Micropublisher camera (Q Imaging, Bumaby, BC). TUNEL (Terminal dUTP transferase nick end labeling) assay for apoptotic cells: Embryos were collected 6 and 16 hours after bead implantation, fixed in 4% paraformaldehyde overnight and embedded in wax. Near adjacent, 7 u,m, sections were placed TESPA coated slides (Fisher, Superfrost) and stained for apoptosis using the Apoptag kit (Chemicon). Sections were counter-stained with 0.5 % methyl green and briefly rinsed in distilled water, and mounted in Entellan. Apoptotic bodies in near adjacent sections were counted in the same regions that were used in the BrdU analysis plus an additional region in the medial frontonasal mass. Since apoptosis results in loss of cell integrity it is often difficult to determine the exact number of apoptotic cells in a specific area, therefore we placed specimens into one of three categories: 0-5; 6-10; 11-50 apoptotic bodies. 2.2.3 Whole mount in situ hybridization and immunohistochemistrv : In situ hybridization: Embryos collected for wholemount in situ hybridization, were fixed overnight in 4% paraformaldehyde in PBS (phosphate buffered saline). Whole mount in situ hybridization (WISH) was performed in the Intavis InsituPro Robot, with DIG-labeled antisense probes using protocols previously published (Song et a l , 2004). Section in situ hybridization was performed with antisense 3 5S-labeled antisense probes using the protocols published in (Wilke et a l , 1997). We wish to acknowledge the following individuals who provided avian probe constructs for our in situ analyses: G. Martin, Sprouty 1,2; P. Francis-West, Bmp4; E. Frolova, Wnt5a, Wnt9b; M . Kessel, Dlx5; S. Wedden, Msxl, 2; O. Pourquie, intronic and exonic FgfS; S. Noji, FgflO; S. 38 Keyse, Pystl. The Spry4, probe came from the M R C Geneservice (UK, Clone ID 603786019F1). Immunohistochemistry: Embryos for wholemount phosphorylated M A P K staining were stained as previously published (Corson et al., 2003) with the polyclonal Phospho-p44/42 Map Kinase antibody (Cell Signaling #9101). 2.2.4 High Density facial micromass culture: Culture preparation and staining: Procedures were followed as described (Weston et al., 2000). Briefly, medial nasal processes of the E l 1.5 mouse embryos were dissected in Puck's Saline A and dissociated in 3 ml of 12 U/ml of Dispase for 45 to 60 minutes with shaking at 37°C until a single cell suspension was present. These preparations were strained through Falcon cell strainer to remove any remaining clumps of mesenchymal cells and the epithelia. The cells were washed in 7 ml of media and counted on a haemocytometer. The cells were centrifruged (500G for 5 min) and resuspended in media to a final high density concentration of 2 x 107 cells/ml. They were plated as 10 u.1 droplets in tissue cultures dishes (Nunc, Nunclon surface) and left to adhere for 1 hour, after which the plates were flooded with media (60:40 D M E M :F 12, 10% fetal calf serum, ascorbic acid, IX antibiotic/antimycotic, IX glutamine). Some plates were treated with SU5402 (750 ng/ml or 2.5 p.M, S U G E N or E M D Biosciences) or FGF8b protein (5 ng/ml, Peprotec). The media and treatment were replaced after 24 hours and then every other day after that. The cultures were grown for 8 days in the presence of 5 m M beta-glycerol phosphate in order to assess bone and cartilage differentiation. Mineralized skeletal tissue (primarily bone) was detected with positive staining for alkaline phosphatase. For the alkaline phosphatase staining the cells were rinsed with PBS and fixed for 30 minutes in 10% formalin, and then incubated for one hour with a filtered solution of Fast Red (0.1 M Tris pH 8.3; 0.01% Naphtol AS-Mx-phosphate in dimethyl formamide; 0.06% Fast Red Violet Salt) (Sigma). Following the alkaline phosphatase reaction, cartilage nodules were stained with acidic alcian blue. For cartilage staining the cultures were rinsed in water, acidified with 0.1 M hydrochloric acid (HC1) for 15 minutes, and stained with 4:1 alcian blue (0.1 M HC1: 0.5% Alcian blue in 95% ethanol) overnight in a humidified chamber. The excess stain was washed off with 70% ethanol and the cultures were photographed. 39 Micromass cell counts: After 8 days in culture, three spots, each plated in a single well (24 well size), were counted and averaged to determine the mean cell number per spot. In order to separate the cells from the matrix, the micromass cultures were removed from the culture dishes with 0.1% Trypsin, 0.001 M E D T A (ethylene diamine-tetra acetic acid). The cultures were further treated for 30 minutes with 700 ul of 137.8 U of collagenase II (Worthington Biochemical Company) (dissolved in 0.7% NaCl ; 0.04% KC1, 0.52 % K 2 H P 0 4 , 0.14% C a C L 2 , 0.6% HEPES Buffer, 0.55% Mannitol, 0.2% Glucose and 0.2% B S A ) to dissociate cells from a differentiated matrix. Cells were resuspended in 200 uJ of media and cell number was counted with a haemocytometer. Quantitative Realtime PCR: For quantitative realtime PCR (Q-PCR), we collected R N A from 10 spots in one 35 mm dish after 24 hours of culture. We used the RNA-Easy Miniprep (Qiagen) for R N A preparation. Briefly, the plates were flooded with 350 u.1 of the R L T buffer including P-mercaptoethanol and aspirated into the QIAshredder column and spun for 1 minute. The flow-through was stored at -80°C until R N A preparation and c D N A synthesis. We prepared c D N A using random primers with the High-Capacity c D N A Archive kit (Applied Biosystems 4368814) with 0.5 u.g of total R N A . Q-PCR was performed on the A B I PRISM 7500 system using 25 ng of R N A per reaction. We used the mouse Assay on Demand Taqman Primer probe sets to characterize Sprouty! expression (Applied Biosystems: Sprouty2: Mm00442344_ml). Each biological replicate was normalized to 18S R N A and the results are expressed as fold change compared to 24 hour D M S O treated carrier-control. 40 2.3 RESULTS: Our study uncovered differential gene expression patterns within the frontonasal mass and other regions of the face that led us to hypothesize there were differences in FGF signaling within the developing face. In order to understand the endogenous FGF signals taking place at the time of fusion we used bead implantation to locally block F G F receptor signaling. Our findings show a surprising degree of dependence on FGF signals in a region of the frontonasal mass, not normally thought to play a role in lip fusion. 2.3.1 Sprouty 1, 2, and 4 are expressed in partially overlapping domains in the frontonasal mass Several studies have detailed the role of signaling derived from both B M P and S H H signaling in inducing cleft lip (Ashique et a l , 2002; Hu and Helms, 1999). Inducing cleft lip by altering B M P levels results in altered FgfS expression, however no study has looked at the role of FGF signaling explicitly in cleft lip (Ashique et a l , 2002). Several FGFs and FGFRs are expressed near the zone of fusion and we wanted not only to detail the expression of FGFs in the fusing upper lip, but to characterize where active FGF signaling is occurring during fusion. To identify the tissues where active FGF signaling is occurring we characterized the expression of Sprouty}, 2 and 4 in the upper lip. Members of the Sprouty family have been identified as inhibitor of FGFR signaling by preventing the downstream phosphorylation cascade leading to activation of M A P K of growth factor signaling (Hanafusa et a l , 2002). Spryl expression does not require protein synthesis for increased expression with the application of FGF8 (Liu et a l , 2003), and Spry2 is induced within 25 minutes of FGF application (Chambers et a l , 2000), both of these studies suggest that the Spry family members are potent indicators of the level of FGF signaling. Previous papers generalized Spry2 expression in both mouse and chicken embryos to the mesenchyme that underlies ectodermal FgfS expression domains, (Chambers and Mason, 2000; Minowada et a l , 1999; Zhang et a l , 2001). These studies reported that Spry2 is found in the facial mesenchyme but was lacking detailed analysis of the expression within the facial prominences. At stage 20, two days prior to lip fusion, (Fig. 2.1G,J,M) and at stage 28, when lip fusion is occurring (Fig. 2.1H,K,N), Spry 1, 2 and 4 transcripts overlapped in the cranial mesenchyme of the frontonasal mass and were adjacent to the olfactory and respiratory epithelial expression domains of both FgfS and FgflO (Fig. 2.1 A ,B ,D,E) . We observed differential 41 expression of each of the Spry genes within the frontonasal mass. In the stage 28 frontonasal mass, Spry 1, 2, and 4 are expressed in overlapping domains in the mesenchyme near the olfactory epithelium at the cranial apex of the nasal pit, while only Spry2 expression extends caudally into the zone of fusion (i.e. the corner of the frontonasal mass or the globular process) and the expression extends more medially along the caudal edge of the frontonasal mass (Fig. 2.1H-I). Interestingly, the FGF ligands and Spry family members are not expressed in the medial maxillary prominence within the zone of fusion (Fig. 2.1K-N). The Spry expression data provides clues to where the highest requirement for FGF signaling in the frontonasal mass is located. However, functional studies were required to determine if there is an FGF-dependent growth centre in the cranial-caudal axis of the frontonasal mass. This expression data also suggests that the frontonasal mass contribution to the zone of fusion requires FGF signaling, whereas the maxillary component is independent of FGF signaling. 2.3.2 Loss of F G F signaling in the FNM induces a cleft beak phenotype: We functionally tested whether FGF-induced signaling is required in the frontonasal mass and maxillary prominence during lip fusion. We used beads soaked in the FGFR pan-antagonist, SU5402, to specifically reduce the levels of FGF signaling in individual facial prominences. We targeted the face at stage 26, so that peak SU5402 release would take place just prior to fusion between the frontonasal mass and maxillary prominence (Tamarin et al., 1984; Wi l l and Meller, 1981). We began by determining the lowest soaking concentration of SU5402 that would induce facial defects (Table 2.1). Embryos treated with beads soaked in 10 mg/ml of SU5402 and implanted into the lateral edge of the frontonasal mass developed notches in the tomium (the edge of the chicken beak; 29% n = 13/45; Table 2.1). This cleft beak morphology resembles the soft tissue appearance of human cleft lip. We found significant variability in the external notch phenotype in the embryos treated in the lateral frontonasal mass. We therefore refined our experiment to reflect the differential expression of the Spry genes along the lateral edge of the frontonasal mass. We placed beads in one of two positions: 1) caudal, within the globular process (the point of contact between the frontonasal mass and maxillary prominence), and 2) cranial, near the cranial margin of the nasal slit. A l l embryos treated in the caudal bead position have normal external upper beaks (n=16/16; Table 2.2); while a significant proportion of embryos treated with the cranial bead position, adjacent to the olfactory epithelia, develop a notch or an external cleft (40%, n= 18/45; Table 2.2; Fig. 2.2C). Embryos treated with the 42 carrier control, D M S O , in the cranial position all developed normally (Table 2.2, n=6/6). The incidence of clefting was higher than in our first experiments due to homogeneity in bead position. The lack of effect in caudally-treated embryos was unexpected because B M P signaling in the same region has been shown previously to be required for lip formation (Ashique et a l , 2002). From these earlier studies, it was hypothesized that growth factor signaling in the globular process was the key to lip fusion. We next tested whether the maxillary prominence required FGF signaling for normal outgrowth and fusion of the upper lip. These embryos developed normally, even though they had been treated with beads soaked in 10 mg/ml SU5402 (100%, n=15; Table 2.2). The lack of effect on the maxillary prominence is consistent with our hypothesis that FGF signaling is required in the frontonasal mass but not in the maxillary prominence during lip fusion. The cleft phenotype in embryos treated with beads in the cranial position may indicate that the frontonasal mass growth is inhibited, which prevented contact with the maxillary prominence. Moreover, our bead position data showed rather unexpectedly that FGF signaling within the cranial frontonasal mass, and distant from the zone of fusion, mediates upper lip development. 2.3.3 Skeletal effects underlying the cleft beak phenotype The external notch observed in cranially treated embryos suggested that there are underlying changes in bone and cartilage morphology. Indeed we found that all externally affected embryos and 30% of the embryos that were normal externally had a skeletal defect in one or more of the following upper beak bones: the premaxilla, maxilla or palatine. The premaxillary and maxillary bones articulate with each other to provide skeletal support for the edge of the upper beak, and we observed a space between these two bones that is coincident with the external notch (Fig. 2.2D-F, arrowhead). The maxillary process of the premaxillary bone, a frontonasal mass derivative, is absent in cranially-treated embryos (40%; n=18/45; Table 2.2). In addition the maxillary bone is displaced proximally (44%; n = 20/45; Table 2.2; Fig. 2.2D-F). The nasal chonchae are cranial to the maxillary process of the premaxillary bone and are misshapen in the cleft embryos (n= 25/45; Table 2.2; Fig. 2.2D-F). The cleft extended medially from the lateral support of the tomium, to the normal, cleft secondary palate and resulted in a separation between the premaxilla and palatine bone. Internally, the skeletal support for both the upper lip and palate are reduced, including the palatine process of the maxillary bone (60%, n=27/45; Table 2.2), and the palatine process of the premaxillary bone (60%, n=26/45; Table 2.2). The maxillary process of the palatine bone was 43 truncated in a majority of specimens (58%; n= 26/45; Table 2.2), although the truncation is likely a secondary response to the dislocation of the maxillary bone (Fig. 2.2E). The upper beak skeleton develops normally in caudally-implanted-SU5402 and DMSO-treated-control embryos (Fig 2.2A,B). We found reductions in the premaxilla, maxilla and palatine bones occur more frequently (Chi-square, p<0.0001) in cranially-treated embryos compared to the caudally-implanted SU5402 embryos and cranially implanted carrier control embryos. From the differential response within the frontonasal mass to decreased FGF signaling, we hypothesize that FGF ligands, perhaps secreted from the adjacent olfactory epithelium, have the ability to influence morphogenesis along the cranial-caudal axis of the F N M . In the cleft phenotype just described, the main body of the bones developed to their normal size, and we assumed that this occurs because most skeletal patterning had already taken place by the stage that the beads were placed. Grafting experiments have showed that facial prominences were specified to develop into specific skeletal elements by stage 20 (Richman and Lee, 2003). To further explore the temporal requirement for FGF signaling during facial patterning, we treated embryos at stage 20, when FGF ligands are very abundant throughout the frontonasal mass epithelium (Schneider et al., 2001; Song et al., 2004; Young et al., 2000). We predicted that FGF signaling would play a more fundamental role in patterning the upper beak outgrowth at these earlier stages. We implanted beads soaked in 10 mg/ml SU5402 into the cranial frontonasal mass mesenchyme of stage 20 embryos. Unlike embryos treated at stage 26, we almost never observe clefts of the beak (n=l/17), instead the tomium is smooth, and occasionally the beak is deviated towards the treated side (21%, n=4/18; Table 2.2; Fig. 2.2G,H). Internally, loss of FGF signaling results in nasal bone reductions (n = 11/22), a derivative of the lateral nasal prominence. Frontonasal mass derivatives are also affected including the prenasal cartilage (deviation, n= 12/22), as well as the body and nasal process of the premaxillary bone (shortened, 19/22). The maxillary and palatine bones were unaffected. Previous work in our lab suggested that the inhibition of retinoid synthesis at stage 20 induces a cleft beak, resulting in part from a precocious downregulation of FgfB expression. The cleft phenotype can be partially rescued by the application of Fgf8b protein (Song et al., 2004). Our stage 20 embryos are less severely affected in the same bones as those treated with Citral and confirm that FGF signaling is downstream of retinoid signaling at stage 20. 44 2.3.4 Loss of F G F signaling in the F N M inhibited the differentiation of osteogenic and chondrogenic precursors: To fully characterize which tissue is affected by the SU5402 treatment, we used the high density micromass experimental paradigm to characterize the role of FGF signaling in the differentiation of bone and cartilage from facial mesenchyme. We chose to work with mouse tissue for these experiments for two reasons: a) several knockout mouse models suggested that FGF signaling within the face is important for bone patterning, so it is a control for our in ovo work and b) the reagent availability for mouse analysis. Mouse lip fusion occurs between the median nasal prominences (equivalent to the lateral edges of the chicken F N M ) and lateral nasal prominences and the maxillary prominences at E l l , in a similar fashion to chicken lip fusion. We used E M . 5 mouse medial nasal prominences (equivalent to chicken stage 26 lateral frontonasal mass) as a source of tissue for our micromass spots. We performed a second dose response to SU5402 in these cultures, and chose 750 ng/ml SU5402. This dose did not decrease the number of cells attaching to the plate and still allowed the cultures to differentiate into bone and cartilage. We included treatment with 5ng/ml FGF8b protein as a positive control for these experiments. After 8 days in culture, we observed decreased levels of alkaline phosphatase and alcian blue staining in response to SU5402 treatment (Fig. 2.3A,B). There was no difference in the total cell number, between the SU5402, carrier control or Fgf8b protein cultures (Fig. 2.3C). As expected, within 24 hours of culture, Spry2 transcript is reduced in SU5402 treated cultures and increased in FGF8b treated cultures using one-tailed t-test (p<0.05), compared to the carrier control cultures using quantitative realtime PCR. This suggests that the cleft phenotype in ovo, resulted from the inhibition of differentiation of the osteogenic and chondrogenic progenitor cells. 2.3.5 SU5402 directly affects downstream FGF signaling within the frontonasal mass In order to confirm at the molecular level that the SU5402 decreased FGF signaling we examined expression of several markers known to be directly regulated by FGF ligands. Spry2 is induced within twenty-five minutes of the application of a bead soaked in FGF8, near the midbrain hindbrain isthmus and the ectopic expression domain is maintained for at least 16 hours (Chambers et a l , 2000). We characterized the loss of FGF signaling in the frontonasal mass by looking at the expression of Spry4, Pystl and Spry2 (Table 4). Spry4 expression is only found near the position of the cranial bead (Fig. 2.1 J). We observed a reduction of Spry4 expression surrounding the cranial bead within 6 hours of treatment (cranial: n=4/5; Fig. 2.4B) while there was no change in Spry4 expression with caudal bead treatment (n= 4/4; Fig. 2.4D). Sixteen 45 hours after cranial bead implantation, the Spry4 expression is recovering near the cranial bead (n=3/3; Fig. 2.4C), and remains unchanged with caudal bead implantation (n= 10/10). ' Decreased expression of Pyst 1VMkp3/DUSP6 in the frontonasal mass, extended from the cranial-most portion of the nasal slit to the globular process (ie along the entire lateral edge of the frontonasal mass; n=6/7, 6 hours). Pystl expression is still reduced in the cranial-caudal axis of the F N M in response to the cranial SU5402 bead (n=5/5, 16 hours; Table 4; Fig. 2.4H). Surrounding the caudal bead, we observed decreased Pystl expression 6 and 16 hours after implantation (6 hours, n=6/6; 16 hours, n=4/4; Fig. 2.41, J; Table 4). Next we tested the expression of Spry2 which extends from the superior edge of the nasal pit caudally to the globular process within the zone of fusion and medially along the caudal edge of the frontonasal mass (Fig. 2.11). Within six hours, the entire expression domain within the frontonasal mass downregulated the expression of Spry2 in response to the cranial SU5402 bead (n= 7/7; Fig. 2.4L) in contrast to the localized decrease in Spry2 expression surrounding the caudally-implanted bead (n=7/7; Fig. 2.4N). At 16 hours, Spry2 expression has recovered between the location of the bead and the superior edge of the nasal slit, but is still absent between the cranial bead and the zone of fusion (n=9/9; Fig. 2.4M, arrow). Spy2 expression is still absent near the caudal bead, 16 hours after implantation (n=6/6; Fig. 2.40, arrow). The partial recovery of gene expression indicates that FGF signaling in some regions of the frontonasal mass has returned to normal levels 16 hours after SU5402 treatment. We have shown that the inhibition of FGF signaling from the cranial frontonasal mass controls the expression of FGF signaling modulators extending into the zone of fusion. Spry family members and Pystl work by decreasing the amount of activated M A P K in response to a FGF signal. Work in the limb and neural plate suggests that Pystl works specifically by reducing the amount of phosporylated E R K (Smith et al., 2006). We hypothesized that SU5402 treatment was also reducing the amount of phosphorylated E R K in the frontonasal mass. Within 6 hours of cranial bead implantation, embryos have decreased levels of phosphorylated M A P K along the lateral edge of the frontonasal mass (n = 4/4, Fig. 2.5B), compared with a localized decrease surrounding the caudal bead position (n = 4/4, Fig. 2.5C). The external notch phenotype and underlying skeletal malformations in cranially-implanted embryos resulted from the loss of FGF signaling through E R K in the lateral edge of the frontonasal mass and confirmed the essential role that endogenous FGF signaling plays in the F N M . The differential effects of bead position on Spry2 and Pystl expression showed that it was not sufficient to block FGF signaling within the globular process itself for a cleft phenotype, 46 because these embryos all developed normally. Rather it was necessary to treat embryos in such a way that the FGF signals along the full length of the nasal slit are reduced. Taken together, the decreased levels of phospho-ERK and the decreased expression of Spr2 and Pystl showed that loss of FGF signaling from the cranial frontonasal mass preceded abnormalities in lip fusion in cranially-implanted embryos. 2.3.6 F G F signaling controls levels of cell proliferation and cell death in the frontonasal mass Organogenesis is a well orchestrated balancing act where tightly controlled levels of cell proliferation and cell death are provided by FGF signaling (Goldfarb, 1996). If the balance is tipped in one direction, the resulting form will be abnormal. Since FGFs are known stimulators of proliferation and cell survival factors, we hypothesized that the mechanism underlying the cleft phenotype results from decreased cell proliferation and increased cell death. Several studies have carefully described cell proliferation in the frontonasal mass of chicken embryos close to the time of lip development. The mesenchyme of the upper face requires epithelia for normal morphogenesis (Richman and Tickle, 1989; Wedden, 1987), and the sub-epithelial facial mesenchyme had the highest rate of proliferation (MacDonald et a l , 2004; McGonnell et a l , 1998; Minkoff and Kuntz, 1977; Wu et a l , 2004). The lateral edge of the frontonasal mass has the highest rate of proliferation near the nasal slits while the medial region has fewer proliferating cells. Several studies have detailed the proliferation rate for stage 24 (McGonnell et a l , 1998; Minkoff and Kuntz, 1977), 27 (Wu et a l , 2004), and 28 (MacDonald et a l , 2004; McGonnell et a l , 1998; Minkoff and Kuntz, 1977; Peterka and Jelinek, 1983), by characterizing the number of cells in S-phase using either pulse B r D U labeling (MacDonald et a l , 2004; McGonnell et a l , 1998; Wu et a l , 2004) or 3H-thymidine (Minkoff and Kuntz, 1977) or the number of mitotic cells using colchicine (Peterka and Jelinek, 1983). The lateral third of the F N M is also an area that is undergoing extensive expansion and cell movement at the time of upper lip development in order to contact with the maxillary prominence (Hu et a l , 2003; McGonnell et a l , 1998; Patterson and Minkoff, 1985). Following a two-hour pulse of BrdU, we collected our embryos 12 hours after bead implantation. We chose to collect the embryos 12 hours following bead implantation, an intermediate time point so that the frontonasal mass will be approximately normal sized, while the irreversible signaling changes have occurred. We assume that the changes are irreversible at 12 hours because in just 4 hours (the 16-hour timepoint) the morphological changes are noticeable (Fig. 2.4C,H,M). We also chose 12 hours as the timepoint so that the changes in 47 proliferation were likely irreversible. Work in this lab has shown that generally changes within 6 hours of bead implantation may be reversible. Nearly every BrdU labeled section was cut directly through the globular process and we noted a striking absence of proliferating cells in the mesenchyme directly adjacent to the zone of fusion (Fig. 2.6A-C, arrowheads; n=21/21). We also noted qualitatively that maxillary mesenchyme directly opposite from the globular process also had low proliferation (n=l7/17). We observed this area of reduced proliferation on either the treated or untreated side of the F N M , and it was not expanded on the treated side of the frontonasal mass (n=5/7). Furthermore, we propose that mesenchyme in the zone of fusion has a lower proliferation index and instead of actively proliferating and acting as the lead edge for contact, expansion is driven by the proliferation of other regions of the frontonasal mass. Indeed treated embryos with beads placed in the cranial position had significantly decreased proliferation. We found that the cranial SU5402 bead position reduced the proliferation index in the cranial two-thirds of the frontonasal mass (p<0.05; Fig. 6C,D). SU5402 did not affect the cell density of the mesenchyme, since similar numbers of cells were counted in the treatment and control groups. We noticed that in D M S O embryos, the superior region had greater proliferation than the inferior region (Fig. 2.6D). The caudally implanted SU5402 did not alter proliferation, likely because proliferation was already relatively low in that region (Fig. 6B,D). The results from these loss-of-function experiments show there is an FGF-dependent growth centre located near the superior nasal slit. These results suggested that the lateral edge of the frontonasal mass in the cranially treated embryos did not reach optimum size, and prevented the tissues connected to it (i.e. the globular process) from contacting the maxillary prominence resulting in the cleft. Normal regions of apoptosis in the developing facial prominences have been described previously and include the groove between the lateral nasal prominence and the maxillary prominence, the groove in the midline of the mandibular prominence and the globular process of the frontonasal mass (Ashique et al., 2002; McGonnell et al., 1998). We characterized changes in cell death in wholemount embryos, using Nile blue sulfate, and in tissue sections, using the T U N E L method. With Nile blue staining, we observed increased cell death exclusively on the treated side of the F N M , confirming that the effects of SU5402 are unilateral. At 3 and 6 hours after bead implantation, the cranial bead position increased cell death from the cranial margin of the nasal slit down to the caudal corner (globular process) of the frontonasal mass (3 hrs: n=5/7 (not shown); 6 hrs: n=6/6, Fig. 2.7A). At nine hours, the area of increased cell death extends 48 cranial to the nasal slit just ventral to the telencephalon and caudally to include the globular process (n=3/4, Fig. 2.7C). In contrast, the caudal bead position slightly enlarged the normal area of cell death found in the globular process of the frontonasal mass (3 hours: n=6/7 (not shown); 6 hours: n=5/5, Fig. 2.7E; 9 hours n = 4/4, Fig. 2.7G). There was no change in the D M S O treated embryos demonstrating that the surgery itself did not increase cell death. T U N E L analysis on tissue sections which allowed us to determine whether apoptosis was occurring in the epithelium and/or mesenchyme of the frontonasal mass. We chose comparable time points to the Nile blue assay and added in a later timepoint in order to determine whether there was recovery from the initial spike in cell death caused by SU5402. After 6 hours of treatment we observed increased apoptosis in the lateral mesenchyme of the F N M in cranially-implanted embryos (Table 2.3; Fig. 2.7B,D). However, 16 hours after cranial bead implantation, the mesenchyme in the superior regions has returned to normal levels of cell death, while the levels of apoptosis was still higher than caudally or D M S O -treated embryos in the middle and inferior region of the F N M (Table 2.3; Fig. 2.7D,H). In cranially treated embryos we also observed increased apoptosis in the medial region of the frontonasal mass, extending from the caudal edge of the frontonasal mass into the future central chondrogenic region (MacDonald et a l , 2004; Matovinovic and Richman, 1997). The increased apoptosis should predict a truncation of the prenasal cartilage, however we never observed this phenotype. This suggested that the increased level of cell death was not severe enough to impact prenasal cartilage development. We did not observe increased apoptosis in the epithelia surrounding the frontonasal mass (Table 2.3). 2.3.7 Position specific effects on expression of signals required for facial fusion We expected to see decreased proliferation, and increased cell death, but the magnitude of these changes were much smaller than we had observed previously in our lab that were also associated with morphological defects (Ashique et a l , 2002; Song et a l , 2004). Therefore we hypothesized that the cleft phenotype caused by cranial SU5402 treatment is caused by the accumulated effects of decreased proliferation, as well as alterations in specific downstream differentiation signals. To characterize the molecular mechanism underlying cleft phenotype, we decided to look at the expression of other epithelial signals to determine which other pathways may be affected by antagonism of FGF signaling. We characterized gene expression changes 6 hours, (before the bead is exhausted and morphological changes become apparent) and 16 hours (after the bead is exhausted, and to determine if early gene expression loss recovers) after bead 49 implantation. We initially looked at the expression of FgfS and found that there was no change in the expression of the intronic or exonic form of FgfS; 6 or 16 hours after bead implantation (Table 2.4; Fig. 2.8A). No change in the expression of FgfS suggested that the level of FGF signaling in the F N M mesenchyme does not maintain a feedback loop with FGF ligands in the epithelium. The role of epithelial to mesenchymal signaling is necessary for normal facial development, based on work from our lab and others we hypothesized that we had also affected the B M P and SHH signaling pathways. Removal of ectoderm that expresses Shh in the globular process or sequestering Shh using beads soaked in anti-Shh antibody at stage 26 induced a cleft lip (Hu and Helms, 1999). Irregardless of bead position, SU5402 bead treatment had no effect on the stomodeal ectoderm expression domain of Shh either 6 or 16 hours after bead implantation (Table 2.4; Fig. 2.8B). The application of beads soaked in Noggin (a B M P antagonist) in the globular and maxillary process induced cleft lip phenotype (Ashique et al., 2002). Similarly, the Nestin-cre driven excision of BMP4 leads to the development of a cleft upper lip in mouse embryos (Liu et al., 2005b). Together, these studies suggested that the loss of B M P signals induced a cleft lip. Dmp4 expression was slightly less abundant in the zone of fusion at the corner of the F N M in cranially treated embryos within 6 hours of bead implantation (n=5/5; Fig. 2.8D), we also observed decreased Bmp4 expression near the bead, but not reduced within the whole domain in caudally treated embryos, (n=5/6; Fig. 2.8E). The Bmp4 expression in cranially treated embryos has recovered to normal levels by 16 hours with both bead positions (n=5/5; Fig. 2.8F,G). Although other studies have implicated B M P and Shh signaling as causative in C L / P in chicken and mouse embryos, we concluded that they are not contributing to our cleft phenotype. Thus we looked at other signaling molecules that are strongly linked to clefting and also expressed in the epithelium near the fusing upper lip. Non-syndromic C L / P in A/WySn mice has been linked to the clfl loci. The clfl locus is located on mouse chromosome 11 and includes the coding region of Wnt9b, a non-canonical Wnt. Wnt9b is expressed in the fusing upper lip in mouse embryos (Lan et al., 2006). The Wnt9b knockout exhibits incompletely penetrant non-syndomic C L (Carroll et al., 2005). Crossing a Wnt9b heterozygous A/WySn null sire with a A/WySn dam, significantly increased the prevalence non-syndromic clefting in embryos (Juriloff et al., 2006). With this strong link to non-syndromic C L / P in mice we characterized the expression of Wnt9b, in our chicken C L / P model and found that there is no change in Wnt9b transcript in cranially treated embryos (n= 15/15). In caudally treated embryos Wnt9b expression was normal 6 hours after treatment 50 (n=5/5) however at 16 hours, we observed decreased Wnt9b expression (n=5/8). Since we only observed a change in caudally-treated embryos, that do not go on to develop a cleft, suggested that the effects on Wnt9b expression are indirect, and do not play a role in our cleft phenotype. A non-canonical WNT, Wnt5a, is heavily expressed in the mesenchyme underlying areas of FGF expression including the limb, maxillary, mandibular, and the lateral nasal prominences (Yamaguchi et a l , 1999). The face is severely truncated in WntSa knockouts, including the loss of the premaxillary and maxillary bones (Yamaguchi et a l , 1999). We observed that the F N M in cranially implanted embryos downregulated the expression of Wnt5a near the bead (6 hr, n=5/6; 16 hr, n=3/3, Fig. 2.9B,D). In response to the caudal bead, the mesenchyme near the bead has reduced Wnt5a expression (Fig. 2.9C,E). M s x l and Msx2 are transcription factors that are downstream of both FGF and B M P signaling. Individual knockouts of Msxl or Msx2 have defects in craniofacial development, including skull and face abnormalities resulting from smaller bones while the compound homozygous double knockouts of Msxl and Msx2 have even more severe facial phenotypes (Ishii et a l , 2005; Satokata et a l , 2000; Satokata and Maas, 1994). The Msxl/Msx2 double knockouts, have severe defects in the facial development including severe reduction in the size of facial bones and cleft lip. The cleft lip results from the failure of fusion of the lateral nasal, medial nasal and maxillary prominence, because the prominences failed to attain the appropriate size for fusion to occur (Ishii et a l , 2005). Msxl/2 is induced by the application of BMP4 soaked beads to mandibular cell culture (Vainio et a l , 1993); similarly the expression of Msx2 is absent in the mandible of Nkx2.5 c r e driven Bmp4 knockout mouse, while Msxl is still expressed in distal areas of the mandible (Liu et a l , 2005a). The Nkx2.5 c r e driven Bmp4 knockout mouse also has a cleft lip (Liu et a l , 2005b). FGF is required to maintain the expression of M s x l in the limb after A E R removal (Vogel et a l , 1995). FGF8- and Bmp2-soaked beads induced ectopic expression of Msxl (data not shown) (Barlow and FrancisWest, 1997). Cranially-treated SU5402 embryos decreased the expression of Msxl, 6 and 16 hours after bead implantation (6 hours, n= 4/4; 16 hours, n= 4/4; Fig. 2.10B,C). Surprisingly, we only observed a subtle decrease in Msxl expression in the globular process with the caudal bead position (6 hr n= 6/10; 16 hr n=5/5; Fig. 2.10D,E). The cranial bead position decreased the globular process expression domain of Msx2 (6 hrs, n=8/8; 16 hours, n= 5/5; Fig. 2.10G,H), while smaller decreases are observed with the caudal bead position, even when the bead was within the expression domain (6 hrs, n=7/7; 16 hours, n= 7/7; Fig. 2.10I,J). This indicated that other signals are maintaining expression of Msx2 in the globular process. These positional data suggest the expression of 51 Msxl and Msx2 in the cranial regions of the frontonasal mass is regulated by FGF signals, and the globular process expression domain is regulated by B M P signals. We observed that the cranially-treated SU5402 embryos also decreased Bmp4 expression, which may have resulted in a more significant loss of Msxl transcripts. Bmp4 has recovered in the globular process by 16 hours, when we still observed decreased Msxl, and Msx2 expression in cranially-treated embryos. The differential changes in gene expression in the globular process with respect to the position of the SU5402 bead suggested that FGF signaling directly in the zone of fusion is functionally different than the signals in the cranial frontonasal mass. 2.3.8 Dlx5, an olfactory marker, is decreased in cranial treatment. The cranial bead is placed in the mesenchyme adjacent to the olfactory epithelia. We have shown that the olfactory epithelium expresses FGF ligands, and are targeted by cranial SU5402 treatment, which results in malformed the nasal conchae. We hypothesized that we were affecting the development of the nasal pits with our cranial bead treatment. We chose Dlx5 as a marker for the nasal pit because it is strongly expressed in the olfactory epithelia, the olfactory bulb, and it also defines the mesenchyme within the globular process. We observed a subtle decrease in both the olfactory epithelium and globular process domains within 6 hours of treatment, the decrease was even more obvious 16 hours after treatment (6hr: n=3/5; 16hrs: 6/6 Fig. 2.1 IB,C). While the caudal bead only disrupted the globular process expression domain (Fig. 2.1 ID, E). The loss of Dlx5 expression in the globular process likely reflects its transcriptional control by FGF signaling. These data suggest that we have targeted the olfactory epithelium with our cranial bead implants and that signals from the olfactory epithelium are required for gene expression in the zone of fusion. 52 2.4 DISCUSSION: Here we have shown a novel role for FGF signaling in the frontonasal mass, and provided evidence that the size and volume of the frontonasal mass is controlled by FGF signals in the cranial F N M , and this is disrupted in our cranially-treated embryos preventing the physical association of the facial prominences which resulted in a cleft lip. This also implies that FGF signals from areas outside the zone of fusion have essential roles in upper lip development. We have documented crosstalk between FGFs, BMPs, and non-canonical Wnt signaling and suggest that alterations in any of these players contributed to abnormal upper lip development. 2.4.1 FGF signals are required in the frontonasal mass contribution to the upper lip. Our data provides the first experimental evidence that FGF signaling in the frontonasal mass is required for the development of a continuous upper lip. First we showed the nested expression domains of FGF ligands (FgfS and FgflO), and FGF signaling modulators (Spryl, Spry2, Spry4 and Pystl) within frontonasal mass contribution to the zone of fusion, while the same molecules are absent from the maxillary component. Secondly we showed that beads soaked in the FGF signaling inhibitor, SU5402, and implanted in the lateral frontonasal mass induced a cleft lip while those implanted in the maxillary prominence developed normally. Furthermore we showed that the FGF signals from the cranial frontonasal mass are required for normal upper lip development. Importantly, these data suggested that tissues not directly within the zone of fusion contribute to normal lip fusion. Loss of FGF signaling in the cranial-bead treated embryos not only increased cell death and decreased proliferation, but changed gene expression within the zone of fusion, approximately 600 microns caudally. Our results compliment the increased apoptosis within the entire limb mesenchyme observed with the apical ectodermal ridge specific conditional knockout of FgfS (Moon and Capecchi, 2000). These results suggest that the signaling from FGF ligands act over a long distance from where they are produced. It also suggests that the likely source of FGF ligands in the face, the invaginating nasal pit, has dual roles, first to develop into a functional olfactory epithelia, and secondly to provide patterning information to the face. 2.4.2 Role of the nasal pit in the context of upper lip development The olfactory placode develops from non-neural ectoderm and requires the interactions of multiple transcription factors, and the presence of frontonasal mass neural crest cells (Bailey et al., 2006). Our cranial bead position lies in the mesenchyme adjacent to the olfactory 53 epithelium. The olfactory epithelium expresses at least two FGF ligands, and maintains the proliferation of a niche of stem cells that are partially defined by the co-expression of intronic FgfS and Sox2 (Kawauchi et a l , 2005). Using FoxGl-cre to knock out the olfactory and facial epithelial expression domain of FgfS results in embryos that have malformed nasal cavities and reduced expression of olfactory markers, at early stages of olfactory development (Kawauchi et a l , 2005). The embryos that survive until birth, have a severely misshappen and reduced upper jaw, with agenesis of the mandible and external ear (Kawauchi et a l , 2005). Careful examination of the development of the olfactory epithelia suggests that the abnormal olfactory development resulted from increased apoptosis in the neurogenic stem cell domains that are defined by the Fgf8 morphogenetic center (Kawauchi et a l , 2005). We hypothesized that the FgfS expression domain near the olfactory epithelia is acting as a morphogenetic centre for the frontonasal mass mesenchyme as well as providing stem cells to the olfactory epithelia. Together, our evidence suggests that FGF derived signaling from the nasal pit acts over a long distance (600 microns) to pattern F N M derivatives. Although we did not observe any changes in the expression of FgfS itself, in the cranially treated embryos, the loss of FGF-mediated signaling induced the loss of another survival factor: Wnt5a. Concomitantly, we observed increased apoptosis and decreased proliferation in the same area which also lost Wnt5a expression, ultimately leading to reduced number of cells within the lateral frontonasal mass. Decreased cell number could lead to one of two consequences, one being the globular process unable to meet the maxillary process resulting in a cleft, and second a loss of progenitor cells for the premaxillary bone, a frontonasal mass derivative. 2.4.3 Frontonasal mass and maxillary derivatives are affected in the cleft embryos: The fate map of the face has been created using two experimental paradigms: by grafting facial prominences to the limb bud and following which cartilages and bones undergo nearly normal morphogenesis (Lee et a l , 2004; MacDonald et a l , 2004; Richman and Tickle, 1989) or by the injection of a fluorescent-fixable dye, C M - D i l , into the maxillary prominence and observing which bones and cartilages are labeled (Lee et a l , 2004). When grafted to the limb bud, frontonasal mass mesenchymal cells differentiate into the prenasal cartilage, while the cells of the lateral nasal prominence gave rise to the nasal conchae. However, the data is incomplete because the grafts were only stained to show cartilage morphology (Lee et a l , 2004; MacDonald et a l , 2004; Richman and Tickle, 1989). Injections of Dil into the maxillary prominence labeled the body of the palatine bone, the maxillary bone, the jugal and the quadratojugal, while the 54 premaxillary bone and the prenasal cartilage were not labeled (Lee et.al., 2004). In the cleft embryos, we observed reductions in the processes of the premaxillary bone, the maxillary bone and the palatine bone, while the body of the bones developed to approximately normal size. The size of the bones was unaffected because the shape of the body of the bones is determined before the SU5402 treatment. Together with the bead implants at stage 20, we would like to suggest that pattern is likely established before stage 20. Regardless of bead position, in nearly every embryo treated with a SU5402-soaked bead in the frontonasal mass (93%; n=52/56), we observed reductions in the nasal process of premaxillary bone, while we never observed premaxillary bone defects with the maxillary prominence implants. Our frontonasal mass implant data supports the fate mapping experiments in the origin of the premaxillary bone. We collected some embryos 48-hours after bead implantation (data not shown), observed a failure of fusion and a cleft between the frontonasal mass and maxillary prominence. Often in cleft embryos, the maxillary bone is proximalized, a direct result of the failure of fusion between the frontonasal mass and maxillary prominence. The proximalized maxillary bone results from decreased forces in the zone of fusion, which we hypothesize should have pulled the maxillary bone distally, and perhaps sculpted the mesenchymal condensation into the correct shape. The connective tissue between the maxillary and premaxillary bones was partially labeled with D i l , suggesting the maxillary prominence provides some of the connections between the frontonasal mass and maxillary prominence (Lee et al., 2004). The palatine bone was also affected in cleft embryos, we suggest that the differentiation and development of the maxillary process of the palatine bone is tied closely to the articulation with the maxillary bone, and defects in the maxillary process of the palatine bone arise secondarily to the cleft. FGF signaling is required within the frontonasal mass (and is affected equally by either the cranial or caudal bead position) for the patterning of the nasal and body of the premaxillary bone. The mechanism underlying the FGF signaling derived patterning events may rely on cell survival cues within the mesenchyme. We observed increased cell death and decreased cell proliferation, which may have decreased the survival of skeletogenic precursors. We propose that the osteogenic precursors in the frontonasal mass provide enough cells to make up the mesenchymal condensations, so that correct form can be elucidated, and secondly, the correct complement of cells provided the correct volume to the frontonasal mass. When both of these requirements are met, then normal upper lip development occurs. However, in the cranially treated embryos, we observed a smaller frontonasal mass by 16 hours of treatment, decreased proliferation in the cranial two-thirds of the frontonasal mass and decreased Wnt5a expression. 55 The area of decreased proliferation coincides with the area that we observed decreased Wnt5a expression and the area designated as a 'facial proliferative' centre (Peterka and Jelinek, 1983). The original Wnt5a knockout phenotype was described as the loss of osteogenic precursors resulting in the shortening of structures that needed to grow out (Yamaguchi et a l , 1999). We would like to propose that the cleft phenotype resulted from the loss of the facial proliferative center and prevented the frontonasal mass from attaining the correct size. The reason that embryos treated in the globular process developed normally is that FGF signals in the F N M are required for cell proliferation, and the globular process is naturally devoid of proliferating cells. For lip fusion to occur normally, you might suppose that the regions of the face that must fuse would be proliferating to ensure that they make contact, this is not the case however. We propose that the cranial two-thirds of the frontonasal mass displaces the globular process so that the fusing facial prominences will meet within the zone of fusion. We propose that the size of the FGF dependent zone is controlled by crosstalk between FGF and non-canonical Wnt signaling. The SU5402 treatment induced apoptosis of skeletogenic precursors resulting in reduced numbers of cells contributing to the nasal and body of the premaxillary bone. Taken together, our data suggested that it in this lip fusion model there is crosstalk between FGFs, and non-canonical W N T signaling that cooperate for normal lip fusion. We observed decreased bone and cartilage staining in the facial micromass cultures, which are in contrast to those found in chicken limb micromass cultures treated with a slightly higher concentration of SU5402 (800 ng/ml vs. 750 ng/ml) (Montero et a l , 2001). The limb micromass cultures were initiated from stage 25 progress zone chicken limb buds, and cultured for 5 days in serum free media. These cultures do not include any epithelial cells, so there is no source of Fgf8 in them. At stage 25, FgflO is expressed quite highly in the mesenchyme of the progress zone, and to a lesser extent in the mesenchyme of chicken or mouse face (Fig. 2.1D,E; (Bachler and Neubuser, 2001; Havens et a l , 2006)). The presence of FgflO expressing cells in the limb bud micromass cultures may account for the different response to SU5402. The other technical difference between the two cultures is that we treated the cultures after an hour of spotting them, while the limb cultures were treated the 24 hours later. We cultured our facial micromass spots for 8 days in serum-containing media and stained for both alkaline positive and alcian blue positive cells, while the limb cultures were cultured in serum-free media and only stained for cartilage differentiation. These technical differences likely explain the differences in culture staining. 56 2.4.4 Chicken as a model for CL/P The embryonic chicken face develops in a similar way to mouse and human embryos, allowing it to be used for comparative studies. The chicken genome has been published and chicken cDNAs are available for purchase (Boardman et al., 2002; Consortium, 2004). Our experiments presented here took advantage of the easily accessible chicken embryo face to alter the signals specifically within the fusing upper lip, and allowed us to test the requirement of FGF signaling within an individual prominence. We then characterized the resulting morphological changes. Although the chicken embryo face has a slightly different conformation of facial prominences, the part that was most affected by our treatments: the lateral edges of the frontonasal mass, has very high cell density and higher proliferative rate than the medial portions of the F N M making it equivalent to the medial nasal prominences of mouse embryos. The maxillary prominence, contributes to the same bones as in other amniotes (Cerny et al., 2004; Lee et al., 2004; Richman et al., 2006). In the data that we presented here, the cleft phenotype ranged in severity, suggesting that the chicken embryos is modeling human C L / P and can recapitulate the morphological variations in human C L / P In humans, the autosomal dominant version Kallmann syndrome is caused by a loss of function mutation in FGFR1 (Dode et al., 2007; Dode et al., 2003). FGFR], is expressed while FGFR2, and 3 are not expressed in the fusing facial prominences in mouse and chicken embryos (Hebert et al., 2003; Wilke et al., 1997). The data presented here suggested that loss of FGF-mediated signaling in the F N M elicits a cleft phenotype. In the cleft chicken embryos, we predominantly created a loss of function of FGFR1 because Fgfrl is primarily expressed where the SU5402 bead was placed (Wilke et al., 1997). The data presented here provides the first experimental evidence that the cleft lip within autosomal dominant Kallmann syndrome could result from the loss of FGFR function. We have shown here that loss of FGF signaling in the cranial regions of the F N M resulted in decreased abundance of transcript in the expression domains of Msxl, Msx2, and Spry2. Screening populations of humans that have non-syndromic or typical C L / P found loss of function variants in each of these three genes, and we have provided here experimental evidence to link these genes to a cleft-lip (Jezewski et al., 2003; Suzuki et al., 2004; Vieira et al., 2005). Not only have downstream genes been implicated by human population screening, a second study has identified missense mutations in FGF8, FGFR1, FGFR2, and FGFR3 in cleft populations (Riley et al., 2007). Extrapolating from our work, would suggest that these mutations likely do play a causative role in the development of non-syndromic C L / P , and 57 judging by our variability in the cleft population, we propose that FGF signaling is required for normal development of the upper lip in both chicken and human embryos. 2.4.5 Model for facial fusion: We propose that facial fusion occurs through orchestration of several signaling pathways that are controlled by the cranial FGF signals. Furthermore, the FGF signals, from the olfactory epithelia are more active in the cranial frontonasal mass, while they are not required in the globular process or the maxillary prominence (Fig. 2.12A). In the cranial frontonasal mass FGF signals controlled the expression of transcription factors that allow proliferation to occur, while preventing aberrant cell death, which then enabled the F N M to attain the correct size for fusion (Fig. 2.12B). In the globular process, application of a Noggin-soaked bead, induced a cleft phenotype, and decreased proliferation in the frontonasal mass mesenchyme, while maintaining epithelial cell survival (Ashique et a l , 2002). This suggests that proliferation in the frontonasal mass, is controlled cranially by FGFs and caudally by BMPs (Fig. 2.12B). We showed that FGF signaling regulated the size of the 'proliferative centre' of the frontonasal mass rather than the tissues within the globular process. This suggested that the globular process does not act as the leading edge for contact and fusion. Instead, the globular process sits on top of the 'proliferative centre' and contacts the maxillary prominence passively, if the frontonasal mass is the correct size (Fig. 2.12C). This model suggests that growth centres outside of the zone of fusion contribute to clefting. 58 59 Figure 2.1: Spry J, 2, and 4 are expressed overlapping domains in the mesenchyme underlying the epithelial FgfS and FgflO expression within stage 20 and Stage 28 frontonasal mass. (A,B) Wholemount in situ hybridization to FgfS ( C - N ) Radioactive section in situ hybridization. Stage 20 frontal sections (D, G, J, M ) , stage 28 (E, H , K, N), and stage 28 globular process (C, F , I, L). (A, B, C) FgfS expression: (A) At stage 20, FgfS is expressed in the commissural plate and the frontonasal mass ectoderm including the medial edges of the. invaginating nasal pit. FgfS is also expressed in the caudal maxillary prominence and the cranial mandibular prominence, but not the lateral nasal prominence. (B) FgfS expression becomes restricted to the surface ectoderm surrounding the nasal pits just prior to lip fusion at stage 27, while FgfS is maintained in the caudal maxillary prominence and caudal mandibular prominence. (C) Within the zone of fusion FgfS is not expressed in the epithelium of the fusion zone including the globular process contribution or the maxillary prominence contribution. (D, E , F) FgflO expression: (D) At stage 20, FgflO is expressed in the invaginating nasal pit, and the neuroepithelium. (E) At stage 28 FgflO is expressed in the nasal pit epithelium, and the lateral mesenchyme of lateral nasal prominence and maxillary prominence. (F) FgflO is not present in the epithelium of the globular process in the zone of fusion (from box in E). (G, H, I) Spryl expression: (G) Spryl is expressed in the mesenchyme of the lateral edge of the frontonasal mass and the medial edge of the lateral nasal prominence, and the neuroepithelium of the dorsal telencephalon at stage 20. (H) At stage 28, Spryl is expressed in the medial lateral nasal prominence, extending from the cranial nasal pit to the groove between the lateral nasal prominence and maxillary prominence. In the stage 28 frontonasal mass, Spryl expression extends from the cranial nasal pit to the cranial margin of the globular process. Spryl is absent from the cranial maxillary prominence. (I) As the maxillary and frontonasal mass approximate with each other, Spryl expression is excluded from the globular process of the F N M ; and the most cranial portion of the mxp (box from H). (J, K, L) Spry2 expression: (J) At stage 20, Spry2 is expressed in the mesenchyme of the frontonasal mass and the nasal pit epithelia. (K) At Stage 28 the frontonasal mass mesenchyme expresses Spry2 from the cranial nasal to the globular process. At stage 28, Spry2, extends along the caudal edge of the frontonasal mass almost to the midline. (L) The globular process strongly expresses Spry2, while the apposed mxp has significantly less expression (box from K). (M, N ) Spry4 expression: (M) At stage 20 Spry4 is expressed in the lateral nasal and frontonasal mass mesenchyme. ( N ) . Spry4 expression is present near the cranial nasal pit and is excluded from the zone of fusion. 60 Key: Scale bars: A , B = 0.5 mm; C, D, F, G, J, I, L , M , = 100 urn; E, H , K, N , = 200 u.m. cp, commissural plate; fnm, frontonasal mass; gp, globular process; lnp, lateral nasal process; mdp, mandibular prominence; mxp, maxillary prominence; np, nasal pit; tel, telencephalon; 61 Figure 2.2: Loss of F G F signaling in the frontonasal mass induces a cleft beak. Skeletal morphology of embryos treated with 1G mg/ml SU5402 at stage 26 (A-F) and stage 20 (G-I). (A, B) Stage 26 embryos implanted with beads soaked in lOmg/ml SU5402 (or D M S O soaked, carrier control) beads in the globular process of the frontonasal mass have normal skeletal morphology. (C-F) Stage 26 embryos implanted with beads soaked in 10 mg/ml SU5402 and placed in the cranial bead position. (C) The embryo has an external notch in the upper beak (arrowhead). (D) Underlying the notch, the cleft occurs between the premaxillary and maxillary bone (arrowhead). The maxillary bone is also proximalized, and the nasal conchae are malformed. (E) The palatal view of the same embryo, truncations in the processes of the premaxillary, maxillary (arrowhead) and palatine bone. (F) Schematic view of the palate. The upper half matches the typical cranially-implanted SU5402 morphology, while the lower half has normal morphology. (G-H) SU5402 implantation in the cranial frontonasal mass of stage 20 embryos. (G) The nasal bone is reduced (asterisk). (H) The palate develops normally. Key: Scale bars: A = 5 mm and applies to B, D-H; C = 5 mm. e, eye; ios, intraorbital septum; j , jugal; md, mandible; mpp, maxillary process of palatine bone; mppmx, maxillary process of the premaxillary bone; mxb, maxillary bone; n, nasal bone; nc, nasal conchae; p, palatine; ppmx, palatine process of maxillary bone; pppmx, palatine process of premaxillary bone; pmx, premaxillary bone; pnc, prenasal conchae; 63 Figure 2.3: Reduced staining in micromass cultures treated with SIJ5402. Tota l ce l l n u m b e r af ter 8 d a y s of m i c o m a s s cu l t u re « 400 § 350 J 300 1 250 i (A S 200 IM x 150 2 100 CL <fl 1 50 a U n Control Media 2.5 jiM SU5402 Treatment 5 ng/ml FGF8b Sprouty2 is d e c r e a s e d in m i c r o m a s s c u l t u r e s t rea ted w i t h S U 5 4 0 2 180 160 c 0 « w o a X o © > 80 a 3 a: 140 120 100 6 ; 40 J 20 | I 01 Control media D 2.5 M M SU5402 Treatment 5 ng/ml FGF8b Figure 2.3: Reduced staining in micromass cultures treated with SU5402. (A, B), Alcian blue and alkaline phosphatase staining of 8-day high density micromass cultures treated with carrier control media (A) and 2.5 u M SU5402 (B). (C) Total cell number within each treatment is the same regardless of treatment (Students, t-test, p <0.05). (D) Spry2 expression is reduced in the SU5402 treated cultures compared to the carrier treated controls. The realtime data has been normalized to 18S R N A . Scale bar = 1 mm 64 Figure 2.4: The expression of FGF signaling modulators in F N M responds are decreased differentially in response to the bead positions. (A-E) Sprouty4, (F-J) Pystl, (K-O) Sprouty2. 16 hour D M S O treated embryos (A, F, K), 6 hours after beads soaked in 10 mg/ml SU5402 are implanted into the caudal and cranial F N M (B, D, G, I, L , N) 16 hours after beads soaked in 10 mg/ml SU5402 are implanted into the caudal and cranial F N M (C, E , H, J , M , O). Spry4 is only expressed in the cranial F N M , fails to be maintained both 6 and 16 hours after cranial bead implantation (B,C). Sprouty4 is unaffected in the caudal bead implantation (D, E). Pystl expression is decreased in the lateral F N M (from the cranial nasal pit to the globular process) in response to cranial beads soaked in 10 mg/ml SU5402 (G, H). Compared to the small decrease in expression near the caudal bead (I, J). Spry2 is expressed in the greatest area in the frontonasal mass of the FGF signaling modulators, and fails to be maintained in the lateral frontonasal mass from the cranial nasal pit, caudal to the globular process and extending medially along the caudal edge 6 hours after cranial 10 mg/ml SU5402 (L). Expression is recovering by 16 hours of cranial bead treatment (M). Only the globular process and caudal edge expression zone fails to be maintained with the caudal bead treatment 6 and 16 hour after surgery (N, O). Key: Scale bar = 0.5 mm. fnm, frontonasal mass; Inp, lateral nasal process; mdp, mandibular process; mxp, maxillary process. . 66 Figure 2.5: SU5402 reduced the levels of phosphorylated M A P K 6 hou r Figure 2.5: SU5402 reduced the levels of phosphorylated M A P K E m b r y o s w e r e s t a ined u s i n g the p r o t o c o l ( C o r s o n et a l , 2 0 0 3 ) . T h e s i g n a l w a s de tec ted u s i n g h o r s e r a d i s h p e r o x i d a s e w i t h D A B de t ec t i on . N o p r i m a r y a n t i b o d y (A); C r a n i a l S U 5 4 0 2 (B); C a u d a l S U 5 4 0 2 ( C ) . 67 Figure 2.6: Cel l Proliferation is reduced in the cranially treated frontonasal mass Region of the frontonasal mass counted Figure 6: Cell proliferation is reduced within 12 hours of bead implantation in the cranial 2/3 of the F N M in cranially treated embryos. (A-C) The images of the BrdU labeled cells (green) were placed over the total cells (blue) in a representative composite sections used for the BrdU analysis. D M S O treated embryo (A); Caudally-implanted SU5402 treated embryo (B); Cranially-implanted SU5402 embryo (C). The zone of fusion has reduced proliferation (black arrowheads, and white lines). (D) The proliferation index of three regions of the frontonasal mass, compared using M A N O V A with Fisher's LSD (p<0.05) post hoc testing. We found that the cranial two-thirds of the frontonasal mass (i.e. middle and superior regions of the lateral F N M ) in cranially treated embryos have decreased cell proliferation compared to D M S O and caudally treated embryos. The caudal bead treatment did not reduce cell proliferation in the inferior region of the frontonasal mass. The caudal region of the D M S O treated frontonasal has less proliferation than the superior region of the D M S O treated frontonasal mass. Key: Scale bar:= 0.5 mm; fnm, frontonasal mass; lnp, lateral nasal prominence; mxp, maxillary prominence 69 Figure 2.7: Cell death is increased in the cranially treated frontonasal mass 70 Figure 2.7: Increased cell death with SU5402 treatment. (A, B, E , F): Cell death was assayed in whole embryos using Nile Blue vital dye staining. We compared the treated side to both D M S O treated control embryos and the untreated contralateral side. Cranial bead treatment increased cell death in the frontonasal mass from the cranial nasal pit into the globular process (A, white arrowheads). Caudal bead treatment induced increased cell death near the bead (B, white arrowheads). Normal areas of cell death are present in the groove between the lateral nasal prominence and maxillary prominence (black arrow). Few apoptotic cells are normally observed in the frontonasal mass (contralateral side). Nine hours after cranial bead treatment, cell death is induced in the same area as observed at 6 hours, but also extends cranially into the mesenchyme ventral to the telencephalon (E, arrowheads). Caudal beads induced cell death near bead (F). (C, D, G, H): Apoptotic cells were quantified in tissue sections using T U N E L (Table S3). Boxes in C, D, G, and H , are shown magnified in C , D ' , G ' , H ' . Many apoptotic cells were found in the mesenchyme 6 hours after bead implantation (C, C , white arrowheads). Few apoptotic cells are observed in the mesenchyme of the frontonasal mass after caudal bead treatment (D). Increased apoptosis is still observed in the malformed fnm 16 hours after cranial bead treatment (G). Caudal bead treatment does not increase apoptosis in the frontonasal mass mesenchyme (H). Key: Scale bars: A = 0.5 mm, applies to B, E, F; C= 0.1 mm and applies to D, G, H; C =0.1 mm and applies to D ' , G ' , FT; fnm, frontonasal mass; lnp, lateral nasal prominence; np, nasal pit; 71 Figure 2.8: FgfS and Shh expression are not affected in cranially treated embryos, while Bmp4 is downregulated for a short time DMSO 6 hours 16 hours Figure 2.8: FgfS and Shh expression are not affected in cranially treated embryos, while Bmp4 is downregulated for a short time W h o l e m o u n t in situ h y b r i d i z a t i o n for the e x p r e s s i o n o f e p i t h e l i a l l y secre ted s i g n a l i n g m o l e c u l e s , FgfS (A), Shh (B) a n d Bmp4 ( C - G ) . N e i t h e r FgfS n o r Shh e x p r e s s i o n are c h a n g e d w i t h the c r a n i a l o r c a u d a l bead t reatment at e i ther 6 o r 16 hours after i m p l a n t a t i o n (A, B). Bmp4 e x p r e s s i o n fa i l s to be m a i n t a i n e d at 6 hour s after bead treatment. D M S O treated e m b r y o 6 hours after bead i m p l a n t a t i o n (C). C r a n i a l bead treatment reduces the a b u n d a n c e of t ranscr ip t i n the g l o b u l a r p rocess e x p r e s s i o n d o m a i n (D), w h i l e e x p r e s s i o n is absent near the c a u d a l b e a d p o s i t i o n , but is e xp r e s se d at n o r m a l l e v e l s c r a n i a l to the bead , and the m a x i l l a r y p r o m i n e n c e (F). Bmp4 e x p r e s s i o n has r e c o v e r e d to n o r m a l l eve l s 16 hour s after bead i m p l a n t a t i o n (E, G) . K e y : S c a l e bar =0.5 m m . f n m , f ron tonasa l mass ; l n p , la tera l nasa l p r o m i n e n c e ; m d p , m a n d i b u l a r p r o m i n e n c e ; m x p , m a x i l l a r y p r o m i n e n c e . 72 Figure 2.9: The expression of a non-canonical Wnt, WntSa, is decreased in response to SU5402 treatment Figure 2.9: The expression of a non-canonical Wnt, WntSa, is decreased in response to SU5402 treatment WntSa is h e a v i l y expressed in the f ron tonasa l mass , a n d its e x p r e s s i o n is not m a i n t a i n e d i n response to the loss o f F G F s i g n a l i n g . D M S O t reatment has no affect o n WntSa e x p r e s s i o n ( A ) . C r a n i a l bead t reatment reduces WntSa i n an area s u r r o u n d i n g the bead 6 a n d 16 hours after i m p l a n t a t i o n , ( w h i t e a r r o w s ; B , C ) . T h e c a u d a l bead o n l y reduces WntSa e x p r e s s i o n c lo se to the bead ( a r r o w s ; D , E ) . K e y : S c a l e bar = 0.5 m m . f n m , f rontonasa l mass ; l n p , la tera l nasa l p r o m i n e n c e ; m d p , m a n d i b u l a r p r o m i n e n c e ; m x p , m a x i l l a r y p r o m i n e n c e . 73 Figure 2.10: Msxl and Msx2 expression fails to be maintained in response to SU5402 treatment. 74 Figure 9: Msxl and Msx2 expression fails to be maintained in response to SU5402 treatment. Whole mount in situ hybridization with antisense probes to Msxl (A-E) and Msx2 (F-J). D M S O treatment does not affect Msxl expression (A). Msxl expression is not maintained in the area from the nasal pit to the globular process within 6 hours of bead implantation (B). The expression domain is still decreased 16 hours after cranially-bead treatment (C). The caudal bead decreases expression near the bead 6 and 16 hours after implantation (D, E). The cranial bead position reduced the globular process expression domain of Msx2 within six hours of treatment and it failed to recover by 16 hours (G, H). Caudal bead treatment reduced regions of the expression of Msx2 near the bead, but not throughout the whole expression domain (I, J). The cranial bead treatment induced more significant downregulation of Msx2 compared to the caudal bead, even though the caudal bead was placed directly in the expression domain. Key: Scale bar = 0.5 mm, applies to all; fnm, frontonasal mass; gp, globular process; lnp, lateral nasal process; mdp, mandibular prominence; mxp, maxillary prominence. 75 Figure 2.11: Dlx5 is decreased in the olfactory epithelial domain Cran ia l Caudal Figure 2.11: DlxS is lost in the olfactory epithelial domain Dlx5 is e x p r e s s e d i n the o l f ac to ry b u l b , o l f a c t o r y e p i t h e l i a , a n d is used as a m a r k e r for the g l o b u l a r p rocess . W i t h i n s ix hours o f t reatment w e o b s e r v e d a subt le decrease i n the o l f a c t o r y e p i t h e l i a d o m a i n ( B , a r r o w ) , the d o w n r e g u l a t i o n is m o r e s i g n i f i c a n t w i t h i n 16 hour s o f t rea tment (C, a r r o w ) . T h e c a u d a l bead treatment o n l y affects the g l o b u l a r p rocess d o m a i n o f Dlx5 ( D , E). 76 F i g u r e 2.12: M o d e l for F G F s ignal ing in the fusing facial p rominences i k p r e s s i o n of F G F s igna l i ng modu la to r s FGF signals Msx1/2, WntSa BMP signals Msx1/2 C r a n i a l reg ions of the F N M are F G F - d e p e n d e n t a n d mainta in prol i ferat ion in the growth cen te r B Prol i ferat ion in the growth cen te r d r ives outgrowth of the g lobu la r p r o c e s s Most active FGF signals Most active B M P signals Proliferative centre Areas of lowest mSm proliferation Spry4, 1, 2 and Pystl Spryl, and Pystl, Spry2 m Pystl and Spry2 | \ Spry2 F i g u r e 2.12: M o d e l for F G F s igna l ing i n the fusing facial p rominences : ( A ) T h e nes ted e x p r e s s i o n o f F G F s i g n a l i n g m o d u l a t o r s (Spry], 2, 4 a n d Pystl) i n g reen , is greatest near the nasa l pi t . (B) T h e c r a n i a l r e g i o n s o f the f ron tonasa l mass are m o r e s ens i t i ve to F G F s igna l s t han the c a u d a l F N M , w h i l e the d i s t a l m a x i l l a r y p r o m i n e n c e does no t r equ i r e F G F s i g n a l i n g for upper l i p d e v e l o p m e n t (g reen r e g i o n s ) . In the c r a n i a l f ron tonasa l m a s s , F G F s i g n a l s m a i n t a i n e d the e x p r e s s i o n of Msxl/2 a n d Wnt5a, a n d the appropr ia te l e v e l o f c e l l p r o l i f e r a t i o n , w h i l e p r e v e n t i n g c e l l death. In the c a u d a l f ron tonasa l mass , B M P s i g n a l s m a i n t a i n e d the e x p r e s s i o n o f Msxl/2, and regula tes e p i t h e l i a l s u r v i v a l w i t h i n the z o n e o f fus ion ( A s h i q u e et a l , 2002). (C) In n o r m a l e m b r y o s , the c o m b i n a t i o n o f F G F a n d B M P s igna l s m a i n t a i n the co r rec t l e v e l o f p r o l i f e r a t i o n i n the m i d d l e o f f ron tonasa l mass to create e n o u g h v o l u m e i n the g r o w t h centre (green) w h i c h pushes the g l o b u l a r p roces s , an area o f l o w p r o l i f e r a t i o n (o range) w i t h l i t t le e x p r e s s i o n o f F G F s i g n a l i n g m o d u l a t o r s ( A ) , t o w a r d the s e c o n d area o f l o w p r o l i f e r a t i o n (peach) i n the d i s ta l m a x i l l a r y p r o m i n e n c e w i t h i n the z o n e o f fu s ion . 77 Table 2-1: Dose response to SU5402 implanted into the lateral frontonasal mass External Notch Maxil lary and palatine process of Premaxillary bone Palatine Process of the Maxi l lary bone Palatine bone Palatal view C o n c e n t r a t i o n o f S U 5 4 0 2 % w i t h externa] cleft % absent ( abnormal / to t a l ) % r e d u c t i o n in the s ize o f P P M X ( r e d u c e d / t o t a l ) a % r e d u c e d i n the l e n g t h o f the p rocess ( abnormal / to ta l ) 0.1 m g / m l 0 % (0 /12 ) 0 % (0 /12) 0 % (0 /12) 0 % (0 /12) 1.0 m g / m l 0% ( 0 /11 ) 0 % (0 /11) 9 % (1 /11) 0 % (0 /11) 5.0 m a / m l 0 % (0 /10 ) 0 % (0 /10) 3 0 % (3 /10) 2 0 % (2 /10) 10 m g / m l 2 9 % ( 1 3 / 4 5 ) 4 2 % (19 /45 ) 3 7 % ( 1 7 / 4 5 ) 3 1 % (14 /45) 2 0 m a / m l 6 7 % (6 /9) 7 8 % (7/9) 1 0 0 % (9 /9) 5 5 % (5/9) D M S O 0 % (0 /6) 0 % (0/6) 0 % (0/6) 0 % (0/6) a a l l o f the affected P P M X are a lso d i s l o c a t e d in this 78 Table 2-2: Cranial bead positioninduces a cleft beak Stage of surgery Initial location of bead External Notch Nasal Bone Nasal Conchae 26 26 26 A B C % with an external cleft 40% (18/45) 0% (0/16) 0% (0/15) % with reduction 0 % (0/45) 0 % (0/16) nd % misshapen 56% (25/45) 0 % (0/16) 0% (0/15) 20 lateral FNM 8% (1/13) 50% (11/22) nd Premaxillary bone % with reduction in the % missing either of size of the nasal process the maxillary or palatal and body of premaxilla process 26 26 26 20 A B C lateral FNM 93% (40/42) 86% (12/14) 0%(0/15) 86% (19/22) 62% (28/45)3 0%(0/16) 0 % (0/15) 36% (8/22) Palatine process of maxillary bone % with reduction % dislocated 26 26 26 26 20 A B C DMSO lateral FNM 60% (27/45)b 0 % (0/16) 0 % (0/15) 0 % (0/6) 36% (8/22) 44% (20/45) 0 % (0/16) 0 % (0/15) 0 % (0/6) 5% (1/22) ' i linn—IIM^ • — " r ..T :• Palatine bone _ ™ C ^ a ^ " ^ ' - ^ % with reduction 26 26 26 20 A B c lateral FNM 58% (26/45)c 0 % (0/16) 0 % (0/15) 18% (4/20) a - 6 are moderately reduced;b - 70% (20/27) of these ppmx are dislocated;0 - 10 are severely truncated 79 Table 2-3: mass Quantification of increased apoptosis in the frontonasal Region in FNM number of apoptotic cells 6 hours Cranial SU5402 bead position (n = 5) Caudal SU5402 bead position (n = 3) DMSO (n = 3) + Untreate d side (n = 10) 16 hours Cranial SU5402 bead position (n = 6) Caudal SU5402 bead position (n = 3) DMSO (n=2) + Untreate d side (n = 10) superior 0-5 cells 6-10 cells 11-50 cells 2 2 1 13 12 lateral edge of FNM mid 0-5 cells 6-10 cells 11-50 cells 1 2 2 12 1 11 1 inferior 0-5 cells 6-10 cells 11-50 cells 1 2 7 L 11 2 2 4 10 2 Medial FNM 3 0-5 cells 6-10 cells 11-50 cells 3 1 2 I Olfactory Epithelia 0-5 cells 6-10 cells 11-50 cells 3 2 11 3 1 a combined DMSO with Caudal SU5402 specimens for medial region, therefore n= 6 for 6 hour; n=5 for 16 hour 80 Table 2-4: Gene expression decreases along the cranial-caudal axis in response to the cranial bead position Expression Domain Expression Domain Gene Time I T x n a b c Gene Time I t x n a b c Cranial 5 -(4) Cranial 5 4(5) 6 hours Cauda l 4 + (4) 6 hours Cauda l 6 J. (6) Spry4 D M S O 4 + (4) Bmp4" D M S O 4 + (4) 16 hours Cranial Cauda l D M S O 3 10 7 - (3) + (10) + (7) o i c 16 hours Cranial Cauda l D M S O 5 4 3 + (5) + (4) + (3) + (5) + (4) + (3) Cranial 7 - (7 ) - (7 ) "(7) s Cranial 6 + (6) + (6) 6 hours Cauda l 7 + (7) + (7) - (7) t/i m 6 hours Cauda l 5 + (5) + (5) Spry2 D M S O 4 + (4) + (4) + (4) a. Wnt9b D M S O 3 + (3) + (3) 16 hours Cranial 10 1(10) - (10) - (10) Oi « c Ui 1 6 Cranial 7 + (6) + (8) Cauda l D M S O 6 5 + (6) + (5) + (6) + .5) 1(6) + (5) hours Cauda l D M S O 10 2 + (8) + (2) - (5) + (2) Cranial 9 J (8) K8) 0) Cranial 6 -(5) + (6) 6 hours Cauda l 6 + (6) - (6 ) 6 hours Cauda l 5 + (5) - (5) Pystl D M S O 2 + (2) + (2) 5 Wnt5a D M S O 4 + (4) + (4) 16 hours Cranial 5 J (5) ! (5) 16 hours Cranial 3 - (3) + (3) Cauda l D M S O 5 6 + (5) + (6) 4(4) + 16) Cauda l D M S O 3 1 + (3) + (3) - (3) + (3) Cranial 16 + (11) + i.V.) 6 hours Cauda l 20 + (20) + (20) FgfS D M S O 3 + (8) + (8) 16 hours Cranial 8 + (6) + (6) Cauda l 17 + (17) + (14) D M S O 2 + (2) + (2) Cranial 4 J 4 1 4 6 hours Cauda l 10 + (10) 4(6) Msxl D M S O 4 + (4) + (4) 16 hours Cranial 4 4 (4) 1(4) 1(4) Cauda l D M S O 6 6 + (6) + (6i 1(5) + (6) + (6) + (6) Cranial 8 . (8) 6 hours Caudal 7 1(7) Msxl D M S O 4 + (4) 16 hours Cranial 5 - (5 ) Caudal D M S O 7 6 1(7) + (6) Cranial 8 4(5) 4.(5) 6 hours Caudal 8 + (8) 4(5) Dlx5a D M S O 5 + (5) + (5) 16 hours Cranial Cauda l 6 7 J (6) + (7) 1(6) 1(7) D M S O 4 + (4) + (4) All embryos treated with beads soaked in 10 mg/ml SU5402 or D M S O vehicle control. 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Critical Reviews in Oral Biology & Medicine 11, 304-317. Zhang, S. B., Lin, Y. F., Itaranta, P., Yagi, A. and Vainio, S. (2001). Expression of Sprouty genes 1, 2 and 4 during mouse organogenesis. Mechanisms of Development 109, 367-370. Zhang, X., Ibrahimi, O. A., Olsen, S. K., Umemori, H., Mohammadi, M . and Ornitz, D. M . (2006). Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. Journal of Biological Chemistry 281, 15694-700. 88 Chapter 3: Tissue interactions between the nasal pit and facial mesenchyme suggest new roles for the nasal pit in craniofacial skeletal patterning Heather L. Szabo Rogers and Joy M . Richman Department of Oral Health Sciences, Life Sciences Institute, The University of British Columbia, Vancouver B C , Canada 2 A version of this chapter has been submitted for publication. Szabo Rogers H L , Whiting C, Fu K K and Richman J M Tissue interactions between the nasal pit and facial mesenchyme suggest new roles for the nasal pit in craniofacial skeletal patterning Development. 89 3.1 INTRODUCTION: 3.1.1 Development of the cranial placodes: At the boundaries of the neural plate in neurula stage embryos, two very different cell populations are being induced: the cranial neural crest cells and the cranial placode cells. The cranial neural crest cells are induced by ectodermal canonical W N T signaling at the border between neuronal ectoderm and surface ectoderm (Garcia-Castro et al., 2002). The cranial neural crest cell migrate in a stereotypical pattern (Lumsden et al., 1991) and then give rise to ectomesenchyme, which fills the facial prominences (Le Lievre and Le Douarin, 1975; Noden, 1983). Once in the head, the ectomesenchyme wil l differentiate into skeletogenic cells and other neural crest cells give rise to peripheral neuronal and pigment cell types (Abzhanov et al., 2003; Le Douarin et al., 2004; Le Lievre and Le Douarin, 1975). During migration, the neural crest cells are patterned by the FGF (Fibroblast growth factor) signals from the midbrain hindbrain isthmus (Creuzet et al., 2004; Noden, 1983; Trainor et al., 2002) as well as Sonic hedgehog inputs from the underlying endoderm (Brito et al., 2006; Couly et al., 2002). At later stages of differentiation the skeletal derivatives of the upper face rely on the juxtaposition of the FGF and Shh (the frontonasal ectodermal zone (FEZ)) signals near the stomodeal ectoderm to pattern form and direction of growth of the frontonasal mass derivatives (Hu et al., 2003). The orientation of the endoderm (Couly et al., 2002) and the F E Z (Hu et al., 2003) determine the direction of the duplicated outgrowth. In the face, a region analogous to the apical ectodermal ridge has not been identified, although localized centers of gene expression have the ability to provide permissive signaling. The cranial placodes make up the paired sensory organs of the head and include the olfactory, lens, otic, trigeminal, hypophyseal, lateral line and epibranchial placodes. The placodes are first recognizable by localized thickening of the surface ectoderm. The ectodermal cells surrounding the anterior neural plate contribute to the olfactory placodes and have been mapped using either quail-chick chimeras (Couly and Le Douarin, 1985) or vital dye labeling (Bhattacharyya et al., 2004). The placodes are induced within the non-neural ectoderm outside of the neural plate but within a pre-placodal domain (Bailey et al., 2006; Bhattacharyya et al., 2004; Schlosser, 2005; Schlosser and Ahrens, 2004). The pre-placodal region has lens and olfactory precursors intermingled (Bhattacharyya et al., 2004; Streit, 2002). Reducing the amount of FGF signaling from the anterior neural ridge prevents the induction and formation of 90 the olfactory placode, and the olfactory placode requires cranial neural crest cells to repress the lens placodal program (Bailey et a l , 2006). The expression of Dlx5 defines the olfactory placode (Bhattacharyya et a l , 2004). The cranial placodes differentiates into sensory neuronal cell types, and non-neuronal lineages such as the lens of the eye, but never skeletal tissue like the cranial neural crest cells. The olfactory placode gives rise to the typical derivates of the sensory placodes including the olfactory and gonadotrophic releasing hormone neurons as well as olfactory ensheathing cells. The olfactory placode is atypical in that it is the only placode to differentiate into glia to support the olfactory nerve (Schlosser, 2005). The two placodes in the upper face are the optic and olfactory, with the olfactory placode developing much later than the optic placode. The olfactory placode is first visible at the end of cranial neural crest cell migration and when the pharyngeal arches are beginning to form. In chicken embryos, the nasal placode is first recognizable at stage 14 (Hamburger and Hamilton, 1951), and 24 hours (stage 20) later it invaginates to form a nasal pit. At stage 20, gonadotrophin-releasing neurons begin migrating to the telencephalon (Drapkin and Silverman, 1999) . The invaginating epithelial of the nasal pit expresses several transcription factors, including Dlx5, a member of the Distalless family (Acampora et a l , 1999; Brown et a l , 2005; Long et a l , 2003; Simeone et a l , 1994). The position of the placode within the upper face and timing of formation suggest that it may be exerting patterning influences on the adjacent facial mesenchyme. The function of FgfS from the anterior neural ridge in inducing the olfactory placode (Bailey et a l , 2006) suggests that Fgf8 could also be important at later stages of facial morphogenesis. Other work on Fgf8 in facial patterning has focused on the genetic deletion of FgfS in mouse facial epithelium which results in foreshortened faces including the loss of the premaxillary and maxillary bones, olfactory epithelium (Kawauchi et a l , 2005; Macatee et a l , 2003; Trumpp et a l , 1999), while scant data exists on what exogenous levels of Fgf8 can do in facial patterning (Shigetani et a l , 2000; Song et a l , 2004). Much of the work on later stages of nasal pit development has focused on the interactions of the olfactory bulbs and the olfactory pit (Wang et a l , 2001). For the olfactory placode to begin to thicken and invaginate it must be adjacent to the frontonasal mesenchyme (LaMantia et a l , 2000) . In stage 20 chicken (Hu and Helms, 1999; Song et a l , 2004) and El0.5 mouse embryos (LaMantia et a l , 2000) the nasal pit is surrounded by the asymmetric expression of several secreted signaling molecules. The lateral edge of the nasal pit expresses RALDH2, a retinoic acid synthetic enzyme (Blentic et a l , 2003; LaMantia et a l , 2000) while the expression of FgfS 91 defines the medial epidermal ridge near the nasal pit, while Bmp4 and Shh expression is caudal to the nasal pit (LaMantia et al., 2000; Song et al., 2004). Numerous studies suggest that all of these molecules are required for normal facial development (Francis-West et al., 1998), and since their expression domains are adjacent to the nasal pit then one could hypothesize that FGF, SHH, and B M P signaling could alter early nasal pit development. This was tested by challenging explants of the nasal pit epithelium and mesenchyme in organ culture with altered levels of retinoids, FGFs, BMPs and SHH and then characterized the response of the tissues by the presence of markers of the lateral nasal mesenchyme (PaxT) and the mediolateral epithelium (neural cell adhesion molecule (NCAM)) (LaMantia et al., 2000). As expected, retinoid treatment increased the number of lateral nasal cells, and decreased the neuronal differentiation, while Fgf8 reduced the amount of lateral nasal cells, and increased neuronal cells (LaMantia et al., 2000). Neither sonic hedgehog nor B M P signaling altered the mediolateral patterning of the nasal pit; Shh acts as a growth promoting signal while B M P decreased the size of the explant, and altered the trajectory of the olfactory neuron (LaMantia et al., 2000). Thus, we have a role for FGF, Shh, B M P , and retinoid signaling in short-term development of explanted frontonasal ectoderm and mesenchyme, but there is no information regarding the role of each of these molecules in the development of the nasal capsule. 3.1.2 Tissue interactions in the developing upper face The role of the nasal placode in patterning the face has been studied in amphibians, by extirpating the olfactory placode in Amblystoma larvae. After extirpation, the premaxillary and maxillary bone are enlarged with loss of the tectum nasi, and a reduction of the telencephalon (Burr, 1916). Other workers have extirpated and grafted the nasal pit of Xenopus laevis but focused the analysis on the neuronal phenotype rather than facial morphogenesis (Stout and Graziadei, 1980). Similar extirpations done in stage 18 chicken embryos prevented the formation of olfactory epithelium, however no skeletal analysis was performed (Wang et al., 2001). One study explored the morphogenetic capacity of nasal pits by grafting nasal pits to the flank of Triton taeniatus which resulted in ectopic limbs (Balinsky, 1933). In contrast, grafting chicken embryonic nasal pits with frontonasal mass mesenchyme into a region rostral to the hindlimb resulted in the development of a nasal capsule but did not induce an ectopic limb (Street, 1937). In these experiments the ectopic nasal capsule was most likely derived from donor mesenchyme. Instead of telling us about the inductive capacity of the nasal pit, it showed that the frontonasal region is capable of autonomous growth in an ectopic location as long as 92 epithelium and mesenchyme are included. The question still remains if the nasal pit epithelium has instructive capabilities and can pattern other non-nasal mesenchyme to make a nasal capsule. Here we analyze the role of the nasal pit epithelium in patterning the skeleton of the upper face in chicken embryos. Using a series of nasal pit extirpations and grafting of supernumerary nasal pits we examined the role of the nasal pit in patterning the upper beak skeleton. In addition we tested the function of one of the signals close to the nasal pit, Fgf8. We conclude that the nasal pit has a role in patterning the facial mesenchyme. 93 3.2 METHODS: 3.2.1 Embryological Manipulations: Embryos: Fertile white leghorn chicken eggs {Gallus gallus) were obtained from the Poultry Research Unit at University of Alberta (Edmonton, A B ) and Japanese quail eggs (Coturnix coturnix japonica) from the Poultry Center at Oregon.State University (Corvalis, OR). Quail embryos were incubated approximately 12 hours after chicken embryos so that they reached the appropriate developmental stage to compensate for the quail embryos developing more quickly than chickens (Schneider and Helms, 2003). A l l embryo work was performed under the approval of the U B C Animal Care Committee. Stage 20 nasal pit extirpation: Stage 20 embryos were stained with neutral red to identify the nasal pit and then a small quantity of 0.1% nile blue sulfate in phosphate buffered saline was applied to the right nasal pit using a gel loading tip (Microloader, Eppendorf). A sharpened tungsten needle was used to tear the surface epithelium surrounding the nasal pit, following that the needle was manoeuvered under the nasal pit epithelia to lift it off of the underlying mesenchyme. The nasal pit appeared to come off as a single sheet of epithelia. Fgf8b protein bead implantation: Approximately 50 heparan acrylic beads (Sigma Aldrich H5263) were soaked on ice in 2 ul of 1 mg/ml Fgf8b protein (Peprotech) for a minimum of 1 hour. Stage 15 and 26 chicken embryos were stained with neutral red to highlight the facial prominences, a small incision was made in the presumptive maxillary region (stage 15) or the lateral maxillary prominence (stage 26) and the bead was placed into the mesenchyme. Skeletal analysis: We compared changes to the nasal capsule of the Stage 20 extirpation embryos to the un-manipulated contralateral side and used three categories to describe the changes in bone and cartilage morphology: normal, reduced and absent. Embryos were scored as reduced when the bony process was less than 50% of its normal length. Morphological change resulting from the grafts was compared to the untreated, control side of the embryo. 94 Wholemount in situ hybridization: Embryos were collected in cold PBS (phosphate buffered saline) and fixed in 4% paraformaldehyde overnight in the fridge. Whole mount in situ hybridization (WISH) was performed in the Intavis InsituPro Robot, with DIG-labeled antisense probes using protocols previously published (Song et al., 2004). Following detection, representative embryos were photographed with the Qimaging Micropublisher camera. A subset of the embryos were dehydrated to 100% ethanol, cleared in toluene and embedded in wax. Consecutive 7 p.m sections were placed on plain sides (Superfrost, Fisher), dewaxed in xylene and mounted in Entellen. The images were captured with a Sony Video Camera on a Ziess Axioskop. We wish to acknowledge the following individuals who provided avian probe constructs for our wholemount in situ analyses: M . Kessel, Dlx5; and O. Pourquie, exonic FgfS. 3.2.2 Grafting Experiments Donor tissue preparation: Stage 20 or 26 donor (quail or chicken) embryos were collected in Hanks Balanced Salt solution without C a 2 + and M g 2 + (HBSS). The donor embryos were dissected into three different pieces: lateral nasal prominence and lateral third of the frontonasal mass for the nasal pit grafts, the medial region of the F N M excluding the caudal edge of the F N M , (Fig. 3.1). The pieces were placed on ice in 2% trypsin made in HBSS for approximately 30 minutes for stage 20 nasal pits and 60 minutes for stage 26 nasal pits. The end point of trypsin is chosen as the point when the edges of the epithelia are lifting up cleanly from the mesenchyme. The trypsin was stopped by transferring the tissue pieces to a second dish with HBSS + 10% FBS. The epithelia and mesenchyme were separated using forceps. At this point, the donor epithelium included surface epithelia from the lateral nasal prominence, frontonasal mass and the nasal pit itself. In most cases the axonal projection from the nasal pit was still attached. Further cuts were made to isolate the nasal pit and the remaining epithelia were discarded. The donor epithelia were then stained with 0.1% C M - D i l (chloromethylbenzamido 1, 1 ;-dioctadecyl-3, 3, 3', 3'-tetramethylindocarcocyanine perchlorate, Invitrogen C-7001) and 0.5% neutral red in HBSS to enable visualization in the host embryos. Host embryo preparation: Stage 15, 20 or 26 host embryos were used for grafting experiments, and stained with neutral red which enabled visualization of the host site (Fig. 3.1). A sharpened tungsten needle (0.1 mm diameter, Goodfellow; catalogue no. 005138) was used to peel the epithelia away from 95 the underlying mesenchyme. The piece of donor epithelia was positioned over the graft site, with the basement membrane side contacting exposed mesenchyme. The grafted was pinned in place with platinum pins or staples (0.025 mm diameter, Goodfellow; cat. no. PT005113). Bone and cartilage staining: To study bone and cartilage morphology in wholemount, stage 39 embryos were collected, fixed in 100% ethanol, permeabilized with acetone and then stained with alcian blue and alizarin red (Plant et a l , 2000), neither the epidermis nor the eyes were removed in these embryos, until after the embryo had cleared. We also collected other embryos for microscopic analysis. Embryos were collected at stage 28, 29 and 30, fixed in acidic formalin (60% ethanol, 10% formaldehyde (diluted from 37% stock, 10% acetic acid) (Creuzet et a l , 2002) and embedded in paraffin. The embryos were sectioned at 7 urn. For bone and cartilage staining on sections, they were dewaxed and rehydrated, acidified in 1% acetic acid, stained for 30 minutes in 1% Alcian Blue in 1% acetic acid followed by a rinse in 1% acetic acid and stained in Picrosirius red (0.001% Sirius Red F3B in saturated picric acid) for 1 hour. The slides were rinsed in 1% acetic acid and dehydrated through a graded series of ethanol passed through xylene and coverslipped using Entellen (Ashique et a l , 2002). Immunohistochemistry: Embryos were collected at appropriate time points after the grafting experiment and fixed overnight in 4% paraformaldehyde in phosphate buffered saline. The chicken-quail chimeras were fixed in acidic formalin overnight. The embryos were embedded in paraffin, and 7 um sections were placed on TESPA (3-aminopropyltriethoxysilane, Sigma) coated slides (Superfrost, Fisher). The processing of the slides was identical for either the QeTN or TuJl antibody. The slides were dewaxed, rehydrated and then endogenous peroxidases were blocked with 3.0 % hydrogen peroxide. Q^PN: We detected the quail cells in heterospecific grafted embryos with the QjziPN antibody (Developmental Studies Hybridoma Bank, University of Iowa). Briefly, following published protocols (Creuzet et a l , 2006), the sections or wholemount embryos were incubated with neat QeTN supernatant overnight, and washed off, then incubated with goat anti mouse horseradish peroxidase conjugated secondary antibody (1:100) (Jackson Labs), and detected with D A B substrate (ES005 diluted in ES010, Millipore), and counterstained in 0.1% methyl green. TuJl antibody: Antigen retrieval was performed with 0.05% trypsin (2 minutes at room temperature in PBS), rinsed in PBS and incubated overnight with 1:500 TuJl mouse monoclonal antibody which recognizes neuronal class III P-Tubulin (Covance, MMS-435P). The primary 96 antibody was rinsed off and the slides were incubated with horseradish peroxidase conjugated goat anti-mouse secondary antibody (Jackson Labs) at 1:100 for 2 hours and detected. The slides were rinsed briefly in PBS and detected with D A B substrate (ES005 diluted in ES010, Millipore) and counterstained with 0.1% Methyl Green. 97 3.3 RESULTS: Here, we addressed the role of the nasal pit in skeletal development. We show that once the nasal pit has begun to invaginate into the frontonasal mass mesenchyme, it has pro-skeletogenic influences in competent facial mesenchyme. 3.3.1 Exogenous FGF8 provides incomplete patterning information We chose to implant beads soaked in FGF8b into two stages of embryos either stage 15 embryos, when the presumptive maxillary region mesenchyme is plastic enough to transform into a duplicated frontonasal mass (Lee et al., 2001), or stage 26 embryos, when the maxillary mesenchyme is committed to form the maxillary skeleton (Richman and Tickle, 1989). We chose the location of bead implants to be within an area that was Fg/S-negative. This bead position in the post-optic mesenchyme provides a more rigorous test of the instructive effects of the FGF8b protein on cranial neural crest derived mesenchyme. We first confirmed the bioactivity of Fgf8b protein in the chicken embryo. The expression of Msxl, a downstream target of Fgf signaling, was induced within six hours of treatment (stage 26, n=3/4; data not shown). The FGF8b protein was also capable of changing skeletal pattern. In stage 15 embryos treated in the presumptive maxillary mesenchyme, the majority of specimens had ectopic bones and cartilages (n=7/10; Fig. 3.2A-B). The ectopic bone and cartilage elements were small and could not be identified. A n additional skeletal change observed was ectopic processes induced on already existing bones (quadratojugal, n=3/10). At stage 26, FgfS is expressed along the caudal edge epithelium of the maxillary prominence so we implanted the beads as distant as possible from this domain, at the cranial edge of the prominence. The results were similar to those obtained with younger embryos. Small pieces of ectopic bone and cartilage were formed that could not be identified (n=9/13; Fig. 3.2C-D). Therefore the age of the mesenchyme did not affect the ability of the facial mesenchyme to respond to the Fgf8b protein since both ectopic bone and cartilage were induced in either stage tested. From this we concluded that Fgf8b is able to direct skeletal patterning to a limited extent, but it is likely that other signals from the nasal pit may be required. 98 3.3.2 Extirpation of the nasal pit at stage 20 leads to mild patterning changes in the upper face Previous studies detailing tissue interactions in the upper face have focused on the surface epithelium of the frontonasal mass (Hu et a l , 2003; Richman and Tickle, 1989), maxillary and mandibular prominences (Richman et a l , 1989; Richman et a l , 1997; MacDonald et a l , 2004), but have not looked at tissue interactions with the nasal pit epithelium. We decided to test the patterning capabilities of the nasal pit epithelium since it was clear that examining the effects of one signal at a time would give results that were inconclusive. We hypothesized that the nasal pit epithelium was required for patterning the adjacent facial prominences: the lateral nasal prominence and the frontonasal mass. We therefore predicted that nasal pit removal would result in deletions of the frontonasal mass and lateral nasal prominence derivatives likely including loss of the nasal conchae, premaxillary bone and the nasal bone. Mechanical extirpation of the nasal pit at stage 20 resulted in a mild external phenotype. Malformations of the external nares (n= 10/15), and a slightly shorter beak were usually observed. However, underlying the almost normal external morphology, we often observed reductions of the bones and cartilages surrounding the nasal capsule. The nasal capsule consists of the nasal conchae which are surrounded laterally by the nasal and lacrimal bones (all of which are most likely lateral nasal prominence derivatives), distally by the premaxillary bone (a frontonasal mass derivative) and ventrally by the vomer, maxillary and palatine bones. While the maxillary and palatine bones clearly originate from the maxillary prominence (Lee et a l , 2004; MacDonald et a l , 2004) the origin of the vomer bone has not been determined. Based on the anatomical position of the developing vomeronasal organ in Python sebae (Buchtova et a l , 2007), man (Smith and Bhatnagar, 2000), and rat (Garrosa et a l , 1998) we propose that the vomer is derived from the frontonasal mass. In almost all specimens, the vomer bone was reduced (n=12/14; Fig. 3.3B), however other frontonasal mass derivatives such as the prenasal cartilage and premaxilla were unaffected. We observed a significant reduction in the size of the nasal conchae and the nasal bone (n= 9/14; Fig. 3.3A). The centre of the lacrimal bone was also lost (n=6/14; Fig. 3.3A). Therefore all derivatives of the lateral nasal prominence were affected to some degree. In embryos with deficient nasal conchae we observed a concomitant reduction in the palatine process (n=7/14; Fig. 3.3B) and an enlargement of the premaxillary process of the maxillary bone (5 of the 7 specimens with a reduced palatine process of the maxillary bone; Fig 3.2A). The effect of the nasal pit on maxillary bone morphology, was unexpected and could indicate that the nasal 99 capsule is required for the development of all the adjacent bones that surround the nasal conchae. From these results we concluded that the stage 20 nasal pit is necessary for patterning the external nares, derivatives of the lateral nasal and maxillary prominences. We cannot exclude a greater role for the nasal placode in patterning the upper beak skeleton at younger stages. Some of the variation in the skeletal morphology in these embryos may result from incomplete epithelial removal. To assess the thoroughness of epithelial removal we collected embryos immediately after the nasal pit was extirpated. Most of the epithelium was removed however there were still some small regions of thicker epithelium, similar to that found in the contralateral nasal pit (n=3/3, 6h or less after surgery). We also collected embryos 24 hours following extirpation to characterized wound-healing and assess if re-epithelialization took place, we found that 24 hours after extirpation the embryos had grown a layer of thin epithelia over the mesenchyme (Fig 3.3E-E"), while there were still some areas of pseudo-stratified columnar epithelium typical of the nasal pit (n=3/3; Fig. 3.3F-G"). The residual nasal pit epithelium does not continue to form the nasal slit as determined by external morphology (24 hours, n = 15/15; Fig 3.3I,J). We next wanted to look at gene expression of markers for the nasal pit and adjacent epithelium following extirpation. These data would indicate which epithelium was left after extirpation. At stage 20 FgfS is only expressed along the medial edge of the nasal pit, however by stage 24, the end of our experiment, expression would also have initiated in the lateral edge (Song et al., 2004). In contrast, Dlx5 is expressed in the nasal pit at stage 20 and 24. We found that there was no change in medial FgfS expression at 0 and 6 hours after extirpation (0 hours = stage 20, n=5/5; 3 hours = stage 21 n=2/2; Fig. 3.3H). The continued expression of FgfS was expected because we had tried to remove only the nasal pit, not the frontonasal mass epithelium. A small foci of FgfS expression was also present at later timepoints (16 hours, n=2/2; 24 hours, n=4/5; Fig. 3.31). We observed a complete epithelial covering the wound-site at 24 hours, and this epithelium wound site did not express FgfS. However, small patches of FgfS expression were detected where the nasal pit should be (Fig. 3.31), and seem to be expressed in columnar epithelium (Fig. 3.3P-I"). We observed decreased or absent expression of the internal nasal pit marker, Dlx5 on the treated side (24 hours, n=2/3 decreased; Fig. 3.3J). The Dlx5 expression and sections confirmed that the mechanical removal was incomplete, and likely contributed to the variability in the skeletal phenotype. Furthermore at stage 20, the source of the wound-healing epithelium surrounding the nasal pit is no longer able to express either FgfS or Dlx5 suggesting 100 that either the mesenchymal inductive signals are no longer present near the wound site or the healed epithelium is not competent to respond to these signals. 3.3.3 Nasal pits can organize ectopic structures of ectopic bone and cartilage Rationale for grafting study design The position of the nasal pit between the frontonasal mass and lateral nasal prominences may mean that signals from this epithelium pattern the upper beak skeleton. We tested the skeletogenic inductive capacity by grafting a supernumerary nasal pit to competent facial mesenchyme of a second host embryo. We placed the donor nasal pits in an ectopic location to test i f they contain an instructive capacity that can pattern mesenchyme (Fig. 3.1). We chose the maxillary region over the frontonasal mass or lateral nasal prominences because the maxillary prominence forms only bony elements (Lee et a l , 2004; Richman and Tickle, 1989) and any induced ectopic cartilages will be obvious. We expect that if the nasal pit has a patterning capacity that it should be able to induce both bone and cartilage based on the derivatives of the adjacent facial prominences. The ability of the nasal pit to elicit patterning changes will also be influenced by the ability of the host mesenchyme to respond to the signals. The first mesenchymal condensation in the maxillary region can be identified at stage 15 (Richman et a l , 2006), by stage 20 the fate of the maxillary mesenchyme is determined to give rise to maxillary bones (Richman et a l , 1989) but still requires epithelial signals for outgrowth. By stage 26 the maxillary prominence has grown considerably and contains peanut agglutinin positive pre-osteogenic condensations (Dunlop and Hall , 1995). Facial mesenchyme at stage 26 no longer requires epithelium for outgrowth or patterning (MacDonald et a l , 2004). We chose to focus our studies on the nasal pits from donors of two stages, stage 20 and stage 26. We chose to work mainly with stage 20 donors because the nasal pit has only slightly invaginated and can easily be separated from the mesenchyme. Stage 20 is also when other facial epithelia can induce ectopic skeletal elements to form (Hu et a l , 2003). We also tested selected stage 26 donors, because the cartilage of the nasal passages wil l start to differentiate shortly thereafter. By selecting two stages for donor tissues we could test the effect of epithelial specification on the ability to induce patterning changes in the host mesenchyme. Nasal pits rarely induced skeletal changes in stage 26 hosts In all our grafts of nasal pits, we expected that small pieces of bone and cartilage would be induced, partly based on our experiments with FGF8b beads (Fig. 3.2) but also because of the 101 nasal pit epithelia is normally surrounded by cartilaginous conchae. We first tested the ability of the stage 26 mesenchyme to respond to the signals provided by stage 20 or 26 nasal pit. The supernumerary nasal pit, induced ectopic processes on the quadratojugal or jugal bone (n=4/ll; Table 1); while another two embryos had formed a small ectopic bone (n=2/ll). The ectopic processes that developed in response to the stage 20 nasal pit suggested that the maxillary pre-osteogenic condensation has not changed its fate but can be influenced by epithelial interactions. In contrast, a small piece of cartilage was induced in three homochronic grafts of stage 26 nasal pits onto stage 26 hosts, (n = 3/6; Table 1). The majority of stage 26 host embryos developed externally normal (stage 20 and 26 nasal pits, n= 16/18). Overall, only limited instructive capability of the nasal pit has been demonstrated in the stage 26 hosts. Stage dependent induction of ectopic cartilage and bone in stage 20 host embryos We next tested whether younger hosts would be more competent to respond to the signals from the ectopic nasal pit. A homochronic graft of stage 20 nasal pits to stage 20 host embryos induced changes in the eyelid or enlarged the external auditory meatus (the usual location of the grafted epithelium; n=5/14: Fig. 3.4A), the remaining 9 cases developed normally externally (n=9/14). Internally, skeletal analysis showed that more than half of the stage 20 nasal pits induced ectopic processes on the quadratojugal or squamosal bone (n=7/12; Fig 3.4B, C), four of these ectopic processes also had separate pieces of ectopic bone articulating with the process, but no ectopic cartilage. Out of the remaining five cases, the stage 20 nasal pit induced completely separate pieces of ectopic bone (n=4/5; Table 3.1). Stage 26 donors gave slightly different results than stage 20 donors. We observed more obvious external changes to the eyelid morphology including ectopic ridges surrounding what appeared to be a central opening (n=16/17; Fig. 3.4D) along with partial loss of the eyelid and nictating membrane (n=17/26). One of the roles the stage 26 nasal pit epithelium may be to pattern the external nares. Still other embryos had changes to the external auditory meatus (n=7/26) and 3 developed normally. The changes to the eyelid may have occurred because the lateral maxillary prominence epithelium originates from the same region of superficial cephalic ectoderm as the inferior eyelid (Couly and Le Douarin, 1990). There are several skeletal changes underlying the epithelial changes that the stage 26 nasal pit induced. These included ectopic processes that were contiguous with existing intramembranous normal bones (n= 20/24; Fig. 3.4E,F; Table 3.1), and these were often accompanied by ectopic bone (n=9/20) and 102 cartilage (n=10/20). Thus the stage 26 donors were able to induce more significant epithelial changes and larger cartilages compared to the stage 20 nasal pits. We hypothesize that younger host mesenchyme would reveal more about the inductive potential of the nasal pit. Groups of ectopic bones are induced by nasal pits grafted to stage 15 host embryos We found that the phenotypes produced in stage 15 embryos were consistent with some instructive patterning information being provided to both the mesenchyme and adjacent epithelium. Strikingly we observed changes to the external morphology of the embryo including ectopic outgrowths proximal to the eye (stage 20 nasal pit, n=6/17, Fig. 3.5A; stage 26 n=7/22), and abnormalities in the eyelid and/or external auditory meatus (stage 20 nasal pit, n= 7/17, Fig. 3.5D; stage 26 nasal pit, n= 12/22, Fig 3.5G). The stage 20 nasal pit grafts induced skeletal changes in the joint region of the skull. The results can be divided into two categories based on the morphology of the induced elements: one that consists of an organized collection of skeletal elements (n=4/14; Fig. 3.5A-F), or another that included isolated ectopic bones. The organized elements consisted of a minimum of 6 ectopic bones that articulated with each other in a circular pattern (n=4/4). In the second category, the nasal pits induced small rods of cartilage (n=6/10), and ectopic bones (n= 7/10). These ectopic bones often articulated with the proximal end of the quadratojugal bone (Fig. 3.5B,E). The patterning of the quadrate was occasionally affected in these grafts with the squamosal process of the quadrate being reduced and displaced cranially (n= 5/14). We also observed a reduction in the size of the quadrate process of the squamosal bone, along with reductions of post orbital cartilage. Stage 26 nasal pits induced similar intramembranous bones to those induced by the stage 20 nasal pits, however the older nasal pit was able to induce qualitatively larger pieces of ectopic cartilage (at least twice the size of those induced by stage 20 grafts). These embryos could also be classified into two categories one where the induced cartilage was covered with at least three intramembranous bones (n = 5/15; Fig. 3.5H, I) or the second category that consisted of isolated ectopic bones and cartilages. The organized grafts consisted of a plate of cartilage that is surrounded by the ectopic bones, the cartilage that is induced is often lobular, rather than a flat sheet of cartilage (n=5/5; Fig. 3.51). In the remaining embryos, the induced bones and cartilages were much smaller (n=10/10) and consisted of ectopic process (n=3/10) or isolated combinations of ectopic rods of bone and/or cartilage (n= 7/10). 103 In all grafts placed in stage 15 hosts, the skeletal elements that were induced were much smaller than normal facial bones, and only a few had recognizable features. The normal quadratojugal articulates with the quadrate bone, and at the point of articulation there is the formation of a characteristic small secondary cartilage. We observed a piece of intramembranous bone with a similar secondary cartilage in all 4 of the organized grafts from a stage 20 nasal pit (Fig. 3.5C). The form of the bones in the remaining 32 grafts was not recognizable. With the schema for the dissection of the nasal pits, we may have inadvertently included frontonasal mass or lateral nasal prominence surface ectoderm (Fig. 3.1). In order to determine whether the induced elements resulted from the presence of the ectopic nasal pit, rather than contaminating surface epithelia, we grafted surface epithelium from the frontonasal mass of stage 26 donors to stage 15 and 20 hosts. No ectopic skeletal elements were observed in these grafts (stage 15 hosts n = 6/6; stage 20 hosts, n = 15/15; Fig. 3.4 G-I). As a second active control, we included a series of experiments where we prepared a graft site but added no exogenous epithelia. The graft site preparation and re-epithelialization may have altered the signals in the mesenchyme and induced patterning changes, however all of these embryos developed normally (stage 15: 9/9; stage 20, 15/15, data not shown). Therefore, we have shown that the stage 15 mesenchyme is more plastic to respond to ectopic nasal pits, and younger nasal pits induced a more organized skeletal structure. The stage 26 nasal pit epithelia contained more of the signals that influence the development of the external nares and is more pro-chondrogenic than the stage 20 nasal pits. Grafted nasal pits retain their olfactory character and differentiate into neurons The previous series of experiments had determined the temporal conditions that promoted the greatest response in the mesenchyme. Since the nasal pit was being placed in an area outside of its normal inductive signals there is a possibility that the grafted nasal pits had changed their fate in response to signals from the underlying mesenchyme. To characterize the development of the nasal pit epithelia we collected a series of embryos from stage 20 nasal pit grafts placed on stage 15 host embryos. We collected embryos 24 hours after the surgery to determine whether the nasal pits invaginated and maintained their epithelial character. We collected older stages so that the induced skeletal elements would also be apparent near the grafted nasal pits. In some of these experiments quail donors were used to distinguish the donor epithelium from that of the host and to show if the grafts included any mesenchymal cells. Following staining with the 104 Q0PN antibody in wholemount (n = 9; Fig. 3.6A) or in sections (n= 5/5; Fig. 3.6B, F) we found quail cells only in the epithelia of the graft, and not in the mesenchyme surrounding the grafts (Fig. 3.6B, F). The stage 20 nasal pits invaginated into the mesenchyme of the stage 15 graft site within 24 hours (n=5/5; Fig. 3.6B) and underwent extensive invagination and expansion at a rate similar to normal in situ development (Fig. 3.6C). We sectioned the embryos collected between the stages 28 to 33 in the horizontal plane and found that the grafts developed nasal cavities with a lumen (Figs. 3.6C, 3.7). We observed increased alcian blue staining surrounding the graft demonstrating that cartilage was being induced (n=6/7; Figs. 3.6C, 3.7F, J). We observed clear evidence of external nasal openings in 2 embryos (n=2/7; Fig. 3.7A,B). The grafts in remaining embryos always approached the surface epithelium, but because we did not collect serial sections we can not say definitively i f these grafts made external nasal openings. We next tested whether the nasal pit epithelia underwent neuronal differentiation by looking at a neuronal specific marker. Neuronal class III P-tubulin (TuJl) is expressed in differentiated post-mitotic neurons, and is expressed in embryonic olfactory epithelia, and olfactory nerves, but not the facial ectoderm (Roskams et a l , 1998). A l l of the grafts had differentiated neurons within the olfactory epithelia and there were neurons extending from the grafted nasal pit (n=8; Fig 3.7C-K). There were regions of the grafted nasal pit epithelium that did not stain with the TuJl antibody, which is likely respiratory epithelium (Fig. 3.7E,H,I,L,M). TuJl antibody allowed us to characterize the responding mesenchyme to a certain extent. In one case, we observed one graft that gave rise to a neuron that made an ectopic connection to the trigeminal ganglion. In three cases, a distinct ectopic outgrowth was observed and the mesenchyme of the outgrowth contained TuJl positive cells suggesting that the presence of the ganglion may have facilitated the ectopic outgrowth (Fig. 3.7F-I). In one embryo stained with QsiPN, we observed neuronal-like cells that are of quail origin in the mesenchyme surrounding the graft (n=l/5; Fig. 3.6F). 105 3.4 DISCUSSION: Here we have shown that the nasal pit can induce skeletogenesis in the adjacent mesenchyme. By using gain- and loss-of-function experiments, we have shown that the nasal pit epithelium is required to pattern the lateral nasal derivatives. Furthermore, we have shown that stage 20 nasal pit epithelium is irreversibly determined to undergo normal differentiation in ectopic locations. This work suggests that the role of the nasal pit in skeletogenesis needs to be examined more fully. 3.4.1 Autonomous differentiation of the nasal pit epithelium was supported in an ectopic location We observed highly keratinized tissue that was similar to the external nares, with external nasal apertures which were most obvious in the stage 26 donor nasal pits. The differentiation of the external nasal passages are one of the later portions of the nasal capsule to be patterned, since it was induced more readily from stage 26 grafts. We also observed differentiation and migration of neurons from the grafted nasal pit. The olfactory placode gives rise to both the olfactory nerve and G n R H (Gonadotrophin releasing hormone) neurons. The GnRH neurons migrate from the olfactory pit by stage 21, but do not enter the telencephalon until stage 26 (Drapkin et al., 2002). The stage 20 donor grafts underwent neuronal differentiation in situ, as indicated by the presence of TuJl positive cells within and extending from the grafted epithelia. We concluded that the epithelia differentiated into presumptive olfactory and G n R H neurons, but we can not identity them specifically because we did not stain for olfactory marker protein or for GnRH markers. The connectivity of ectopic nasal pit neurons to the central nervous system has been demonstrated in larval Xenopus (Stout and Graziadei, 1980). The ectopic nasal pits were placed in three different regions in the head, they always connected to the brain, however the grafts that were placed near the normal olfactory placode often fused to the in situ olfactory placode and graft-derived nerves migrated to the telencephalon (Stout and Graziadei, 1980). Similarily, we observed one case where neurons derived from the grafted nasal pit epithelium connected with the trigeminal ganglion. Neuronal pathfinding, and differentiation into post-mitotic neurons show that the nasal pit graft is capable of autonomous differentiation in an ectopic location. Thus we have shown that by stage 20 the nasal pit neuronal fate is already determined and no longer requires signals from the lateral nasal or frontonasal mesenchyme. 106 3.4.2 The nasal pit secretes a combination of permissive and instructive signals We placed the grafts in the maxillary region of the face which normally only contributes intramembraneous bones to the skull so that we could test the inductive influence of the nasal pit with the response of the mesenchyme. A permissive signal permits the duplication of the mesenchymal derivatives that are normally present in that region, while an instructive signal wil l induce mesenchymal derivatives that are not normally present in the facial prominences. Through permissive signaling, the nasal pit was able to organize the growth of an ectopic limb in the flank of Triton taenatius (Balinsky, 1933). We presented evidence, most clearly in the stage 15 hosts, that the nasal pits are providing instructive signals,by the formation of ectopic cartilage in the maxillary region and permissive signals by the formation of an ectopic quadratojugal bones (a derivative of the maxillary region). However, we were unable to recognize any other of the ectopic bones, which complicates the analysis of the mesenchymal response and possible fate change. This, combined with the induction of ectopic cartilage in the maxillary region, suggests that the nasal pit is providing instructive and permissive patterning to maxillary prominence, that we have not been able to characterize on the basis of gross morphology. 3.4.3 Stage 26 host mesenchyme is the most restricted in its response to nasal pit epithelium Our experimental design allowed us to test the commitment of the maxillary prominence mesenchyme by grafting nasal pits to three stages of hosts. With stage 20, and 26 host embryos we showed that the ability to respond to ectopic skeletogenic inductive cues is severely reduced in older maxillary prominences. This restriction of the mesenchymal response is observed qualitatively by the number of elements and size of the elements that were induced. Often, in stage 15 host embryos we observed the loss of the squamosal bone concomitantly with the gain of ectopic skeletal processes. Fewer skeletal elements were induced in the mesenchyme of stage 20 and 26 hosts and there was no effect on the squamosal or quadrate bones. Cells from these regions were not recruited to make the ectopic structures because the condensations were already committed to making the squamosal and quadrate bones. Thus we have shown that the maxillary region is undergoing a lineage restriction from stages 15 to 26. 3.4.4 Older nasal pit epithelia can induce a larger variety of structures. We observed that the nasal pit grafts were able to induce the maxillary region to form cartilage, a tissue that it would not have normally formed. In the least committed mesenchyme 107 of the stage 15 hosts we observed the induction of both cartilage and bone in response to a supernumerary nasal pits. The stage 26 nasal pit grafts induced bone, cartilage and external nasal apertures in stage 20 hosts and only cartilage in the stage 26 hosts. The stage 20 nasal pits were only capable of inducing bone in later staged host mesenchyme. The stage 26 nasal pit induced three different tissues in the graft site which suggests that as nasal pit invagination and differentiation occurs, the inductive capacity of the nasal pit broadens. Furthermore, during nasal pit development, the epithelia provides a "pro-chondrongenic" signal at stage 26 and while acting as an organizer at stage 20. These data also suggests that the determination of the nasal capsule occurs in a step-wise fashion: 1) the epithelia induced the mesenchyme to make osteogenic condensations, 2) the epithelia induced the mesenchyme to make the cartilage of the nasal conchae, and 3) the epithelia induced the external features of the nares. 3.4.5 FGFs in the nasal pit are not the only organizing molecule in the nasal pit Grafting a nasal pit to the flank mesoderm of Triton taenialus induced ectopic limbs that sometimes included the pelvic girdle (Balinsky, 1933). Analogous experiments have been published in chicken embryos using beads soaked in Fgf l , Fgf2, Fgf4 and Fgf8 proteins and implanted into the flank of chicken embryos resulted in the induction of ectopic supernumerary limbs (Cohn et al., 1995; Vogel et al., 1996). Taken together these two papers suggest that FGF induced signals are likely eliciting the skeletal patterning (Slack, 1995). In these trunk regions, the grafted nasal pit and Fgf8 protein are providing signals to a Hox-positive mesodermal population. In the face, the nasal pit is signaling to a Hox-negative ectomesenchymal population. The Hox-negative population of cranial neural crest cell derived ectomesenchymal cells differentiate into the facial bones and cartilages, while the Hox-positive trunk neural crest must first downregulate Hox gene expression before chondrogenesis occurs in culture (Abzhanov et al., 2003). The chondrogenic potential of cranial neural crest cells can be abolished by overexpressing HoxDIO in culture (Abzhanov et al., 2003). At very early stages of development, the extirpation of the facial skeletogenic domain of cranial neural crest cells prevents the formation of the facial bones and cartilages (Creuzet et al., 2004). The application of Fgf8b beads wil l rescue the development of the face by replacing the anterior neural ridge signal and by inducing the Hox-negative neural crest cells that normally do not contribute to the face to migrate and develop into the facial bones and cartilages (Creuzet et al., 2004). Based on these experiments, and the expression pattern of FgfS in the frontonasal mass, we expected that Fgf8 was providing the patterning information to the ectomesenchymal cells of the face. 108 Unexpectedly we found that FGF8b-soaked beads only induced small spurs of cartilage and bone. The response of the mesenchyme can be explained by the response of FGF independent, B M P dependent mesenchyme. This suggests that skeletogenesis induced by the nasal pit results from the signaling derived from FGFs, and other secreted molecules expressed in the nasal pit. 3.4.6 In situ development: lessons from extirpation We showed that the extirpation of the stage 20 nasal pit induced mild malformations of the cartilaginous conchae and the bones of the nasal capsule. One possible reason for the mild effects is that the nasal pit epithelium regenerated. The removal of the olfactory placode in Xenopus larval stages (equivalent to a chicken stage 20 nasal pit) resulted in the replenishment of the denuded epithelium and a smaller olfactory placode (Stout and Graziadei, 1980). In an analysis of previously published work on Xenopus placodes, Burr (1916) suggested that the regeneration observed was the result of the incomplete removal of the nasal pit (Burr, 1916). Complete removal of the nasal pit in Amblystoma embryos resulted in the overgrowth of the tectum nasi, and these animals were unable to react to odors (Burr, 1916). The mild phenotype in our extirpated embryos results from the incomplete removal of the olfactory epithelia rather than regeneration of the nasal epithelium. Previous work by others showed that nasal pit removal in stage 16-18 chicken embryos had variable results, in some embryos the treated side had no olfactory epithelium (Lutz et al., 1994; Wang et al., 2001). In these studies the extirpated embryos were not followed immediately after surgery and the skeleton was not described, (Wang et al., 2001) however from a single section presented that does not have any olfactory epithelium, it appears that most of the nasal capsule has also been lost. We observed wound healing after extirpation with a thin layer of epithelia that is not typical of the olfactory epithelium. Removal of facial ectoderm at stage 20 resulted in the wound site being healed over by 15 hours and able to re-express Sonic hedgehog (Shh) 24 hours after the extirpation allowing normal development (Hu and Helms, 1999). In contrast, the removal of the same region of ectoderm at stage 26, which healed in the same time frame as the stage 20 removals, but did not reinitiate the expression of Shh, resulting in embryos with defects in upper beak development (Hu and Helms, 1999). We do not observe the replenishment of the olfactory epithelium in these experiments, which suggests that the small residual population of olfactory progenitor cells that were not removed from the nasal pit, maintains the expression of FgfS, which may partially rescue nasal capsule development. 109 In this study we have shown through gain and loss of function experiments that the nasal pit epithelia has both instructive and permissive signals that influence skeletogenesis. Extirpation of the nasal pit caused mis-patterning of the normal nasal capsule, while supernumerary nasal pits induced ectopic skeletal elements. We characterized the response of the mesenchyme to the supernumerary nasal pits, and showed that between stage 15 and 26 mesenchymal fate restriction occurs. We tested two stages of nasal pit epithelia, however we showed evidence that the stage 26 nasal pit epithelium can induce at least three kinds of tissue (bone, cartilage and external nasal apertures), while the younger stage 20 nasal pit primarily induced ectopic bones. By stage 20, the nasal pit epithelium has the ability to develop autonomously. Thus we conclude that Fgf8 together with other secreted molecules from the nasal pit epithelia work together to pattern the facial skeleton. 110 Table 3-1: Skeletal structures induced by supernumerary nasal pits grafted into maxillary mesenchyme Stage 15 host Stage 20 nasal pit (n=14) Stage 26 nasal pit (n=15) Stage 26 F N M epithelia (n=6) Graft site only (n=9) organized outgrowth ectopic bone ectopic cartilage Normal 4 7 6 1 5 8 6 0 0 0 0 6 0 0 0 9 Stage 20 host Stage 20 nasal pit (n=12) Stage 26 nasal pit (n=24) Stage 26 F N M epithelia (n=15) Graft site only (n=15) ectopic bony processes ectopic bone ectopic cartilage Normal 7 8 3 0 22 9 10 2 0 0 0 15 0 0 0 15 Stage 26 host Stage 20 nasal pit (n=ll) Stage 26 nasal pit (n=6) ectopic bony processes ectopic bone ectopic cartilage Normal 4 2 0 . 5 0 0 3 3 111 Figure 3.1: Methods of grafting A. Donor embryos: surface ectoderm Figure 3.1: Methods of grafting. (A) Donor tissue preparation: Stage 20 and 26 donor embryos were dissected into the area surrounding the nasal pit (box) and shown in the middle panel. These pieces were placed in trypsin to separate the epithelium from the mesenchyme and the neuroepithelium. Further dissection to isolated nasal pits yielded the pieces that were grafted. Stage 26 frontonasal mass surface ectoderm was dissected in the same way, but the area that was dissected is shown in B . (B) Host embryo preparation: The graft site on stage 15, 20 and 26 embryos were prepared in the area represented in yellow (middle panel). Homo- and hetero-chronic grafts were performed as illustrated by the arrows. Representative grafts from stage 20 host embryos are presented in Fig. 4, and stage 15 host embryos are presented in Fig. 5. Key: fnm, frontonasal mass; lnp, lateral nasal pit; md, mandibular prominence; mxp, maxillary prominence; np, nasal pit; pal , pharyngeal arch 1; pa2, pharyngeal arch 2; 113 Figure 3.2: F G F 8 b induced ectopic bone and cartilage Figure 3.2: F G F 8 b induced ectopic pieces of bone and cartilage. F g f 8 b beads i m p l a n t e d into f ac i a l m e s e n c h y m e that is not e x p o s e d to FgfS e x p r e s s i o n i n d u c e d an ec top i c ske l e togen ic p r o g r a m . A , C ) L a t e r a l v i e w B, D) V e n t r a l v i e w : A , B) B e a d s p l a c e d in the p r e s u m p t i v e m a x i l l a r y p r o m i n e n c e at stage 15 i nduce ec top ic bone and ca r t i l age ( a r r o w h e a d ) . C , D) B e a d s p l a c e d i n the stage 2 6 m a x i l l a r y p r o m i n e n c e i nduce a s m a l l p i e c e o f ca r t i l age a r t i c u l a t i n g w i t h the l a c r i m a l bone . K e y : S c a l e bar : 1 m m a n d app l i e s to a l l . ios , in t raorb i t a l s e p t u m ; 1, l a c r i m a l ; m x , m a x i l l a r y b o n e ; n , nasa l b o n e ; n c , nasa l c o n c h a e ; p , pa la t ine b o n e ; p m x , p r e m a x i l l a r y b o n e ; q j , quadra to juga l ; v , v o m e r 114 115 Figure 3.3: The nasal pit extirpation at stage 20 results in small morphological changes (A-D), Stage 20 nasal pit extirpation changes the pattern of some bones in the nasal capsule. (E-G"), 24 hours after nasal pit extirpation at stage 20; (H-J), Wholemount in situ hybridization of extirpated embryos. (A-D) Alcian blue and alizarin red staining of an extirpated embryo 10 days after nasal pit extirpation at stage 20. (A) Lateral view of the skull, showing a reduction of the nasal bone and nasal conchae on the treated side (arrowheads). The lacrimal bone is split into two (arrow). (B) View of the palate of the same embryo. The palatine process of the maxillary bone is severely reduced (arrowhead), and the vomer is reduced to a small spur of bone (arrowhead). (C) Dorsal view of the nasal capsule. The nasal conchae (arrowhead) and nasal bone (asterisk) are severely reduced on the treated side. (D) The nasal bones have been removed in this dorsal view of the nasal capsule. The treated side nasal conchae are reduced. (E-G), The mesenchyme is covered by twenty-four hours after nasal pit extirpation at stage 20. Sections are presented in a cranial-caudal sequence (left to right) to show that some areas have thinner epithelia, and others have typical nasal pit epithelia. (E'-G') High power view of the extirpated area in the section above. The woundsite in the cranial-most section has been covered by a thin layer of epithelia (arrowhead), which is not typical of the normal nasal pit epithelia (E'); while other sections further caudally, shows some typical nasal pit epithelia (arrowhead) (F', G') . (E"-G") High power view of the contralateral, control nasal pit which shows typical nasal pit epithelia. (H-J) Nasal pit morphology assessed by wholemount in situ hybridization to exonic FgfS (H,I), and Dlx5 (J). (H) FgfS expression is maintained in the surface ectoderm immediately following (0 hours) surgery because only nasal pit ectoderm was removed. (I) Twenty-four hours following extirpation, FgfS expression is reduced to a small foci of expression (arrowhead). The contralateral nasal pit is surrounded by FgfS expression and shows the morphology of the nasal pit. (I', I") After photography, the embryo in (1) was sectioned coronally and shown here. FgfS expression is found in pseudostratified columnar epithelia on the treated side. (J) Twenty-four hours following extirpation, a small foci of Dlx5 expression remained confirming that the extirpation was not complete (arrowhead). Key: Scale bars: all are 0.1 mm except I" is 0.05 mm; A applies to B; E applies to F-G; E ' applies to E " - G " ; cp, commissural plate; e, eye; fnm, frontonasal mass; lnp, lateral nasal process; mx, maxillary bone; n, nasal bone; nc, nasal conchae; np, nasal pit; p, palatine; pmx, premaxillary bone; pnc, prenasal conchae; tel, telencephalon; v, vomer 116 Figure 3.4: Ectopic pieces of bone and cartilage are in induced by nasal pits grafted to Figure 3.4: Ectopic pieces of bone and cartilage are induced by nasal pits grafted to Stage 20 hosts (A-C) S tage 20 d o n o r nasal pi ts . (D-F) S tage 26 d o n o r nasa l p i t s . (G-I) Stage 26 c o n t r o l f ron tonasa l e p i t h e l i u m . (A) C h a n g e s to the ex te rna l a u d i t o r y mea tus in response to the stage 20 nasal p i t grafts. ( B ) Stage 20 nasal pi t i n d u c e d e c t o p i c p rocess o n the p r o x i m a l end o f the quadra to juga l ( a r r o w h e a d ) . ( C ) S q u a m o s a l a r t i c u l a t i o n is e n l a r g e d in response to the graft (as ter isk) . ( D ) K e r a t i n i z e d t i ssue has been i n d u c e d in the e y e l i d in response to a stage 26 nasal p i t ( a r rowheads ) . M u l t i p l e e c t o p i c r idges and o p e n i n g s are present . ( E ) E c t o p i c p rocess o n p r o x i m a l q u a d r a t o j u g a l , a n d ec top i c p iece o f ca r t i l age in e y e l i d ( a r r o w h e a d s ) . ( F ) E c t o p i c p i ece o f ca r t i l age i n e y e l i d ( a r r o w h e a d ) . ( G ) C o n t r o l , f ron tonasa l mass e p i t h e l i u m a l l o w e d n o r m a l d e v e l o p m e n t e x t e r n a l l y . ( H ) T h e s k u l l a l so d e v e l o p e d n o r m a l l y i n f ron tonasa l mass e p i t h e l i u m grafts. (I) N o r m a l m o r p h o l o g y o f the j a w j o i n t . K e y : S c a l e bars A = 5 m m a n d app l i e s to B , D , E , F , G , H ; C = 1 m m a n d app l i e s to C , F , I. e, eye ; earn, ex te rna l a u d i t o r y mea tus ; ; pt, p t e r y g o i d ; q, quadrate ; q j , q u a d r a t o j u g a l ; s, s q u a m o s a l 117 Figure 3.5: Organized outgrowth of bone and cartilage is induced by nasal pits into Stage 15 hosts 118 Figure 3.5: Organized outgrowth of bone and cartilage is induced by nasal pits grafted into Stage 15 hosts Ectopic skeletal elements induced in Stage 15 host embryos by stage 20 donor nasal pits (A-F) or stage 26 donor nasal pits (G-I) (A,D,G) External view of embryos, (E-H) Graft development in situ (C,F,I) Induced grafts; (A) outgrowth near the external auditory meatus (arrow); (B) Graft forms an ectopic process on the quadratojugal with several other skeletal elements organized around a central pit. (C) Lateral of the graft, attached to the quadrate. The presence of secondary cartilage of the normal quadratojugal, is duplicated in the induced skeletal structures. Several other skeletal elements were induced. (D) Minor changes in the epithelium of the eyelid (arrowhead). (E) Graft is located at the proximal end of the quadratojugal directly underlying the epithelial changes in D. Triangular-shaped skeletal element outlined in yellow. (F) Multiple pieces of ectopic elements (G), Ectopic external nasal aperture with keratinous tissue induced in response to stage 26 nasal pits. Inset is view of whole head. (H) Ventral view of graft, showing that is has grown out of the skull. (I) Large amount of induced cartilage surrounded by several bones. Key: Scale bars, A = 5 mm, applies to D,E,G; inset in G = 5mm; B = 1mm; C = 1 mm applies to F , l ; H = 1 mm; e, eye; ena, external nasal aperture; eena, ectopic external nasal aperture; esc, ectopic secondary cartilage; f, frontal; md, mandible; q, quadrate; qj, quadratojugal; s, squamosal; sc, secondary cartilage 119 120 Figure 3.6: The grafts consist of epithelium only. A series of embryos had quail stage 20 or 26 nasal pits grafted into chicken hosts to determine the origins of the induced structures. (A) Twenty-four hours after placement, the graft (asterisk) is present in the maxillary prominence. The pin is also present in this specimen. (B) Within 24 hours of grafting, the stage 26 nasal pit has invaginated into the host mesenchyme. We observed quail cells only in the grafted epithelium. (C-F) Embryo collected 6 days after surgery, upon collection, an ectopic outgrowth is present in the maxillary prominence (inset, arrowhead). The plane of section is indicated by the line in the inset. Section stained for gone and cartilage encompassing the graft. The graft is present in the outgrowth (arrowhead). (D) Detection of post-mitotic neurons with the TuJl antibody. There are differentiated neurons near the graft (box and arrowhead). (E) Magnified view of box in D that shows neurons extending from the graft. (F) Nearly adjacent section to D, labeled with the QeTN antibody shows that the grafted epithelium is quail in origin, and no evidence of quail mesenchymal cells were observed. We observed QeTN positive cells, outside the immediate area of the graft, and those are very close to the area that is labeled with TuJl in E. Key: Scale bars: (A): 1 mm; (C,D): 0.2 mm; (B,E,F): 0.1 mm. be, basii cranium; e, eye; en, entoglossum; mb, manibular branch of the trigeminal nerve; mc, Meckel's cartilage; mdp, mandibular prominence; mxp, maxillary prominence; np, nasal pit; tg, trigeminal nerve body 121 Figure 3.7: The nasal pit grafts undergo normal morphogenesis and differentiate into nasal structures A l c i a n B l u e and P i c ros i r i us R e d TUJ1 D en q-be -O C oc me h. ^ ' . > Ibc P C oc -v B D K u $ m i ' M / -122 Figure 3.7: The nasal pit grafts undergo normal morphogenesis and differentiate into neurons. Stage 20 chicken nasal pits were grafted into stage 15 host embryos, and collected at stage 28 to stage 30. Close-ups of grafts were rotated 90 so that the presumptive external nasal aperture is oriented towards the bottom of the page. ( A - B ; F-I) Embryo collected at stage 30. (A) The grafted nasal pits induced an outgrowth of chondrogenic cells and the graft undergoes normal morphogenesis by making an external nasal aperture. (B) External nasal aperture is closed at this stage of development. (C-E) Embryo collected at stage 28. (C) Graft is present near the mandible (arrowhead). (D) Graft is positive for differentiated neurons. (E) Box in D. Graft has many TuJl positive cells within it. (F) The embryo has an outgrowth that is separate from the head, the grafted nasal pit lies at the base of the outgrowth and wil l make an external opening (A, B). (H) Box in G. The grafted nasal pit in this section, has few TuJl positive cells, (I) Nearly adjacent section of the graft has many TuJl positive cells, and a nerve exiting the grafted epithelium. (J-M) A stage 30 embryo graft that showed different morphology than the embryo in (F-I). (K) This graft is representative of a more cranial section of an in situ nasal cavity. (L) Box in K . 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JNeurobiol 49, 29-39. 128 Chapter 4 - Discussion 4.1 The chicken embryo is an appropriate model for cleft lip Directly within the zone of fusion, the embryonic chicken, and mouse upper lip share similarities including spatio-temporal gene expression patterns, such as the expression of FgfS in the frontonasal mass epithelium that becomes increasingly restricted to the edges of the nasal pit, (Bachler and Neubuser, 2001; Song et al., 2004), increased apoptosis in the epithelium (Chapter 2; (Jiang et al., 2006; Sun et al., 2000), and evidence for epithelial to mesenchymal transformation (Fitchett and Hay, 1989; Sun et al., 2000) at the time of l ip fusion. An advantage of using the chicken embryo to model cleft lip is that we are be able to manipulate signals that are required for apoptosis or epithelial to mesenchymal transformation and follow the morphogenesis in three dimensions. M y results in chicken embryos showed that FGF signaling is required for lip fusion have recently been validated in human genetic studies. Screening large populations of humans with cleft lip in both the Phillipines and Iowa, identified several missense mutations in FGFs and FGFRs (Riley et al., 2007). In a child with bilateral cleft lip, they described a novel, D73H mutation in FGF8, which prevents FGF8 from binding its cognate receptor (Riley et al., 2007). The D73 moiety is conserved in chick, zebrafish, mouse, rat, human, and pufferfish (Riley et al., 2007). FGFR1 is the gene underlying the autosomal dominant version of Kallmann syndrome, where cleft lip is part of the phenotype (Dode et al., 2003). Mutations have been found in exon 8 of FGFR1 which is important for ligand binding and is invariant in either the epithelial (FGFRlb) or mesenchymal (FGFRlc ) splice variant (Dode et al., 2007). The pathological basis of the mutations of the FGFR1 are loss function, either by loss of tyrosine kinase activity or misfolding in the endoplasmic reticulum, resulting in reduced FGF signaling (Pitteloud et al., 2006). Thus mutations in FGF receptors or ligands could be one of the predisposing factors that increase the likelihood for developing a cleft. Thus the chicken model is able to identify similar pathways to human genetic studies but can add further insight into the exact mechanism by which a particular signal causes a cleft. 4.2 The cleft results from reduced FGF signaling in the upper face Analysis of wholemount in situ hybridization in Chapter 2 showed that within 6 hours of bead implantation we observed decreased expression of Spry2 and Spry4, intracellular inhibitors of FGF signaling. This could mean that in fact we have increased FGF signaling, through loss of 129 its inhibitor. Loss of Spry expression has been shown to result in overactivity of the breathless (FGF) signaling in Drosophila, resulting in increased tracheal buds (Hacohen et a l , 1998). 1 wil l now go through the evidence to show that despite decreased Spry transcripts, SU5402 did in fact reduce FGF signaling. The most direct way of assessing FGF signaling activity was to look for molecular evidence of decreased phosphorylation of intracellular signal transduction proteins or decreased expression of downstream gene targets of FGF signaling. We observed both decreased abundance of Pystl transcripts: a dual specificity phosphatase that controls levels of activated E R K , and decreased abundance of activated phospho-ERK protein in cranially-treated embryos. The caudal position, which did not result in clefts, only had slight decreases in Pystl and phospho-ERK. Moreover three transcription factor known to be activated by exogenous FGFs, but not directly in a FGF functional feedback loop, Msxl (Ferguson et a l , 2000; Mina et a l , 2002), Msx2 (Mina et a l , 2002) and Dlx5 (Ferguson et a l , 2000), were also downregulated. Thus the gene expression and other molecular data are consistent with a decrease in FGF signaling. Our next line of evidence that we temporarily inhibited FGF signaling with SU5402 treatment comes from cellular dynamics assays in micromass culture. In comparison to the maxillary and mandibular prominence, the frontonasal mass mesenchyme has more proliferating cells in response to exogenous FGF after 48 hours in micromass culture, suggesting that proliferation in the frontonasal mass mesenchyme is driven by FGF signaling (Richman and Crosby, 1990). In the cranially treated embryos, we showed that SU5402 decreased proliferation by twelve hours in the frontonasal mass, consistent with decreased FGF signaling. FGF8 is a cell survival factor (Trumpp et a l , 1999) and as predicted, apoptosis was increased with SU5402 treatment. It also suggests that the cleft phenotype results from the loss of proliferation and decreased cell survival leading to a failure of the frontonasal mass to reach optimal size. We can also compare our results to conditional knockout mice in which FGF ligand or receptors were removed during upper lip development. If FGF signaling is required for skeletal development, then deletions of facial bones should be observed in the mouse knockouts. Conditional knockouts of Fgf8 in the facial epithelium, driven by Nestin-cre (Trumpp et a l , 1999), AP2a-cre (Macatee et a l , 2003) or FoxGl-cre (Kawauchi et a l , 2005) or hypomorphic alleles of Fgf8 (Abu-Issa et a l , 2002; Frank et a l , 2002) have severe facial defects. These embryos have reduced maxillary and premaxillary bones (Abu-Issa et a l , 2002; Frank et a l , 2002; Trumpp et a l , 1999), coincident with the foreshortening of the upper face (Kawauchi et 130 al., 2005; Macatee et al., 2003). Dual conditional knockouts driven by FoxGl-cre FGFR1 and FGFR2 in the neural and facial ectoderm also have severely hypoplastic medial nasal prominences by El2 .5 (Gutin et al., 2006). Although no cleft lips were observed in the various FGF knockout mice, the losses of distal facial bones are similar to our SU5402 treated embryos. In contrast, Sprouty knockout mice in which there would be predicted to be a gain in FGF signaling for the most part do not have deletions or reductions of the facial skeleton. There are not many effects on facial morphogenesis in either Spry] (Basson et al., 2005), Spryl (Shim et al., 2005) or Spry4 (Taniguchi et al., 2007) knockouts which are all fertile and viable. Each Spry knockout mouse develops overgrowth of the systems reported: the Spry2 knockout has ectopic cells in the cochlea (Shim et al., 2005), and supernumerary teeth in the diastema (Klein et al., 2006), and hyperganglionosis in the colon (Taketomi et al., 2005). Each of these supernumerary structures induced in the single knockouts were accompanied by elevated levels of phospho-ERK in the structures at early stages (Klein et al., 2006; Shim et al., 2005). As is the case with many transgenic mouse lines, changing the background can reveal phenotypes that were not observed in the original strain. The one exception to the general lack of deleterious effects on facial skeleton by Spry gene deletion was the reduced mandibles produced by changing the genetic background of Spr4 knockouts (Taniguchi et al., 2007). In addition to the effect of genetic modifiers present in different strains of mice, redundancy of function together with overlapping expression domains of the Spry family members could explain the absence of facial phenotype in the single knockouts. Double knockouts of Spr2, and Spr4 have severe craniofacial abnormalities, including cyclopia, holoprosencephaly and exencephaly, these embryos were embryonic lethal at El2 .5 preventing skull analysis, and unfortunately were not analyzed for the amount of FGF signaling in the embryos (Taniguchi et al., 2007). I expect that further.analysis of conditional, compound knockouts will clarify the role of Spry genes in facial morphogenesis Taken together, the molecular, cellular and skeletal defects we have produced demonstrate that loss of FGF signaling is the main effect of SU5402 and that the decrease in just this one pathway is enough to predispose the embryos to cleft lip. 4.3 The upper face is sculpted by the coordinated actions of specific regions of epithelia Our extirpation experiments confirmed that the bones that surround the nasal conchae are patterned by the nasal pit. We also showed that loss of FGF signals from the cranial frontonasal mass prevent normal patterning, and we propose that it is the nasal slit epithelium that is the 131 endogenous source of FGFs for the frontonasal mass. Thus, we propose that the coordinated outgrowth and patterning of the skeleton of the upper face depends on epithelial signals from the nasal pit and frontonasal epithelial zone (Fig. 4.1 A) . The nasal pit signals first to prime the osteogenic and chondrogenic precursor cells in the mesenchyme, probably between stage 15 and 20. Thereafter the already committed mesenchymal cells know which part of the face they belong to and details of cartilage and bone patterning are imparted by signals from the frontonasal mass epithelial zone (Hu et a l , 2003). Following these axis determination steps, then from stage 22-27 the upper face enlarges which is facilitated by the growth centers in the lateral, cranial frontonasal mass, the caudal maxillary prominence, and. the middle of the lateral nasal prominence (Chap.2; (Peterka and Jelinek, 1983)). These growth centres ensure that the facial prominences are the correct size for normal development. We showed that when the growth centres are disrupted lip fusion fails. After fusion, the external nasal apertures are induced by the nasal pit, and sculpting of the bones of nasal capsule ensues. We showed that the nasal capsule was malformed and the external nasal apertures were absent in the stage 20 embryos that had the nasal pit extirpated. The external nasal aperture was induced by stage 26 nasal pit grafts. Therefore, the upper face is sculpted by the interaction of the frontonasal ectodermal zone and the nasal pit. 4.4 Morphogenesis in the face is driven by distinct proliferation patterns Our analysis has identified regional differences in proliferation in the frontonasal mass and maxillary prominences. On the untreated contralateral side of the embryo, we identified areas with decreased proliferation on either side of the zone of fusion. Detailed analysis of arrested metaphases and prophases in 5-day old colchicine treated chicken embryo faces identified two 'proliferative centres' in the frontonasal mass; one in the lateral edge of the frontonasal mass, in an area that we have identified as most sensitive to FGF signaling and one in the caudal edge of the medial frontonasal mass (Peterka and Jelinek, 1983). In addition, these authors observed a third proliferative center in the caudal region of the maxillary prominence, which has a lower proliferation index than the lateral frontonasal mass (Peterka and Jelinek, 1983). The proliferative centre of the maxillary prominence is also very close to the maxillary FgfS expression zone. A re-examination of the data in recent studies that characterized the proliferation in the cranial or caudal half of the lateral frontonasal mass in the stage 26 chicken and duck faces (Wu et a l , 2004) and stage 28 chicken (MacDonald et a l , 2004; Wu et a l , 2004) comes to slightly different conclusions than ours. They show that the caudal 132 half of the lateral frontonasal mass has more proliferating cells than the cranial half (MacDonald et al., 2004; Wu et al., 2004), which likely reflects the precise section depth. Proximal or distal sections wil l not include the zone of fusion. Within the zone of fusion we observed decreased proliferation in both the globular process and cranial maxillary prominence. We are better able to resolve regional differences in the frontonasal mass because we used three regions from the cranial edge of the nasal slit to the globular process and fluorescence detection. When the frontonasal mass is divided only in a cranial and caudal regions (MacDonald et al., 2004; Wu et al., 2004), it divides the lateral frontonasal growth centre between both regions (Peterka and Jelinek, 1983). Both studies used a D A B (diaminobenzidine) as a detection method, so it is more difficult to see the contrast between labelled and unlabelled cells, and prevents automated counting. From proliferation studies, we propose a new mechanism for how the growth patterns in the frontonasal mass and maxillary prominence promote fusion. The frontonasal mass growth centre proliferates and increases the frontonasal mass volume which pushes or displaces the globular process towards the maxillary prominence. In addition to discovering how proliferation contributes to fusion, we have also found a molecular pathway that mediates this process. The proliferation within the frontonasal mass (and probably maxillary) growth centre is driven by FGF signaling. We placed the cranial SU5402 beads near the growth center of the frontonasal mass and this manipulation decreased proliferation directly within the most active proliferative region. Embryos treated with SU5402 in areas of mesenchyme that had instrinsically lower proliferation have no effect on upper lip morphogenesis. These FGF-independent regions lie immediately adjacent to the fusion zone in the globular process and cranial edge of the maxillary prominence. 133 4.5 Three FGF independent regions in the face The idea of areas of FGF independence in the developing face had been proposed first for the mandibular prominence (Trumpp et a l , 1999). However in my study 1 have uncovered two more areas that are also less dependent on FGF signals. The regions that we identified as independent of FGF signaling are the cranial edge of the maxillary prominence and the caudal region (globular process) of the lateral frontonasal mass. I wi l l now discuss each region and relate them to each other. Distal region of the mandible The mandible develops from paired pharyngeal arches that merge in the midline with increased cell death in the central furrow. Bmp4 is expressed in midline epithelium of the mandibular prominence and determines incisor identity (Tucker et a l , 1998). In Nkx2.4 ere driven Bmp4 excision deleted the distal mandible, and incisors, while the proximal mandible still formed (Liu et a l , 2005). FgfS is expressed in a complementary domain, lateral to the Bmp4 transcripts. The nestin-cre driven excision of FGF8 in facial epithelium, caused the loss of all bones within the mandible, however, looking at younger stages of embryos, the gene expression of normal markers of the distal mesenchyme are still present (Trumpp et a l , 1999). Mice that are hypomorphic for Fgf8, have less severe phenotypes, the proximal region is mostly deleted but the distal region of the mandible including the incisors still forms (Abu-Issa et a l , 2002). Together these mice suggest that distal region of the mandible can develop independently of FGF8 though there is a requirement for B M P 4 signaling. As we shall see FGF and B M P signaling are also acting in complementary regions of the upper facial prominences. Cranial edge of maxillary prominence and globular process of the frontonasal mass The upper lip zone of fusion consists of the frontonasal mass-derived globular process and the cranial region of the maxillary prominence. Interestingly at the time of fusion both regions are covered in epithelia that express Bmp4 but not FgfS (Ashique et a l , 2002; Song et a l , 2004). The role of endogenous B M P signaling has been directly tested in the two sides of the fusion zone in chicken embryos. Application of Noggin (a B M P antagonist) soaked beads in both the cranial maxillary prominence and the globular process of the frontonasal mass induced a cleft phenotype (Ashique et a l , 2002). The beads induced a significant reduction in the size of the maxillary prominence and frontonasal globular process, through reduced proliferation, suggesting that endogenous B M P signaling is required to maintain the proliferation that is taking 134 place in the fusion zone (Ashique et al., 2002). Additionally, the Noggin-soaked beads increased epithelial cell survival and thickness of the epithelia within the zone of fusion (Ashique et al., 2002). Thus endogenous BMPs are also regulating the removal of the epithelial seam after fusion has started. In the non-manipulated chicken embryo the patterns of cell death are also highest under areas where Bmp4 is expressed, eg. the cranial maxillary prominence and globular process(Ashique et al., 2002; McGonnell et al., 1998; Song et al., 2004). Thus the normal role of B M P signaling may be to remove epithelial cells in areas that are going to merge or fuse. We have tested directly whether or not FGF signaling is active in the zone of fusion and have shown that it is not required. Instead our bead implants show that endogenous FGF signals are required in the cranial frontonasal mass near the cranial edge of the nasal slit. We did not test the caudal maxillary prominence since this area is not directly involved in lip fusion, although we hypothesize that FGF signals here are required for the jaw joint. We have presented evidence for B M P and FGF pathways operating in the frontonasal mass and maxillary prominence, and reviewed the evidence in the mandible. In each of these prominences, FGF is associated with controlling growth and B M P is required for controlling apoptosis. Moreover, within the developing upper lip, the FGF maintains increased proliferation in the cranial frontonasal mass, while proliferation in the globular process is under the control of B M P signaling. Such that the BMPs provide a cell proliferation cue in the globular process, and FGFs do the same in the cranial frontonasal mass. In summary, normal development occurs through two signal pathways controlling the morphogenesis in distinct regions of the facial prominences. The complimentarity of FGF and B M P signaling is a recurring theme in developmental biology and imparts a greater significance to our findings that extends beyond lip fusion. 135 4.6 FUTURE DIRECTIONS Additional experiments could be done to analyze proliferation the fusing upper lip, the results of which would provide a significant advance for the field. To fully analyze proliferation in the face, I recommend BrdU labelling three stages of embryos, one before fusion, during fusion, and following fusion focusing on serial sections that include the globular process. Then the frontonasal mass should be divided into three cranial-caudal regions. This will provide definitive data for the dynamics of cell proliferation within the face, and how that contributes to lip fusion. Although there is much data already published on the facial prominences, this study will provide focused data directly on lip fusion, and together with already published morphometric analysis wil l provide a framework for assessing the mechanical contribution to facial clefting. We showed that the stage 20 or 26 nasal pit has an inherent capacity to induce elements that are reminiscent of portions of a nasal capsule. To truly test the capacity of the nasal pit to induce an ectopic nasal skeleton, I propose that the grafting experiments be performed with stage 17 nasal pits. The stage 17 nasal pits need to be grafted to stage 15 and stage 20 hosts, so that a comparison can be made between the data presented in Chapter 3. I propose grafting to a younger host to target migrating neural crest. To further test the possibly that the nasal pit epithelia is able to instruct the facial mesenchyme to become nasal like, we should look at the localization of Pax7 to determine if a lateral nasal phenotype was induced. If Pax7 is induced then the nasal pit is providing instructive signals to the responding mesenchyme, and may suggest that the placodes are providing cues that regionalize head ectomesenchyme. Is the ability to respond to the instructive signals from the placode only present in the head? The mesenchyme specialization can be tested by grafting an isolated nasal pit to the flank region. I predict that these grafts wil l have nasal cavities within an ectopic limb. Such a result would then suggest that the in situ olfactory placode, in particular, is providing a cue to be anterior facial mesenchyme, and the facial mesenchyme is able to respond by creating a nasal capsule. However in the flank, the epithelia can induce the outgrowth of the mesenchyme, but the mesenchyme can only make a limb based on its expression of Hox genes. 136 Figure 4.1: FGF dependent and independent regions control outgrowth and patterning of the face 137 Figure 4.1 F G F dependent and independent regions control outgrowth and patterning of the face (A) Epithelial signaling domains that pattern facial development. Stage 15: The nasal pit primes the osteogenic and chondrogenic precursors in the upper face; Stage 20: The frontonasal ectodermal zone induces outgrowth (Hu et a l , 2003); Stage 26: The nasal pit patterns the nasal conchae and the external nares. (B) Coordination of FGF independent and dependent regions of the face. In the frontonasal mass, we identified a FGF dependent growth center (green circle) that drives the increased volume in the frontonasal mass. 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