Dentistry, Faculty of
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UBCV
Hui, Jenny Nga Jen
Master of Science - MSc
University of British Columbia
Retinoids have been known to influence cell migration, interaction, and differentiation, but their role in skeletal development is still unclear. We investigated the role of retinoic acid receptor (RAR) signaling in facial patterning in chicken embryos. Beads soaked in RAR pan-antagonist, AGN 193109, and implanted into the right nasal pit of stage 15 to 20 embryos induced mild skeletal abnormalities. In addition, Msx-1, Msx-2, Shh, and Fgf-8, expression were not altered, implying that AGN 193109 was not potent enough to completely inhibit all endogenous retinoid signaling in vivo. We indirectly studied the in vivo effectiveness of AGN 193109 by implanting embryos with both RAR antagonist and exogenous retinoic acid (RA) soaked beads to determine if the RAR antagonist could antagonize the RA induced teratogenic effect. We demonstrated that a low concentration of exogenous RA could result in failure of outgrowth of the prenasal cartilage and premaxilla. Moreover, the RA induced defects could be rescued by the cotreatment of RAR antagonist, suggesting that AGN 193109 was effective in antagonizing retinoid signaling in vivo for 18 hours and could not be dislodged from RARs by exogenous RA. RA treated embryos did not show any changes in Fgf-8 and Shh expression, implying that Fgf-8 and Shh were independent of retinoid signaling. However, ectopic expression of Msx-2 was induced by RA treatment and was not downregulated by the cotreatment of the RAR antagonist. This data indicated for the first time that Msx-2 was regulated by retinoid signaling but was not important for the patterning of the chicken upper beak. Most importantly, we demonstrated that a temporary downregulation of Msx-1 in stage 24 RA treated embryos resulted in failure of outgrowth of the prenasal cartilage and premaxilla, but this downregulation was recovered by the cotreatment of RAR antagonist. Thus, our study revealed that maintenance of Msx-1 between stage 20 and stage 24 was essential for the outgrowth of the chicken upper beak.
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THE EFFECTS OF ENDOGENOUS A N D EXOGENOUS RETINOIDS ON F A C I A L PATTERNING by J E N N Y N G A JEN HUI B.Sc , The University of British Columbia, 1999 A THESIS SUBMITTED IN P A R T I A L FULFJMENT OF REQUIRMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Oral Health Sciences, Faculty of Dentistry) We accept thisJJiesis as conforming to. the reqiffred standard THE UNIVERSITY OF BRITISH C O L U M B I A SEPTEMBER 2001 © Jenny Nga Jen Hui, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date DE-6 (2/88) Abstract Retinoids have been known to influence cell migration, interaction, and differentiation, but their role in skeletal development is still unclear. We investigated the role of retinoic acid receptor (RAR) signaling in facial patterning in chicken embryos. Beads soaked in R A R pan-antagonist, A G N 193109, and implanted into the right nasal pit of stage 15 to 20 embryos induced mild skeletal abnormalities. In addition, Msx-1, Msx-2, Shh, and Fgf-8, expression were not altered, implying that A G N 193109 was not potent enough to completely inhibit all endogenous retinoid signaling in vivo. We indirectly studied the in vivo effectiveness of A G N 193109 by implanting embryos with both R A R antagonist and exogenous retinoic acid (RA) soaked beads to determine i f the R A R antagonist could antagonize the R A induced teratogenic effect. We demonstrated that a low concentration of exogenous R A could result in failure of outgrowth of the prenasal cartilage and premaxilla. Moreover, the R A induced defects could be rescued by the cotreatment of R A R antagonist, suggesting that A G N 193109 was effective in antagonizing retinoid signaling in vivo for 18 hours and could not be dislodged from RARs by exogenous R A . R A treated embryos did not show any changes in Fgf-8 and Shh expression, implying that Fgf-8 and Shh were independent of retinoid signaling. However, ectopic expression of Msx-2 was induced by R A treatment and was not downregulated by the cotreatment of the R A R antagonist. This data indicated for the first time that Msx-2 was regulated by retinoid signaling but was not important for the patterning of the chicken upper beak. Most importantly, we demonstrated that a temporary downregulation of Msx-1 in stage 24 R A treated embryos resulted in failure of outgrowth of the prenasal cartilage and premaxilla, but this downregulation was recovered by the cotreatment of RAR antagonist. Thus, our study revealed that maintenance of Msx-1 between stage 20 and stage 24 was essential for the outgrowth of the chicken upper beak. ii Table of Contents Page Abstract i i Table of Contents iii List of Tables iv List of Figures vii Acknowledgments viii CHAPTER 1 INTRODUCTION 1.1 Facial patterning and morphogenesis 1 1.1.1 Human and chicken facial development 1 1.1.2 Skeletal development in chicken embryos 5 1.2 Retinoids 10 1.2.1 Retinoid production and degradation 10 1.2.2 Teratogenic effects of excess retinoids and retinoid deficiencies 13 1.2.3 Effects of retinoic acid on skeletal cell differentiation 14 1.2.4 Retinoid transduction 16 1.2.4.1 Retinoid receptor signaling 19 1.2.4.2 Genes regulated by retinoid signaling 20 1.2.5 Distribution and formation of RARs and RXRs 22 1.3 R A R antagonists 25 CHAPTER 2 2.1 Purpose of the study 30 iii C H A P T E R 3 METHODS A N D MATERIALS 3.1 Bead treatments 32 3.2 Implantation of beads in chicken embryos 34 3.3 Skeletal staining of chicken embryos 35 3.4 Preparation of embryos for whole mount insitu-hybridization 36 3.5 cDNA preparation 37 3.6 DIG-labeled R N A probe synthesis 39 3.7 Whole-mount insitu hybridization 40 C H A P T E R 4 RESULTS 4.1 External phenotype of DMSO, RA, and R A R antagonist treated embryos 42 4.2 Skeletal structure of DMSO and R A R antagonist treated embryos 46 4.3 Effectiveness of A G N 193109 54 4.3.1 External phenotype of R A treated embryos 54 4.3.2 External phenotype of R A - R A R antagonist treated embryos 59 4.3.3 Skeletal structure of R A and R A - R A R antagonist dual treated embryos 64 4.4 Whole mount insitu-hybridization 70 4.4.1 Msx-1 expression 70 4.4.2 Msx-2 expression 73 4.4.3 Fgf-8 expression 76 4.4.4 Shh expression 76 iv C H A P T E R 5 DISCUSSION 5.1 Endogenous R A was required for morphogenesis of lateral nasal, and maxillary primoridal derivatives 81 5.2 The antagonist rescued R A induced truncation of the upper beak 82 5.3 A G N 193109 was more effective than LG100815 85 5.4 Is signaling via RXRs important for outgrowth of the chicken upper beak? 86 5.6 Msx-1 expression at stage 24 was important for the outgrowth of chicken upper beak 87 5.7 Ectopic expression ofMsx-2 was induced by exogenous retinoids butMsx-2 was not important for the patterning of the upper beak 88 5.8 Fgf-8 and Shh expression were retinoid-independent in facial morphogenesis 89 C H A P T E R 6 CONCLUSION 90 References 91 Appendix 109 v List of Tables Page Table 1 Different treatment of R A R antagonist beads 33 Table 2 cDNA preparation from plasmids 38 Table 3 External phenotype of chicken embryos treated with DMSO beads 43 Table 4 External phenotype of chicken embryos treated with R A R antagonist beads 44 Table 5 Skeletal analysis of chicken embryos treated with R A R antagonist beads 48 Table 6 Defects of palatine process of maxillary bone in R A R antagonist treated embryos 50 Table 7 Extra cartilage and bone in R A R antagonist treated embryos 52 Table 8 Categories of severity scores of defects in treated embryos 55 Table 9 Significant differences between R A and R A - R A R antagonist treatments 60 Table 10 Msx-1 expression in chicken embryos with different treatments 71 Table 11 Msx-2 expression in chicken embryos with different treatments 74 Table 12 Fgf-8 expression in chicken embryos with different treatments 77 Table 13 Shh expression in chicken embryos with different treatments 79 v i List of Figures Page Figure 1 Facial prominences in human embryo 3 Figure 2 Facial prominences in chicken embryo 4 Figure 3 Membranous bones and cartilage in chicken upper beak 9 Figure 4 Retinoic acid metabolism and signaling in cell 11 Figure 5 Structure of R A R 18 Figure 6 Chemical structure of R A and R A R antagonist, A G N 193109 26 Figure 7 External phenotype of normal, DMSO, RA, R A R antagonist treated embryos 45 Figure 8 R A R antagonist affected regions in chicken upper beak 49 Figure 9 Defects of palatine process of maxillary bone in R A R antagonist treated embryos 51 Figure 10 Ectopic cartilage and bone in R A R antagonist treated embryos 53 Figure 11 External phenotype of chicken embryos in each score category 56 Figure 12 External phenotype of R A treated embryos 58 Figure 13 External phenotype analysis of RA, and R A - R A R antagonist, treated embryos 62 Figure 14 Skeletal structure of RA, R A - R A R antagonist treated embryos 66 Figure 15 Skeletal analysis of RA, R A - R A R antagonist (1 bead), R A - R A R antagonist (2 beads), treated embryos 67 Figure 16 Msx-1 expression 72 Figure 17 Msx-2 expression 75 Figure 18 Fgf-8 expression 78 Figure 19 Shh expression 80 v i i Acknowledgements I would like to thank Dr. Joy Richman for teaching me technical skills, providing me with supervision throughout the project, and helping me to bring this thesis to completion. I would like to sincerely thank Dr. Virginia Diewert for providing me with many important ideas and suggestions when I encountered difficulties in the project. I would also like to thank Dr. Douglas Waterfield, Dr. Tim O'Connor for their input, helpful advice, and review of this thesis. I have learned a lot throughout these two years, and without the support of my supervisor and committee members, finishing this project would not have been possible. I would like to thank my lab members for providing me with their generous assistance. Special thanks to Dr. Sang-Hwy Lee for interesting discussion, Katherine Fu for her expertise in technical skills, and Mary MacDonald for help and advice. Finally, I am very grateful to my parents for supporting me all the time and my brother for important advice. Their care and understanding encourage me not to give up during difficult times but try my best to reach my goals. I would like to express my deepest thanks to them for always being there to support me. viii C H A P T E R 1 I N T R O D U C T I O N 1.1 Facial patterning and morphogenesis 1.1.1 Human and chicken facial development Face development is a complex program that involves proper fusion and merging of facial prominences, which are buds of mesenchyme encased in epithelium. The major prominences in the human face are the frontonasal prominence, maxillary prominences, lateral nasal prominences, medial nasal prominences and mandibular prominences (Figure 1). The frontonasal, lateral nasal, medial nasal, and maxillary prominences form the primary palate, which gives rise to the upper jaw; the mandibular prominences form the lower jaw. In human embryos, primary palate is formed by the end of the fourth week of development, when the nasal pits deepen and the mesoderm of the nasal pit intervenes with mesoderm of the stomodeum. As a consequence, lateral and medial nasal prominences arise and are located between the frontonasal prominence. Fusion of the maxillary prominences with the medial nasal prominences is completed by the end of the sixth week of development. As development proceeds, the maxillary prominences also merge with the lateral nasal prominences. Hence, proper merging and fusion of the maxillary prominences with the medial and lateral nasal prominences is critical for the development of the upper face. Failure of these events results in cleft lip and primary cleft palate. In the early seventh week of development in the human embryos, palatal shelves extend from the maxillary prominences and direct downward on either sides of the tongue. When the tongue drops, the palatal shelves ascend above the tongue and fuse with each other, forming the secondary palate. Failure of fusion between the palatine shelves gives rise to secondary cleft palate. In our study, we used chicken embryos as a model to study face development. Unlike mouse embryos which are inside the mother's womb, experiments can be performed on chicken embryo during embryonic development and the treated eggs can be incubated until the desired embryonic stage. In addition, most of the developmental 1 processes of the face in chicken embryos are similar to those in humans. However unlike in humans, the frontonasal prominence merges with the medial nasal prominences to form one continuous prominence (Figure 2). The frontonasal prominence grows out at stage 24 and fuses with the lateral nasal and maxillary prominences at stage 28 to form the upper beak (Will and Miller, 1981). Failure of outgrowth of the frontonasal prominence affects fusion of the facial prominences, resulting in severe bilateral clefting of the primary palate. This phenotypic defect is often observed in chicken embryos treated with retinoic acid, the model system used in the present study. 2 Eye Nasolacrimal groove Stomodeimv Nasal pit Lateral nasal prominence Medial nasal prominence Figure 1. Facial prominences in human embryo (Langman, 1981). The major prominences in a six week embryo are the frontonasal prominence, lateral nasal prominences, medial nasal prominences, maxillary prominences, and mandibular prominences. Figure 2. Facial prominences in chick embryo. The major prominences in a stage 20 chicken embryo are the frontonasal mass (fnm), lateral nasal prominences (lnp), maxillary prominences (mx), and mandibular prominences (md). np, nasal pit; sto, stomodeum. 4 1.1.2 Skeletal development in chicken embryos Previous experiments indicate that most skeletal elements in the chicken skull, which include the premaxilla, nasal, and maxilla bones, as well as the prenasal cartilage, nasal conchae, and interobital septum are all derived from neural crest cells (Le Lievre, 1978; Noden, 1978; Couly et al, 1993). Neural crest cells are a group of ectodermal cells that migrate from the neural tube to different parts of the body. Trunk neural crest cells give rise to neurons and supporting glial cells of the sensory, sympathetic, and parasympathetic nervous systems, or pigment cells of the epidermis, whereas cranial neural crest cells also give rise to cartilage, bones, and connective tissues of the face and jaw (Le Douarin, 1982; Noden, 1973; 1978). Generally, skeletal development in the face of chicken embryos can be divided into four main processes: (1) migration of neural crest cells (2) interaction of mesenchymal and epithelial cells, (3) proliferation and condensation of mesenchymal cells, (4) chondrification and ossification. Any interference during these processes may not only disrupt fusion of facial prominences, but will also result in abnormal development of the facial skeletal system. (1) Migration of neural crest cells The facial skeletal elements in chicken embryos are produced by the cranial neural crest and the first five somites (Noden, 1978, 1982). While neural crest cells from the prosencephalon, mesencephalon, rhombomeres r l and r2 contribute to the first branchial arch, those from r4 and r6 contribute to the second and third branchial arches respectively (Couly et al, 1996; Kontges and Lumsden, 1996). The anterior mesencephalic neural crest cells also contribute to the frontonasal prominence to form the prenasal cartilage as well as the premaxilla (Couly et al, 1996; Kontges and Lumsden, 1996). The first branchial arch later develops into the maxillary prominences, which gives rise to the maxilla, palatine, jugal, and quadratojugal, and the mandibular prominences which form the lower beak structures. The more caudal branchial arches give rise to cartilages of the neck. Is the fate of the neural crest cells predetermined before migration? Data from 5 grafting experiments have been inconsistent. Some show that the neural crest cells are prepatterned prior to migration; others indicate that once the cranial neural crest cells arrive at their destination sites, they are immediately modified by factors in the new environment. For example, in 1983, Noden shows the formation of an ectopic beak in the second arch when presumptive second and third arch neural crest cell are replaced with neural crest from the mid-metencephalon. This experiment suggest that the fate of the cranial neural crest cells may be predetermined before migration. However, in 1978, Noden demonstrates that replacement of chicken neural crest cells from the posterior mesencephalon and anterior hindbrain with quail neural crest cells from the posterior diencephalon and anterior mesencephalon, results in normal facial morphogenesis. A possible explanation of the result is that neural crest grafts from the mid-metencephalon in the 1983 experiment includes the mid-hindbrain isthmus. There may be prepatterning in the neural crest cells from this region, but not in more anterior populations (P. Trainor, personal communication). These experiments by Noden are verified by a recent experiment which demonstrates that grafts from mesencephalic and rl/r2 neural crest cells transposed to r4/r6 only differentiate into an ectopic lower jaw if the grafts include the lateral wall of the neural tube, but not if the grafts only include the dorsal edges of the neural tube (Couly et al, 1998). Presumably, grafts that made ectopic beaks also include a part of the isthmus. In addition, Couly et al. (1998) show that r4/r6 neural crest cells do not produce first branchial arch skeletal elements when transposed to rl/r2 and mesencephalon region, suggesting that the fate of these more caudal neural crest cells is not predetermined before migration. (2) Interaction of mesenchymal and epithelial cells, and outgrowth and proliferation of mesenchymal cells When the neural crest cells arrive at their destination sites, they interact with the epithelium. Many experiments have shown that epithelial signaling is required for the activation of mesenchymal genes. Grafts of frontonasal mass in the wing buds of chicken embryos produce upper beak like structures; but removal of the ectoderm before grafting inhibits outgrowth completely and only small fragments of cartilage differentiate 6 (Wedden, 1987; Richman and Tickle, 1989). In addition, recombinations of epithelium and mesenchyme from different primordia of the chicken face reveal that facial epithelia are interchangeable and control the morphogenesis of the mesenchyme (Richman and Tickle, 1989). Moreover, Fibroblast growth factor 8 (Fgf-8), a signaling protein expressed in some facial epithelia, is able to substitute epithelial signaling and activate mesenchymal genes such as the BarH like homeobox (Barxl) in the chicken maxillary primordia (Barlow et al., 1999). Other Fgfs can also substitute for facial epithelium and can promote outgrowth of the mesenchyme (Richman et al., 1997). Recombination of retinoic acid-treated frontonasal mass mesenchyme with untreated frontonasal ectoderm yields truncated prenasal cartilages that mimic the effect of removing the ectoderm (Wedden, 1987). Thus, both epithelial signaling and teratogens, like retinoids, can influence the patterning of neural crest derived mesenchyme. (3) Condensation, proliferation, and differentiation of mesenchymal cells Skeletal condensations begin when a dispersed population of mesenchymal cells gathers together to proliferate and grow. The size and shape of condensations are critical for initiation of skeletogenesis. The boundaries of condensation are controlled by extracellular matrix molecules (reviewed in Hall and Miyake, 2000, Koyama et al, 1995), homeobox genes (Kanzler et al, 1998), as well as growth factors (Kim et al, 1998). Once the size of condensation reaches a critical threshold, genes that promote proliferation are downregulated and genes that are associated with differentiation of mesenchymal cells are upregulated. Consequently, differentiation of chondroblasts and osteoblasts occur. (4) Sequence of formation of craniofacial skeletal elements In the chicken skull, there are two kinds of bones: membranous bones and cartilaginous bones (Figure 3). Membranous bones in the facial region include lachrymal, nasal, premaxilla, maxilla, jugal, quadratojugal, pterygoid, palatine, vomer, and parasphenoid bone. These bones are formed through direct ossification of extracellular matrix. On the eighth day of incubation in chicken embryos, small areas of 7 calcification appear near the articular end of the jaw (Romanoff, 1960). A day later, the jugal bone starts to form; and at the end of ninth day, the quadratojugal, jugal, and maxillary bones begin to fuse into a rod that make up the lateral side of the upper jaw. In addition, the prefrontal and lachrymal bones are formed and fused with each other. By the tenth day of incubation, the palatine process of maxillary bone appears, then projects medially and posteriorly to form a 30° angle with the jugal process and extends medially to abut the distal tip of palatine bone in the following days (Figure 9a). The chicken upper beak is well-formed by the middle of eleventh day. The palatine bones, the palatine process of maxillary bones, as well as the pterygoid are important elements for the formation of palate; however, the palate does not fuse in chicken embryos. Cartilaginous bones in the chicken facial region include the basisphenoid, interorbital septum, palato-quadrate cartilage that forms the quadrate bone. These bones are formed through ossification of cartilage. By the tenth to eleventh day of incubation in chicken embryos, the vestibular nasal conchae is chondrified; and replacement of the cartilage begins in the otic process of the quadrate but is not complete until the fourteenth day of incubation (Romanoff, 1960). Other cartilages in the chicken chondrocranium, including the prenasal cartilage and Meckel's cartilage, develop similarly but don't go through the final ossification stage. 8 Figure 3. Membranous bones and cartilages in a stage 3 8 chick upper beak. Membranous bones are premaxilla (Pmx), nasal (Nas), maxilla (Max), prefrontal (Pfr), vomer (Vom)Jugal (Jug), palatine (Pal), quadratojugal (Qju), ptyergoid (Ptr), quadrate (Qd), and frontal (Fr) bone. Cartilages are interorbital septum (lo) and nasal conchae (Nc). 1.2 Retinoids 1.2.1 Retinoid production and degradation Retinoids are vitamin A metabolites synthesized in cells. The precursor of retinoic acid (RA) is retinol, which is taken up by cells and converted into retinaldehyde through retinol dehydrogenase (ROLDH). R A is produced when retinaldehyde is oxidized by retinal dehydrogenase (RALDH). Once R A is released from tissues, it is rapidly metabolized by cells and degraded to 4-hydroxyl R A and 4-oxo-RA by Cytochrome P450, 26 (CYP26), a member of a novel cytochrome P450 enzyme family (White et al, 1996; Fujii et al, 1997). R A metabolism and signaling in cells are illustrated in Figure 4. Currently, many naturally occurring, biologically active forms of retinoic acids have been isolated from embryonic tissues, such as all-trans R A , 9-cis R A , 3,4-didehydroretinoic acid, and 4-oxo R A . Studies have indicated that different forms of retinoids present in different organisms (Costaridis et al, 1996; Creech-Kraft et al, 1994; Chen et al, 1992; Thaller and Eichele, 1990). For example, all-trans R A , but not 9-cis R A or 4-oxo R A is present in zebrafish at all stages from fertilization (Costaridis et al., 1996); but all three forms of retinoic acids (all-trans, 9-cis, and 4-oxo) are detected in Xenopus (Azuma et al, 1990). Moreover, the major retinoids in chicken embryos are retinol and retinyl esters that serve as precursors for all-trans R A and 3,4 didehydro-RA (Thaller and Eichele, 1990; Maden et al, 1998). In fact, 3, 4 didehydro-RA is the major form of retinoic acid present in the neural tube, somites, eyes, tail bud, frontonasal mass, branchial arches, and limb buds of stage 24 chicken embryos (Maden et al, 1998). This study (Maden et al, 1998) also indicates that different tissues contain a characteristic relative levels of retinols and retinoic acids. Thus, each tissue has a unique retinoid profile that may affect retinoid signaling. 1 0 Figure 4. Retinoic acid metabolism and signaling in cell. Retinol is metabolized into all-trans retinoic acid (RA) through retinol dehydrogenase (ROLDH) and retinaldehyde dehydrogenase (RALDH). All-trans R A is degraded by cytochrome P450, 26 (CYP26) into 4-hydroxyl R A and 4-oxo RA. All-trans R A also binds to RARs or RXRs, and 9-cis R A binds to RXRs, in the nucleus. Four different combination of retinoid receptors have been proposed to activate gene transcription: Liganded R A R forms heterodimer with (1) unliganded R X R or (2) liganded RXR; liganded R X R forms dimer with (3) orphan receptors or (4) themselves. Thus, RARs and RXRs both act as transcription factors and different combination of retinoid receptors are formed to activate gene transcription. R A R E , retinoic acid response element; R X R E , retinoid X response element; RAR, retinoic acid receptor; RXR, retinoid X receptor. In chicken embryos, retinoids have been purified from Hensen's node and its surrounding area pellucida (Chen et al, 1992), dorsal neural tube, ventral floor plate of the neural tube (Wagner et al, 1990), and limb bud mesenchyme (Thaller and Eichele, 1987). In addition, RALDH-2 protein is detected in the somitic mesoderm, lateral mesoderm, kidney, heart, spinal motor neurons, and developing limb bud, suggesting that these tissues synthesize retinoic acids (Berggren et al, 1999). Interestingly, neural crest cells do not express RALDH-2; but the mesoderm through which the neural crest cells migrate in the head expresses RALDH-2 (Berggren et al, 1999). Moreover, cells expressing RALDH-2 are detected in the cranial mesoderm dorsal to the eye, the first branchial groove, nasal placode, lateral region of the otic vesicle, and on the inferior surface of the presumptive maxilla in stage 17 to 18 chicken embryos (Berggren et al, 1999). Similarly, RALDH-2 is first detected in the posterior half of mouse embryos on embryonic day 7 (Niederreither et al, 1997). Later, R A L D H is localized in Hensen's node, forebrain and eye region, mesenchyme surrounding the spinal cord, and subpopulation of motor neurons in the spinal cord that innervate the limbs in the mouse embryos (Zhao et al, 1996; Niederreither et al, 1997). Thus, in both mouse and chicken embryos, the node is a site of retinoid production and retinoids are suggested to specify anterior-posterior patterning. Retinoids are also used to promote the outgrowth and survival of both neurons and neural crest cells (Maden et al, 1996). This is supported by a recent study which indicates that mouse embryos with a homozygous targeted disruption of RALDH-2 die at midgestation without undergoing axial rotation (Niederreither et al, 1999). Moreover, they show truncated frontonasal region, severely reduced otocysts, heart defects, and no limb buds (Niederreither et al, 1999), suggesting that the production of retinoids is essential for limb bud initiation and upper facial morphogenesis. The activities of retinoid degrading enzyme, CYP26, have also been investigated in the early chicken embryos (Swindell et al, 1999). Transcripts of CYP26 are detected in the anterior ectoderm, mid/forebrain, rhombomere 2 and distal limb bud ectoderm. The nonoverlapping expression of RALDH-2 and CYP26 implies that the uptake and metabolism of retinoic acid are spatiotemporally regulated throughout development (Swindell et al, 1999). For example, a study suggests that R A specifies head to tail axial 12 formation by regulating the transcription of CYP26, which is expressed in the anterior end, and the transcription of RALDH-2, which is expressed in the trunk (Maden, 1999). In addition, RALDH-2 is expressed in the developing chicken limb bud mesoderm. Once the limb bud is formed, RALDH-2 is downregulated and CYP26 is activated in the limb bud ectoderm. The spatiotemporal regulation of retinoids in the limb bud suggests that retinoids are required for limb morphogenesis. Moreover, RALDH-2 expression in the presomitic and lateral plate mesoderm and CYP26 expression in the mid/forebrain are separated by a gap that encompasses the hindbrain. A R A concentration gradient is suggested to form in the gap and regulates the Hox gene expression in the hindbrain to specify the establishment of anterior-posterior axis in the embryo (Maden, 1999). Hence, it is the localized concentration and distribution of retinoids, rather than the ubiquitous presence of retinoids, that is essential to direct proper morphogenesis in embryonic development. 1.2.2 Teratogenic effects of excess retinoids and retinoid deficiencies Since retinoid production and degradation are regulated carefully during development, any changes with the level of endogenous retinoids may affect normal embryonic development. Indeed, retrospective studies on pregnant women who have taken Accutane, \3-cis retinoic acid, for the treatment of severe cystic acne, reported spontaneous abortions and malformed infants with a characteristic pattern of anomalies, such as absent or defective ears, absent or small jaws, cleft palate, aortic arch abnormalities, thymic deficiencies, and abnormalities of the central nervous system (Lammer et al, 1985). Similarly, experiments on exogenous application of high dose of retinoic acid in mouse embryos induce a range of malformations, including abnormalities of the face, cleft palate, malformation of the hindbrain, truncations of the limb, and digit deletions (Sulik et al, 1988; Sulik and Dehart, 1989). In chicken embryos, local application of retinoic acid to the wing bud between stage 18 to 21 results in absence of the upper beak in 100% of treated embryos (Tamarin et al, 1984; Wedden and Tickle, 1986; Wedden et al, 1988). Retinoic acid-induced truncation of the chicken upper beak originates from a failure of outgrowth of the frontonasal mass, which fails to fuse with 13 the lateral nasal and maxillary prominences, resulting in bilateral clefting of the primary palate (Wedden, 1987). Thus, exogenous retinoids have teratogenic and systemic effects that cause malformations in the embryo. Interestingly, a deficiency of retinoids also affects embryonic development. Fetuses from rat mothers reared on vitamin A-deficient diets exhibit severe congenital abnormalities (Wilson et al, 1953). The malformations include defects of the eyes, respiratory tract, heart and great vessels, urogenital system, as well as the diaphragm (Wilson et al, 1953). Cleft palate has also been reported in vitamin A-deficient pigs (Hale, 1933). Vitamin-A deficient quails die early from heart defects. The phenotype includes a shortening of the hindbrain (Maden, 1996) and abnormal axis formation in the limbs (Stratford et al, 1999). Facial morphogenesis only can be studied until stage 21-22 in such embryos, therefore the effects on skeletal development are unknown. 1.2.3 Effects of retinoic acid on skeletal stem cell differentiation Retinoids have been proposed to affect migration of neural crest cells. Many studies suggest that retinoids modify the distribution and composition of extracellular matrix, on which neural crest cells migrate (Sauer and Evans, 1980; Kapron-Bras and Trasler, 1988). This is demonstrated in chicken embryos injected with 1.5 (ig R A beneath the germinal disk which show an alteration in mesencephalic and rhombencephalic neural crest cells migration and an increase in concentration of non-sulphated glycosaminoglycans (Moro Balbas et al, 1993). In addition, the pattern of neural crest cell migration changes depending on the timing of exogenous R A administration before and during neural crest cell migration (Moro Balbas et al, 1993). It has also been shown that R A blocks D N A synthesis and lengthens the proliferative activity of neural crest cells in early chicken embryos, thereby inhibiting proliferation of neural crest cells and modifying their distribution (Salvarezza et al, 1997). In support of this study, exogenous retinoic acid has been demonstrated to cause a dose-dependent inhibition of proliferation of some normal (Zouboulis et al, 1991) and tumoral (Lotan et al, 1981) cell types. Other experiments indicate that retinoic acid alters the spatial expression of homeobox-containing genes, such as Hoxb-1, consequently affecting the migration of a 14 subpopulation of neural crest cells that gives rise to neurogenic derivatives in rhombomere 4 (Gale et al, 1996). Thus, these studies have shown that a retinoid microenvironment may be essential to maintain the composition of the extracellular matrix, as well as to regulate cell proliferation and gene expression, thereby directing proper migration of neural crest cells to different parts of the body. Later in development, retinoids have been suggested to interfere with epithelial-mesenchymal interactions and neural crest cell differentiation. Recombination of retinoic acid treated mesenchyme and non-treated epithelium of chicken frontonasal mass results in reduced outgrowth of the cartilage rod in the graft (Wedden, 1987). Retinoic acid is proposed to primarily affect mesenchyme and alter the interaction between epithelium and mesenchyme to reduce outgrowth of the chicken upper beak (Wedden, 1987). Moreover, a recent study has demonstrated that implantation of retinoic acid soaked beads in the mid-hindbrain isthmus of stage 9 chicken embryos affects patterning of skeletal derivatives of migrating hindbrain cranial neural crest cells (Plant et al, 2000). The R A treated embryos show mismigration of r4 neural crest cells into the first branchial arch, and anteriorly shifted expression of Hoxa2 and Hoxbl in the mesencephalon (Plant et al, 2000). Consequently, ectopic cartilage elements are induced and the quadrate is displaced laterally to fuse with the retroarticular process (Plant et al., 2000). Thus, many experiments have suggested that retinoids affect many different processes of embryonic development, such as neural crest cell migration, interaction of epithelium and mesenchyme, and differentiation of neural crest cells. In addition, the effects of retinoids on development depend on the different cellular and tissue types, as well as the different dose and duration of retinoids present in the environment. 15 1.2.4 Retinoid transduction Retinoids move into cells by diffusion since they are lipid soluble. Once they are inside the cells, they are transported into the nucleus and bind to nuclear receptors. The nuclear hormone receptor family is comprised of hormone receptors and orphan receptors that regulate gene expression in a wide variety of biological processes. Retinoid signaling is mediated by two classes of nuclear hormone receptors, RARs (Retinoic acid receptors; Petkovich et al, 1987; Giguere et al, 1987) and RXRs (Retinoid X receptors; Manglesdorf et al, 1991). Both RARs and RXRs comprise of three isotypes (a, p\y), and each isotype has different isoforms. While all-rrans-RA only activates RARs, 9-cis-RA is a bifunctional ligand that activates both RARs and RXRs. The structure of all three subtypes of RARs (a, p\ y) is similar among mice, humans, amphibians (Ragsdale et al, 1989), and chicken (Smith and Eichele, 1991), suggesting that conservation of RARs exists throughout the evolution of higher vertebrates. RARs comprise of six domains, designated A to F (Figure 5). Region C, which represents the D N A binding domain, is the most conserved region among different types of RARs. Region E is the ligand binding domain and exhibits high affinity to all-trans R A and 9-cis RA. While region B , C, and E are highly conserved among different species, region F and D exhibit less conservation (Lohnes et al, 1992). Variant isoforms of RARs are generated by alternate splicing of the N-terminal A regions and differential usage of promoter. To date, two main isoforms have been discovered for mRARa (Leroy et al, 1991), four isoforms for mRAR/3 (Zelent et al, 1991), and two isoforms for mRARy(Kastner, 1990). Metabolism and availability of retinoid ligands have been suggested to be regulated by two types of cellular retinoid-binding proteins: Cellular retinol binding protein type I and II (CRBPI and II), and Cellular retinoic acid binding protein type I and II (CRABP I and II). CRBP I, which binds retinol, is suggested as a substrate carrier in the generation of retinal from retinol (Posch et al, 1991). In contrast, C R A B P I, which binds R A , is suggested to sequester R A in the cytoplasm, preventing R A from binding to nuclear receptors (Maden et al, 1989). In mouse embryos, CRBP I expression and retinol accumulation are detected in the ectoderm and endoderm of craniofacial structures and limbs. CRABPI is detected only in the migrating neural crest cells and branchial arch 16 mesenchyme (Gustafson et al, 1993; Maden, 1994). The nonoverlapping distribution of these cellular retinoid-binding proteins implies that ectoderm and endoderm are the source of retinoic acid, and mesenchyme is the target of retinoids (Gustafson et al, 1993). Studies on the role of cellular retinoid-binding proteins in development have been controversial. For example, overexpression of CRABPI in teratocarcinoma cells added with a low exogenous R A concentration results in a reduction in expression of differentiation-specific genes, but this reduction is eliminated at higher exogenous R A concentration, suggesting that greater expression of CRABPI reduces the potency of R A in cell differentiation (Boylan and Gudas, 1991). However, CRABPI null mice are normal in external morphology and skeletal formation (Gorry et al, 1994; de Brujin et al, 1994). Although CRABPII null mice display an additional postaxial digit in the forelimb, no other anomalies in development are reported (Faweett et al, 1995). CRABPI/CRABPII null mice have been generated and found to exhibit no defects in external morphology and cochlea development (Romand et al, 2001). CRBPI null mice are morphologically normal; however they exhibit lower levels of retinyl ester in hepatic stellate cells. Interestingly, CRBPI-null mice fed with vitamin A deficient diet for five months show severe syndrome of vitamin A deficiency, suggesting that CRBPI is only essential for development when vitamin A level is low. Overall there is no critical requirement for CRABPs or CRBPs during embryonic development. 17 D N A binding Ligand binding A B C D E F Figure 5. Structure of RAR. R A R has six major domains. C is the D N A bindin region and E is the ligand binding region. 1.2.4.1 Retinoid receptor signaling To induce signaling, RARs form heterodimers with RXRs, but RXRs also form homodimers themselves. Three pathways have been proposed for retinoid receptor signaling (Figure 4): (1) R A R - R X R heterodimer signaling, (2) R X R - R X R homodimer signaling, and (3) RXR-other receptor signaling. (1) R A R - R X R heterodimer signaling Two different models have been suggested. In the first model, RXRs function as unliganded auxiliary proteins which form heterodimers with RARs, and the R X R - R A R heterodimer complex is bound to a retinoic acid responsive element (RARE) that regulates gene transcription. Binding of all-trans or 9-cis R A to R A R in a R A R - R X R heterodimer complex induces conformational change in RAR. As a result, the ligand is prevented from binding to RXRs, and gene transcription is activated. This is demonstrated in the R X R - R A R heterodimer complex, which binds to a DR5 responsive element (direct repeat with a spacer of five nucleotides) (Baniahmand et al, 1995). Initially, a corepressor, such as the nuclear receptor co-repressor (N-CoR) (Horlein et al, 1995), or the silencing mediator for retinoid and thyroid-hormone (SMRT) (Chen and Evans, 1995), masks the transcriptional activation of the heterodimer. When a ligand activates the R A R moiety, the conformation of R A R changes. The corepressor is released and transcription occurs. In the second model, RXR, like RAR, binds its respective ligand, 9-cis RA; and the dual occupancy of both retinoid receptors induces synergism and regulates gene expression (Lu et al, 1997). (2) R X R - R X R homodimer signaling and (3) RXR-other receptor signaling Besides forming heterodimers with RARs, RXRs also form homodimers with themselves and bind to R X R E (retinoid X-responsive element) within the promoter of specific genes (Mangelsdorf et al, 1991). For example, the transcription of CRBPII is activated by the binding of liganded R X R homodimer to the retinoid responsive element 19 within its promoter (Kliewer et al, 1992; Tran et al. 1992). However, CRBPII gene transcription is repressed when R X R heterodimerizes with COUP-TF1 (Chicken ovalbumin upstream promoter transcription factor), a nuclear hormone receptor (Kliewer et al., 1992; Cooney et al, 1992, 1993; Tran et al, 1992). Thus, RXRs regulate gene transcription by forming homodimers or heterodimers with other nuclear receptors. RXRs have also been shown to heterodimerize with orphan receptors, such as Liver X receptor (LXR, Willy et al, 1995), or peroxisomal proliferator-activated receptor (PPAR, Yang et al, 1999). PPAR has been suggested to compete with R A R for dimerization with R X R to activate gene transcription. When PPAR dimerizes with RXR, it not only permits the binding of R X R ligand to RXR, but also allows the binding of a coactivator, to activate gene transcription. This data reveals that R X R signaling is actually not controlled by the abundance of R X R ligands, but by the presence of RARs and other nuclear orphan receptors, which inhibits or permits binding of the R X R ligand to R X R . For all three pathways, both RARs and RXRs act as transcriptional factors, that activate upstream responsive elements to direct gene transcription. Since both retinoid receptors comprise of different types and isoforms, different combinations of receptor heterodimers and homodimers can be formed. The diversity through varied combinations of heterodimers not only allows differential regulation of gene transcription, but may also contribute to the pleiotrophic effects of retinoids in controlling embryonic development. In addition, the complex mechanism of retinoid signaling implies that gene transcription is specifically regulated at each ligand-receptor, receptor-receptor, and receptor-gene level by the presence of ligands, the distribution of retinoid receptors, and the specific combination of retinoid receptors. 1.2.4.2 Genes regulated by retinoid signaling Retinoids have been suggested to directly regulate expression of homeobox-containing genes that control patterning in the embryo. Genes that contain retinoic acid response element (RARE) in their promoter and thus are directly regulated by retinoid signaling include RAR(32 (de The et al, 1990), RARa2 (Leroy et al, 1991), RARy2 20 (Lehmann et al, 1992), cellular retinol-binding proteins type I (CRBPI; Smith et al, 1991), cellular retinoic acid binding proteins type JJ (CRABPII; Durand et al, 1992), and the Hox genes (al, b l , d4 (Popperl and Featherstone, 1993). For example, treatment of pregnant mice with a teratogenic dose of R A (100 mg/kg) on day 11 of gestation (Harnish et al, 1992) results in embryos with elevation of RAR|32, RARa2, and R A R y l mRNA levels. However, R A R a l , RAR(3l, and RAR(33 mRNA levels are unaffected by R A treatment. Interestingly, while the limb buds show a twelve fold increase in RARJ32 expression, the head and craniofacial region demonstrate a four fold elevation of RARf32, suggesting that RARP2 mRNA displays a region-specific pattern in response to R A treatment and may mediate the teratogenic effects of R A in its distributed regions. In support of this result, retinoic acid treatment of stage 20 chicken embryos by implanting a all-trans retinoic acid (10 mg/ml) soaked bead beneath the A E R of the right wing bud also induces upregulation of RAR(3 in the posterior part of both right and left maxillary prominences (Rowe et al, 1991), suggesting that RARp is regulated by exogenous retinoic acid and may play an important role in facial teratogenesis. Retinoids also indirectly regulate the expression of many genes that are important for embryonic patterning. For example, stronger expression of Transforming growth factor p 2 (Tgf|32), and decreased expression of Bone morphogenetic protein 2 and 4 (Bmp-2 and Bmp-4) are detected in multipotent stem cell lines that differentiate into osteoblasts when treated with 10"6 M retinoic acid (Gazit et al, 1993). In addition, chondrocytes treated with 3 |ig/ml retinoic acid display weaker expression of chondrocyte markers, such as collagen II and the precursor protein of the cartilage proteoglycan, and stronger expression of collagen III and fibronectin (Horton et al, 1987). Hence, the direct regulation of homeobox containing genes, as well as the indirect regulation of non-homeobox containing genes, by retinoids, imply the pleiotrophic effects of retinoids in controlling embryonic development and patterning. 21 1.2.5 Distribution and function of RARs and RXRs RARal is the most abundant of all R A R isoforms and is expressed in most cell types (Leroy et al, 1990). However, targeted disruption of coding region of RARal produces morphologically normal mice mutants (Li et al, 1993; Lufkin et al, 1993). In addition, disruption of the whole RARa gene in mice results in early postnatal lethality and testis degeneration, but no other anomalies in development are observed (Lufkin et al, 1993). Thus, RARal is suggested to play some general housekeeping function, as implied by its ubiquitous distribution in embryonic tissues and its function can probably be substituted by other retinoid receptors (Brand et al, 1990; Leroy et al, 1991). On the other hand, RARp expression are detected in specific localized regions of the face (Osumi-Yamashita et al, 1990; Mendelsohn et al, 1991; Smith and Eichele, 1991; Rowe et al, 1992., 1991). In chicken embryo, RARp is strongly expressed in the anterior part of the maxillary prominences but weakly expressed in the posterior part (Rowe et al, 1991). In addition, higher concentration of RARR transcripts is found in the lateral nasal processes, at the edges and corners of the frontonasal mass. However, in the mandibular processes, RARfi is uniformly and weakly expressed. Similar to RARa, disruption of RAR/32 in mice embryos does not result in any morphological anomalies, implying that RAR(32 does not possess any unique function (Mendelsohn et al, 1994a). Mice with disruption of all isoforms of RAR(3 have also been studied. The RARp null mice are viable and fertile, with no craniofacial and limb malformations, suggesting that RAR(3 is not essential for embryonic development (Luo et al, 1995) In contrast to the specific expression of RARp, RARy expression is more widely expressed in early development, then becomes progressively restricted to regions of chondrogenesis (Ruberte et al, 1990). RARy transcripts are first expressed in all three germ layers in the presumptive posterior region of 8.0 dpc mice embryo, but by 13.5 dpc, they become specifically restricted to all cartilages (Osumi-Yamashita et al, 1990). At 14.5 dpc, i?A/?ytranscripts are detected in all three skin layers (Dolle et al, 1990; Ruberte et al, 1990).. RARy2 knock out mice are apparently unaffected and disruption of whole RARy induces squamous metaplasia of the seminal vesicles and prostate gland, 22 incomplete penetrance of agenesis of ocular Harderian gland and homeotic transformations of the axial skeleton, but no anomalies are observed in the face (Lohnes et al, 1993). Thus, RARy is proposed to play a role in chondrogenesis, and differentiation of squamous epithelia (Lohnes et al, 1993). Although RARa null mice and RARy null mice display reduced viability, growth deficiency, and male sterility, none of the R A R knock out mutants display any major congenital malformations as observed in fetal vitamin A deficiency (VAD) studies (Wilson et al, 1953). These major congenital anomalies include squamous metaplasia of epithelia in the respiratory tract, alimentary tract, genitourinary tract, eyes and related gland. Thus, the individual RARs disrupted to date suggest they are functionally redundant (Lohnes et al, 1993, 1994; Mendelsohn etal, 1994b). Studies of double knock outs of RARs in mouse embryos implies that certain retinoic acid receptors are more important than others for embryonic morphogenesis and patterning. Double knock outs of RARal''fiZ*', a'fil'', al'1"/a2+/', p/'y2'/' did not result in embryonic lethality, but these mutants die shortly after Cesarean delivery at 18.5 dpc (Lohnes et al, 1994). Similarly, craniofacial defects are not observed in RARap2, alfi2, or (32y mutants. In contrast, RAR ay mutants exhibit severe craniofacial skeletal abnormalities (Lohnes et al, 1994). The abnormalities include median facial cleft, exencephaly, and missing of frontonasal mass, first, second and third branchial arch-derived bones (Lohnes et al, 1994). The distribution of RXRs has also been investigated. Studies in rat show that the RXRs are ubiquitously expressed in metabolic organs, such as the liver, kidney, and intestine (Mangelsdorf et al, 1990; 1992). RXRa is strongly expressed in liver, kidney, lung, muscle, and spleen, but weakly expressed in adrenals, brain, heart, intestine, and testes in rats. Similarly, RXRfl is abundant in all tissues except intestine, liver, and testes. However, RXRy is less abundant and both isoforms are expressed in the heart and muscle. The chicken homolog of RXRyhas also been studied in stage 24 and 27 chicken embryos (Rowe et al, 1991). cRXRy transcripts are highly expressed in liver and in the posterior trunk neural crest cells at stage 16, which differentiate into cells of the peripheral nervous system, such as the dorsal root ganglia, cranial ganglia, and peripheral nerve tracts. 23 Similar to single type of R A R knock out mice, RXRs null mice mutants exhibit some characteristics of the fetal V A D syndrome. RXRa null mutants display cardiac and ocular abnormalities, (Kastner et al, 1994; Sucov et al, 1994); RXR ft null males are sterile (Kastner et al, 1996); and RXRy null mutants are morphologically normal (Krezel et al, 1996). Moreover, triple knock out of homozygous RXRy and RXRfi and heterozygous RXRa yields growth deficient but viable offsprings (Krezel et al, 1996). Thus, the lack of craniofacial abnormalities in the R X R single and compound knock out mutants suggest that RXRs homodimers signaling may not play an important role in facial and skeletal development, but a single R X R a allele can support a near-normal development (Mark et al, 1998) R X R and R A R double knock out mice mutants have also been generated to study the role of R A R - R X R heterodimer signaling in embryonic development. A study has reported that RXRa RARy double mutants present similar craniofacial defects to RARay null mutants (Mark et al, 1998). Although the similarities in craniofacial anomalies are not reported in the study, it is suggested that i£X/?a-/?A/?y heterodimers signaling may be involved in craniofacial skeletogenesis (Mark et al, 1998). In addition, mutants deficient for both RXRa and RARfi demonstrate severe ocular defects, implying that RXRa-RARfi heterodimers signaling is involved in eye morphogenesis in mice (Mark et al, 1998). The studies of double RARs null mice mutants and triple RXRs null mice mutants reveal that RARs are the major mediators of retinoid signaling. Single R A R knock out experiments reveal that the subtypes and isoforms of RARs are functionally redundant; and R A R must operate via forming heterodimers with RXRs to mediate retinoid signaling, thereby controlling embryonic development and morphogenesis. 24 1.3 R A R antagonists Given that the various constituents of R A metabolism and signal transduction are characterized, we can interfere with R A signaling at several different levels. Synthetic retinoids have been made that can bind RARs and RXRs, and inhibit or stimulate R A R dependent gene transactivation. There are several reported R A R antagonists. Some are specific for certain isoforms of RARs and others are pan-antagonists. The R A R pan-antagonist, A G N 193109 (Allergan) is a C - l substituted acetylenic retinoid that inhibits retinoid signaling through RARs (Johnson et al, 1995). Its structure in comparison with all-trans R A is shown in Figure 6. A G N 193109 exhibits very high affinity to all three subtypes of RARs (Binding constants (Kd) for RARoc = 17 nM; RAR(3 = 7 nM; R A R y = 7 nM) (Johnson et al, 1999). It does not bind to any RXRs (Johnson et al, 1995) or affect the ability of R A R / R X R heterodimers to bind D N A in vitro (Klein et al, 1996). Compared to another R A R pan-antagonist, Ro 41-5253 which exerts antagonistic effects on all R A R isoforms but preferentially on R A R a (Kiedel et al, 1994), A G N 193109 has a higher affinity to all three subtypes of RARs and has a nearly 500 fold lower IC5o for RARy (Klein et al, 1996). In addition, transactivation assays indicate that the R A R antagonist has approximately four to six fold higher affinity for RARs than the natural hormone, R A (Johnson et al, 1995). At equimolar concentrations as RA, the R A R antagonist A G N 193109 inhibits R A signaling by approximately 85%, 62%, and 100% at R A R a , RARp, RARy, respectively (Johnson et al, 1995). In addition, when A G N 193109 is administrated with a 10 times higher concentration than RA, it completely inhibits all transactivation activity of R A at all three R A R subtypes (Johnson et al, 1995) . Thus, the R A R antagonist, A G N 193109, is a potent and effective antagonist of R A function at the RARs. Moreover, A G N 193109, has been shown to repress the basal activity of all three RARs, suggesting that the R A R antagonist acts as an inverse agonist that inhibits the activation of R A R in the absence of exogenously added retinoid agonist (Klein et al, 1996). It is proposed that A G N 193109 may change the conformation of RARs to increase interaction between R A R and other corepressor molecules (Klein et al, 1996) . Consequently, activity of unliganded RARs is inhibited. 25 0) Figure 6. Structure of R A and RAR antagonist, A G N 193109 (Johnson et al., 1995). (A) RA. (B) RAR antagonist. 26 Experiments using R A R antagonists to inhibit retinoid signaling have been performed in vitro and in vivo. In vitro organ culture of fetal rat metatarsal bones added with 100 nM or 1000 n M of A G N 193109 yields accelerated bone growth (De Luca et al., 2000). Moreover, 100-fold molar excess of the antagonist completely reverses the growth inhibiting effects of 100 nM R A on metatarsal bone culture (De Luca et al., 2000). This is supported by another in vitro study of micromass cultures of mouse embryo limb bud cells which demonstrates that coadministration of both R A R antagonist, A G N 193109, and R A R agonist, A G N 190121, reverses the inhibitory effects of R A on chondrogenic differentiation (Kochhar et al, 1998). However, application of 0.1 to 1.0 ng/ml A G N 193109 alone to the culture does not affect chondrogenic differentiation (Kochhar et al., 1998). On the other hand, addition of 1.0 X 106 ng/ml A G N 194301, a R A R a specific antagonist, into mice limb mesenchyme cultures causes an increase in cartilage nodule formation (Weston et al, 2000). Interestingly, A G N 194301 has actually a lower affinity to all three subtypes of RARs when compared to A G N 193109, as indicated by the effectiveness of both R A R antagonists in inhibiting RARs signaling when coadministrated with a 10 fold lower concentration of R A in vitro (Teng et al, 1997; Johnson et al, 1995). Thus, the induction of chondrogenesis by the antagonist is probably attributed to the use of a higher dose of the antagonist. R A R antagonists have also been tested for their ability to inhibit transcriptional activity of the potent R A R agonist TTNPB in vitro. At one half the concentration of the R A R agonist, A G N 193109 and another R A R antagonist, A G N 194310, display very significant inhibition of TTNPB activity, suggesting that both antagonists can compete with TTNPB in binding and activation of RARs (Johnson et al, 1999). Moreover, A G N 194310 has been demonstrated as having a nearly six fold lower Kd for R A R a , 3.5 fold lower K d for RARp, and 1.4 fold lower Kd for RARy (Johnson et al, 1999). Since A G N 194310 is a highly effective R A R antagonist, it is currently used in pre-clinical development as a topical agent for the treatment and prevention of the mucocutaneous toxicity produced by Accutane (Johnson et al, 1999). Although facial mesenchyme has not been cultured in the presence of any R A R antagonists, R A R antagonists have been used in vivo to inhibit endogenous retinoid 27 signaling in craniofacial development. Mice fed with a daily oral dose of 1 mg/kg A G N 193109 on 8 to 8.5 dpc (day post coitum) produce embryos with defects in frontonasal mass, eyes and cervical vertebrae, but no defects are observed in the limbs or the rest of skeleton (Kochhar et al, 1998). In addition, none of the embryos exposed to A G N 193109 (1 mg/kg to 100 mg/kg) prior to or after 8 to 8.5 dpc exhibit any craniofacial anomalies (Kochhar et al, 1998). Similarly, mice fed with 2.0/kg citral, an alcohol dehydrogenase antagonist that diminishes the availability of R A (Conner and Smit, 1987), at E8.5 produce smaller embryos with reduced number of somites, opened posterior neuropore, and no tail (Anchan et al, 1997). Several craniofacial structures, such as the eye, the incipient maxillary prominences, the branchial arches, and the otic placode, can still be identified but the olfactory pit cannot be recognized both morphologically and histologically (Anchan et al, 1997). Moreover, citral treatment at E9, 9.5 of 10.5, results in less or no gross anomalies in the olfactory pit (Anchan et al, 1997). Thus, both experiments suggest that endogenous R A signaling is only critical for craniofacial development during a narrow and specific temporal window. Other in vivo experiments using locally released R A R antagonists demonstrate position specific effects. R6 41-5253 or A G N 193109 soaked beads were implanted into the anterodorsal proximal portion of the right limb bud in stage 21 to 22 chicken embryos (Koyama et al, 1999). R A R antagonists treated limbs display shorter and abnormally bent humeri and A G N 193109 is found to be more effective than Ro 41-5253 in vivo (Koyama et al, 1999). R A R antagonist treated limbs also exhibit downregulated gene expression of hypertrophic chondrocytes and increased gene expression of prehypertrophic chondrocytes, suggesting that inhibition of endogenous retinoid signaling prevents chondrocyte maturation (Koyama et al, 1999). In agreement with this result, another lab implants A G N 193109 soaked beads proximal to the stylopod-zeugopod (S-Z) boundary in the chicken limb bud at stage 19 and 20 resulting in loss of proximal skeletal elements (Mercader et al, 2000). In contrast, antagonist soaked beads incorporated distal to the zeugopod-autopod (Z-A) boundary at stage 19 and 20 yield no alterations in patterning or growth of limb (Mercader et al, 2000). Thus, the specific implantation site of antagonist bead is important for the inhibition of endogenous retinoid signaling in the limb. In addition, the antagonist downregulates expression of Meis 1 and 28 Meis 2, which have been implicated as determinants of proximal limb compartments and are expressed in the lateral plate mesoderm and later in the proximal limb region (Mercader et al, 2000). Thus, in both studies, endogenous retinoid signaling is proposed to be critical for inducing chondrogenesis in the proximal limb region. In support of this suggestion, RALDH2 transcripts are detected in the lateral plate mesoderm and most proximal region of the chicken limb bud, implying that endogenous retinoids are synthesized in these regions for initiating limb bud outgrowth (Swindell et al, 1999). Consistent with this hypothesis, R A L D H 2 knock out mice do not have limbs (Niederreither et al, 1999). Another in vivo study on effects of R A R antagonist at later stages of development produces different results, implantation of A G N 193109 soaked beads in the interdigital regions of stage 28 chicken limb prevents interdigital cell death and induces ectopic cartilage (Rodriguez-Leon et al, 1999). Moreover, sequential application of the R A R antagonist at stage 28 and 20 later downregulates interdigital expression of Muscle segment homeobox 2 (Msx-2), which has been demonstrated to be upregulated by exogenous R A (Rodriguez-Leon et al, 1999). Although in vitro studies demonstrate the effectiveness of R A R antagonists, in binding to RARs and preventing R A R signaling, in vivo studies indicate that the administration method, timing, as well as location of the R A R antagonists and stage of embryonic development could affect the outcome. 29 C H A P T E R 2 Purpose of the study The purpose of our study was to determine whether the inhibition of endogenous R A R signaling in vivo, through the local application of R A R antagonist, A G N 193109, affected facial morphogenesis and patterning in chicken embryos. Previous experiments on retinoid receptors knock-out mice have implied that R A R signaling plays an important role in craniofacial skeletal patterning. However, the inactivation of retinoid receptors in these mice occurs very early in embryogenesis and the observed phenotype could result from the progressive development of primary effects of absence of retinoid receptor signaling. To study the specific effect of inhibition of endogenous R A R signaling in facial development, a pan R A R antagonist, A G N 193109, was used in our study. This antagonist was employed because it exhibits a very high affinity to all three subtype of RARs, and a four to six fold higher affinity to all three subtypes of RARs than all trans R A in vitro (Johnson et al., 1995). In addition, it is only R A R specific, and does not bind to RXRs; thus was used in our study to inhibit endogenous retinoid signaling through RARs. In chicken embryos, the nasal placode first appears at stage 15. The enzyme that synthesizes retinoic acid, retinaldehyde dehydrogenase2 (RALDH-2), has been localized to the nasal placode of stage 17 chicken embryos (Berggren et al., 1999). Since the presence of endogenous retinoids in these regions may imply its role in facial patterning, the R A R antagonist, A G N 193109, was applied into the right nasal pit of chicken embryos from stage 16 to 23 to inhibit endogenous retinoid signaling. We first determined the concentration at which A G N 193109 would completely dissolve in dimethylsulfoxide. Then AG1-X2 ion exchange beads were soaked in antagonist solutions implanted into the nasal pit of chicken embryos. In order to maximize the potency of the R A R antagonist, we soaked the beads in a range of concentrations, varied the soaking and rinsing times of the beads, varied the size of the bead and finally varied the number of beads applied. 30 Since both the R A R antagonist and all trans-retinoic acid have similar chemical structures, others have assumed that the R A R antagonist exhibits similar uptake and release rate from the AG1-X2 beads as RA. We designed several experiments to determine indirectly whether AG1-X2 beads release A G N 193109 into the tissue in a similar manner to RA. Beads soaked in R A R antagonist and R A were implanted simultaneously into the right nasal pit of stage 16 to 20 chicken embryos to determine i f the antagonist could rescue the defects induced by RA. As a control, beads soaked in the same dose of R A were implanted into chicken embryos. Although previous experiments demonstrate the teratogenic effect of high concentration of retinoic acid in chicken embryos (Tamarin et al, 1984; Wedden and Tickle, 1986; Wedden et al., 1988), experiments using a minimal dose of RA, which is just potent enough to induce truncated upper beaks and reduce secondary effects on adjacent facial structures, have never been performed. In addition, we analyzed the bone in R A and R A - R A R antagonist treated embryos, which has never been studied in previous experiments (Richman and Delgado, 1995; Tamarin et all, 1984). The expression of genes that may be important for facial and skeletal patterning in RA, R A R antagonist, and R A - R A R antagonist treated embryos was examined. These genes include Muscle segment homeobox 1 and 2 (Msx-1 and Msx-2), which have been implicated i n . epithelial mesenchymal signaling and R A teratogenesis, Fibroblast growth factor 8 (Fgf-8), an epithelial signaling molecule, and Sonic hedgehog (Shh), a secreted protein expressed on epithelium essential for the morphogenesis of frontonasal and maxillary prominences (Hu and Helms, 1999). Furthermore, the duration of the R A R antagonist was determined by implanting one or more R A R antagonist soaked beads into the nasal pit of chicken embryos, followed by treatment of R A soaked bead into the same nasal pit 18 hours later. 31 C H A P T E R 3 METHODS A N D M A T E R I A L S 3.1 Bead treatments In the beginning of this study, a series of methods were used to test the effectiveness and potency of the R A R antagonist, A G N 193109 (Allergan). Hence, different treatments of R A R antagonist soaked beads were implanted into chicken embryos. Table 1 lists the different concentrations, soaking time, rinsing time, and size of the R A R antagonist soaked beads prior to implantation. For embryos dual-treated with all-trans R A (0.75 mg/ml, Sigma) and R A R antagonist, R A R antagonist soaked beads were treated only with the most effective and potent method to maximize the effectiveness of the antagonist. AG1-X2 ion exchange resin beads (BioRad, formate form) of 100 |im or 200 p:m diameter (dry size) were selected using the Leica Wild M3C dissecting microscope, equipped with a graticule and illuminated with a fibre optic light, at 40 X magnification. After selection, beads were soaked in Dimethylsulfoxide (DMSO) containing 0.6 mg/ml to 5.0 mg/ml of R A R antagonist, A G N 193109. Although a range of concentration of the antagonist was used, the highest concentration that could be absorbed into the beads was probably about 1.4 mg/ml. This is because the antagonist was not completely dissolved in DMSO. Increasing dilutions with DMSO showed no remaining precipitate at a concentration of approximately 1.4 mg/ml. Thus, even though some beads were soaked in higher concentrations than 1.4 mg/ml, as indicated in Table 1, probably only 1.4 mg/ml of the antagonist was taken up by the beads. Control beads were soaked in DMSO for 20 minutes, followed by rinsing for 20 to 40 minutes until implantation. A l l beads were rinsed in Minimum Essential Medium ( M E M , Invitrogen Life Technologies) containing 10% Fetal Calf Serum (Invitrogen Life Technologies) until implantation to make them more visible. Since retinoids are light sensitive compounds, the whole experiment from soaking beads to bead implantation was carried out with as little ambient light was possible. 32 Table 1. Different treatment of R A R antagonist beads. Treatment (Stage) Concentration Bead size Soaking time Rinsing time before implantation R A R antagonist (stage 15 to 17) 0.6 mg/ml to 5.0 mg/ml 100 urn 20 min. 20 to 40 min. R A R antagonist (stage 16 to 22) 5.0 mg/ml 200 urn 60 min. 30 seconds R A - R A R antagonist (stage 16 to 20) RA: 0.75mg/ml R A R antagonist: 1.4 mg/ml 200 urn RA: 20min R A R antagonist: 60 min. RA: 20 to 40 min. R A R antagonist: 30 seconds 33 3.2 Implantation of beads in chicken embryos White Leghorn eggs (Coastline chicks, Abbotsford and University of Alberta, Poultry Unit) were incubated at 99.5°F for two and a half to three and a half days. Eggs were then windowed and examined under the Leica Wild M3C dissecting microscope at 16 X magnification, illuminated with a fibre optic light. Serosa and amnion membranes were gently torn away using forceps and 2 | i l of neutral red was added to the face of the embryo to reveal the nasal placode. To implant beads on the ectoderm of the right nasal placode, staples, made from platinum wire of 0.025 mm diameter (Goodfellow), were used to hold beads against the ectoderm of the right nasal pit in stage 15 to 17 (Hamburger and Hamilton, 1951) chicken embryos. In order to implant beads into the mesenchyme of the nasal placode, an incision was made with a tungsten needle in the right nasal placode and beads were inserted into the mesenchyme using forceps. For embryos dual treated with all-trans-RA and R A R antagonist, beads were implanted into the mesenchyme of nasal placode not only because they were more likely to stay in place, but also this method might increase the potency of the beads, since the major target of retinoids is the mesectoderm (Wedden, 1987, Mark et al., 1998 ). Since the nasal pits are deeper in stage 19 to 23 chicken embryos, beads were inserted into the ectodermal groove of right nasal pit using forceps and no incision was made. After implantation, eggs were tape-sealed and incubated at 99.5°F. The day following implantation, the treated embryos were observed to verify the presence of the beads in the face. Then the eggs were tape-sealed and reincubated until the desired stage. 34 3.3 Skeletal staining of chicken embryos Chicken embryos were decapitated at stage 38 (12 days of incubation). Skin and eyes were removed to allow better staining and faster degradation of tissues. The heads were fixed in 100% ethanol for 4 days, followed by 100% acetone for another 4 days. Subsequently, embryos were briefly rinsed with distilled water, and stained in 1.5% Alcian blue (Sigma), 0.5% Alizarin Red S (Sigma), 5% acetic acid, in 70% ethanol for 7 days with mild shaking at room temperature. Embryos were then briefly rinsed again with distilled water and cleared in a solution of 2% potassium hydroxide and 2% glycerol for another 7 days with mild shaking. Once the soft tissues were degraded and cleared, the skeletal structures were stored in 50% glycerol/water and analyzed with a Leica Wild M3C dissecting microscope, illuminated with a fibre optic light, at 10 X magnification. 35 3.4 Preparation of embryos for whole mount insitu-hybridization Embryos at stage 24 (4.5 days of incubation) and 28 (5.5 days of incubation) were removed from the eggs. Serosa and amnion membranes were removed. Embryos were washed in PBS (137 m M NaCl, 8.1 m M Na 2 HP0 4 , 2.7 m M KC1, 1.5 m M K H 2 P 0 4 ) and fixed in 4% paraformaldehyde (Fisher 0-4042-500) at 4°C overnight. After fixing, embryos were washed twice in PTW (0.1 % Tween-20 in PBS) at 4°C for 30 minutes, once in 50% methanol in PTW for another 30 minutes, and twice in 100% methanol for 30 minutes. Then, specimens were stored in 100% methanol at -20°C. 36 3.5 cDNA preparation The cDNAs were kindly provided by the following people: chicken Msx-1 and Msx-2 (S.E. Wedden), chicken Fgf-8 (J.C. Izpisua-Belmonte), and chicken Shh (C. Tabin). Large-scale plasmid D N A preparation was performed, using the QIAfilter maxiplasmid kit (Qiagen 12262). Table 2 shows the vector, size of insert, enzymes for the antisense RNA, and R N A polymerase for antisense RNA. Following restriction digest, 1 jul of each cDNA samples was fractionated on 1 % agarose gel to determine the size of the fragments. Samples were then purified by combining with an equal volume of phenol and an equal volume of chloroform. Subsequently, samples were mixed and centrifuged at 14,000 rpm for 5 minutes. The top aqueous layer was taken and washed with an equal volume of chloroform to remove residual phenol. This was followed by another centrifugation at 14,000 rpm for 5 minutes and the top aqueous layer was combined with 2.5 X volume of 100% ethanol to precipitate at - 80° C for 1 hour. Next, D N A was collected by centrifugation at 14,000 rpm for 10 minutes and the resultant pellet was washed with 70% ethanol. Subsequently, the purified D N A pellet was air dried and resuspended in deionized distilled water containing 0.1% DEPC (Diethyl pyrocarbonate; Sigma). 37 Table 2. Preparation of cDNA from plasmids. Gene Vector Size of insert (kb) Enzymes for antisense R N A R N A polymerase for antisense R N A Msx-1 Bluescript SKII 0.9 Bam HI T7 Msx-2 Bluescript SKII 0.4 Bam HI T7 Fgf-8 Bluescript SKII 1.2 Hind III T7 Shh Bluescript SKII 1.5 Hind III T3 38 3.6 DIG-labeled R N A probe synthesis 1 p:g of linearized cDNA was mixed with 2 jil of a 10 m M nucleotide mix (100 m M ATP, 100 m M CTP, 100 m M GTP, 100 m M UTP, all supplied by Invitrogen Life Technologies, and 10 m M dig-UTP, Roche Diagnostics), 2 ul of 10X transcription buffer, 2 ul of 0.1 M DTT, and 1 ul of RNAse guard, with a total volume of 20 ul in 0.1% DEPC water. The combined mixture was then denatured at 80°C for 2 minutes and cooled on ice for 2 minutes. Transcription occurred when 1 ul of R N A polymerase was added to the mixture and incubated at 37°C for 2 hours. Next, 1 JLXI of each sample was fractionated on 1 % agarose gel to determine the size of the synthesized RNA. The R N A sample was used for insitu hybridization if its intensity under U V was about ten times the intensity of the D N A fragment on the agarose gel. Subsequently, 8.8 ul of 5 M Lithium chloride and 360 ul of 100 % ice cold ethanol were added to the R N A samples. This was followed by the precipitation of R N A at -80°C for 30 minutes and centrifuged at 14,000 rpm for 10 minutes. Then, the R N A pellet was washed with ice cold 70% ethanol in 0.1% DEPC water, air dried, and resuspended in 100 ul of 0.1 % DEPC water. 39 3.7 Whole-mount insitu hybridization Before the embryos underwent whole mount insitu-hybridization, forceps were used to decapitate and make a small tear in between the mesencephalon and diencephalon to avoid trapping of the R N A probe inside the head. Embryos were rehydrated through a series of methanol/ PTW (75%, 50%, 25%) until settled and washed twice in PTW for 5 minutes each. Subsequently, stage 24 and 25 embryos were treated with 10 |ig/ml proteinase K for 30 minutes, while stage 28 embryos were treated with 10 |lg/ml proteinase K for 35 minutes. The specimens were then fixed in 4% paraformaldehyde containing 0.1% glutaraldehyde (Sigma) for 20 minutes. After fixing, embryos were rinsed in PTW and equilibrated in 1: 1 PTW/ hybridization mix solution until settled. The hybridization mix solution consists of 50% deionized formamide, 1.3 X SSC (150 mM Sodium chloride, 150 m M Sodium citrate, pH 4.5), 5 m M E D T A (Disodium Ethylenediamine Tetraacetate pH 8.0), 50 |ig/ml Yeast R N A (Sigma R-6625), 0.2% Tween-20, 0.5% CHAPS (Roche Diagnostics), and 100 ng/ml Heparine (Sigma H-7005) in 0.1% DEPC H 2 0 . Next, embryos were rinsed with hybridization mix until settled and prehybridized at 70°C for a minimum of three hours with rotation. Hybridization was achieved by adding 10 |lg of R N A probe to the specimens and incubating them overnight at 70°C with rotation. Next day, the specimens were rinsed two times with prewarmed hybridization mix at 70°C and washed twice in prewarmed (70°C) hybridization mix for 30 minutes each. These washes removed the probes that did not anneal to target RNA. Next, the embryos were washed with prewarmed (70°C) 1: 1 hybridization mix / M A B (100 m M Maleic acid, 148.8 m M NaCl) with 0.1% Tween -20 (MABT) for 10 minutes. Subsequently, the samples were rinsed twice with M A B T and washed in M A B T for 15 minutes at room temperature. Blocking was performed by incubating the embryos for 1 hour in M A B T containing 2% Blocking Reagent (Boehringer), and another hour in M A B T containing 2% Blocking Reagent (Boehringer) and 2% heat inactivate sheep serum (Invitrogen Life Technologies). Then, the hybridized RNAs were primary detected by incubating the samples at 4°C overnight in M A B T containing 2% Blocking Reagent, 2% heat 40 inactivated sheep serum, and 1/2000 anti-DIG AP Ab fragment (Boehringer). On the third day of whole-insitu hybridization, specimens were rinsed three times and washed three times in M A B T containing 1% Tween-20 for one hour each with rotation to remove unbound antibodies. The embryos were then equilibrated in freshly made NTMT (10 m M NaCl, 100 m M Tris-HCl pH 9.5, 50 m M M g C l 2 , and 0.1% Tween-20 in 0.1% DEPC H 2 0) . R N A transcript signals were detected by incubating the samples in N T M T containing 4.5 ul /ml NBT and 3.5 ul / ml BCEP (Invitrogen Life Technologies) in a dark film box until signal could be observed under the microscope. Subsequently, detection was stopped by rinsing the embryos with PTW and fixing them in 10% formalin. 41 CHAPTER 4 RESULTS 4.1 External phenotype of DMSO, and RAR antagonist treated embryos A series of treatments was used to maximize the effectiveness and potency of the R A R antagonist, A G N 193109. Embryos were implanted with beads into their right nasal pits from stage 15 to 23 and fixed at stage 38. Since all the D M S O treated embryos with the same implantation procedures as R A R antagonist treated embryos exhibited a normal phenotype (Table 3), the implantation procedures themselves did not affect development of the nasal placode. Table 4 shows the different treatments that were applied to the chicken embryos and the external phenotype of the antagonist treated embryos at stage 36. Embryos were treated with one or two antagonist soaked beads in the right nasal pit at stage 15 to 17, and stage 21 to 22. Moreover, antagonist soaked beads were applied not only in the right nasal pit, but also in the right maxillary, that attributes to part of the formation of the upper beak, in stage 19 and 20 embryos. Different concentrations of the R A R antagonist, ail-trans-RA ranging from 0.6 mg/ml to 5.0 mg/ml, were also tested but the highest concentration of A G N 193109 that could be completely dissolved was approximately 1.4 mg/ml, similar to that used by Kobayama et al. (1999). Since the ion exchange beads only absorb negatively-charged dissolved molecules in solution, concentrations higher than 1.4 mg/ml of the antagonist solution should not induce any additional effects. Furthermore, two antagonist soaked beads were applied sequentially into the right nasal pit of the embryos at stage 16 and 20 to increase the duration of antagonist treatment. Interestingly, regardless of the implanting site, size, concentration, and number of antagonist beads, all R A R antagonist treated embryos exhibited a normal external phenotype (n = 56/56). Figure 7 shows the external phenotype of DMSO and R A R antagonist treated embryos. 42 Table 3. External phenotype of chicken embryos implanted with DMSO beads. Treatment Stage External phenotype 1 bead (100 um) in nasal pit with staple 16, 17 11/11 normal 2 beads (100 urn) simultaneously in nasal pit with staple 16 4/4 normal 1 bead (200 um) in nasal pit and maxillary without staple 19, 20 10/10 normal 2 beads 18 hours apart (200 um) in nasal pit with staple 17, followed by 20 3/3 normal 1 bead (200 um) in nasal pit without staple 21,22 3/3 normal 43 Table 4. External phenotype of chicken embryos implanted with R A R antagonist beads. Treatment Stage External phenotype 1 bead (100 um) in nasal pit with staple 15, 16, 17 28/28 normal 2 beads (100 um) simultaneously in nasal pit with staple 15, 16 5/5 normal 1 bead (200 um) in nasal pit and maxillary without staple 19, 20 10/10 normal 2 beads 18 hours apart (200 um) in nasal pit with staple 16, followed by 20 5/5 normal 1 bead (200 um) in nasal pit without staple 21,22 8/8 normal 44 Figure 7. External phenotype of normal, DMSO, all-trans-RA, and R A R antagonist treated embryos. (A) Normal untreated embryo. (B) DMSO treated embryo. (C) R A R antagonist treated embryo with a normal external phenotype. (D) All-trans-RA (0.75 mg/ml) treated embryo exhibited a truncated upper beak. 45 4.2 Skeletal structure of DMSO, RAR antagonist treated embryos Although all R A R antagonist treated embryos had a normal external phenotype, they might exhibit some skeletal anomalies, as previous experiments of retinoid receptor knock out mice imply that inhibition of R A R signaling should affect skeletal development. Thus, the skeletal structures were analyzed in stage 38 DMSO and R A R antagonist treated embryos. While all embryos treated with DMSO had normal skeletal structures, embryos treated with R A R antagonist at stage 15 to 20 exhibited mild skeletal malformations. Table 5 shows the skeletal structures of stage 15 to 17 chicken embryos implanted with one antagonist bead and a staple in the right nasal pit, as well as stage 19 to 20 embryos implanted with one bead in the right nasal pit and one bead in the right maxillary. Embryos treated with R A R antagonist at stage 22 to 23 were almost normal, only 3 out of 8 embryos exhibited very mild skeletal anomalies in the palatine process of the maxillary, and the palatine bone. Thus, the antagonist was more effective in stage 15 to 20 chicken embryos. Embryos treated with R A R antagonist mainly had anomalies on the membranous bones and cartilage of the treated side only (Figure 8). The lower beak of the embryo was not affected at all. The final positions of the beads were mainly in the nasal conchae or between the nasal bone and prefrontal bone (Figure 8). More defects were detected in areas proximal to the bead, such as the prenasal cartilage, nasal conchae, premaxillary, maxillary, palatine process of maxillary, prefrontal and lachrymal bones, and fewer defects in areas distal to the bead, which include the palatine bone and the rostrum of parasphenoid. These defects ranged from smaller size, abnormal shape, to total absence of the affected skeletal structures. In general, most of the antagonist affected skeletal areas were smaller than the untreated left side of the embryo, and very few structures were completely missing. Also, occasional ectopic cartilage and bones were observed near the palatine process of maxillary bone region. Since a defective palatine process of the maxillary bone, as well as ectopic bones and cartilages, were the most common anomalies in R A R antagonist treated embryos, these structures were further analyzed in more detail. The palatine process of maxillary bone on the treated side was usually smaller than the untreated side (Table 6 and Figure 46 9) . Less frequently, abnormal shape and absence of the palatine process were observed. Ectopic cartilages and bones were also detected around this region (Table 7 and Figure 10) . The ectopic cartilage seemed to extend from nasal cartilage near the right palatine process of maxillary bone. Ectopic bones were also detected around this region. 47 Table 5. Skeletal analysis of chicken embryos implanted with R A R antagonist. Affected bones/cartilage Stage 15 to 17 (n = 28) 19 to 20 (n = 10) Premaxillary-maxillary bones 13 2 Nasal-maxillary bones 4 3 Prefrontal bone 6 2 Lachrymal bone 12 5 Palatine process of maxillary bone 23 7 Palatine bone 13 9 Rostrum of parasphenoid bone 0 3 Extra cartilage or bone 23 3 48 (A) (B) Figure 8. Schematic drawing of the chicken upper beak. (A) Lateral view. (B) Inferior view. The darkened areas represent regions affected by the R A R antagonist. The usual final position of the bead is indicated. 49 Table 6. Defects of the palatine process of maxillary bone in R A R antagonist treated embryos. Bead implant location Nasal pit Nasal pit and Stage of treatment 15 to 17 maxillary 19 to 20 Defects of the palatine process of maxillary Abnormal shape 0 4 Smaller 16 2 Absent 7 2 Affected embryos / total 23/28 7/9 50 Figure 9. Defects of the palatine process of maxillary bone in R A R antagonist treated embryos. (A) Normal. (B) Abnormal shape. (C) Smaller. (D) Absent, ec, ectopic cartilage; inc, inferior nasal conchae; j , jugal; mx, maxillary; ns, nasal septum; p, palatine; pmx, premaxillary; ppmx, palatine process of maxillary; qj, quadratojugal; snc, superior nasal conchae; v, vomer; vnc, vestibular nasal conchae. Astrisks indicate position of missing structures. Arrows point to smaller, absent, or deformed structures. Scale bar, 1 mm. 51 Table 7. Ectopic cartilage and bones in R A R antagonist treated embryos. Bead implant location Nasal pit Nasal pit and maxillary Stage of treatment 15 to 17 19 to 20 Ectopic cartilage 15 2 Ectopic bone 3 1 Ectopic cartilage and bone 5 2 Affected embryos / total 23/28 5/9 52 Figure 10. Ectopic cartilages and bones in R A R antagonist treated embryos. (A) Lateral view. (B) Inferior view, b, bead; eb, ectopic bone; ec, ectopic cartilage; j , jugal; 1, lachrymal; mx, maxillary; n, nasal; pmx, premaxillary; vc, vestibular conchae. Scale bar, 1 mm. 53 4.3. Effectiveness of the RAR antagonist, AGN 193109 4.3.1 External phenotype of all-trans-RA treated embryos In order to determine the stage and the lowest dose of exogenous retinoic acids that would induce consistent and severe defects in the chicken upper beak development, embryos were implanted with all-trans retinoic acid soaked beads in the right nasal pits from stage 15, when the nasal placode first appeared, to stage 23, when the embryos were no longer sensitive to application of exogenous retinoids. Previous experiments by Richman and Delgado (1995) indicated that embryos treated with 5 or 10 mg/ml all-trans-RA at stage 20 gave rise to consistent truncation of the chicken upper beak, suggesting that stage 20 embryos were most sensitive to exogenous retinoids. In the beginning of the present experiments, 0.5 mg/ml all-trans-RA was implanted into the right nasal pits of embryos at stage 20; however, embryos were found with a range of external phenotypes from normal to absence of upper beak (data not shown). A slightly higher dose, 0.75 mg/ml all-trans-RA, was used in the present study because it was the lowest dose that could induce a consistency of defects in the upper beak of chicken embryos. The range of external phenotypes of embryos was classified into five categories based on the severity of defects on the upper beak (Table 8). Score 0 represented embryos that were morphologically normal, and score 4 represented embryos with the most severe defects, truncation of the upper beak (Fig. 11). At stage 19 to 20, 80% of all-trans-RA treated embryos had truncated upper beaks, suggesting that embryos at these stages were most sensitive to exogenous retinoids (Figure 12 and Figure 13). Sensitivity to exogenous retinoids decreased at stage 16 to 18 and at stage 22 to 23. At stage 16 to 18, 12.5% of all-rra/u-RA-treated embryos exhibited a truncated upper beak phenotype, 56.25% had deviation or deviation and clefts, and 31.25% had mild deviation. At stage 22 to 23, only 25% of all-trans-RA treated embryos exhibited truncation of the upper beak and 37.5% of the treated embryos had a normal external phenotype. 54 Table 8. Categories of severity scores of defects in all-trans-RA, R A R antagonist treated embryos. Score Phenotype 4 Truncated upper beak Almost truncated upper beak 3 Cleft and deviation Very short upper beak and deviation Shorter upper beak and deviation Big deviation 2 Cleft and slight deviation Shorter upper beak and slight deviation Shorter upper beak Mi ld cleft and slight deviation Slightly shorter upper beak and slight deviation Deviation 1 Slight deviation Very slight deviation Slightly shorter upper beak 0 Normal 55 Figure 11. External phenotype of chick embryos in each score category. A n example of lateral and upper view of chick embryos in each score is shown. (A) to (Ft) are schematic drawings of the upper view of chick embryos. The upper beak was considered as (A) normal, if its midline is aligned with (B) very slight deviated, i f its midline is 1 to 2° off, (C) slight deviated, i f its midline is 3 to 5° off, (D) deviated, i f its midline is 6 to 15° off, (E) big deviated, i f its midline is greater than 15 0 off, the midline of the lower beak. In addition, the upper beak was considered as (F) slightly shorter, i f it is not aligned with, (G) shorter, it its length is less than 50% shorter, (H) very short, i f its length is more than 50% shorter than, the length of the mandible. Scale bar, 2 mm. 56 04 EJ3 E32 • I MO 15 to 18 (n= 16) 19 to 20 (n= 10) 22 to 23 (n = 8) Stage Figure 12. External phenotype of R A treated embryos. Embryos were most sensitive to 0.75 mg/ml R A at stage 19 to 20, as shown by the 80% of embryos with truncated upper beak. Stage 16 to 20 embryos exhibited much less sensitivity to RA; and in stage 22 to 23 embryos, 3/8 embryos were morphologically normal. 58 4.3.2 External phenotype of R A R antagonist dual-treated embryos In order to determine the effectiveness of the R A R antagonist in inhibiting retinoid signaling, both all-trans-RA and R A R antagonist beads were implanted into the same nasal pit of chicken embryos. Embryos were implanted simultaneously with both all-trans-RA and R A R antagonist beads or sequentially with R A R antagonist bead first, followed by implantation of all-trans-RA bead 3 or 18 hours later. If the R A R antagonist is effective in binding to R A R in vivo, it would inhibit exogenous all-trans-RA signaling and rescue the defects induced by all-trans-RA. Ch i 2 statistics (Appendix ) indicated that there were significant differences for embryos dual treated with all-trans-RA and R A R antagonist simultaneously at stage 19 to 20 (Figure 13b), and for embryos treated with R A R antagonist at stage 16 to 18 first, followed by all-trans-RA 18 hours later (Figure 13d, Table 9). However, there were no significance for embryos dual treated with all-trans-RA and R A R antagonist simultaneously at stage 19 to 20 (Figure 13 a) and for embryos treated with R A R antagonist at stage 16 to 18, followed by all-trans-RA 3 hours later (Figure 13c). Since embryos treated with all-trans-RA at stage 19 to 20 showed a significant rescue of all-trans-RA induced defects when R A R antagonist was applied, the insignificant results of embryos dual treated with all-trans-RA and R A R antagonist at stage 16 to 18 is attributed to the decreased sensitivity of stage 16 to 18 embryos to exogenous all-trans-RA (Table 9). Thus, the result indicated that the R A R antagonist, A G N 193109, was effective in binding to RARs, and protecting against exogenous all-trans-RA, for at least 18 hours. 59 Table 9. Significant differences between control (RA treatment alone) and trial treatments of both all-trans-RA and R A R antagonist beads. Stage Trial treatments X2 P 95% significant 16 to 18 R A + A G N X 1 (simultaneously) 0.34 >0.25 No R A + A G N X 2 (simultaneously) 13.08 <0.02 Yes 19 to 20 R A + A G N X 1 (simultaneously) 17.36 < 0.0025 Yes R A + A G N X 2 (simultaneously) 16.32 < 0.005 Yes 16 to 18 A G N X 1 + all-trans-RA (3 hours later) 9.38 <0.10 No A G N X 2 + all-trans-RA (3 hours later) 7.51 <0.15 No 16 to 18 A G N X 1 + all-trans-RA (18 hours later) 12.09 <0.02 Yes A G N X 2 + all-trans-RA (18 hours later) 13.92 <0.01 Yes 60 Figure 13. External phenotype analysis of all-trans-RA, R A R antagonist treated embryos. 13(b) and 13(d) showed a significant rescue of external defects induced by all-trans-RA. However, 13(a) and 13(c), with the exception of 2 beads of antagonist in 13(a), were not significantly rescued by the antagonist since the all-trans-RA treated embryos exhibited variable external phenotypic defects. all-rrans-RA + A G N X 1, one bead of all-trans-RA and one bead of R A R antagonist; all-trans-RA + A G N X 2, one bead of all-trans-RA and two beads of R A R antagonist. 61 14 S 3 E32 • ! HO oN 100 90 80 70 60 50 40 30 20 10 A. 0 '///; '///; '//// '//// RA(n=16) R A + A G N X 1 (n = 9) R A + A G N X 2(n = 9) Figure 13(a). External phenotype of all-toms-RA, R A R antagonist (simultaneously) at stage 16 to 18. • 4 0 3 E32 • ! MO •//// \//// //// //// RA(n=10) R A + A G N X 1 (n= 16) R A + A G N X 2 (n = 9) Figure 13 (b). External phenotype of all-/ra«s-RA, R A R antagonist (simultaneously) at stage 19 to 20. 62 14 S 3 E32 • ! MO 100 90 80 4. 70 60 £ 50 '////. '////. '////. '////. '///A • J j | RA(n=161 R A + A G N X 1 Cn = 91 R A + A G N X 2 (n = 10) Figure 13 (c). External phenotype of alWrara-RA, R A R antagonist (3 hours apart) at stage 16 to 18. 14 S 3 02 • ! MO '////\ '///A '////< '////, RA(n=10) R A + A G N X 1 (n= 11) R A + A G N X 2 (n = 4) Figure 13 (d). External phenotype of all-trans-RA, R A R antagonist (18 hours apart) at stage 16 to 18. Antagonist beads can no longer be seen because they are incorporated deep in the nasal slit. 63 4.3.3 Skeletal analysis of all-trans-RA and R A R antagonist dual treated embryos Although the skeletal defects caused by retinoic acid were not examined in previous experiments (Wedden and Tickle, 1986; Richman and Delgado, 1995), the skeletal structures of embryos treated with all-trans-RA and those dual treated with all-trans-RA and R A R antagonist were analyzed in this experiment, to determine if the R A R antagonist rescued the skeletal defects caused by all-trans-RA. Since embryos dual treated with all-trans-RA and R A R antagonist simultaneously at stage 19 to 20 showed a highly significant rescue defects in their external phenotype, their skeletal structures were examined and compared with embryos treated with all-trans-RA only. Almost all embryos treated with all-trans-RA at stage 20 (n = 8/10) exhibited truncated upper beak. Premaxilla, nasal, prefrontal, and lachrymal bones were usually absent on both sides of the embryos (Figure 14a and b, 15a). In addition, the nasal conchae, and maxilla were present but deformed. In the truncated upper beak embryos, the palatine bones on both sides were much shorter and wider; and the head width of these embryos seemed to be wider. Embryos dual treated with one bead of all-trans-RA and one bead of R A R antagonist, as well as two beads of all-trans-RA antagonist, showed an obvious rescue of skeletal structures that were absent in all-trans-RA treated embryos (Figure 14c and d, 15b and c). For embryos dual treated with one bead of all-trans-RA and one bead of R A R antagonist, nasal, prefrontal, and lachrymal bones were still commonly absent; however, more embryos had normal structure of nasal conchae, nasal, prefrontal, lachrymal, and maxillary bones (N = 16). Embryos dual treated with one bead of all-trans-RA and two beads of R A R antagonist retained premaxilla, and had almost normal nasal, prefrontal, lachrymal, and palatine process of maxillary bones (N = 9). Thus, two beads of R A R antagonist were more effective than one bead in rescuing the skeletal defects caused by all-rrans-RA. Despite the excellent protective effect of the antagonist on prenasal cartilage and premaxillary bones, none of the R A - R A R antagonist dual-treated embryos exhibited completely normal skeletal structures. These structures include the nasal conchae, palatine process of maxillary, and the palatine bones. Thus the antagonist was best at preventing defects in frontonasal derivatives, such as the 64 premaxilla, but had little effect on derivatives of the maxillary and lateral nasal prominences. 65 Figure 14. Skeletal structure of R A and R A - R A R antagonist treated embryos. (A) Lateral view and (B) inferior view of R A treated embryos. Upper beak is truncated and nasal conchae is very deformed that it cannot be individually identified (arrow). (C) Lateral view and (D) inferior view of R A - R A R antagonist dual-treated embryos. This embryo was treated with one bead of R A and two beads of R A R antagonist simultaneously at stage 19 to 20. It showed a rescue of skeletal structures, such as premaxilla (pmx), maxilla (mx), nasal conchae (nc), prefrontal (pf) and lachrymal (1) bones. The right palatine (p) bone proximal tip is missing (asterisk); and the palatine processes of maxilla (ppmx) on the treated side (right side of embryo) seemed to be smaller than the untreated side (arrow), eth, ethmoid; ios, interorbital septum; md, mandible; n, nasal; pt, ptyergoid. Scale bar, 2 mm. 66 Figure 15. Skeletal analysis of all-trans-RA, R A R antagonist treated embryos. 15 (a). Embryos treated with one bead of a\\-trans-RA exhibited truncated upper beak (8/10) with absence of premaxilla, nasal, prefrontal, and lachrymal bones. Also, nasal conchae, maxilla, and palatine bones were deformed. 15(b). However, embryos dual treated with one bead of all-trans-RA and one bead of R A R antagonist simultaneously (n = 16) showed a decrease in absence of nasal, prefrontal, and lachrymal bones; and more embryos exhibited normal premaxilla, nasal conchae, nasal, prefrontal, lachrymal, maxilla, and palatine bones. 15(c). Embryos dual treated with one bead of a\\-trans-RA and two beads of R A R antagonist simultaneously (n = 9) retained nasal, prefrontal, and lachrymal bones. Moreover, they developed normal premaxilla and maxilla bones on both sides. Interestingly, the application of exogenous all-trans-RA may rescue the mild skeletal defects caused by R A R antagonist, since all of these embryos retained the palatine process of maxillary bone and 7/28 R A R antagonist treated embryos showed an absence of the palatine process of maxillary bone on the treated side. Premx, premaxilla; N . conchae, nasal conchae; Pf, prefrontal; lac, lachrymal; Mx, maxilla; PpMx, palatine process of maxilla; Pal, palatine. 67 M Normal E3 Defective El Absent 100 90 80 70 60 £ 50 40 30 20 10 0 ET y y y y ¥ y y y y y y y y y y y y y y Nasal Pf lac Mx PpMx Pal Premx N . conchae Figure 15 (a). Skeletal analysis of all-frans-RA (0.75 mg/ml) treated embryos at stage 19 to 20. M Normal • Defective 0 Absent 0s Premx N . conchae Nasal Pf lac M x PpMx Pal Figure 15 (b). Skeletal analysis of all-/ra«s-RA and R A R antagonist (1 bead) simultaneously treated embryos at stage 19 to 20. 68 H Normal E3 Defective El Absent Figure 7 7 7 7 7 7 7 6 7 Pf lac M x PpMx l Pal Premx N . Nasal conchae 15 (c). Skeletal analysis of all-trans-RA and R A R antagonist (2 beads) simultaneously treated embryos at stage 19 to 20. 69 4.4 Whole mount in-situ hybridization 4.4.1 Msx-1 expression To determine whether Msx genes are regulated by R A R signaling and involved in facial outgrowth, beads soaked in all-trans-RA or R A R antagonist were implanted into the right nasal pit of chicken embryos at stage 19 to 20. Whole mount in situ hybridization was performed 24 hours after the treatment (stage 24), when retinoic acid was completely depleted from the beads (Eichele et al., 1984), and 48 hours (stage 28) after the treatment (Table 10 and Figure 16). A l l DMSO treated embryos exhibited normal expression of Msx-1 at stage 24 (n = 4/4) and stage 28 (n = 4/4). In addition, all DMSO treated embryos had normal nasal pits, showing that implantation of beads did not alter facial patterning in chicken embryos. For embryos treated with 0.75 mg/ml all-trans-RA, Msx-1 expression was completely absent in the right lateral nasal prominence and downregulated in the right lateral edge of frontonasal mass at stage 24 (n = 4/4). However, at stage 28, Msx-1 expression on the treated right side was identical to the untreated left side (n = 3/4). Both stage 24 and 28 embryos treated with all-trans-RA had smaller and abnormal right nasal pits (n = 8/8). R A R antagonist treated embryos did not show any alterations in Msx-1 expression at stage 24 (n = 4/4) and stage 28 (n = 4/4). Although 2 out of the 4 antagonist treated embryos had a more pinched nasal pit on the treated side, all stage 28 embryos (n = 4/4) had normal nasal pits. Since previous results in our study indicated that two beads of R A R antagonist were most effective in antagonizing 0.75 mg/ml RA, Msx-1 expression was also examined in these dual-treated embryos. Although all 4 of the dual-treated embryos exhibited smaller nasal pits on the treated side, 3/4 had normal Msx-1 expression. Moreover, all dual-treated embryos at stage 28 had normal expression of Msx-1, and 5 out of 6 had normal nasal pit morphology. 70 Table 10. Msx-1 expression in chicken embryos with different treatments. Stage Stage 24 Stage 28 Treatment Normal Downregulated Normal Downregulated DMSO 4/4 0/4 4/4 0/4 AM-trans-RA 0/4 4/4 3/4 1/4 R A R antagonist 4/4 0/4 4/4 0/4 R A - R A R antagonist 3/4 1/4 6/6 0/6 71 24 h 48 h Figure 16. Msx-1 expression. A l l embryos treated on right side of face. DMSO treated embryos at stage 24 (A) and stage 28 (B). R A treated embryos showed downregulation of Msx-1 expression (arrows) at stage 24 (C). R A treated embryos at stage 28 exhibited normal Msx-1 expression (D). R A R antagonist treated embryos at stage 24 (E) and stage 28 (F) had normal expression. R A and R A R antagonist (2 beads) simultaneously dual treated embryos had a rescue of Msx-1 expression (arrows) at stage 24 (G), and no change in expression at stage 28 (Ff). b, bead; fnm, frontonasal mass; lnp, lateral nasal prominence; np, nasal pit; mx, maxillary prominence; md, mandibular prominence. Scale bar, 0.5 mm. 72 4.4.2 Msx-2 expression Since Msx-2 has been shown to be regulated by exogenous all-trans-RA (Brown et al., 1997), it may play a role in directing proper craniofacial morphogenesis. We studied Msx-2 expression in embryos treated with all-trans-RA and dual treated with all-trans-RA and R A R antagonist to determine if the truncation of upper beak was associated with Msx-2 and cell death. In DMSO treated chicken embryos at stage 24 (n = 4/4) and 28 (n = 4/4), Msx-2 expression was normal (Table 11 and Figure 17). However, embryos implanted with 0.75 mg/ml all-trans-RA bead showed some ectopic Msx-2 expression at the top of the treated abnormal nasal pit at stage 24 (n = 5/6) and stage 28 (n = 4/5), in addition to Msx-2 expression at the corners of frontonasal mass and lateral nasal prominence. Similar to the Msx-1 result, the R A R antagonist did not induce any changes in Msx-2 expression in stage 24 (n = 4/4) and stage 28 (n = 4/4) embryos. Application of two antagonist beads simultaneously with one all-trans-RA bead did not rescue the expression changes caused by all-trans-RA and 50% of the embryos at stage 24 (n = 3/6) still exhibited ectopic Msx-2 expression around the whole abnormal nasal pits. A l l stage 28 embryos dual treated with R A R antagonist exhibited Msx-2 expression around the whole treated nasal pit. Thus, ectopic Msx-2 expression was induced by all-trans-RA, and it was not rescued by the R A R antagonist in stage 24 and 28 embryos. 73 Table 11. Msx-2 expression in chicken embryos with different treatments. Stage Stage 24 Stage 28 Treatment Normal Down-regulated Ectopic expression at top of nasal pit Normal Down-regulated Ectopic expression at top of nasal pit DMSO 4/4 0/4 0/4 4/4 0/4 0/4 R A 0/6 1/6 5/6 0/5 1/5 4/5 R A R antagonist 4/4 0/4 0/4 4/4 0/4 0/4 R A - R A R antagonist 3/6 3/6 0/6 0/5 0/5 5/5 74 24 h 48 h B ft* l l F 11 * Figure 17. Ms.x-2 expression. DMSO treated embryos at stage 24 (A) and stage 28 (B). R A treated embryos at stage 24 (C) and stage 28 (D) showed ectopic expression (arrows) at top of right nasal pit. R A R antagonist treated embryos exhibited normal expression at stage 24 (E) and stage 28 (F). Msx-2 expression in embryos dual treated with R A and R A R antagonist (2 beads) simultaneously were not rescued by the antagonist (arrows) in 50% of stage 24 embryos (G) and all of stage 28 embryos (H). Scale bar, 0.5 mm. 75 4.4.3 Fgf-8 expression Since Fgf-8 is expressed highly in the nasal pits and it has been implicated as an epithelial signal that controls the patterning of facial prominences (Barlow et al, 1999), its expression was examined (Table 12 and Figure 18). Similar to DMSO control treated embryos, all all-trans-RA, RAR antagonist, treated embryos had normal expression of Fgf-8 around the nasal pits and at the cleft between the maxillary and mandibular prominences. All-trans-RA treated embryos had abnormal and smaller nasal pits, but Fgf-8 expression was not affected by all-trans-RA and the R A R antagonist (n = 6/6). Thus, the results indicated that Fgf-8 expression was not influenced by changes in retinoid signaling. 4.4.4 Shh expression Shh has been suggested as a signaling protein that plays a role in morphogenesis of craniofacial complex. Shh null mice have cyclopia and loss of branchial arch structures (Ahlgren et al., 1999), and transient loss or excess of Shh signaling in chicken embryos results in severe craniofacial anomalies (Hu and Helms, 1999). To determine if Shh signaling is involved in retinoid-regulated craniofacial patterning, Shh expression was examined in embryos treated with all-trans-RA, R A R antagonist, or R A R antagonist at stage 20. A l l embryos did not exhibit any changes in Shh expression at stage 24 (Table 13 and Figure 19). Shh transcripts were expressed on the roof of the stomadeum and medial side of maxillary in all embryos and no ectopic expression was detected. Although the nasal pits of all-rrans-RA treated embryos were abnormal and smaller, no other morphological defects were observed. Thus, Shh transcription was not be regulated by retinoids in the face. 76 Table 12. Fgf-8 expression in chicken embryos with different treatments. Treatment Stage of treatment Stage 24 Normal expression on treated side DMSO 16, 17 2/2 R A 16, 17, 19, 20 4/4 R A R antagonist 16 2/2 R A - R A R antagonist 16 2/2 77 Figure 18. Fgf-8 expression. A l l embryos treated with DMSO (A), R A (B), R A R antagonist (C), R A and R A R antagonist (1 bead) simultaneously (D), exhibited normal expression of Fgf-8 at stage 24. Scale bar, 0.5 mm. 78 Table 13. Shh expression in chicken embryos with different treatments. Treatment Stage 24 Normal expression DMSO 2/2 R A 2/2 R A R antagonist 2/2 R A - R A R antagonist 2/2 Figure 19. Shh expression. A l l embryos treated with DMSO (A), R A (B), R A R antagonist (C), R A and R A R antagonist (1 bead) simultaneously (D), exhibited normal expression of Shh at stage 24. Scale bar, 0.5 mm. 80 CHAPTER 5 DISCUSSION In this study, the role of endogenous retinoids in patterning the upper facial morphology was investigated. Beads soaked in R A R antagonist, A G N 193109, were implanted into chicken embryos from stage 15 to 23. Results indicated that all antagonist treated embryos exhibited a normal external phenotype; however, embryos treated at stage 15 to 17 and stage 19 to 20 showed more severe skeletal anomalies than those treated at stage 22 to 23, which had almost no skeletal abnormalities. R A R antagonist treated embryos exhibited a normal external phenotype and mild anomalies of skeletal structures derived from the frontonasal mass, lateral nasal prominences, and maxillary prominences. We suggest that A G N 193109 was not able to completely inhibit all endogenous retinoid signaling in vivo. R A R antagonist treated embryos did not show any changes in gene expression of Msx-1, Msx-2, Fgf-8 or Shh. We then determined the effectiveness of the R A R antagonist. Embryos were dual treated with R A and R A R antagonist simultaneously or sequentially with an 18 hour gap. Our study showed that A G N 193109 prevented the RA-induced truncation of the prenasal cartilage and premaxilla and was effective for up to 18 hours. The antagonist was also effective at rescuing the expression of some genes but not others, revealing that some genes are required for normal outgrowth of the prenasal cartilage but others are not. Taken together, we demonstrate that endogenous retinoid signaling, through RARs, regulates skeletal morphogenesis of the upper beak. 5.1 Endogenous RA was required for morphogenesis of lateral nasal, and maxillary primordial derivatives Implantation of R A R antagonist beads in the nasal placode of stage 16 to 20 chicken embryos affected the skeletal patterning but not the surface morphology of the upper beak. This result was quite unexpected, since experiments from several labs have implicated the role of endogenous retinoids in craniofacial patterning in mice (Lohnes et al, 1994; Mark et al, 1998; Anchan et al, 1997; Kochhar et al, 1998). Despite our 81 attempts to increase the effectiveness and potency of the R A R antagonist, embryos implanted with R A R antagonist beads at stage 15 to 20 appeared morphologically normal and exhibited only mild skeletal anomalies. The effect of R A R antagonist was nonetheless specific since skeletal defects were only observed in derivatives of lateral nasal, and maxillary prominences (nasal conchae and palatine process of the maxillary bone). Ectopic cartilage and bones were also usually detected around the palatine process of the maxilla, suggesting that patterning was also affected in addition to morphogenesis. Since all bones and cartilage in the chicken facial region are derived from neural crest cells (Noden, 1978; Le Lievre, 1978; Couly et al, 1993; Kontges and Lumsden, 1996; Couly et al, 1996), the differences in sensitivity to endogenous retinoids might be related to the localization of the R A R antagonist soaked bead, as well as the differences in sequence of chondrogenesis and ossification of each bone and cartilage. Chondrogenesis and ossification of membranous bones follows at stage 28 and 32 respectively (Murray, 1963). Since chondrogenesis of the nasal cartilage and ossification of maxilla, premaxilla, lachrymal, and palatine bone occur on the sixth to eighth day of incubation (Romanoff, 1960; Murray, 1963), the R A R antagonist probably did not directly affect skeletal cell condensation but may affect earlier patterning of these structures. We tried to verify the effect of the R A R antagonist in epithelial-mesenchymal interaction by combining R A R antagonist treated epithelium with nontreated mesenchyme; however, the experiment was not successful due to the small size of the grafts. We also tried to study the effect of the R A R antagonist in older embryos (stage 22 to 25), but almost no skeletal anomalies were observed, suggesting that the R A R antagonist was only effective in stage 15 to 20 embryos. 5.2 The antagonist rescued RA induced truncation of the upper beak Although the ability of the antagonist to rescue the effects of R A has been studied in vitro (Johnson et al, 1995), the in vivo effectiveness of this particular antagonist has never been investigated. In our study, embryos were treated with 0.75 mg/ml exogenous all-trans retinoic acid soaked beads of 200 um because it was the lowest concentration that could induce consistent defects in the chicken upper beak yet reduced the secondary 82 effects on adjacent facial structures. In contrast to the data from Richman and Delgado (1995), who found variable external phenotypes in stage 19 to 20 chicken embryos implanted with 1 mg/ml R A soaked beads of 100 |im diameter, our study demonstrated that a lower soaking concentration and a larger bead produced consistent phenotypes. In addition, similar to their results, our data indicated that stage 20 embryos were most sensitive to exogenous R A , and embryos at stage 16 to 18 and at stage 22 to 23, exhibited much less sensitivity. Moreover, our study indicated, for the first time, that at a lower dose, R A treated embryos exhibited truncated upper beak due to lack of premaxilla and prenasal cartilage and had minimal secondary effects on adjacent skeletal structures. The specificity of the lower concentration R A treatment allowed us to show an equally specific rescue with the antagonist. Furthermore, embryos dual treated with one bead of R A and two beads of R A R antagonist displayed a higher rescue of normal skeletal structures than embryos dual treated with one bead of R A and one bead of R A R antagonist, indicating a dosage response of the R A R antagonist. We were also able to show that structures affected by the antagonist were not rescued by the presence of a large dose of retinoic acid. This supports the idea that the antagonist cannot be dislodged from the receptor once it is bound. These rescue experiments clearly show that the R A R antagonist was able to significantly inhibit exogenous R A signaling. The mild skeletal phenotype with antagonist treated embryos is not explained by a lack of activity of the antagonist. Another explanation for the mild skeletal phenotype induced by the antagonist was that the antagonist was not effective enough to completely antagonize all endogenous retinoid signaling in vivo. Although our experiment did not directly determine the amount of endogenous R A that A G N 193109 could antagonize, it is possible from the available literature a) to estimate the amount of exogenous R A released into the mesenchyme from an ion exchange bead and b) to make an estimation of the endogenous R A concentration in the face. High-performance liquid chromatography (HPLC) analysis of 3H-all-fra«s retinoic acid in stage 20 chicken wing bud indicates that 1 mg/ml 3 H-all-trans-RA released from AG1-X2 beads of 200 |im diameter after 14 hours results in 100 pg 3H-all-rra«5 ,-RA per limb bud (Tickle et al., 1984). The same size bead soaked in 0.75 mg/ml would release 75 pg/ limb bud. The amount of endogenous R A in a limb bud of 83 the same stage is 6.3 pg/ limb bud (Thaller and Eichele, 1987). Therefore, approximately ten times as much exogenous retinoid is present compare to endogenous retinoid. Since the limb bud and frontonasal mass have similar amounts of endogenous R A (Maden et al., 1998) and the release characteristics of the bead are similar throughout the chicken embryo, we presumed the same ratio of endogenous to exogenous all-trans R A exists in our study. Thus, the antagonist was able to antagonize all the endogenous R A in the vicinity of the bead. The mild effect of A G N 193109 in vivo might also attributed to the concentration, as well as the uptake and release properties of the antagonist from AG1-X2 beads. Previous studies have demonstrated that 20 minutes of beads soaking in all-trans-RA loads up to one-third or less of the total capacity of the bead (Eichele et al, 1984) and 100 pg of 1.0 mg/ml all-trans-RA is release in tissue after 40 minutes (Eichele et al, 1984). We cannot comment on the uptake and release properties of A G N 193109 from beads, however we have made other observations on solubility that may explain the mild phenotype of antagonist-treated embryos. All-trans-RA can be completely dissolved at a maximum concentration of 10 mg/ml in dimethylsulfoxide. In contrast, our study showed that A G N 193109 could only be completely dissolved at a maximum concentration of 1.4 mg/ml. Thus, the ability of the antagonist to diffuse through the tissues was probably poorer than all-trans-RA. Consequently, the concentration gradient of antagonist in the local tissues was probably very steep and endogenous R A signaling, even a small distance away from the bead was not affected. Cells expressing RALDH-2 are not only detected in the nasal placode, but also in the cranial mesoderm dorsal to the eye, and on the inferior surface of the presumptive maxilla of stage 17 to 18 chicken embryos (Berggren et al, 1999). These more distant sources of R A were probably unaffected by the antagonist soaked beads in the nasal pit. Our results of gene expression analysis of A G N 193109 treated embryos also supported the idea that all endogenous R A signaling was not completely blocked by the antagonist. A l l embryos implanted with A G N 193109 soaked beads at stage 19 to 20 did not exhibit any changes in Msx-1, Msx-2, Shh, or Fgf-8 expression at stage 24 and 28. We can estimate the maximum distance from the bead at which the antagonist was effective in blocking R A activity from experiments where 84 antagonist beads were placed first and then 18h later, an R A bead was placed. Continued development of the embryo resulted in the antagonist beads being on average at least 200 (am deep to the R A bead. However even if the antagonist could diffuse as much as 200um, this distance was clearly not enough to affect morphogenesis of the frontonasal region in a major way. A final explanation for the mild phenotype of antagonist treated embryos may be that A G N 193109 was less effective at binding to RARs than endogenous R A . This explanation is not likely however since it has been shown the affinity of A G N 193109 for RARs is greater than the affinity of all-trans-RA (Johnson et al, 1995). Furthermore the gene transactivation pathway is the same for exogenous all-trans-RA and endogenous R A . Both exogenous and endogenous retinoids can bind to RARs and activate gene transactivation via a R A R E (Rossant et al, 1991). 5.3 AGN 193109 was more effective than LG100815 During the writing of this thesis, a paper using another R A R pan antagonist on the chicken embryo head was published. LG100815 (Ligand Pharmaceuticals) soaked-beads placed at the anterior end of stage 10 produces embryos with severe hypoplasia in the forebrain and frontonasal prominences (Schneider et al., 2001). In our study, we did not implant A G N 193109 soaked beads in stage 9 embryos, therefore direct comparisons cannot be made. However L G 100815 treatment was also carried out after stage 18 and even a fourfold increase in dose of both L G 100815 and co-application of another R X R specific antagonist (LG100849) produced no dysmorphology. The in vivo effectiveness of LG100815 was also determined in this study (Schneider et al., 2001). We were therefore able to compare the in vivo effectiveness of the R A R antagonist, L G 100815 to the one used in our study, A G N 193109. A G N 193109 has a very high and specific affinity to all three subtypes of RARs in vitro (Johnson et al, 1995). The in vitro effectiveness of L G 100815 has not yet been reported. In the study by Schneider et al. (2001), administration of 100 ng/ml of R A R antagonist (LG100815) and R X R antagonist (LG 100849) for 8 to 10 hours induce dysmorphologies in stage 10 chicken embryos; however, these defects could be rescued by the removal of antagonist beads and the 85 application of bead soaked in 25 |ig/ml of exogenous all-trans-RA. These data show that all-trans RA can dislodge L G 100815 from the RARs. In our study, AGN193109 was able to rescue the effects of beads soaked in 0.75 mg/ml of all-trans-RA. Since the amount of retinoid released from beads is directly proportionate to the soaking concentration (Tickle et al, 1985), A G N 193109 is able to antagonize at least 30 times as much all-trans-RA as L G 100815. Moreover, A G N 193109 was demonstrated in our study to inhibit exogenous R A signaling even 18 hours before application of exogenous R A , suggesting that exogenous R A could not dislodge A G N 193109 once the antagonist was bound to the RARs. From the above comparisons it would seem that A G N 193109 is a more potent and effective R A R antagonist than L G 100815, however we will need to confirm by performing the stage 10 experiments with A G N 193109. 5.4 Was signaling via RXRs important for outgrowth of the chicken upper beak? Due to limitations in obtaining a pan-specific R X R antagonist from Allergan, we were not able to inhibit R X R signaling in vivo. We assumed that the inhibition of retinoid binding to R A R would also prevent the formation of a liganded R A R - R X R heterodimer. Thus, only signaling through liganded R X R homodimers (Lu et al, 1997), or liganded RXR-other nuclear receptors were permitted (Willy et al, 1995; Yang et al, 1999). Many previous experiments suggest that R X R signaling is not important for skeletal patterning in embryonic development, instead skeletal development depends on R A R signaling (Kastner et al, 1996; Krezel et al, 1993). Moreover, the craniofacial skeletal anomalies in RXRaRARy double knock out mice are identical to those present in RARaRARy null mice mutants (Mark et al, 1998), suggesting that RARy is the major receptor in heterodimers that regulates craniofacial skeletal patterning. The recent study by Schneider et al. (2001) implanted stage 10 chicken embryos with a pan-specific R X R antagonist (LG 100849) and embryos developed normally. A combination of a pan-specific R A R and R X R antagonists gives the same phenotype as with the R A R antagonist alone (Schneider et al, 2001). Thus, their results provide some of the strongest evidence that R A R signaling, rather than R X R signaling, plays a major role in craniofacial patterning in chicken embryos . Our results also showed indirectly that RAR, but not 86 RXR, signaling was important for skeletal patterning and outgrowth of the upper beak. Embryos treated with all-trans-RA, which activates both RARs and RXRs, exhibited truncation of the chicken upper beak. However, embryos dual-treated with all-trans-RA and R A R specific antagonist demonstrated outgrowth of the upper beak, suggesting that the inhibition of exogenous R A signaling through RARs restored the normal external phenotype in chicken embryos. Thus, R A R signaling was important for the outgrowth of the chicken upper beak. 5.6 Msx-1 expression at stage 24 was important for the outgrowth of chicken upper beak Msx-1 is a homeobox-containing gene that has been implicated in outgrowth of facial prominences. In chicken embryos at stage 24, Msx-1 is expressed around the nasal pits, in maxillary and mandibular prominences (Barlow and Francis-West, 1997; Brown et al, 1997). At stage 20, the time at which we implanted our beads soaked in all-trans-RA, Msx-1 transcripts are already present around the nasal pit (Richman lab data). Similar to the study of Brown et al. (1997), which implants 1 mg/ml R A beads in the right nasal pit of chicken embryos at stage 20, our results showed a downregulation of Msx-1 expression at stage 24. However, unlike the data of Brown et al. (1997), which showed a sustained downregulation of Msx-1 48h after bead application, our study indicated that there was a recovery of expression at 48h (stage 28). This difference in expression of Msx-1 at stage 28 probably attributed to the higher dose of exogenous R A used in Brown et al. study. Our data shows that the first 24 h of decreased Msx-1 expression are critical for truncation of the upper beak. This timeframe is further supported by our rescue experiments which showed that embryos dual treated with R A and R A R antagonist recovered normal expression of Msx-1 expression at stage 24. Since R A - R A R antagonist dual treated embryos specifically restored the outgrowth of the premaxilla and prenasal cartilage, the result implied that proper retinoid regulation of Msx-1 expression at stage 24, but not at stage 28, was critical for the outgrowth of the frontonasal mass in chicken embryos. Interestingly, the human Msx-1 promoter has been characterized and found to contain positive and negative R A regulatory cis-elements 87 upstream and downstream of the transcription start site, suggesting that Msx-1 is directly regulated by retinoid signaling (Shen et al, 1994). In addition, our results are supported by the phenotype of Msx-1 knock out mice which display among other facial abnormalities, abnormalities of the nasal and frontal bones (Satokata and Maas, 1994). These data further illustrate the important role of Msx-1 signaling in frontonasal outgrowth. 5.7 Ectopic expression of Msx-2 was induced by exogenous retinoids but Msx-2 was not important for the patterning of the upper beak Msx-2 expression was also investigated in embryos at stage 24 and 28. Our study showed that implantation of 0.75 mg/ml R A at stage 20 induced ectopic Msx-2 expression at the top of the treated nasal pit in stage 24 and stage 28 embryos. This result was contradictory to the data from Brown et al. (1997), which demonstrated decreased expression of Msx-2 in the treated nasal epithelia and treated side of the frontonasal mass at stage 24 and 28. Moreover, an examination of non-treated specimens in our lab indicated, for the first time, that Msx-2 was not expressed in the upper facial prominences in stage 20 embryos. Stage 22 was the first stage at which expression could be detected. Thus, the results of Brown et al. (1997) should be reinterpreted to say that all-trans-RA prevents upregulation of Msx-2 expression. In addition, the results in our study revealed, for the first time, that a lower dose (0.75 mg/ml) induced ectopic expression at the top of the treated nasal pit. Another interesting finding in our study was that 50% of the embryos dual treated with R A - R A R antagonist continued to have ectopic expression of Msx-2 at the top of the treated nasal pit at stage 24, and Msx-2 was expressed around the whole abnormal nasal pit at stage 28. Thus, we can eliminate a role for increased Msx-2 expression in mediating the truncation of the frontonasal mass. Further support for our data comes from an examination of the Msx-2 deficient mice (Satokata et al, 2000). Msx-2~'~ embryos show enlarged frontal foramen, smaller interparietal and supraocciptial bones, but no specific defects in derivatives of frontonasal mass (Satokata et al, 2000). Combining the results of Brown et al. and our study together, Msx-2 transcription might be differentially regulated by the level of retinoids. Although Msx-1 promoter has been 88 characterized and found to be directly regulated by R A (Shen et al., 1994), the presence of R A induced enhancer element in Msx-2 has not been reported. 5.8 Fgf-8 and Shh expression were retinoid-independent in facial morphogenesis Our study indicated that expression of Fgf-8 and Shh was not influenced by treatments of exogenous all-trans-RA, or R A - R A R antagonist. Two explanations are suggested. The first explanation is that Fgf-8 and Shh signaling is not required for craniofacial patterning. Several lines of evidence allow us to refute this explanation. First, Fgf-8 epithelial signaling is required to activate the underlying mesenchymal gene expression (Barlow et al. 1999). Second, conditional inactivation of Fgf-8 in mice leads to hypoplasia of proximal mandibular skeletal elements (Trumpp et al., 1999). Third, transient loss of Shh signaling in the frontonasal mass or maxillary prominences of stage 20 to 21 chicken embryos by anti-SM antibody inhibits outgrowth of these facial primordia and results in clefting of the upper beak (Hu and Helms, 1999). Fourth, a recent study has demonstrated that retinoids mediate both Fgf-8 and Shh expression in the forebrain and frontonasal mass during neural crest cell migration in chicken embryos (Schneider et al, 2001). Furthermore, Fgf2 and Shh treatments reverse the R A R and R X R antagonist-induced forebrain and frontonasal defects (Schneider et al, 2001). Our second suggestion was that both Fgf-8 and Shh expression patterns were not regulated by retinoid signaling in later stages of development (stage 16-20). This is supported by the results of Helms et al. (1997), which demonstrate that application of 10 mg/ml R A in stage 20 chicken embryos does not downregulate Shh expression until 30 hours after the treatment and Fgf-8 expression is not affected at all. However, by 30 hours after the treatment, the downregulation of Shh may be a secondary effect due to some loss of frontonasal mass tissue. We did not analyze Shh expression in stage 28 embryos so it is possible that Shh expression may be downregulated when facial abnormalities become apparent. 89 C H A P T E R 6 Conclusion In this study, we demonstrated that retinoid signaling was important for the outgrowth of the prenasal cartilage and premaxilla in chicken embryos. R A R antagonist, A G N 193109, treated embryos exhibited anomalies in skeletal derivatives of lateral nasal and maxillary prominences, suggesting the endogenous retinoid signaling has a role in patterning of these structures. We also showed, for the first time, that 200 | im diameter beads soaked in a low concentration of exogenous R A could induce a consistent truncation of the prenasal cartilage and premaxilla with reduced the secondary effects on adjacent facial structures. Moreover, these R A treated embryos exhibited only a temporary downregulation of Msx-1 24 hours after the treatment, implying that proper Msx-1 expression in the 24h between stage 20 and stage 24 was important for the outgrowth of the prenasal cartilage and premaxilla. This was supported by the rescue of R A induced truncation and recovery of Msx-1 expression, in embryos dual treated with R A and R A R antagonist. Interestingly, ectopic expression of Msx-2 was detected in R A treated embryos but was not rescued by the dual treatment of R A and R A R antagonist, suggesting that Msx-2 was regulated by retinoid signaling but was not important for the patterning of the frontonasal mass. 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Dermatol. 96, 793. 108 Appendix Stage 16 to 18.chick embryos implanted with one bead of R A and one R A R antagonist. bead simultaneously Observed counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 2 7 3 4 0 16 R A + A G N X I 1 3 2 3 0 9 Total 3 10 5 7 0 25 Expected counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 1.92 6.40 3.2 4.48 0 16 R A + A G N X I 1.08 3.6 1.8 2.52 0 9 Total 3 10 5 7 0 25 X 2 = Sum of (observed - expected)2 / expected = 0.003 + 0.056 + 0.0125 + 0.1 + 0.006 + 0.02 + 0.051 + 0.09 = 0.34 D f = ( r - 1 ) ( c - l ) = ( l ) ( 4 ) = 4 P > 0.25 109 Stage 16 to 18 chick embryos implanted with one bead of R A and two R A R antagonist beads simultaneously Observed counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 2 7 3 4 0 16 R A + A G N X2 0 0 0 7 2 9 Total 2 7 3 11 2 25 Expected counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 1.28 4.48 1.92 7.04 1.28 16 R A + A G N X2 0.72 2.52 1.08 3.96 0.72 9 Total 2 7 3 11 2 25 X 2 = Sum of (observed - expected)2 / expected = 0.41 + 0.545 + 0.608 + 1.31 + 1.28 + 0.72 + 2.52 + 1.08 + 2.33 + 2.28 = 13.08 D f = ( r - l ) ( c - l ) = ( l ) ( 4 ) = 4 P < 0.02 1 1 0 Stage 19 to 20 chick embryos implanted with one bead of R A and one R A R antagonist bead simultaneously Observed counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 8 2 0 0 0 0 R A + A G N X I 2 1 3 6 4 16 Total 10 3 4 6 3 26 Expected counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 3.85 1.15 1.54 2.31 1.15 10 R A + A G N X I 6.15 1.85 2.46 3.69 1.85 16 Total 10 3 4 6 3 26 X 2 = Sum of (observed - expected)2 / expected = 4.47 + 0.63 + 1.54 + 2.31 + 1.15 + 2.8 + 0.39 + 0.12 + 1.45 + 2.50 = 17.36 D f = ( r - l ) ( c - l ) = ( l ) ( 4 ) = 4 P < 0.0025 i l l Stage 19 to 20 chick embryos implanted with one bead of R A and two R A R antagonist beads simultaneously Observed counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 8 2 0 0 0 10 R A + A G N X I 0 1 2 2 4 9 Total 8 3 2 2 4 19 Expected counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 4.21 1.58 1.05 1.05 2.11 10 R A + A G N X I 3.79 1.42 0.95 0.95 1.89 9 Total 8 3 2 2 4 19 9 0 X = Sum of (observed - expected) / expected = 3.41 +0.11 + 1.05 + 1.05 + 2.11 +3.79 + 0.12 + 1.16 + 1.16 + 2.36 = 16.32 D f = ( r - l ) ( c - l ) = ( l ) ( 4 ) = 4 P < 0.005 112 Stage 16 to 18 embryos with one bead of R A R antagonist bead first, followed by one bead of R A 3 hours later Observed counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 2 7 3 4 0 16 R A + A G N X I 0 0 2 6 1 9 Total 2 7 5 10 1 25 Expected counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 1.28 4.48 3.2 6.4 0.64 16 R A + A G N X I 0.72 2.52 1.8 3.6 0.36 9 Total 2 7 5 10 1 25 X 2 = Sum of (observed - expected)2 / expected = 0.41 + 1.42 + 0.013 + 0.9 + 0.64 + 2.52 + 0.02 + 1.60 + 1.14 = 9.38 D f = ( r - l ) ( c - l ) = ( l ) ( 4 ) = 4 P<0.1 113 Stage 16 to 18 embryos with two beads of R A R antagonist bead first, followed by one bead of R A 3 hours later Observed counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 2 7 3 4 0 16 R A + A G N X2 0 1 3 4 2 10 Total 2 8 6 8 2 26 Expected counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 1.23 4.92 3.69 4.92 1.23 16 R A + A G N X2 0.77 3.08 2.31 3.07 0.77 10 Total 2 8 6 8 2 26 X 2 = Sum of (observed - expected)2 / expected = 0.48 + 0.88 + 0.13 + 0.17 + 1.23 + 0.77 + 1.40 + 0.21 + 0.28 + 1.96 = 7.51 D f = ( r - l ) ( c - l ) = ( l ) ( 4 ) = 4 P<0.15 114 Stage 16 to 18 embryos with one bead of R A R antagonist bead first, followed by one bead of R A 18 hours later Observed counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 8 2 0 0 0 10 R A + A G N X I 1 4 3 1 2 11 Total 9 6 3 1 2 21 Expected counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 4.29 2.86 1.43 0.48 0.95 10 R A + A G N X I 4.71 3.14 1.57 0.52 1.05 11 Total 9 6 3 1 2 21 X 2 = Sum of (observed - expected)2 / expected = 3.21 + 0.26 + 1.43 + 0.48 + 0.95 + 2.92 + 0.24 + 1.30 + 0.44 + 0.86 = 12.09 D f = ( r - l ) ( c - l ) = ( l ) ( 4 ) = 4 P < 0.02 115 Stage 16 to 18 embryos with two beads of R A R antagonist bead first, followed by one bead of R A 18 hours later Observed counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 8 2 0 0 0 10 R A + A G N X2 0 0 1 3 0 4 Total 8 2 1 3 0 14 Expected counts for treatment and score: Treatment Score 4 3 2 1 0 Total R A 5.71 1.43 0.71 2.14 0 10 R A + A G N X2 2.28 0.57 0.29 0.86 0 4 Total 8 2 1 3 0 14 X 2 = Sum of (observed - expected)2 / expected = 0.92 + 0.23 + 0.71 + 2.14 + 2.28 + 0.57 + 1.74 + 5.33 = 13.92 D f = ( r - l ) ( c - l ) = ( l ) ( 4 ) = 4 P < 0.01 116
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The effects of endogenous and exogenous retinoids on facial patterning
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