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Role of TGIF in cell cycle control and establishment of laterality Mar, Lynn 2006

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ROLE OF TGIF IN CELL C Y C L E CONTROL AND ESTABLISHMENT OF LATERALITY  by LYNN MAR M.Sc, University of Toronto, 1999  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (MEDICAL GENETICS)  THE UNIVERSITY OF BRITISH COLUMBIA August 2006  © Lynn Mar, 2006  ABSTRACT  Holoprosencephaly (HPE) is the most common structural anomaly of the human brain, resulting from incomplete cleavage of the developing forebrain during embryogenesis. Haploinsufficient mutations in TG-Interacting Factor (TGIF) were previously identified in a subset of HPE families and sporadic patients, and this gene is located within a region of Chromosome 18 that is associated with non-random chromosomal aberrations in HPE patients. TGIF is a transcription factor that contains a three amino acid loop extension (TALE) homeodomain and functions both as a corepressor of the TGF-f3 pathway and as a competitor of the retinoic acid pathway. Mice made deficient for 7gz/exhibited laterality defects and growth retardation, and developed kinked tails. Analysis of Tgif' ' mouse embryonic fibroblasts (MEFs) in 1  vitro demonstrated that Tgif regulates proliferation and progression through the Gi cell cycle phase. Wild-type human TGIF was able to rescue this proliferative defect in MEFs. In contrast, a subset of human Tgif mutations detected in HPE patients was unable to rescue the proliferative defect. However, an absence of Tgif did not alter the normal inhibition of proliferation caused by treatment with TGF-fi or retinoic acid. Developmental control of proliferation by Tgif may play a role in the pathogenesis of HPE.  ii  TABLE OF CONTENTS Page No. Abstract  ii  Table of Contents  iii  List of Tables  vi  List of Figures  vii  List of Abbreviations  viii  Acknowledgements  ix  Dedication  x  1. CHAPTER 1 INTRODUCTION 1 1.1. Developmental genetics and embryology 1 1.1.1. Holoprosencephaly 2 Multifactorial etiology 3 Candidate genes in human 4 Forebrain development 5 Anterior visceral endoderm induces anterior neural fate 7 Node derived anterior definitive endoderm reinforces and further elaborates forebrain patterning 9 Node derived prechordal plate provides ventral midline patterning centre 11 Regional pattering of the forebrain 12 Potential disruptions by mutations in HPE genes 15 1.1.2. Asymmetric body plan 16 Breaking of symmetry 19 Asymmetric gene expression in the node and lateral plate mesoderm... 21 Asymmetric gene expression maintained by the embryonic midline 22 Organ positioning and morphogenesis 23 Retinoic acid and symmetrical development of somites 23 1.2. TG-Interacting Factor 24 1.2.1. TGIF is a TALE HD 24 1.2.2. TGIF is a RA antagonist 26 1.2.3. TGIF is a TGF-0 co-repressor 28 1.2.4. TGIF mutations identified in HPE patients 31 1.2.5. Evolution conservation 32 1.3. Mammalian cell cycle 33 1.4. Hypothesis 35 1.5. Objectives 35 in  2. CHAPTER 2 MATERIALS AND METHODS..; 36 2.1. Generation of 7gz/mutant mice 36 2.1.1. Isolation of murine Tgifgenomic clone 36 2.1.2. Construction of the Tgif targeting vector 36 2.1.3. Embryonic stem cell line targeting and identification of homologous recombinant clones 37 2.1.4. Identification of Tgif"mutant alleles by Southern blot analysis 37 2.1.5. Identification of Tgif'mutant alleles by PCR 39 2.1.6. Production of chimeras and Tgif mutant mice from targeted ES cells 39 2.1.7. Mouse breeding, genetic backgrounds, and congenic strains 39 2.2. Expression analysis of Tgif using in situ RNA hybridization 40 2.3. Analysis of Tgif mutant mice 41 2.3.1. Determination of the rate of viability and fertility 41 2.3.2. Morphological examination of adults 42 2.3.3. Morphological examination of embryos 42 2.4. Cell cycle analysis of Tgif mutant mouse embryonic fibroblasts 42 2.4.1. Generation of mouse embryonic fibroblasts 42 2.4.2. Expression of Tgif in mouse embryonic fibroblasts 43 2.4.3. Growth curve analysis 43 2.4.4. Cell cycle analysis by BrdU incorporation and flow cytometry 43 2.4.5. Proliferation assay by H-thymidine incorporation 44 2.4.6. Proliferation assay of synchronized mouse embryonic fibroblasts 44 2.5. Analysis of in vivo proliferation by somite staging 44 2.5.1. Analysis of in vivo proliferation by BrdU incorporation 45 2.6. Expression of human TGIF by retrovirus in mouse embryonic fibroblasts 45 2.6.1. Expression of wild-type human TGIF by retrovirus 45 2.6.2. Expression of human TGIF containing mutations identified in HPE patients.46 2.6.3. Expression of transduced TGIF protein 46 2.7. Analysis of Proliferation response TGF-0, retinoic acid and U0126 46 3  3.  CHAPTER 3 GENERATION AND CHARACTERIZATION OF TGIF MICE 3.1. 7g7/expression during embryogenesis 3.2. Generation of Tgif mutant mice 3.2.1. Isolation of 7g7/genomic clone 3.2.2. Construction of Tgif targeting vector 3.2.3. Screen for clones in which the Tgif locus was targeted 3.2.4. Targeted Tgif alleles are null alleles and produce a fused lacZ transcript 3.2.5. Production of chimeras and Tgif'mutant mice from targeted ES cells 3.3. Analysis of Tgif mutant mice 3.3.1. Analysis of Tgif mutant adult mice on mixed 129/Sv/CDl genetic background 3.3.2. Analysis of the Tgif mutant adult mice with a 129/Sv congenic genetic background 3.3.3. Analysis of the Tgif mutant adult mice with a C57BL6 congenic genetic background 3.3.4. Morphological examination of embryos 3.3.5. Abnormal laterality determination 3.4. Summary iv  48 48 51 51 51 53 54 54 56 56 57 61 61 62 64  4. CHAPTER 4 ROLE OF TGIF IN CELL CYCLE CONTROL.... 4.1. Generation of Tgif mouse embryonic fibroblasts 4.2. Cell cycle defects in mouse embryonic fibroblasts 4.2.1. Growth curve analysis ; 4.2.2. Cytometric analysis of mouse embryonic fibroblasts cell cycle 4.2.3. Proliferation assay by [H]-thymidine incorporation 4.2.4. Proliferation of synchronized mouse embryonic fibroblasts 4.2.5. Rescue of the proliferation defect by re-expression of human TGIF in mutant mouse embryonic fibroblasts 4.3. Proliferation defects in vivo 4.3.1. Growth retardation in embryonic day 8 embryos 4.3.2. BrdU incorporation in embryonic day 8 embryos 4.4. Assaying the proliferative function of human TGIF mutations in mouse embryonic fibroblasts 4.5. Proliferation response to signaling pathways 4.5.1. Proliferation in response to TGF-R 4.5.2. Proliferation in response to retinoic acid 4.5.3. Proliferation in response to Ras/MAPK inhibitor, U0126 4.6. Summary  65 65 65 65 69 69 72  5. CHAPTER 5 DISCUSSION 5.1. Mutant mice did not exhibit HPE, but did exhibit HPE related defects 5.1.1. Lack of HPE phenotype Genetic background 5.1.2. HPE-related defects 5.1.3. Future directions 5.2. Mutant mice exhibited laterality defects 5.2.1. Molecular basis of laterality defects... 5.2.2. Future directions 5.3. Role of TGIF in cell cycle control 5.3.1. Proliferation during neural development 5.3.2. Future directions 5.4. Final comments  86 86 87 88 89 92 94 96 97 98 100 103 105  3  References  72 73 73 73 77 80 80 81 82 85  :  107  V  LIST OF T A B L E S Table 3.1 Table 4.1  Phenotypes resulting from rgz/heterozygote matings on 129Sv/CDl genetic background at 3 weeks of age  58  Phenotypes resulting from rgz/heterozygote matings on 129Sv/CDl genetic background at 3 weeks of age  76  vi  LIST OF FIGURES  Figure 1.1  Forebrain patterning in the mouse embryo  6  Figure 1.2  Molecular network controlling dorsal-ventral patterning of the telencephalon  13  Figure 1.3  Genetic pathway for the determination of left-right asymmetry  18  Figure 1.4  Tgif functional domains  25  Figure 1.5  The retinoic acid signal transduction pathway  27  Figure 1.6  The TGF-P signal transduction pathway  29  Figure 1.7 The cell cycle  34  Figure 3.1  Tgif expression in the early embryo  49  Figure 3.2  Tgi/expression in the developing limbs  50  Figure 3.3  Gene targeting of the mouse Tgif locus  52  Figure 3.4  Targeted ES cells express Tgif-lacZ fusion transcript  55  Figure 3.5  Tgif mutant mice on the 129/Sv/CDl genetic background developed pleiotropic phenotypes Tgif mutant mice on the 129/Sv/CDl genetic background developed  59  laterality defects  60  Figure 3.7  Tgif'mutant embryos displayed abnormal left-right patterning  63  Figure 4.1  MEF cells normally express Tgif but mutant cells do not  66  Figure 4.2  Tgif ' MEFs exhibited reduced growth due to a delay in GI phase  Figure 3.6  1  progression  67  Figure 4.3  Cell cycle analysis of proliferation defect in Tgif ' MEFs  70  Figure 4.4  Tgif ' mutant embryos exhibited growth retardation  74  Figure 4.5  Expression of a subset of mutated human TGIF rescued the proliferative defect of MEFs 78 Analysis of MEF proliferation in response to TGF-P, 9-cis RA, all-transRA and U0126 82  Figure 4.6  1  1  vii  LIST OF ABBREVIATIONS  7 A A D - 7-amino-actinomycin D A N R - anterior neural ridge A V E - anterior visceral endoderm Bmp - bone morphogenetic protein BrdU - bromodeoxyuridine C P M - counts per minute CtBP - carboxyl terminal binding protein En - embryonic day n; e.g. E8, embryonic day 8 Fgf - fibroblast growth factor H D A C - histone deacetylase H P E - Holoprosencephaly I N K - c-Jun N-terminal Kinase L P M - lateral plate mesoderm M A P K - mitogen-activated protein kinase M E F - mouse embryonic fibroblasts O C T - optimum cutting temperature PCR - polymerase chain reaction R A - retinoic acid Shh - sonic hedgehog T A L E H D - three amino acid loop extension homeodomain T F - transcription factor T G F - P - transforming growth factor-(3 TG1F - T G - Interacting Factor V E - visceral endoderm  vm  A C K N O W L E D G E M E N T S  First and foremost, I thank Pamela Hoodless for g i v i n g me the opportunity to w o r k on this exciting project and for always being supportive. Members o f m y committee, Connie Eaves, M u r i e l Harris, and R o b K a y were invaluable and provided excellent advice and guidance. M e m b e r s o f the lab: A n i t a Charters, Rebecca C u l l u m , S a m L e e , R a c h e l Montpetit, K r i s t e n M c K n i g h t , Elizabeth Wederell, and M o n a W u were wonderful people to work with. Special thanks go to R o b i n D i c k i n s o n who always has time to talk about science and experiments, and w i l l always give his best advice. Juan H o u arrived at the lab just i n time to give me sage advice on embryology. It is always fun and stimulating to converse w i t h Pavle Vrljicak. The Terry F o x Laboratories is a wonderful learning environment for me. M a u r a Gasparetto, m y favorite F A C S teacher, 1 can't thank y o u enough. Danny C h u i is a valuable resource o f E S and mouse information. M i k e Hughes and Frann Antignano taught me h o w to do tritium incorporation and counting, and fix the many leaks. Patty Rosten helped me w i t h many molecular biology problems and is always fun to have a laugh with. R e w a G r e w a l performed the crucial blastocyst injections. B o b Argiropoulos and K e i t h Humphries helped w i t h the retroviral transductions. F i n a l l y superb F A C S support was provided by G a r y de Jong, G a y l e Thornbury, L i n d s e y L a y c o c k , and C a m Smith. The B C Cancer Research Centre is also a source o f wonderful people. D a n Doxsee was generous with his time and expertise w i t h the cell counter. B r a d C o e for statistics advice. The J A F and A R C cared for all m y mice. I have been l u c k y to have had great role models i n Sabine Cordes and Bernhard Weber. Thank y o u , T i m o n , Cathie, Irma, Geraldine, K a t , M e l i n d a , and Jon for your friendship. I enjoyed the company o f fellow students Dale Robinson, Iris Cheung, and. Silvia Bakovic. I also w i s h to acknowledge the Effie I. Lefeaux Scholarship i n M e n t a l Retardation and the Albert B . and M a r y Steiner award for travel. M y m o m Sui and m y dad E d d y have always supported through many endeavours. I ' m grateful and hope to make y o u proud. Steven Quayle, m y constant companion, was by m y side through all the ups and downs.  Thank y o u a l l .  ix  DEDICATION  To my parents  x  1.  CHAPTER 1 INTRODUCTION*  1.1.  DEVELOPMENTAL GENETICS AND EMBRYOLOGY Congenital malformations affect 4% of newborns, which equates to more than  150,000 affected newborns a year in the US (U.S. Department of Health and Human Services). After accidents, birth defects are the leading cause of death in children. For these patients, morbidity is also dramatically higher during infancy and childhood than in the average population (Epstein 2003). A major goal of research into congenital malformations is to provide a basis for improved genetic counselling for the families in the short term, and ultimately to create therapies for those directly affected. In addition, such research provides insights into the mechanisms of development. Such discoveries can have significant impacts on the understanding of birth defects and other diseases, thus aiding in the development of drug targets, gene therapy, and cell, tissue and organ transplantation techniques. The pathogenesis of congenital malformations has been investigated in many model organisms, but most of all in the mammalian model, mice (Bier and McGinnis 2003). In mice, technology is available to re-create virtually any mutation in any gene identified in humans (Glaser et al. 2005). Countless studies have demonstrated similarities between mice and humans during embryonic development, as well as between genes that regulate development. Such models demonstrate accurate disease progression, and the accessibility of all developmental stages and tissues makes it possible to investigate the functions of the genes that are altered in heritable human diseases. Significantly, this may also allow testing and development of therapeutic strategies. Nevertheless there are also important differences between mice and human  * A version of this thesis has been published. Mar, L and Hoodless, PA. Embryonic fibroblasts from mice lacking Tgif were defective in cell cycling. Mol Cell Biol. 26(11):4302-4310. 1  since most mouse models fail to reproduce all the features observed in the corresponding human conditions(Watase and Zoghbi 2003). The work described here focuses on two types of congenital defects, holoprosencephaly (HPE), a structural malformation of the forebrain, and laterality defects resulting from a failure to properly establish the left-right axis in the developing embryo. Interestingly, disruptions to forebrain patterning sometimes arise concurrently with laterality defects, thus giving insights into the mechanism of both processes. HPE naturally occurs in lower vertebrates, including zebrafish, Xenopus, sheep and mice (Capdevila et al. 2000; Muenke and Beachy 2000). This is consistent with observations indicating early brain development is similar in vertebrates, although different species acquire unique features later during development (Wilson and Houart 2004).  1.1.1. Holoprosencephaly  HPE is the most common birth defect affecting forebrain development (Cohen 2003). Its frequency is 1 in 10,000 to 20,000 amongst newborns, but a higher frequency of 1 in 250 during fetal development indicates a substantial number of the prenatal embryos are lost (Matsunaga and Shiota 1977). High mortality is associated with this disease during infancy, but a significant number of HPE individuals continue to live for many years (Redlinger-Grosse et al. 2002). The frequency of HPE is actually thought to be higher since mild HPE is believed to be present in asymptomatic individuals with reduced cognitive abilities. The defining hallmark of HPE is the failure of the forebrain to divide into two separate hemispheres and ventricles, resulting from the loss of midline structures (Muenke and Beachy 2000). HPE encompasses a continuum of brain malformations. At the most severe end of the spectrum, alobar HPE is characterized by a single 2  ventricle with no separation between the cerebral hemispheres. Semilobar HPE occurs when the left and right frontal and parietal lobes are fused but the interhemispheric fissure is present posteriorly. Lobar HPE is characterized by the separation of most of the right and left cerebral hemispheres and lateral ventricles, but with a fusion of the most rostral aspects of the telencephalon, especially ventrally. HPE is most commonly accompanied by craniofacial anomalies including cyclopia, cleft lip and palate, and eye defects in about 80% of individuals (Cohen 2003).  Multifactorial etiology  HPE can be caused by exposure to teratogens or other environmental agents (Cohen 2003). For example, maternal diabetes was shown to increase the risk for HPE by 200-fold (OMIM %236100). Low cholesterol is also being investigated as a potential teratogen (Edison and Muenke 2003). Finally, alcohol and retinoic acid (RA) were shown to increase the risk of HPE in humans and in animal models (Lammer et al. 1985; Sulik et al. 1995; Cohen and Shiota 2002). Complex genetic anomalies are also observed in HPE individuals (Cohen 2003). 25-50% of individuals have numerical or structural chromosomal abnormalities that were either inherited or occurred de novo. Mutations in single genes can also cause HPE, or other complex syndromes that include HPE. Nonsyndromic HPE cases that are inherited as a monogenic disease are amenable for genetic investigations. More than 12 chromosomal regions have been mapped in sporadic and familial HPE cases (Cohen 2003). Candidate genes were then identified based on mutations found in patients. Many varieties of mutations were found, including submicroscopic and small deletions, nonsense, missense and frameshift mutations (Cohen 2003; Bendavid et al. 2005a). Currently, 25% of HPE 3  newborns and 22% of HPE fetuses with normal karyotypes have mutations in known candidate genes.  C a n d i d a t e genes i n h u m a n  Heterozygous mutations in the four genes, SHH, SIX3, ZIC2 and TGIF, are most frequently identified in patients (Wallis and Muenke 2000). For instance, SHH (Sonic hedgehog) mutations were identified in 17% of familial cases and 3.7% of sporadic cases (Roessler et al. 1996; Roessler et al. 1997). Mutations were also identified in other members of the SHH pathway. GLI2, a downstream transcription factor, was mutated in 1.8% of patients (Roessler et al. 2003). Four case reports also demonstrated mutations in PTCH, a SHH receptor (Ming et al. 2002). Finally, 2-4% of patients with Smith-Lemli-Opitz syndrome, an HPE related syndromic disease, have mutations in DHCR7, a cholesterol reductase associated with cholesterol modification of SHH (Irons 2003). Six3 mutations were identified in 3-4% of patients (Wallis et al. 1999). Interestingly, mutations were identified at a moderately higher percent (5.3%) in fetuses relative to patients, suggesting Six3 function is critical during the fetal period (Bendavid et al. 2005b). A number of mutations have also been identified in members of the Nodal pathway. 2-6% of patients have haploinsufficient mutations in TGIF, a transcriptional repressor shown to modulate Nodal signaling (Gripp et al. 2000). Additionally, 4-6% of affected newborns and 8.5%) of affected fetuses have mutations in Zic2, a transcription factor that potentially regulates Nodal signaling (Brown et al. 1998; Nagai et al. 2000; Houston and Wylie 2005). Heterozygous mutations in TDGF1, a co-factor critical for Nodal signaling, were identified in 0.5% of patients (de la Cruz et al. 2002).  4  Finally, heterozygous missense variants of FOXH1, a transcription factor that transduces Nodal signals, were also identified in HPE (Ming and Muenke 2002). Intriguingly, HPE demonstrates considerable variability and incomplete penetrance for all known loci; the penetrance for TGIF mutations or deletions is only 10% (Nanni et al. 1999; Aguilella et al. 2003). A discussion of the potential mechanism of these candidate genes during normal forebrain and HPE development will be presented after normal forebrain development is reviewed.  Forebrain development The vertebrate brain arises from the embryonic neural plate. The forebrain, or  prosencephalon, develops from the anterior neural plate and gives rise to the cerebral cortex, basal ganglia, eye, thalamus and hypothalamus, all of which are neural structures affected in HPE patients (Muenke and Beachy 2000). By the time the neural plate arises at the anterior end of the mouse embryo on Embryonic day 7 (E7), many patterning events critical for the neural plate have already occurred. Thus, an understanding of earlier stages is required. At E6 the mouse embryo consists of the epiblast, a radial cup-shaped layer of epithelial cells that will give rise to the entire embryo; a surrounding layer of visceral endoderm (VE); and extra-embryonic tissues located proximal to the epiblast (Figure 1.1). First, neural fate is induced by the anterior visceral endoderm (AVE) located at the future anterior end of the embryo. Simultaneously, gastrulation initiates at the diametrically opposite side to the A V E , marking the future posterior end. Gastrulation, the process whereby epithelial cells ingress and generate mesoderm, initiates and gives rise to the primitive streak. The primitive streak begins at the rim of the epiblast cup and proceeds to the distal tip. The antero-posterior polarity of the embryo is thus established. The 5  PROXIMAL  ANTERIOR  POSTERIOR  primitive streak  anterior visceral endoderm  DISTAL  Figure 1.1 Forebrain induction and patterning in the mouse embryos. Anterior visceral endoderm (red cells; visceral endoderm, pale green) rotates to the future anterior region and induces the overlying epiblast or embryonic ectoderm (blue) to become forebrain. Anterior definitive endoderm and axial mesoderm derived from node (orange) and primitive streak (purple) further refine and pattern the forebrain at later stages. Extraembryonic tissues (white) are located proximal Iy. See text for details. Adapted from Beddington and Robertson, 1998. 6  derivatives of the anterior primitive streak - the node, anterior definitive endoderm, and axial mesoderm - reinforce and further refine the existing patterns within the neural plate. At later stages, additional patterning centres, such as the anterior neural ridge, are generated. Together with the existing patterning centers, they regulate the morphogenesis of the forebrain. A variety of forebrain defects has been characterized in gene disruption experiments, and functional analyses of these genes have given insights to each successive developmental process. Excellent, in depth reviews of this field are available (Beddington and Robertson 1999; Wilson and Houart 2004). Anterior visceral endoderm induces anterior neural fate  One of the most celebrated experiments in developmental biology was published in 1927 by Spemann and Mangold. This experiment revealed the presence of cells that have the ability to pattern the entire vertebrate organism. By grafting a small clump of cells, the organizer, from one amphibian blastula into another, they demonstrated that these cells have the ability to co-opt and organize a complete secondary axis (Gilbert 2003). In mammals this grafting experiment was never possible; graft experiments with the equivalent organizer were able to duplicate an entire trunk, but the resultant secondary axis never included the head (Beddington 1994; Tam and Steiner 1999). However, more recent grafting experiments using the organizer, plus the A V E , have been able to duplicate an entire axis, including the head, in mouse embryos (Tam and Steiner 1999). The converse removal of the A V E resulted in a loss of anterior neural tissues (Thomas and Beddington 1996). Together, these experiments demonstrate that the A V E is essential for inducing anterior neural ectoderm in mammals.  7  The neural plate normally forms by E7.5 and expresses neural markers such as Six3 or Otx2, but mutants with A V E defects never express neural markers, indicating that neurectoderm was never fully induced (Rhinn et al. 1998; Shawlot et al. 1999). The A V E initially forms at the distal tip of the embryo but arrives at the prospective head region through rotation (Figure 1.1). To promote head formation, posterior axis development is suppressed (Piccolo et al. 1999; Robertson et al. 2003; Wilson and Houart 2004); trunk and other posterior structures develop from intricate and complex interactions between the Nodal, Wnt, BMP, and RA pathways. The A V E antagonizes these pathways by inducing the expression of several antagonist genes: Cerrl acts as a multifunctional antagonist against the Nodal, Wnt and BMP signaling pathways; lefty 1 is a Nodal antagonist; and noggin and chordin are both BMP antagonists (Piccolo et al. 1999; Bachiller et al. 2000; Perea-Gomez et al. 2002). Consistent with this model, transplant experiments using posteriorizing factors such as RA, and mutants disrupted for genes in posteriorizing pathways, developed similar neural plate induction defects as A V E mutants (Ang et al. 1994; Liu et al. 1999). One of the key signaling pathways regulating the expression of A V E genes is Nodal, a secreted signaling factor belong to the Transforming Growth Factor - P (TGF^ /?) family of ligands. Nodal signaling directly initiates the expression of lefty 1 and Cerrl in the visceral endoderm at the distal tip of the epiblast (Brennan et al. 2001). Nodal also turns on the expression of Cripto, which directs the orthogonal movement of distal visceral endoderm to the prospective anterior end (Varlet et al. 1997; Ding et al. 1998). Consistent with these observations, disruptions to the Nodal pathway resulted in a failure to activate anterior neural fate (Ding et al. 1998; Brennan et al. 2002; Yamamoto et al. 2004; Takaoka et al. 2006). Interestingly, in Cripto mutants the A V E failed to rotate and remained at the distal tip, and the epiblast cells adjacent to the A V E 8  began to express anterior neural markers (Ding et al. 1998). This result further demonstrated signals emanating from the A V E induced head formation. Node-derived anterior definitive endoderm reinforces and further elaborates forebrain patterning  Concurrent with the rotation of distal visceral endoderm to the anterior end, proximal epiblast rotates to the prospective posterior end to initiate gastrulation and primitive streak formation (Ding et al. 1998; Brennan et al. 2001). The node arises at the anterior end of the primitive streak as it elongates from the rim of the epiblast cup and reaches the distal tip. The node in the mouse embryo is the equivalent structure to the organizer in the frog blastula, even though it lacks head induction activity, as discussed above. However, the function of the node is necessary for head formation as evidenced by forebrain mutants such as Wnt3 and Arkadia that developed a normal A V E and initiated proper expression of neural plate markers, but lacked the node (Liu et al. 1999; Episkopou et al. 2001). This finding is not surprising as the node gives rise to structures that will have critical functions later during forebrain patterning. The ventral cells of the node give rise to the prechordal plate, notochord and definitive endoderm, while the dorsal cells of the node contribute to the floor plate along the ventral midline of the embryo (Placzek and Briscoe 2005). As gastrulation proceeds in the embryo, the A V E is displaced by anterior definitive endoderm (ADE) (Thomas and Beddington 1996). The ADE migrates to the head region and lines the ventral neural plate. Curiously, the ADE expresses many of the same genes as the AVE, including Cerrl, Liml/Lhxl and Hex, prompting the idea that the function of the ADE is to maintain the expression and function previously established by the A V E (Wilson and Houart 2004). Mutants with disrupted ADE 9  generally acquired anterior neural tissue at E7, but by E7.5 - E8 the expression of these neural markers was lost, and, consequently, anterior neural defects developed (Shawlot et al. 1999; Martinez Barbera et al. 2000; Shawlot et al. 2000; Hallonet et al. 2002). Nodal signaling is also critical in generating a wide variety of cells during gastrulation in the anterior primitive streak, including the ADE lineage (Robertson et al. 2000; Tremblay et al. 2000; Vincent et al. 2003; Chu et al. 2004). Consistent with this idea, disruptions to transducers of the Nodal signaling pathway, including ALK4, ActRlIA, ActRIIB, Smad2, Smad3, Smad4, and FoxHl, resulted in anterior primitive  streak defects (Ding et al. 1998; Heyer et al. 1999; Song et al. 1999; Hoodless et al. 2001; Yamamoto et al. 2001; Norris et al. 2002; Vincent et al. 2003; Chu et al. 2004; Dunn et al. 2004). In fact, Nodal plays multiple roles in the establishment of anteriorposterior patterning in the epiblast of the mouse embryo. As a morphogen, various levels of Nodal signaling convey different instructions. To illustrate, high Nodal signaling is necessary for definitive endoderm formation while lower levels are sufficient for A V E and primitive streak formation (Vincent et al. 2003; Dunn et al. 2004). Consequently, a small reduction of Nodal signaling in some mutants listed above caused ADE defects, but a greater reduction in signaling led to A V E defects. Conversely higher than normal levels of Nodal signaling can also cause developmental abnormalities. Ectopic Nodal signaling in mutants that express reduced Nodal antagonists, including Drapl, lefty], and lefty2, caused disruptions to A V E formation (Iratni et al. 2002; Perea-Gomez et al. 2002). It is important to note that the forebrain is the part of the embryo most sensitive to disturbances of Nodal signaling. The biochemistry of the Nodal signaling pathway will be discussed in greater detail below.  10  Node derived prechordal plate provides ventral midline patterning  centre The node also generates axial mesoderm that migrates along the length of the embryo beneath the midline of the neural plate. Some A D E intercalates into the axial mesoderm to form the axial mesendoderm, a rod-shaped structure that later replaces the A D E . Axial mesendoderm underlying the prosencephalon from this stage on is referred to as the prechordal plate, while that underlying the caudal axis is referred to as the notochord. The probable function of the axial mesendoderm, like the A V E and A D E before it, is in the maintenance of neural identity through the continual opposition of posterior signals from the epiblast. As well, the prechordal plate and notochord now produce additional signals that further refine the pattern along the mediolateral axis that will later translate into the dorsoventral axis as the neural plate folds into the neural tube (Shimamura and Rubenstein 1997; Placzek and Briscoe 2005). The anterior neural tube is now called the prosencephalon or forebrain, and will give rise to the telencephalon and diencephalon. The prechordal plate has distinct gene expression and temporal patterns from the notochord. For instance, gsc is expressed by the prechordal plate but not by the notochord and, while chordin and noggin are both expressed in the notochord early at the 3 somite stage, their expression in the prechordal plate appears later at the 5 somite stage (Belo et al. 1997; Anderson et al. 2002). The focus from this point will be on mutants that affect the prechordal plate and the forebrain. Mutants lacking prechordal plate initiate early neural marker expression normally, but the established patterns degenerate over time, indicating prechordal mesendoderm maintains existing expression patterns (Shawlot et al. 1999; Camus et al.  11  2000). In addition, mutants develop expanded dorsal cell fate at the expense of ventral midline and ventral cell fate (Filosa et al. 1997; Nishioka et al. 2005).  Regional patterning of the forebrain  By early somite stages the rostro-caudal and dorsoventral axes within the forebrain are subdivided into the rostral telencephalon and eye field, the caudal diencephalons, which will contribute to the prethalamus and pretectum, and ventrally to the hypothalamus. These divisions arise through expression of region-specific markers: rostral expression of Fgf8, FoxGJ, Rx, Hesx and Six3, caudal expression of Otx2 in the  forebrain-midbrain region, dorsal-medial expression of BMP and Wnt family members (Grove et al. 1998; Lee et al. 2000), and ventral expression ofShh and Foxa2IHnf3B in the notochord and floorplate. Expression of these markers precedes the appearance of morphological structures at E l 1, including the medial ganglionic eminence (MGE) at the ventral-most location, the lateral ganglionic eminence (LGE) at the ventral-lateral location, and the cortex at the dorsal-most location. The mechanism of forebrain patterning is dynamic and complex and is still being investigated. Detailed reviews are available (Hebert 2005; Lupo et al. 2006). Three interdependent regional patterning centres organize and further refine forebrain morphogenesis (Figure 1.2). The anterior neural ridge (ANR) is a morphologically defined structure located at the junction of the anterior neural plate and the non-neural ectoderm and represents the rostral patterning center. Fgf8 signalling from the ANR was discovered to induce and maintain forebrain fate (Shimamura and Rubenstein 1997). Reducing Fgf8 levels caused a smaller telencephalon to develop from reduced rate of neurogenesis, and was preceded by  12  D  T  Figure 1.2 Molecular network controlling regional patterning of the forebrain. Three coordinating patterning centres establish the three axes through the secretion of morphogens: the anterior ectodermal ridge (AER, red) secretes Fgf8, the roof plate (blue) secretes Wnt and BMP, and the prechordal plate (green) secretes nodal and Shh. Tel. telencephalon; Di, diencephalon; mes, mesencephalon; h, hindbrain: d. dorsal; v. ventral; r, rostral; c, caudal. See text for details. Adapted from Hebert, 2005 and Lupo and Lewis, 2005. 13  reduced expression of Foxgl and Six3 (Anderson et al. 2002; Storm et al. 2003; Storm et al. 2006). The activities of the ventral patterning centre are located in the prechordal plate and floorplate, which secrete Shh (Figure 1.2). Mutations in Shh in both mice and humans can cause HPE (Roessler et al. 1996; Zhang et al. 2006). The Shh protein is both necessary and sufficient for the development of ventral telencephalic structures, such as the MGE and LGE, and the expression of associated neural markers (Gunhaga et al. 2000; Watanabe et al. 2005). Interestingly, Shh is required during M G E specification earlier on and during LGE specification at a slightly later stage (Kohtz et al. 1998). A repressive transcription mediator of the dorsal Shh signal in the forebrain is GU3. Mice deficient for GH3 exhibit a dorsal to ventral transformation, consistent with its function as a repressor of Shh signalling. Surprisingly, though, dorsoventral patterning is established normally in the absence of both Shh and GU3 (Rallu et al. 2002). This result showed that Shh and GU3 are crucial for dorsoventral patterning of the telencephalon, but their functions are dispensable when both are eliminated. Shh's function during telencephalon patterning is dependent on timing, but additional mechanisms are clearly also involved. Nodal signalling is also suspected to contribute to ventral patterning, since nodal is expressed in the prechordal mesoderm at this stage (Figure 1.2). In mice, Nodal's role is not yet well understood since many of the mutants are severely affected by disruptions to earlier events. However, in zebrafish, Nodal mutants lack ventral structures in the diencephalons (Mathieu et al. 2002). Currently, detailed understanding of Nodal in this process is limited, but it is thought to function through multiple mechanisms: through direct regulation and through co-operation with the Shh pathway, as well as through pathways that are independent of Shh (Lupo et al. 2006). 14  Dorsal signals from the roof plate and dorsal-medial cells require members of the B M P and Wnt families to mediate patterning of dorsal fates (Furuta et al. 1997; Grove et al. 1998; Lee et al. 2000)(Figure 1.2). B M P has the ability to induce dorsal midline fate as evidenced by its induction of the expression of Msx, a dorsal forebrain marker (Furuta et al. 1997). In contrast, disruption to Bmprla resulted in the loss of the dorsal-medial structure, the choroid plexus (Hebert et al. 2002). In addition, members of the Wnt family are expressed dorsally and were shown to specify dorsal rather than ventral telencephalic identity in chick explants (Braun et al. 2003; Gunhaga et al. 2003). Finally, ES cells induced to form telencephalon neurons can be induced to adopt a dorsal fate by Wnt signalling (Watanabe et al. 2005). It is worth noting again that the local patterning centers and their associated pathways are interdependent, a conclusion that is drawn from multiple studies that witnessed the breakdown of other patterning centres from initial disruptions to one signalling centre (Figure 1.2) (Dou et al. 1999; Camus et al. 2000; Ohkubo et al. 2002; Ribes et al. 2006; Storm et al. 2006). R A has been shown to coordinate these patterning centres. Raldh2, a regulator of steady R A levels, is expressed in the forebrain neuroepithelium and the overlying surface ectoderm, and its loss resulted in defective morphogenesis of various forebrain derivatives (Ribes et al. 2006). It plays a role in maintaining Fg/8 signals from the A N R and Shh signalling from the ventral patterning centre. Thus, R A signalling is integral to the F G F - S H H signalling loop circuit.  Potential disruptions caused by mutations in HPE genes  As discussed earlier, mutations in SHH, SIX3, ZIC2 and TGIF were identified in H P E patients. The Shh pathway is critical as a ventral patterning morphogen; 15  disruptions to this pathway cause dorsoventral transformation in mice, consistent with the involvement in HPE (Cohen 2003). Six3 is an antagonist of the Wnt pathway (Braun et al. 2003; Lagutin et al. 2003). Antagonizing Wnt signaling is essential for establishing the anterior forebrain. Consequently, a reduction in Six3 activity resulted in the loss of anterior forebrain structures (Lagutin et al. 2003). Later, Wnt signals become localized to the dorsal midline and play a role in patterning the dorsoventral axis (Grove et al. 1998). Six3 may also modulate Wnt signaling during this process. Disruptions to anterior forebrain patterning and later dorsoventral patterning can both lead to HPE. Zic2 mutations are found in HPE patients, and interestingly this group of patients did not develop craniofacial abnormalities (Wallis et al. 1999). Mouse models indicate dorsal patterning has been disrupted (Nagai et al. 2000). Disruptions to both Zic2 and Tgif may affect Nodal signaling and cause abnormal forebrain patterning (Hoodless et al. 2001; Houston and Wylie 2005; Lupo et al. 2006). Knowledge of the precise functions played by Nodal during forebrain patterning are not known, but evidence points to disturbances in Shh expression in the prechordal plate (Rohr et al. 2001). Alternatively, Tgif may modulate the RA pathway inappropriately. The RA pathway is now known to be an important coordinator of forebrain patterning centres. Potential molecular mechanisms by which Tgif'may affect Nodal and RA signaling will be discussed below.  1.1.2.  Asymmetric body plan Superficially, mammals appear bilaterally symmetric, with a symmetric face,  head, torso and bilateral appendages. Upon reflection and examination, though, mammals are obviously pseudo-bilateral. Many internal organs possess invariant left16  or right- handedness, known as situs. Specifically, the heart and spleen are predominantly in the left side of the thoracic cavity, the stomach is in the left side of the abdominal cavity, and so on. Furthermore, it is fascinating to discover language, sense of humour, taste and smell, as well as many other aspects of our brain functions, are located in asymmetric CNS organs (McManus 2002; Sun et al. 2005; Sun and Walsh 2006). Congenital laterality disorders are found in humans at a frequency of 1 in 8500 (Capdevila et al. 2000; Purandare et al. 2002; Bisgrove et al. 2003). Situs inversus, a complete reversal of the internal organs, has a natural frequency of 1 in 10,000 but is present in 20% of Kartagener syndrome patients (Capdevila et al. 2000; Bisgrove et al. 2003). While people with this condition have minor sinus and pulmonary infections, and males are sterile, they have a normal life expectancy. However, situs ambiguus, resulting in the reversal of only some of the organs, is a source of significant health problems. There is a wide spectrum of situs ambiguus disorders, including right isomerism, or asplenia syndrome, where bilateral right-sidedness occurs; left isomerism, or polysplenia, where bilateral left-sidedness occurs; as well as a host of complex phenotypes that require careful examination and diagnosis (Bisgrove et al. 2003; Levin 2005). Genetic analysis and experimental perturbations in human and animal models revealed a complex cascade of molecular events that establish laterality early during embryogenesis before the onset of organogenesis. The following review focuses on left-right determination in mice, although the mechanism of left-right patterning is fundamentally conserved in vertebrates, and any significant or relevant differences will be noted (Figure 1.3). Briefly, a break in bilateral symmetry before or around the formation of the node initiates this process. Subsequently, situs-specific gene 17  LEFT  RIGHT midline  barrier  Node  leftness  Figure 1.3 Genetic pathway for the determination of left-right asymmetry. See text for details. Adapted from Hamada, 2002.  18  expression patterns become established within or near the node during gastrulation. Left-right patterning information is then transferred from the node to the lateral plate mesoderm (LPM), where left-sided expression of Nodal becomes stabilized. The establishment of asymmetric expression is unilaterally restricted by midline structures. Finally, patterning information from the L P M is transferred to organ primoridia where asymmetric morphogenesis takes place. Excellent and comprehensive reviews of these events are available (Bisgrove et al. 2003; Hornstein and Tabin 2005; Levin 2005; Raya and Belmonte 2006). Breaking of symmetry One of the most controversial and intellectually captivating questions in the laterality field is what mechanism initiates bilateral asymmetry? There is now little dispute that nodal cilia, located within the node, rotate in a clockwise fashion and generate a right to left fluid flow across the node (see videos at http://www.cell.eom/cgi/content/full/95/6/829/DCl) (Nonaka et al. 1998). The nodal flow model was proposed after the identification of numerous genes involved in human and mouse with left-right patterning defects were found to affect genes encoding dyneins, or motor proteins, and other proteins involved in the ultrastructure and motion of the nodal cilia. Nodal flow and cilia movements can be visualized by electron microscopy and high resolution microscopy (Nonaka et al. 1998; Okada et al. 2005). Sophisticated methods were used to monitor and experiment with the velocity and direction of fluids within the node (Nonaka et al. 2002; Okada et al. 2005). Altogether, these experiments showed a strong correlation between loss of cilia movement, alteration in nodal flow, and resultant aberrant laterality development.  19  The functional effect of the uni-directional fluid flow is still under dispute (Tabin and Vogan 2003). An attractive model postulates the leftward transport of morphogen proteins by nodal flow (Nonaka et al. 1998). Recently, nodal vesicular parcels were visualized as being released within the node, transported by the nodal flow and subsequently fragmented, presumably leading to the release of the contents of the disrupted parcels (Tanaka et al. 2005). These were shown to contain the putative morphogens Shh and RA, and the release of these parcels required Fgf signalling (Tanaka et al. 2005). Consistent with this model, Fg/8, Shh, Ihh, and RA signalling have been shown to regulate asymmetric Nodal expression and early asymmetric patterning (Meyers and Martin 1999; Tsukui et al. 1999; Zhang et al. 2001). An alternative model also starts with a unidirectional fluid flow but is based on a physical deformation of mechano-sensory cilia that provide directional cues, leading to subsequent asymmetric downstream events (McGrath et al. 2003; Tabin and Vogan 2003). This two-cilia model was proposed upon discovery of immotile cilia that surround motile cilia within the node (McGrath et al. 2003; Tabin and Vogan 2003). In contrast to the nodal flow model, which cannot adequately explain why mutants that lacked cilia have the same laterality phenotype as mutants that had immotile cilia, the two cilia model can account for these different defects. The two cilia model predicts mutants with immotile cilia, but functional mechanosensory cilia, such as iv, developed situs inversus and situs solitus (normal situs) because small, stochastic signals randomly defined left from right. Conversely, mutants, such as Kif3B, that completely lacked cilia developed a situs ambiguus phenotype when there was neither directional information nor subsequent signalling activity. Furthermore, mechano-sensory cilia were shown to trigger C a  ++  influx, and asymmetric C a  ++  influxes were previously  associated with symmetry breaking events (McGrath et al. 2003; Raya et al. 2004). 20  Current controversies mostly surround the speculation that cilia-induced flow may only be transducing an earlier asymmetric signal rather than initiating asymmetry de novo. This idea arises from discoveries in zebrafish and chick that asymmetric activities, namely H /K -ATPase channel activity, Ca flux and accumulation, and +  +  ++  Notch signalling, preceded node formation and the appearance of cilia in the node (Hornstein and Tabin 2005). Perhaps these seemingly contradictory observations indicate the initial steps are different in vertebrates, arising from different evolutionary pressures on the different gastrulas. Although nodal flow is universal in all vertebrates (Bisgrove et al. 2000), it may be the primary mechanism used for symmetry breaking in mammals (Kawakami et al. 2005). Other notable differences exist, such as the differences in expression pattern and function of Sonic hedgehog and Fgf8, between mammals and.other vertebrates during laterality determination (Yoshioka et al. 1998; Meyers and Martin 1999; Tsukui et al. 1999). Asymmetric gene expression in the node and lateral plate mesoderm  Regardless of the precise nature of the initial symmetry breaking events, expression of the TGF- P family member Nodal becomes enhanced on the left side of the node during gastrulation in all vertebrates (Figure 1.3)(Brennan et al. 2002; Hamada et al. 2002). The left-sided Nodal signal from the node is likely relayed to the left LPM through diffusion, and is later amplified and stabilized. The expression of Nodal in the LPM is critical for initiating downstream target genes, such as lefty 1, lefty2 and pitx2. Accurate regulation of these expression patterns is achieved through complex positive and negative feedback loops. Nodal expression is maintained by amplification through a positive autoregulatory loop mechanism. The threshold, range and duration of Nodal signalling is restricted by numerous factors, including SPC1, SPC4, Drapl, and lefty2, 21  through negative feedback loops (Meno et al. 1997; King and Brown 1999; Constam and Robertson 2000a; Constam and Robertson 2000b). Regulatory elements participating in these feedback loops have also been identified. For instance, the expression of Nodal, lefty 1 and pitx2 in the L P M were co-ordinately regulated by an asymmetric enhancer that contains binding sites for the forkhead transcription factor FoxHl (Hamada et al. 2002). Ultimately, the precise regulation of Nodal levels and expression pattern is essential for the downstream target genes that orchestrate asymmetric morphogenesis. Asymmetric gene expression maintained by the embryonic midline The importance of the embryonic midline, which includes the notochord and the neural floorplate, during laterality determination, was appreciated early on. Surgical removal of notochords in frogs led to aberrant cardiac looping (Danos and Yost 1996); right-handed looping of the heart tube is the earliest visible manifestation of asymmetry. The notochord is a rod-like structure derived from mesodermal cells that ingress through the node during gastrulation. The neural floorplate is located at the base of the developing neural tube, and is induced by and directly adjacent to the notochord. Many midline mutants in zebrafish and mice also develop situs abnormalities. For example, Shh mutants did not maintain notochord and neural floorplate and developed multiple laterality defects (Meyers and Martin 1999). Experimental results indicate the midline prevents the contra-lateral spread of asymmetric gene expression patterns as they become established. Thus, midline mutants developed randomized Nodal expression and exhibited situs ambiguus. Midline cells provide a barrier between the left and right sides through a number of mechanisms. To prevent Nodal expression from crossing over to the right side of the 22  midline, lefty 1, a homologue of lefty2, and an antagonist of Nodal, is expressed in the floorplate (Meno et al. 1998; Bisgrove et al. 2003; Levin 2005). The midline is also rich in extracellular matrix which have the ability to insulate activities such as leftsided C a  ++  signals from propagating to the right side of the embryo (McGrath et al.  2003). Interestingly, this property of the midline is underlined by the common observation that among two conjoined twins, only the twin on the right developed laterality defects (Levin 2005). Apparently, asymmetric signals from one embryo cannot cross over the midline, but are able to influence the neighboring embryo. Organ positioning and morphogenesis The last steps in laterality morphogenesis are the simplest conceptually, but the precise molecular pathway is not yet well characterized. This step instructs organ morphogenesis that obeys situs information. For example, during late neurulation stages, the linear heart tube, formed from paired cardiac progenitors in the left and right L P M , undergoes right-sided looping. Another fascinating example is embryonic turning in mouse and chick embryos; events that bring the embryo from a dorsally flexed position to a ventrally flexed or fetal position. The only gene known thus far to play a role in these events ispitx2 (Ryan et al. 1998). Retinoic acid and symmetrical development of somites Somites are paired structures that give rise to a number of bilateral structures, including the muscles and the skeleton. In the mouse embryo somites are physically adjacent to tissues that develop in response to asymmetric patterning information. As discussed, the Nodal, RA, Wnt, Notch, Shh, Fgf and BMP pathways play critical roles in laterality determination. These pathways are used repeatedly during many other 23  developmental processes, from limb development to facial patterning. How do tissue progenitors respond to the same signals when they are used both during laterality determination and during somite development? Recently it was discovered that symmetric development of somites is not a default state. While this is not yet well understood, during symmetric somite development RA signalling was shown to buffer and prevent laterality signals from ectopically activating somitogenesis asymmetrically (Kawakami et al. 2005; Vermot and Pourquie 2005). Disruptions to the RA pathway can cause laterality disturbances to both synchronized somite development and other organs. Likewise, inhibition to the RA pathway, using antagonists in embryos at the headfold stage, led to bilateral expression of pitx2 in the L P M (Vermot and Pourquie 2005). In summary, forebrain patterning and laterality determination involve overlapping developmental mechanisms and signalling pathways.  1.2.  T G - INTERACTING F A C T O R (TGIF) The discovery of Tgi/mutations in HPE patients was particularly exciting since  Tgif'has been shown to modulate both the Nodal and retinoic acid pathways (Gripp et al. 2000). As discussed, the Nodal and RA pathways have long been associated with the development of HPE in patients and in animal models.  1.2.1. TGIF is a T A L E HD TGIF is a 272 amino acid transcription factor that belongs to the three amino acid loop extension (TALE) superclass of homeobox genes (Figure 1.4) (Burglin 1997). Homeobox proteins are an evolutionarly conserved family of transcription factors that contain a highly conserved stretch of 60 amino acids that fold into a helix-turn-helix 24  H P E Mutations  u —  C/3  * *  a> **  *  *  272 CtBP Binding Domain  Honieodomain  Smad2/3 & H D A C Interaction Domain  Repressive Domain & Sin3 Interaction Domain ( E G F / M A P K phosphorylation)  Figure 1.4 TGIF functional domains. H P E patient mutations are indicated by asterices above the domains.  25  motif. Members of the TALE family, which includes PBC and ME1S class proteins, form heteromeric complexes with each other to regulate transcription of developmentally important genes, such as the Hox genes and Pax6 (Mann and Affolter 1998). As co-factors, they provide specificity and cooperativity for Hox proteins. TALE proteins can also serve as co-factors to other transcription factors (Sagerstrom 2004). Finally, TALE proteins may also penetrate inactive chromatin and anchor Hox or other transcription factor to regulate activity. Although TGIF has not been shown to bind PBC proteins (Chang et al. 1997; Knoepfler et al. 1997), TGIF was shown to mutually compete with Meis for DNA binding and inhibit D1A dopamine receptor (Yang et al. 2000a). The homeodomain is located between residues 35-97 (Wotton et al. 1999a).  1.2.2. TGIF is a RA antagonist TGIF was first implicated as an antagonist in the RA pathway because it competed for DNA binding with the RXRa retinoic acid receptor (Bertolino et al. 1995). The RA pathway is an important morphogen for multiple processes during development (Chambon 1996; Mark et al. 2006). Signalling of this pathway is initiated by binding of RA to nuclear receptors, the RARs (a, p\ and y), which then regulate the transcription of target genes by heterodimerizing with retinoid X receptor (RXR) (Figure 1.5) (Chambon 1996). TGIF directly competes for DNA binding with RXR responsive elements found within the promoter regions of genes such as the cellular retinol-binding protein II (Bertolino et al. 1995). More recently, TGIF was also shown to function as a transcriptional corepressor independent of direct DNA binding. TGIF directly bound to the ligand binding domain of the RXR family of receptors, and this binding led to the repression 26  Figure 1.5 The retinoic acid signal transduction pathway. See text for details.  27  of downstream transcription via the recruitment of CtBP complexes (Melhuish and Wotton 2000; Bartholin et al. 2006). Furthermore, interaction between TGIF and RXRa could be competed by the addition of RA (Bartholin et al. 2006). It is worth repeating here that the RA pathway has previously been implicated in the development of HPE. Prenatal exposure to RA in humans and model organisms can increase the incidence of this developmental defect (Schneider et al. 2001; Ming and Muenke 2002). Consistent with this, RARa-RARy double mutants, and Raldh2 mutants, display telencephalic abnormalities (Lohnes et al. 1994; Ribes et al. 2006).  1.2.3. TGIF is a TGF-P co-repressor TGIF was also shown to be a transcriptional co-repressor for TGF-P responsive transcription through its interactions with Smad2 (Wotton et al. 1999a). The TGF-P pathway plays important roles during cell cycle regulation, growth, differentiation and other fundamental processes during embryogenesis and in the adult (Massague et al. 2005). Signalling events at the cell surface are initiated by the binding of ligands and the formation of type I and type II receptor complex (Figure 1.6). The serine/threonine kinase domain of the type II receptor subsequently phosphorylates the type I receptor. For example, Nodal binds to Activin Receptor II (ActRII), which phosphorylates Activin Receptor I (ALK4). Nodal then propagates its signal through the Type I receptor by phosphorylation of the receptor (R)-regulated Smad proteins, Smad2 or Smad3, in the cytosol. Upon activation, R-smads form complexes with the common Smad, Smad4. Smad complexes then translocate into the nucleus and regulate transcription by forming heteromeric complexes with DNA binding proteins such as FoxHl. The level of transcriptional activity is regulated by the availability and  28  extracellular space  Figure 1.6 TGFP signal transduction pathway. See text for details. I, type I receptor; II, type II receptor.  29  recruitment of transcriptional activators, such as p300/CBP, or transcription repressors, such as TGIF, that become incorporated into the Smad protein complex. TGIF represses TGF-P responsive transcription through multiple mechanisms: by directly competing with the co-activator p300/CBP for Smad2 interaction, and by recruiting histone deacetylase (HDAC) complex, the carboxyl terminal binding protein (CtBP) complex, and the Sin3 complex (Wotton et al. 1999b; Melhuish and Wotton 2000; Wotton et al. 2001). All three of these complexes lead to modifications of the chromatin in the neighbouring regions, effectively "closing" the DNA and limiting access by transcription activating factors. Consistent with this, the chick orthologue of TGIF, AKR, was also shown to be a transcriptional repressor (Ryan et al. 1995). Two adjacent domains between residues 137-192 that interact with Smad2 and Smad3,  and that recruit histone deacetylase (HDAC) protein complexes (Wotton et al.  1999a). The N-terminal residues 24-28 are capable of recruiting CtBP protein complexes (Melhuish and Wotton 2000). The extreme carboxyl terminus domain between residue 192 and 272 that binds Sin3 protein complexes (Melhuish and Wotton 2000; Wotton et al. 2001). It is relevant to note here that the TGF-p/Nodal pathway has also previously been implicated in the development of HPE: (1) mutations in FOXH1,  a transcription  factor in the TGF-P pathway, have been identified in HPE patients (Ming and Muenke 2002); and (2) mice deficient for genes in the TGF-P pathway, such as the compound mutant Smad2 ~; Nodat ', +/  1  develop HPE (Nomura and Li 1998).  TGF-P also activates the c-Jun N-terminal Kinase (JNK) pathway, which then suppresses Smad2 signaling by recruiting Tgif, which competes with Smad2 for binding to p300 (Pessah et al. 2001). A final interesting property of TGIF is its stability is enhanced by the Ras/MAP kinase pathway (Lo et al. 2001). Increased stability of the 30  TGIF protein can be achieved via phosphorylation of Erk MAP kinase sites, T235 and T239, near the C-terminus. Differential splicing results in two rgz/isoforms that produce proteins differing by 20 amino acids at the N-terminus, but does not affect any known functional domains.  1.2.4. T G I F mutations identified in H P E patients  More than 12 chromosomal regions have been mapped in sporadic and familial HPE cases. One region, mapping to 18pl 1.3 in humans, contains TGIF. Heterozygous deletions and mutations of TGIF have been associated with HPE (Overhauser et al. 1995; Gripp et al. 2000; Chen et al. 2002; Aguilella et al. 2003; Bendavid et al. 2005a; Bendavid et al. 2005b; Chen et al. 2006). The incidence of TGIF microdeletions deletions that encompass the entire gene but are only detectable at the sub-karyotypic level - occurs at the same frequency (1-3%) as point mutations in HPE patients. However, patients at the mildest end of the HPE spectrum do not carry TGIF microdeletions (Bendavid et al. 2005a; Bendavid et al. 2005b). Interestingly, most point mutations affect known functional domains of TGIF and may provide insights into the mechanism of HPE. For example, the nonsense mutation, Y59X, produces a protein that terminates within the homeodomain (Aguilella et al. 2003). Multiple missense mutations, such as S28C, P63R, R90C, T151A, and T162F, probably disrupt the CtBP binding domain, homeodomain, Smad2/3 interacting domain or the HDAC interacting domain, respectively (Gripp et al. 2000; Chen et al. 2002). The mutations Q107L and V126A (Aguilella et al. 2003; Chen et al. 2006) are not located in known domains but may prevent protein interactions or proper folding of the TGIF protein. A series of polymorphisms have also been reported in patients, but their significance is not yet known (Wallis and Muenke 2000). 31  1.2.5. Evolutionary conservation Tgif is conserved in eukaryotes from yeast to mammals (Burglin 1997). TALE proteins are characterized by an extension of three amino acids between a-helices 1 and 2 within the homeodomain. Within the TGIF class, the homeodomain is distinguished by the presence of an Ala residue at the 27 position; in all other members of the TALE th  superclass, a Pro is found at this position. TOS8 is the yeast ortholog of Tgif While its precise function is not known, TOS8 is directly downstream of the Swi-4-Swi6 cell cycle box binding factor (SBF) and Mlul binding factor (MBF), which are transcription factors that regulate the start of the yeast cell cycle, and therefore TOS8 presumably functions during G i / S progression (Horak et al. 2002). The Drosophila genome contains 2 orthologues, achintya and vismay, and both orthologues play redundant roles during meiosis and spermatogenesis (Ayyar et al. 2003; Wang and Mann 2003). A chick orthologue named Avian KnottedRelated was identified as a negative regulator of apoVLDLll transcription (Ryan et al. 1995). Orthologues in Xenopus and zebrqfish have also been identified, but are only well conserved over the homeodomain. There are 3 paralogues in mice and humans: Tgif2 (Imoto et al. 2000; Melhuish et al. 2001), Texl (Lai et al. 2002), and Tgiflx/y (Bianco-Arias et al. 2002). Tgif2 lacks the N-terminus CtBP binding domain, but the HDAC and Sin3 repressive domains were shown to be functionally similar to Tgif (Melhuish et al. 2001). Tgiflx/y is located in a shared region among the sex chromosomes and evolved by retrotransposition of Tgif2. Consequently, Tgif2 and Tgiflx/y contain the same domains (Bianco-Arias et al. 2002). The most divergent paralogue is Texl which functions during spermatogenesis (Lai et al. 2002). The human Texl is not yet identified but is presumed to also be present in the genome. 32  1.3.  MAMMALIAN CELL CYCLE  The importance of proliferation during development is evidenced by the dramatic growth that occurs during embryogenesis. Despite this, our understanding of the mechanisms by which patterning coordinates proliferation with other processes is superficial. The cell cycle generates daughter cells by cyclic progression through 4 phases: G i , when cells are resting in the diploid stage; synthesis (S), when cells replicate DNA to become tetraploid cells; G2, when cells generate sufficient materials for generating two independent cells; and mitosis (M), when cells partition equivalent genetic and other materials into the daughter cells (Figure 1.7)(Hunter 2000; Nurse 2000). Cells can exist in an extended Gi phase referred to as Go when progression into S-phase is blocked (Lodish et al. 2000). Checkpoints ensure that each of these phases proceed in an orderly manner and respond appropriately to both intrinsic signals, such as DNA damage or developmental programs, as well as extrinsic signals, such as TGFp\ retinoic acid and Fgf. The restriction (R) point is arguably the most important checkpoint and regulates the commitment of a cell to undergo another round of cell division. Briefly, cyclic expression of cyclin proteins leads to the formation of a cyclin-dependent kinase (CDK)-cyclin complex, which has the ability to phosphorylate retinoblastoma (Rb) and bypass the R point. The presence of the active CDK-cyclin complex is regulated at multiple levels, including protein stability and a host of cyclin-kinase inhibitor proteins such as p57  Kip2  , p27  Kipl  and p21  Cipl  (Sherr and Roberts 1995).  Many mammalian cell cycle proteins have been identified over the last 30 years. The complex regulation of a multitude of these proteins by intrinsic and extrinsic cues in single cells and cell lines is now better understood. However, the integration of 33  Figure 1.7 Checkpoints in the the cell cycle. The three major checkpoints, G I , G2, and metaphase are indicated by bars. Passing the GI checkpoint or R (restriction) point commits cells to another round of cell cycle. Phosphorylation of the retinoblastoma (Rb) protein by the cyclin (cycDl) - cylin-dependent-kinase (CDK4) complex is regulated by the cyclin kinase inhibitors, p21, p27 and p57. See text for details.  34  intrinsic genetic programs and extrinsic signals is likely more complicated in multicellular organisms. Yet the idea that the function of some cell cycle proteins is vital has been challenged by the viability of mice that are depleted of essential cell cycle genes (Kozar et al. 2004; Malumbres et al. 2004; Hinds 2006). Nevertheless, evidence is emerging that in multicellular organisms related family members previously believed to be functionally redundant may serve specialized functions during development (Dyer and Cepko 2001).  1.4.  HYPOTHESES The hypotheses that were addressed are: (1) the loss of Tgif'will cause HPE or  related forebrain defects in mice; (2) the loss of Tgif will cause other developmental defects that result from abnormal patterning of the primary axes; and (3) Tgif carries out these functions by repressing target genes downstream of the  TGF-P, RA and  Ras/MAPK pathways.  1.5.  OBJECTIVES To investigate these hypotheses, the following experiments were planned: (1) to  create mice with a disrupted Tgif, (2) to examine the mutant mice obtained for forebrain and other developmental defects; (3) to determine potential defects at the cellular level that might explain any developmental abnormalities seen; (4) to determine potential defects at the molecular level; (5) to determine the effect of Tgif disruption on TGF-P, RA and Ras/MAPK signalling.  35  2.  CHAPTER 2 MATERIALS AND METHODS  2.1.  GENERATION OF TGIF MUTANT MICE  2.1.1. Isolation of murine Tgf/genomic clone The murine 129S6/SvEvTac genomic bacterial artificial chromosome (BAC) library (generously donated by K. Humphries) was screened using Tgif'EST BF133915 as a hybridization probe. Multiple independent BAC clones containing Tgif md its surrounding sequences were thus isolated. Mouse genome sequences available through UCSC (http://genome.ucsc.edu/), Ensembl (http://www.ensembl.6rg), and NCBI (http://www.ncbi.nlm.nih.gov) indicated a 7.6 kilobase (kb) EcoRl fragment within BAC 49612 included exons 2 and 3 of Tgif, which contain the majority of the coding sequences. Subsequent cloning generated the genomic plasmid clone used to prepare the Tgif targeting construct (see below). Restriction mapping was performed to confirm that the desired ^//"sequences were present in the subcloned plasmid.  2.1.2. Construction of the Tgif targeting vector The targeting vector was designed to replace exons 2 and 3, which encode nearly all of the open reading frame sequences of Tgif, with the reporter gene lacZ fused in frame to the remaining N-terminal 7 amino acids (Hasty 1993). The residual 7 amino acids are likely not functional. The construct also included a PGK-neo positive selectable marker gene cassette that allowed enrichment of targeting events. The targeting vector consisted of a 5' homology region, which was the 3.4 kb Xhol-SacIL fragment, and a 3' homology region, which was the 1.5 kb EcoRl-Hindlll fragment. Construction of the targeting vector (Figure 3.3) was as follows. First, a 7.0 kb EcoRI-XhoI fragment was subcloned from the 7.6 kb EcoRl plasmid. Next, exons 2 36  and 3, which were contained within a 2.3 kb SacII-Hindlll fragment, were excised and replaced with lacZ and PGK-neo.  lacZ  was constructed such that it inserted in frame  downstream of the remaining exon 2. The targeting vector was sequence verified.  2.1.3. Embryonic stem cell line targeting and identification of homologously recombined clones A brief description, including minor modifications, of the protocols used for ES cell maintenance and targeting protocols are as follows (Wurst 1993). ES cell culture media consisted of Dulbecco's Modified Essential Medium (DMEM) with high glucose, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 uM monothioglycerol, 2 mM L-glutamine, 15 % fetal calf serum, 50 ug/mL each of penicillin and streptomycin, and leukaemia inhibitory factor (LIF) that was prepared by L. Sly. The gene targeting vector was linearized with EcoRl and 20 ug was electroporated using Bio-Rad Gene Pulsar (0.34 kV, 250 mF) into 1 x 10 CCE ES cells 7  (generously donated by E. Robertson). ES cells were then selected with 180 ug/mL of active G418 for neomycin expression for five days. Resistant colonies were plucked into 96-well plates and expanded in culture. Duplicate plates were prepared; one was frozen at -80°C as stock, while the other was used to prepare genomic DNA for identification of homologous recombinants.  2.1.4. Identification of 7g// mutant alleles by Southern blot analysis Southern blot analysis was used as the primary screen for homologous recombination events. First, genomic DNA from individual ES cell lines was prepared. ES cells in 96-well plates were lysed in 10 mM Tris (pH 7.5), 10 mM EDTA, 10 mM NaCl, 0.5 % sarcocyl or SDS, and 1 mg/mL proteinase K (Sigma) at 65°C for 12-16 37  hours. DNA in each well was precipitated with 100 uL of chilled 75 mM NaCl: 100 % ethanol (ETOH) for 1 hour, washed three times with 70 % ETOH, and the DNA pellets were allowed to dry. Second, restriction digested genomic DNA was transferred to nylon membranes. Genomic DNA was digested with EcoRl, and these DNA fragments were separated on a 0.7 % agarose gel at 20 V for 12 hours. DNA in the agarose gel was transferred to Nylon Membrane (Bio-Rad Zeta-Probe Genomic Tested Blotting Membrane) overnight in lOx SSC, and fixed to the membrane by vacuum-drying for 1 hour at 80°C, or by UV cross-linking. Third, radioactive probes were hybridized to genomic DNA that was fixed to the membrane. The probe was labelled with [ P] 32  dCTP by random priming using Ready to Go™ DNA Labelling Beads (-dCTP) (Pharmacia). Membranes were pre-hybridized in 9 mL of hybridization buffer: 1.5x SSPE, 1 % SDS, and 1 % skim milk. Denatured probe was added and hybridized for 16 hours at 58°C. Excess probe was washed off at 65°C for two 30 minute intervals for each wash solution, 0.3x SSC and 0.1 % SDS. Fourth, results of the hybridization experiment were visualized by exposing membranes to a Phosphorlmager cassette for 1 night, scanned on a STORM 860 Phosphorlmager, and the resulting signal analyzed using ImageQuant (Molecular Dynamics, Sunnyvale CA). To demonstrate correct and unique homologous recombination events occurred in the identified clones, homologous recombination on both the 5' and 3' homologous arms was verified by Southern blot analysis. Correct targeting of the 5' arm was demonstrated when the 5' external probe, a 0.3 kb Xhol fragment, detected the endogenous 7.6 kb EcoRl wild-type fragment, and a modified 13 kb fragment. At the 3' arm, the external probe, a 0.5 kb Xbal fragment, detected the endogenous 7 kb Pstl fragment, and the modified 5.5 kb Pstl fragment. Two CCE clones, B2 and C6, were identified after screening 144 clones. 38  2.1.5. Identification of Tgif mutant alleles by P C R  An alternative PCR strategy was devised for ear-punch and visceral yolk sac DNA to rapidly determine the genotype of mice and embryos. A 418 bp fragment within exon 2 was only present on the wild-type allele and was amplified by primers TGIF 105 (5'-ttccctgctggtgaaagcaa-3') and TGIF 207 (5'-tgttcatacagccagtctcg-3')- The mutant allele consisted of the remains of exon 2 fused to the lacZ gene, and this junction was amplified only by TGIF 105 and LacZ 201 (5'-ggcctcttcgatattacgcc-3') to yield a 160 bp PCR product.  2.1.6. Production of chimeras and 7gi/mutant mice from targeted E S cells  Two independently-targeted ES cell lines, CCE derived B2 and C6, were injected into the inner cell mass of embryonic day E3.5 C57BL/6 host blastocysts to generate chimeric mice (Papaioannou 1993). Two independently targeted cell lines were used to generate chimeras in order to eliminate artefacts introduced by gene targeting. Germline transmission from two chimeric males was apparent by coat colour and confirmed by genomic blot analysis. The phenotypes observed in mice derived from both ES cell lines were similar and their combined results are presented. Multiple chimeric mice were generated and used for germline transmission of the mutated Tgif allele to their progenies.  2.1.7. Mouse breeding, genetic backgrounds, and congenic strains  All mice were bred and maintained at the Joint Animal Facility (JAF) at the British Columbia Cancer Research Centre. Inbred strains 129/SV and C57BL/6, and outbred strain CD1 were obtained from the JAF.  39  Routine testing for viral pathogens and mycoplasma was carried out on the mouse colonies by JAF staff. Experiments complied with all relevant federal guidelines and were approved by the institutional animal care and use committee (see Appendix). Chimeric males were mated to CD 1 females to generate mice with a mixed 129/Sv/CDl background. The colony with this mixed genetic background was established by mating of mice of the appropriate genotype within the colony. The same chimeras were also used to mate to 129/Sv female mice to generate mice with the inbred 129/Sv genetic background. Since the mutant allele of the heterozygote originated from ES cells, and the ES cells were derived from 129/Sv mice, a colony with the inbred 129/Sv genetic background was established after one cross to 129/Sv females. This colony was subsequently maintained by sister-brother mating of heterozygote mice. The same chimeras were also used to mate to C57BL/6 female mice to generate mice and embryos of a mixed 129/Sv/C57BL/6 background. A congenic C57BL/6 strain was generated after 10 generations of breeding heterozygote 129/Sv/C57BL/6 mice to C57BL/6 mice.  2.2.  E X P R E S S I O N A N A L Y S I S O F T G I F USING IN SITU R N A HYBRIDIZATION  In situ  RNA hybridization is a standard technique used to characterize RNA  expression pattern in wholemount embryos. The method used is briefly outlined below but has been previously described in detail (Belo et al. 1997). First, embryos at the appropriate stages were prepared for cRNA hybridization. Mouse embryos were fixed in 4 % paraformaldehyde and methanol (MeOH). Embryos were then bleached in 6% 40  H 0 :PBT (PBS + 0.1 % Tween-20) for 1 hour at 4°C. Embryos were finally 2  2  permeated by 10 ug/mL proteinase K digestion. Second, cRNA probes incorporated with digoxigenin were prepared. In situ RNA probes were transcribed using linearized template, digoxigenin-labelled nucleotides, and the appropriate RNA polymerase, SP6, T7 or T3, according to the manufacturer's instructions. RNA probes were purified through ProbeQuant G-50 microcolumns (Amersham) to remove unincorporated ribonucleotides. Hybridization took place at 60°C for over 16 hours. Third, excess probe was washed off. Fourth, digoxigenin was localized using antibody conjugated to alkaline phosphatase (Roche) overnight. Fifth, staining using B M Purple (Roche) substrate was used to localize the RNA probes. The expression of Tgif 'was characterized using antisense RNA probes synthesized from EST BF133915 and subcloned fragments and hybridized to embryos and tissues of interest between E6 - E l 2.  2.3.  ANALYSIS OF TGIF M U T A N T M I C E  2.3.1. Determination of the rate of viability and fertility Experimental genotype and sex ratios were compared to the expected Mendelian ratios using the Chi-squared test. Statistical analyses utilizing the Chisquared tests were performed using the Analyze-It plug-in for Microsoft Excel. Fertility of mutant mice was determined by mating mutant mice to heterozygote or wild-type littermates. Transmission of the mutant allele indicated both mutant male and female mice were fertile.  41  2.3.2. Morphological examination of adults Mouse phenotyping was guided by Necropsy of the Mouse by V. Covelli (http://www.eulep.org/Necropsy_of_the_Mouse/index_2004.php). Examination of weight, eye, and tail were carried out in the JAF in a level II biohazard laminar flow hood. A mouse was considered growth retarded if its weight was 20 % less than the mean weight value of its litter. Fisher's exact test was used to determine the significance of observed phenotypes (Table 3.1)(Zar 1984). Adult mice were sacrificed by asphyxiation in CO2 gas. The sidedness and morphology of internal organs including the heart, lungs, liver, spleen, stomach, gut and brain were macroscopically examined and photographed using a Nikon Coolpix 950 digital camera.  2.3.3. Morphological examination of embryos Embryos were collected from natural timed matings of Tgif' mice. Noon on the day of the vaginal plug was designated as 0.5 days post coitum (dpc). Embryos were dissected at E8.0 - E8.5. Morphology of the embryo was visually inspected, with particular emphasis on the forebrain. The surrounding yolk sacs were used to prepare DNA for genotyping.  2.4.  C E L L C Y C L E ANALYSIS OF TGIF M U T A N T MOUSE E M B R Y O N I C FIBROBLASTS  2.4.1. Generation of mouse embryonic fibroblasts Primary mouse embryonic fibroblasts (MEF) were isolated from Tgif^ , /+  Tgif'  and Tgif' ' E l 3.5 embryos obtained by heterozygous matings (Nagy et al. 2003). 1  Briefly, carcasses were first dissected free of limbs, internal organs, and brain, then 42  minced to a slurry, and finally the minced tissues were digested gently to a cell suspension by trypsinization over a 30 minute interval. Cells were subsequently maintained in D M E M supplemented with 10 % fetal bovine serum (FBS) and antibiotics (Stem Cell Technologies). Experiments were carried out only with early passage (<8) cells from Tgif^ and Tgif ' littermates. /+  1  2.4.2. Expression of Tgif in mouse embryonic fibroblasts  Total RNA was isolated from MEFs using TRIZOL® Reagent (Invitrogen) according to the manufacturer's protocol. Reverse transcription was performed using SuperScriptll (Invitrogen) with 1 ug of template RNA. Subsequent PCR reactions were performed using 1 ug of the resulting cDNA as template. The wild-type transcript was amplified with primers TGIF-RT (5'-atgaaaagcaagaagggtct-3') and TGIF 207 (5'tgttcatacagccagtctcg-3').  2.4.3.  Growth curve analysis  Experiments were carried out by comparing cells at identical passage numbers obtained from littermate embryos. In 12-well culture plates, 2 x 10 MEFs of each 4  genotype were plated, fed, and counted on a daily basis using a Coulter Counter. Two experiments were performed using independently derived MEFs.  2.4.4. Cell cycle analysis using B r d U incorporation and flow cytometry  1 x 10 MEFs at log phase were labelled with bromodeoxyuridine (BrdU) and 76  amino-actinomycin D (7AAD) following the manufacturer's instructions (BD Biosciences Pharmingen). BrdU is a halogenated thymidine analogue that is only incorporated into DNA synthesized during S-phase, while 7AAD is a DNA binding dye 43  used to distinguish cells in all phases of the cell cycle. BrdU labelling time was optimized for 45 minutes, the length of time required for all cells in S phase to have incorporated BrdU. FACS results were acquired on a Cytopeia Influx and analyzed using FlowJo flow cytometry software. Statistical analysis of data used single factor A N O V A using the Analyze-It plug-in for Microsoft Excel. This set of experiments was repeated five times, and each replicate examined a different and independently derived group of MEFs.  2.4.5. Proliferation assay by f H]-thymidine incorporation 3  Cell proliferation assays were performed using 5000 cells/well in 96-well plates in triplicate. 1 uCi/well [ H]-thymidine was added to the cells, incubated for 2 hours, 3  harvested, and [ H]-thymidine incorporation quantified using a p-counter (LKB Wallac, 3  Turku, Finland).  2.4.6. Proliferation assay of synchronized mouse embryonic fibroblasts 5000 MEFs per well were grown in D M E M lacking serum for 2 days to induce quiescence (Herrera et al. 1996). Re-addition of 10 % FBS at defined timepoints stimulated synchronous re-entry into Gi. DNA synthesis was then assessed using [ H]3  thymidine incorporation as described. Two experiments were each performed in triplicate, using cells derived from independent MEF lines.  2.5.  ANALYSIS OF IN VIVO PROLIFERATION BY SOMITE STAGING Embryos were collected as described above. Embryos were dissected at E8.0 -  E8.5. Developmental progression was precisely scored by headfold morphology, early  44  or late, or by counting the number of somites, which was typically between 0-10 somites. The surrounding yolk sacs were used to prepare DNA for genotyping.  2.5.1. Analysis of in vivo proliferation by BrdU incorporation Congenic C57BL/6J Tgif" females pregnant with E8.0 - E8.25 embryos received an intraperitoneal injection of 400 ug BrdU per gram weight and were euthanized 1 hour later, as described previously (Hebert et al. 2002). Embryos were collected and staged as described above, fixed in 4 % paraformaldehyde (PFA) overnight and frozen in optimum cutting temperature (OCT) medium. Frozen 10 um sections were stained with anti-BrdU conjugated alexa 549 (Molecular Probes) and Hoechst. The fraction of BrdU incorporated cells was determined by counting the number of positive cells and dividing by the total number of Hoechst positive cells. Only neuroectodermal cells were counted. Numbers were also compared in dorsal 1/3, ventral 1/3 and mid 1/3 sections. At least three adjacent sections were counted, and three sets of matched mutant and control embryos were analyzed.  2.6.  EXPRESSION OF HUMAN TGIF BY RETROVIRUS IN MOUSE EMBRYONIC FIBROBLASTS  2.6.1. Expression of wild-type human TGIF by retrovirus Human TGIF cDNA was subcloned upstream of an IRES-yFP cassette in the murine stem cell virus (MSCV) retroviral vector to generate MSCV-7/G/F-IRES-7FP (Buske et al. 2001). Tgif was flag-tagged at the N-terminus to allow for western blot analyses. High titer helper-free recombinant MSCV-TGIF-IRES-YFP generated and tested according to protocols developed by G. Nolan 45  retroviruses were  (www.stanford.edu/group/nolan). Briefly, Phoenix Eco cell lines were transfected using FuGENE (Roche). Supernatant from the Phoenix viral producer cells supplemented with 6 ug of ammonium sulphate per millilitre was used for infection of the MEFs. YFP-expressing cells were enriched through a Cytopeia Influx sorter, and proliferation was measured by [ H]-thymidine incorporation as described above. 3  2.6.2. Expression of human TGIF containing mutations identified in HPE patients Site-directed mutagenesis using a modified restriction site protocol generated the 7 point mutations previously identified in patients (Tao 1994). All mutations were sequence verified. Patient mutations were generated in MSCV-retroviral constructs, expressed in Tgif ' MEFs, and proliferation was assessed. Five experiments were each 1  performed in sextuplicate using MEFs derived from independent TgiJ* and Tgif'' /+  embryos.  2.6.3. Expression of transduced TGIF protein The level of transduced TGIF protein expression was assessed by standard Western blot analyses of MEF cell extracts and probed with an anti-flag antibody. TGIF generally migrated as a doublet of 30-32 kDa; the upper band likely represents the phosphorylated form (Lo et al. 2001).  2.7.  ANALYSIS OF PROLIFERATION RESPONSE TO TGF-P, RETINOIC ACID AND U0126  46  5000 MEFs per well were grown in the presence of 0.6 pg/mL - 10 ng/mL TGF-P for 24 hours the day after plating. DNA synthesis was measured by [ H]3  thymidine incorporation as described. Fresh 9-cis-RA and all-trans-RA  were added to  5000 MEFs per well on a daily basis for 5 days. [ H]-thymidine incorporation was 3  performed on a daily basis. U0126, an inhibitor of MEK, ranging from 0.01 - 100 uM, was added to 5000 MEFs per well and [ H]-thymidine incorporation was performed 3  after 24 hours of culture. Three experiments were each performed in triplicate using MEFs derived from independent Tgif  +  and Tgif ' embryos. Normalization was 1  conducted by dividing the incorporation value by the maximum incorporation level.  47  3.  CHAPTER 3 GENERATION AND CHARACTERIZATION OF TGIF NULL MICE  3.1.  TGIF EXPRESSION DURING EMBRYOGENESIS  Expression patterns of genes can lead to informed hypotheses for function. Wholemount in situ RNA hybridization is a standard technique used to visualize RNA expression pattern in relation to the anatomy of the mouse embryo. The ease of performing parallel experiments on numerous embryos at various ages, and from different genotypes allows meaningful comparisons within an experiment. Notably, this technique detects dynamic spatial changes in expression during development or changes to expression by genetic modifications. To examine the expression pattern of Tgif, antisense RNA probe synthesized from EST BF133915 was hybridized to embryos or tissues between the ages of E6 E12. Tgif expression at E6, prior to gastrulation, was ubiquitously low throughout the epiblast (Figure 3.1 A). During head fold stages (E8.0-E8.25), Tgif expression was detected in the anterior neuroectoderm at a low and ubiquitous level and at a slightly higher level along the midline of the developing forebrain and the lateral mesoderm exiting the primitive streak near the tail bud (Figure 3.1B,C,E). 7g//"continues to be expressed throughout the forebrain until neural tube closure (Figure 3.1D,E,G). Later during embryogenesis, Tgif expression was also in the hindbrain and branchial arches from E8 - E9.5 (Figure 3.1F,G). Interestingly, the expression of Tgif'was found in proliferating cells of the developing limbs, including the ectodermal layer and the tips of the digits (Figure 3.2). Similar expression patterns were detected using N and C terminal portions of the gene. Thus, the expression pattern of Tgif is consistent with its potential function in the developing forebrain.  4 8  A  Figure 3.1 rgz/expression in the early embryo. (A) Tgif'xs expressed as early as embryonic day 6; a, anterior; p, posteior. (B, C) Respectively, dorsal and ventral views of the developing forebrain at E8.25 demonstrating Tgif is expressed at a low and ubiquitous level, but at a higher level in the midline cells (arrow). Anterior is at the top. (D,E) Dorsal and ventral views of E8.5 embryos with 7g//expression persistent in the neural plate as it folds into the neural tube; note the lack of expression in the heart (h). Lateral views of E8.5 (F) and E9.5 (G) embryos expressing Tgif in the lateral mesoderm (lm) that exited the primitive streak, developing branchial arches (ba) and hindbrain (hb). 49  Figure 3.2 Tgz/expression in the developing limb. (A) Tgif'xs expressed in the overlying ectoderm of the embryonic day 10 limb pad. (B) Expression is later found in the tips of the growing digits of the embryonic day 11 limb pad. (C) Lateral views of the embryonic day 12 limb pad showing expression in proliferative zone on both dorsal and ventral side of the limb pad.  50  3.2.  GENERATION OF TGIF MUTANT MICE Genetic manipulation in mice is one of the most important experimental systems  to study the in vivo function of genes in mammals. Numerous kinds of mutations have been created for thousands of genes to provide unprecedented insights, and the results have made great contributions to our understanding of development and disease (Bier and McGinnis 2003). Thus 1 undertook the generation of mice that lack Tgif.  3.2.1. Isolation of a Tgif genomic clone Multiple BAC clones containing Tgif and the surrounding sequences were identified by screening a 129S6/SvEvTac genomic BAC library. The gene structure and sequence of Tgif'was available through mouse genome sequence databases. Analysis indicated a 7.6 kb EcoRl fragment from BAC 19612 was sufficient for preparing the Tgif targeting construct because it included exons 2 and 3 of Tgif, which contained the majority of the coding sequences (Figure 3.3A) and the necessary flanking sequences for homologous recombination. This genomic fragment was subcloned into a plasmid and verified by restriction mapping.  3.2.2. Construction of Tgif targeting vector To ensure the function of Tgif'was completely disrupted, nearly all of the coding sequences were removed (Hasty 1993). The targeting vector was designed to replace exons 2 and 3 of Tgif'with the reporter gene lacZ fused in frame to the remaining N terminal 7 amino acids (Figure 3.3A). The remaining 7 amino acids are likely not functional. The construct also included a PGK-neo positive selectable marker gene cassette that allowed for enrichment of targeting events. Sufficient sequence for efficient homologous recombination was provided by 3.4 kb of 5' homology region, 51  A EcoRl X h o l Wildtype allele  Pstl SacII Hindlll  EcoRl Pstl  L___L.  L  ,.«  Psti'X'Hindlll  X h o l . x P s t l SacII iillt 1 VI lacZ-»  EcoRl Xhol Targeted allele  L__J  1  \  ?  Targeting vector  EcoRl  PGK-neo  Pstl SacII  Pstl  Hindlll  EcoRl Pstl  I I  ^---h^mmmmmamamdm^ lacZ -»  PGK-neo  5* probe —  B  1 kb  3' probe  Pst 1  EcoR I x\ x\'y x  \ xVy X  x  -13 Kb  «—7.0 Kb  '7 Kb  |H <«—5.5 Kb  Figure 3.3 Gene targeting of the mouse Tgif \ocus. (A) Gene targeting strategy utilizing homologous recombination in ES cells. The wild-type allele, targeting vector and targeted allele are shown with relevant restriction sites and exons (noncoding sequence, open box; coding exons, grey boxes). The dashed lines depict regions of homologous recombination. (B) Southern blot analysis of mouse M E F cells demonstrated correct targeting of the locus. EcoRI-digested genomic D N A generated a 7 kb band for the wild-type allele and a 13 kb band for the targeted allele using the 5' probe indicated in (A), while Pstl-digested genomic D N A generated a 7 kb band for the wild-type allele and a 5.5 kb band for the targeted allele using the indicated 3' probe. Genotypes are indicated above the lanes.  52  and 1.5 kb of 3' homology region (Figure 3.3A). The design of the targeting vector also allowed for subsequent strategies to unambiguously discriminate recombinant clones by Southern blot analysis.  3.2.3.  Screening for clones in which the 7g/yiocus was targeted  Mouse ES cell lines are derived from pre-implantation embryos. When ES cells are injected back into pre-implantation embryos to create a chimera they can remain multipotent and contribute to all tissues (Papaioannou 1993). Significantly, ES cells can be cultured in vitro extensively in an undifferentiated state and therefore allow gene targeting experiments. This characteristic is the cornerstone of genetic manipulation technology in mice. Equally important is the ability of ES cells to contribute to the germline through a chimera, and thus perpetuate mutations produced by gene targeting in ES cells. The targeting vector was linearized and electroporated into ES cells to allow homologous recombination to occur (Wurst 1993). However, foreign DNA is more likely to integrate randomly into the host genome, and the clones in which the desired genetic change (i.e. homologous recombination at the 7g-//locus) occurs need to be identified. Therefore, colonies that survive the positive selection were then clonally isolated and screened. A number of clones survived G418 selection, and 144 clones were selected for screening. Correctly targeted 7_7/clones were identified by Southern blot analysis using Pstl digestion and a 0.3 kb 3' external probe that hybridized to the wild-type 7 kb allele and the targeted 5.5 kb allele (Figure 3.3B). The 5' arm of homology was also examined for correct targeting using a 0.3 kb Xhol external probe that detected the wild-type 7 kb EcoRl fragment and the mutated 13 kb fragment. This screen identified 53  two homologously recombined clones. The frequency of 1/72 is within the normal range of homologous recombination events. A PCR strategy was designed for ear-punch and visceral yolk sac DNA to routinely and rapidly determine the genotype of mice and embryos. The wild-type allele was detected using primers that amplified a 418 bp fragment within exon 2. The mutant allele was detected using primers that amplified a 160 bp PCR product that included the junction between the remaining exon 2 and the fused lacZ gene.  3.2.4. Targeted Tgif allele is a null allele and produced a fused lacZ transcript  To confirm that 7g//'expression was disrupted, RNA was isolated from wildtype and targeted ES cells and assayed using reverse-transcriptase (RT)-PCR (Figure 3.4). The Tgif-lacZ fusion transcript was detected only in heterozygous ES cells demonstrating Tgif'was disrupted as expected.  3.2.5. Production of chimeras and Tgif mutant mice from targeted E S cells  Multiple chimeric mice were generated from the injection of two independentlytargeted ES cell lines, CCE-derived B2 and C6, into the inner cell mass of E3.5 C57BL6 host blastocysts. Two independently targeted cell lines were used to generate chimeras in order to eliminate mutations that may have occurred accidentally in the ES cells during culture, cloning or gene targeting. Germline transmission from two chimeric males was confirmed by genomic blot analysis. The phenotypes observed in mice derived from both ES cell lines were similar and the combined results are presented below. Experiments were performed using mice derived from two chimeric males to eliminate the possibility that any effects seen were due to mutations unrelated to the targeted locus in the ES cells. 54  Wildtype allele TGIF-RT  Targeted allele TGIF-RT jtcacigggac tcgagucca ga^||  iacZ  Exon 1  Intron 1  CCE  Exon 2  CCE CCE  1 kb  B2  C6  ladder +/+  +/-  +/-  Figure 3.4 Targeted ES cells expressed Tgif-lacZ fusion transcript. Above, relative location of PCR primers for wildtype and targeted alleles. Below, RT-PCR result using the indicated primers demonstrated that Tgif-lacZ transcripts (arrow) were expressed in targeted ES cell clones, B2 and C4, but not in the parental C C E cell line. Genotypes are indicated above the lanes.  55  3.3.  ANALYSIS O F TGIF M U T A N T MICE  3.3.1.  Analysis of Tgi/mutant adult mice on mixed 129/Sv/CDl genetic background  A mixed 129/Sv/CDl genetic background colony was first established. This breeding scheme took advantage of the hybrid vigour of FI mice and is the fastest means of producing mutant homozygotes for analysis. The genotype and sex of mice that were generated from heterozygote matings were examined. Although reduced viability in heterozygote mice was anticipated, as has been observed in humans, a X analysis of 80 F2 offspring at 3 weeks of age 2  demonstrated a normal Mendelian ratio [25 Tgif* (31%), 36 Tgif  (45%), 19 Tgif  (24%); p=0.43], demonstrating Tgif was not essential for embryo viability. Fertility was also examined since the loss of achintya and vismay, two Drosophila orthologs, were important during Drosophila spermatogenesis (Mann and Affolter 1998; Ayyar et al. 2003). In contrast, both mutant male and female mice were fertile. Adult mice were examined using a guide prepared by V. Covelli on the website, Necropsy of the Mouse. Attention was paid to the hallmark of human HPE, varying degree of separation of the cerebral hemispheres, and other accompanying characteristics including craniofacial anomalies, developmental delay, seizures, and perinatal and neonatal lethality. The results of these examinations were as follows. Live mice examined externally for the appearance of fur, face, spacing between eyes, nasal opening, teeth and limbs, showed obvious differences between wild-type and mutant mice. Neither activity nor gait was noticeably different. Examination of the structure and size of the brain in mice did not identify any overt abnormalities. The two hemispheres and nasal bulbs were always present and distinctly separate.  56  However additional analysis of 92 Tgif' mice revealed that 19% were growth retarded (Table 3.1; Figure 3.5A). For this analysis, the weight of a mouse had to be 20% less than the mean weight value of its litter to be considered growth retarded. In addition, some mutant mice exhibited kinked tails and eye defects (Figure 3.5 B-E; Table 3.1). Some mutant mice exhibited microcephaly (Figure 3.5F), but the frequency of this phenotype will have to be examined further in the future. None of these defects were observed in control littermates (Table 3.1). Significantly, 13% of Tgif ' mice developed laterality defects, with both situs 1  inversus and situs ambiguus being observed after examining 67 mutant mice (Figure 3.6 A-D; Table 3.1). Situs ambiguus most often involved the incorrect positioning of the heart and occasionally involved abnormal lung lobe numbers (3 cases) or polysplenia (1 case). Cardiac situs was not examined to determine the isomerism class, but together with polysplenia, it is likely situs ambiguus cases were left isomerism. Overall, 43% of 92 mutant mice examined displayed at least one of the defects listed above (Table 3.1). However, each individual defect was observed in only a limited number of homozygous mutant mice, indicating a low penetrance of each defect identified.  3.3.2.  Analysis of the Tgif mutant adult mice with a 129/Sv congenic genetic background  After initial characterization of the effects of Tgif deletion, this genotype was introduced into different inbred genetic backgrounds. The rationale was that the penetrance of the mutation might be higher in an inbred genetic background (LeCouter et al. 1998; Hide et al. 2002). A Tgif-\29Sv congenic strain was thus created from matings between the chimeras and the 129/Sv strain. 57  Table 3.1 Phenotypes resulting from Tgif heterozygote matings on 129Sv/CDl genetic background at 3 weeks of age Genotype +/+ + /-/Phenotype % with defects 0% 0.4% 46% % with no apparent defects 54% 100% 99.6% Growth retardation*f 0/35 0/73 6/31 (19%) 0/124 10/92(11%) Kinked tail 0/243 Situs ambiguus % 0/72 0/145 6/67 (9%) Situs inversus % 0/72 2/67 (3%) 0/145 Eye defect 0/124 1/243 (0.4%) 3/92 (3%) Kinked tail and situs ambiguus 0/72 0/145 1/67(1%) *31 mutant mice were weighed with littermate controls; {67 mutant mice were dissected with littermate controls; 92 mutant mice were physically examined with littermate controls. fA mouse was considered growth retarded if its weight was 20% less than the mean weight value of its litter. # Fisher's exact test  p-value* 0.000 0.000 0.000 0.055 0.027  Figure 3.5 7g/7 mutant mice on the 129/Sv CI) 1 genetic background developed pleiotropic phenotypes. (A) Some mutant mice were already visibly smaller than littermates 1-week after birth. Tgif mutant mice exhibited kinked tails (B.C), eye defects (D,E), or microcephaly (F). Genotypes are indicated.  59  Figure 3.6 Tgif mutant mice on the 129/Sv/CDl genetic background developed laterality defects. Adult mice, placed on their backs, were dissected to determine the sidedness of their visceral organs. The dashed lines indicate the sternum (s) and the midline of the body axis. (A) In Tgif+/+ mice, the heart (h). stomach (st), and spleen (sp) were on the left side of the body cavity. (B) A Tgif-I- mouse exhibiting situs inversus where the heart, stomach, and spleen were located on the right side of the body cavity. (C, D) Tgif -I- mice exhibiting situs ambiguus where the heart lay within the right chest cavity; liver (1); not shown: the stomach and spleen lay on the left side). 60  The genotype and sex of mice derived from heterozygous mating was determined at 3 weeks of age. x analysis demonstrated a normal Mendelian ratio [21 2  Tgif  +  (21 %), 56 Tgif' (57 %), 22 Tgif (22 %); p=0.43], showing mice inheriting  the homozygous Tgif mutation on the 129/Sv genetic background were also viable. Mutant mice were also fertile on this background. 129 substrains are notoriously poor breeders and difficult to maintain. Therefore, only limited analyses were performed after it became apparent that mutant mice were viable and fertile with no overt defects.  3.3.3. Analysis of the Tgif mutant adult mice with a C57BL/6 congenic genetic background  A congenic C57BL/6 Tgif null strain was also derived in an attempt to increase the penetrance of the observed phenotypes (Zhang et al. 2006). This strain was produced by 10 generations of backcrossing of Tgif'" mice onto C57BL/6J mice. Contrary to expectation, Tgif mutations on the C57BL/6 genetic background did not result in an HPE phenotype. A slight reduction in the expected number of mutants was observed, although the number of mice examined was too small to demonstrate a statistically significant difference [22 Tgif (22 %), 59 Tgif (60 %), 17 Tgif (17 +  %); p=0.10]. All mutant mice displayed normal tails and growth. Examination of 26 mutant mice also revealed normal situs of the internal organs. From this result it was concluded that the phenotypes observed were dependent on the genetic background.  3.3.4. Morphological examination of embryos Developing forebrains were examined for developmental progress. Morphological landmarks including narrow forebrain, width and apposition of bilateral 61  structures such as the optic sulci, optic pits, and thickening of the ANR can be examined as early as E8 (4 - 5 somites) (Juriloff et al. 1985; Hentges et al. 1999; Lagutin et al. 2003). Over 20 mutant embryos between E8 - E10 were examined for these landmarks, but significant differences were not detected. However, mutant embryos were often developmentally delayed as early as E8 (see Chapter 4).  3.3.5.  A b n o r m a l laterality determination  Kinked tails are an indication that midline patterning was disrupted during development (Table 1). Additionally, the occurrence of situs inversus and situs ambiguus suggested that early patterning events in the Tgif ' mice may be disrupted. 1  As described earlier, left-right determination and patterning take place early during embryogenesis. After the breaking of symmetry, expression of the TGF-P family member Nodal becomes enhanced on the left side of the node during gastrulation (Hamada et al. 2002). The left-sided Nodal signal is relayed to the left L P M where downstream target genes such as lefty2, pitx2, and nodal itself are activated. The leftsided signals and pathways are barred from the right side by midline mesodermal cells. These molecular determinants precede overt morphological changes. To determine whether abnormal molecular patterning had led to the observed laterality defects, I examined the expression pattern of Nodal, lefty2 and pitx2 in 20 Tgif + / +  and 20 Tgif ' mice (Figure 3.7). Nodal expression was examined in embryos at the 1  early headfold, late headfold, and 0 - 5 somite stages. Iefty2 was examined in embryos at the 3-8 somite stages, while pitx2 was examined in embryos at the 5-12 somite stages. Nodal and lefty2 were normally expressed in all embryos tested for each marker. However, 1 mutant embryo (out of 20) did exhibit bilateral pitx2 expression,  62  wildtype  mutant  Figure 3.7 7g//mutant embryos displayed abnormal left-right patterning. Both wildtype and mutants displayed normal nodal and lefty2 expression patterns, but 1/20 mutant embryos examined showed bilateral pitx2 expression. *, region of right sided pitx2 expression. Dashed lines indicate the midline of the embryo; 1, left; r, right.  63  while all wild-type embryos displayed pitx2 expression only in the left L P M (Figure 3.7). The expression ofpitx2 is present but weaker on the right. The detection of bilateral pitx2 expression in early embryos is consistent with the situs ambiguous phenotype observed in adult mutant mice. The frequency of bilateral pitx2 (5%) was also similar to the frequency of situs ambiguous in the adult (9%). Although the frequencies of situs ambiguous and bilateral pitx2 may appear low, considering that the natural incidence of laterality defects is 1 in 10,000, the incidence is 300 fold higher than expected. Bilateral expression of pitx2 suggests the loss of Tgif may disrupt laterality determination at a molecular level upstream of pitx2 expression. Unfortunately, the low penetrance of this phenotype precluded further studies to determine the exact mechanism by which this occurred.  3.4  SUMMARY  Heterozygous and homozygous deletions at the Tgif locus were generated in mice derived from multiple genetic backgrounds. Overt evidence of HPE was not observed on any of these backgrounds, and all mutant mice were both viable and fertile. Low penetrance, pleiotropic phenotypes were observed only on the 129/Sv/CDl genetic background, suggesting these defects were dependent on the genetic background. Amongst the phenotypes observed, Tgif'' mice were more frequently growth retarded and exhibited evidence of defects in laterality determination. Such altered laterality patterning signals may cause situs inversus and situs ambiguous observed in the adult mice. These results indicate Tgif'may function early during left-right patterning of the developing embryo.  64  4.  CHAPTER 4 ROLE OF TGIF IN CELL CYCLE CONTROL  4.1.  GENERATION OF TGIF MOUSE EMBRYONIC FIBROBLASTS Immortalized cell lines have been used historically for cell cycle studies, but  these cells acquire spontaneous and undefined genetic alterations during the process of immortalization (D'Abaco and Olson 2000). Mammalian cell cycle studies now commonly use primary cell lines, mostly MEFs (Herrera et al. 1996; D'Abaco and Olson 2000; Malumbres et al. 2004). The advantages to using M E F s include the power of genetic analysis, the ability to generate genetically modified mice disrupted for one or multiple genes, and the ease of M E F preparation. Characterization of M E F s derived from mice with a disruption in Rb (Herrera et al. 1996), cell cycle proteins and regulators (Malumbres et al. 2000; Sotillo et al. 2001; Kozar et al. 2004; Malumbres et al. 2004), and many others have provided valuable insights into cell cycle regulation (Ciemerych and Sicinski 2005). Tgif is normally expressed in MEFs. Using reverse transcriptase (RT)-PCR, Tgif transcripts were robustly detected in wild-type and heterozygous M E F s , while no transcript was observed in mutant M E F s (Figure 4.1).  4.2.  CELL CYCLE DEFECTS IN MOUSE EMBRYONIC FIBROBLASTS  4.2.1. Growth curve analysis To address the role of Tgif in cell cycle regulation, M E F s were generated from Tgif and Tgif ' embryos for functional investigation of 7g//" activity. To compare +  1  their proliferative capability, equal numbers of early passage primary M E F s were plated and counted on a daily basis for 12 days. Mutant cells proliferated at a slower rate and accumulated to a lower density compared with control cells (Figure 4.2A).  65  Wi id type allele  TGJF-RT  ,  *  |tggcaaqaqa aqga^gaqaq .  "  ,.aj*u^tLCACjdtt>: • y  i ( ,  "-  r  L : : ; : ^ : ; : ua^r,;.:/:,;^ <•}  , i;cagagoa gaqaaaggais^tqtccca j  Targeted allele T01F-RT j 'caagqr.jac tcgagrtcca gj|tgaa-)-;g r^gcacjqj  Exon 1  RT  4?tc  intron  Exon 2  _+- + - +  160 bp  Figure 4.1  M E F cells normally express Tgif, but mutant cells do not. RT-PCR result  using the indicated primers above demonstrated that Tgif transcripts were expressed in Tgif+/+ and Tgif +/-, but not Tgif-I- MEFs. RT indicates presence (+) or absence (-) of reverse transcriptase in each sample.  66  B 400(H  7-AAD  c  50454035302520151050-  D wildtype  JL  Apoptotic G0/G1  I mutant  G2/M 67  Figure 4.2 Tgif' MEFs exhibited reduced growth due to a delay in GI phase progression. (A) Equal numbers of MEFs were plated and counted daily for 12 days. Tgif ' cells proliferated at a lower rate and accumulated to a lower density than Tgif 1  +/+  cells. Data are pooled from two experiments using independently derived MEFs. (B) Representative FACS profile of cells labelled with BrdU and 7AAD. Apoptotic cells contained little DNA and stained low for 7AAD; cells in Go/Gi phase were negative for BrdU and diploid for DNA content; cells in S phase were BrdU positive and intermediate between diploid and tetraploid DNA content; cells in M phase were negative for BrdU and tetraploid for DNA content. Cells were gated accordingly and their relative percentages to the total number of cells are indicated. (C) FACS analysis of cells labelled with BrdU and 7AAD described above indicated Tgif ' cells 1  accumulated in GO/GI phase and showed delayed entry into S phase. * />=0.003 and * p=0.002 between Tgif* (n=8) and Tgif' (n=8) cells with regard to the percentage of cells in GO/GI and S phase respectively using single factor ANOVA. A representative result is shown from 5 experiments using independently derived MEFs.  68  4.2.2. Cytometric analysis of mouse embryonic fibroblast cell cycle To investigate the cause of the proliferation defect, flow cytometry was used to characterize cell cycle progression. MEFs were labelled with 7AAD, a fluorescent dye that binds stoichiometrically to DNA and thus allows cell in the Go/Gi, S and G2/M phases of the cell cycle to be distinguished (Tlsty et al. 1995; Herrera et al. 1996; Kozar et al. 2004)(Figure 4.2B). However, the precise number of cells in S phase can be difficult to distinguish from those in Gi and G2 phases of the cell cycle. Therefore, BrdU, a thymidine analog, was incorporated into MEFs undergoing DNA synthesis to selectively label cells in S phase. Cells in S phase could then be precisely distinguished by their binding of fluorescently labelled antibodies specific for BrdU (Figure 4.2B). A higher percentage of Tgif ' MEFs was observed in the G0/G1 phase of the cell 1  cycle (36 % Tgif* vs 44 % Tgif; p-value=0.003, n=8; Figure 4.2C); a similarly reduced percentage of Tgif  cells was detected in the S phase (28 % Tgif* vs 20 %  Tgif'; p-value=0.002, n=8). The percentages of cells undergoing apoptosis or in G2/M phase were comparable between Tgif* and Tgif decreased proliferation of Tgif  cells. These results indicate the  MEFs was due to prolonged Gi progression.  4.2.3. Proliferation assay by [ H]-thymidine incorporation 3  To determine whether Tgif  MEFs were impaired in their ability to respond to  mitogens that regulate the restriction point, proliferation was assessed by measuring [ H]-thymidine incorporation into MEF DNA following stimulation of log phase cells 3  with increasing levels of mitogen (0.1% - 10% FBS). The proliferation rate was directly proportional to the concentration of FBS for both Tgif* and Tgif' cells, suggesting Tgif  cells were able to sense increasing levels of mitogen (Figure 4.3A).  69  6.0  A CD  —  5.0 4.0  Xi  3.0 2.0 1.0 0.0  B  2% 3% 4% 5% Serum concentration  \\  •wild type •mutant  7.5% 10%  a  c  wildtype mutant  u  x  5  C  10 15 20 Hours after serum stimulation  5.0 o Cu  U  I  4.0 3.0 2.0 1.0 0.0  Wildtype  Mutant Heterozygote Mutant 129/Sv  Wildtype  Mutant Wildtype C57BL/6J  5.0 o o  4.0 3.0 2.0  U  1.0 0.0  Mutant  D o o  —  MIY  MlY-TGIF  Wildtype 70  MIY  MIY-TG1F  Mutant  Figure 4.3 Cell cycle analysis of proliferation defects in Tgif ' MEFs. (A) Tgif* and 1  Tgif ' cells were cultured in media supplemented with increasing concentrations of 1  serum. DNA synthesis was measured by [ H]-thymidine incorporation for 2 hours. 3  Proliferation rate was directly proportional to the concentration of serum. Mutants consistently proliferated at a slower rate than wild-type controls. (B) Tgif* and Tgif ' 1  cells were growth arrested by serum starvation, then serum was added to stimulate synchronous re-entry into the cell cycle. Fewer mutant MEFs re-entered the cell cycle at all time points. Consequently, the total number of mutant MEFs entering into cell cycle was lower. Representative results from two experiments are shown, each performed in triplicate, using cells derived from independent MEFs. (C) Analysis of proliferation in MEFs derived from congenic 129/Sv and C57BL/6J embryos. MEF lines derived from two independent litters of each genotype are shown. One litter from the congenic 129/Sv genetic background did not include any Tgif* embryos, and Tgif *'~ MEFs were used for analysis instead. Mutant MEFs displayed lower proliferation relative to wild-type and heterozygote controls (p<0.002). Experiments were performed in triplicate using cells derived from independent embryos. (D) Tgif* * and Tgif' ' 1  1  MEFs were infected with MSCV-retroviruses expressing human TGIF and YFP (MIYTGIF) or YFP alone (MIY). Infected cells were enriched by FACS and DNA synthesis was measured by [ H]-thymidine incorporation as described above. Expression of human TGIF restored the proliferation rate of mutant MEFs to normal levels. Expression of human TGIF in wild-type MEFs induced a small but consistent increase in proliferation rate. Data shown here were obtained with MEFs derived from embryos on the 129/Sv/CDl genetic background. Error bars indicate standard deviation. CPM, counts per minute.  71  4.2.4. Proliferation of synchronized mouse embryonic fibroblasts To further characterize the Gi entry of the mutant MEFs, cells were arrested at Go/Gi and allowed to enter S phase synchronously. Serum starvation is an effective method for inducing Go/Gi arrest in a variety of normal and untransformed cell lines, including MEFs (Krek and DeCaprio 1995; Herrera et al. 1996; Kozar et al. 2004; Malumbres et al. 2004). Serum-starved cells are healthy for many days in this quiescent state. The re-addition of high concentrations of serum then induces them to leave the Go state and progress synchronously through the subsequent cell cycle stages. Wild type and mutant cells were fed media supplemented with 10% FBS and DNA synthesis was subsequently assessed using H-thymidine incorporation as described. 3  Approximately two-fold more quiescent Tgif  +/+  MEFs entered S phase relative  to Tgif'' MEFs at the indicated times after serum stimulation (Figure 4.3B). Consequently, the total number of mutant cells that re-entered the cell cycle over time was lower. Taken together, these results indicate that Tgif'' MEFs do not progress as rapidly through G| as Tgif* MEFs, consistent with the results of the FACS analysis. A proliferation defect was also observed in MEFs derived from congenic 129/Sv and C57BL/6J embryos (Figure 4.3C), indicating the loss of Tgif caused a proliferation defect in multiple genetic backgrounds.  4.2.5. Rescue of the proliferation defect by re-expression of human TGIF in mutant mouse embryonic fibroblasts To determine whether the proliferative defect observed in Tgif  MEFs could be  rescued, a cDNA for human TGIF was expressed in mutant MEFs, which were then tested for proliferative capability. Retroviral constructs were generated to express human TGIF cDNAs upstream of an IRES-YFP sequence. The infection efficiency of 72  MEFs was 50-60%, and cells expressing TGIF were enriched by fluorescence activated cell sorting (FACS) for YFP expression. Expression of human TGIF was able to rescue the proliferation defect in mutant MEFs (Figure 4.3D). Over-expression of TGIF in wild-type MEFs resulted in a small, but consistent, increase in proliferation.  4.3.  P R O L I F E R A T I O N D E F E C T S IN V I V O  4.3.1.  Growth retardation was observed in embryonic day 8 embryos  To assess whether proliferation defects were present in vivo, embryos were dissected between E8.0 and E8.25, the stage during which dorsal-ventral patterning is occurring in the forebrain. Embryos were precisely staged with respect to their morphology and somite number. Upon examination of 15 wild-type and 17 mutant embryos from 7 litters, mutant embryos were determined to be developmentally delayed by 2 - 3 somites (Figure 4.4A, 0.001<p<0.002 using the Mann-Whitney test). Consistent with this observation, some neonates and adult mutant mice were growth retarded (Figure 3.5; Table 1). Together, these observations suggest reduced proliferation in the absence of Tgif led to a growth retardation phenotype in vivo as early as E8.  4.3.2. B r d U incorporation rate in embryonic day 8 embryos  Using FACS analysis, it was determined that 28% of wild-type MEFs were in S phase compared to 20%> of mutant MEFs (Figure 4.2C). To determine whether embryonic cells in vivo also showed reduced proliferation, in vivo BrdU incorporation experiments were conducted to compare the proportion of cells in S phase between the wild-type and mutant embryos at E8. The experiments were conducted on E8 embryos because staging using somite number indicated this was an appropriate stage. 73  74  Figure 4.4 Tgif' mutant embryos exhibited growth retardation. (A) Examination of the developmental stage of Tgif  /+  and Tgif'' embryos (n=15 and 17, respectively, from 7  litters) at E8-E8.25 according to their somite numbers. Mutant embryos were determined to be developmentally delayed by 2-3 somites (0.00l<p<0.002 using the Mann-Whitney test). (B, C) In vivo BrdU incorporation rate was similar between wildtype and mutant forebrains at E8.0-E8.25. Embryos labelled with BrdU were used to determine the percentage of cells in S phase in vivo; all nuclei were counter stained with Hoechst. The fraction of BrdU incorporated cells was determined by counting the number of BrdU positive cells (red) and dividing by the total number of Hoechst positive cells (green). At least three adjacent sections were counted, and three sets of matched mutant and control embryos were analyzed. The boxed regions in (B) were enlarged in (C) and spots were placed over positively stained cells to determine the percentage of BrdU positive cells. The embryos shown here were on the C57BL/6J genetic background.  75  Table 4.1 Proliferation index of developing forebrain WILDTYPE BrdU Hoechst % avg std dev  MUTANT BrdU Hoechst %  avg  std dev  set 1*  22 33 44  48 60 66  46 55 67  56  10.54  58 64 55  98 77 91  59 83 60  67  13.58  set 2  52 61 51  74 72 78  70 86 65  74  10.97  101 83 36  106 112 42  100 74 86  87  13.01  set 3  31 58 63  36 80 89  86 73 71  77  8.14  88 79 71  132 108 95  67 73 75  72  4.16  * sets indicate embryos matched for similar developmental stage progression  Littermate embryos at the same somite stage were carefully matched, and 10 um frontal sections spanning the developing forebrain, midbrain and hindbrain were compared. The proportion of BrdU positive cells within the dorsal, mid and ventral regions of the neural tube was also compared. The sensitivity of this technique was approximately 510% since the proportion BrdU positive cells in adjacent sections generally deviated within this range. A significant difference was not detected in the proportion of cells that incorporated BrdU between wild-type and mutant forebrains (Figure 4.4B, C, Table 4.1). The sensitivity of in vivo BrdU incorporation may be too low to detect the small difference in proliferation predicted from the in vitro MEF assays. Alternatively, it is also possible that in vivo proliferation was not significantly different between wild-type and mutant embryos at the developmental stage examined, as cell cycle is normally tightly regulated during in vivo development. Consistent with these observations, the E8 embryo or telencephalon would be predicted to be significantly smaller if the reduction in proliferation observed in MEFs also affected most embryonic tissues, or the entire telencephalon.  4.4.  ASSAYING T H E PROLIFERATIVE FUNCTION OF H U M A N TGIF MUTATIONS IN MOUSE E M B R Y O N I C FIBROBLASTS To test whether mutations identified in HPE patients (Figure 4.5A) (Gripp et al.  2000; Chen et al. 2002; Aguilella et al. 2003) were able to rescue the proliferation defect, TGIF point mutations were re-created in the retroviral constructs, expressed in Tgif ' MEFs, and proliferation assessed. Not surprisingly, the Y59X mutation did not 1  rescue the proliferative defect, presumably because the protein pre-terminated within the homeodomain (Figure 4.5B). Notably, the other point mutations within the 77  H P E Mutations  272 CtBP Binding Domain  Homeodomain  Smad2/3 & HDAC Interaction Domain  78  Repressive Domain & Sin3 Interaction Domain ( E G F / M A P K phosphorylation)  Figure 4.5 Expression of a subset of mutated human TGIF rescued the proliferative defect of MEFs. (A) Diagram of HPE patient mutations, and their positions relative to the functional domains of TGIF, which were generated in MSCV-retroviral constructs. (B) Tgif ' MEFs were infected with MSCV-retroviruses expressing human TGIF and 1  YFP (MYY-TGIF), YFP alone (MIY) or human TGIF containing the patient mutations from (A). Infected cells were enriched by FACS and DNA synthesis was measured by [ H]thymidine incorporation as described. Expression of human TGIF restored the 3  proliferation rate of mutant MEFs to normal levels. Mutations affecting the homeodomain were unable to rescue the proliferative defect. Representative results from 5 experiments are shown; each experiment was performed in sextuplicate using cells derived from independent Tgif* and Tgif ' embryos. Error bars represent 1  standard deviation of the mean. * indicates p<0.001 between wildtype and mutant TGIF transduction; § indicates p<0.001 between MIY and mutant TGIF transduction using Student's t-test. Cpm, counts per minute. (C) Tgif'~ MEFs were infected with MSCVretroviral constructs containing flag-tagged TGIF as described. Western blot analysis of MEF cell extracts showed similar expression levels for all mutant proteins. TGIF generally migrated as a doublet of 30-32 kDa; the upper band likely represents the phosphorylated form.  79  homeodomain, P63R and R90C, were also unable to compensate for the loss of Tgif. In contrast, other mutations, S28C, Q107L, T151A, and T162F, which are located within the CtBP and SMAD2 and HDAC interaction domains, rescued the proliferative defect in mutant MEFs. These results confirm a proliferation function for Tgif that requires the homeodomain. During construction of the retroviral expression construct, Tgif was flag-tagged at the N-terminus to allow for western blot analyses. The level of TGIF protein expression for all mutant proteins was assessed using MEF cell extracts and standard Western blot using antibody to the flag epitope. The level of protein expression was similar between wild-type and mutant TGIFs, with the exception of Y59X, which did not produce a detectable protein at the expected size (Figure 4.5C). This suggests the ability of each mutant protein to rescue the proliferative defect is likely due to the modifications to their activity resulting from the point mutations.  4.5.  PROLIFERATION RESPONSE TO SIGNALLING PATHWAYS  4.5.1. Proliferation in response to TGF-P TGF-P is a cytokine known to exhibit potent anti-proliferative activity (Massague et al. 2000). As discussed earlier, Tgif'was previously shown to repress the transcriptional response of the TGF-P pathways; the relative level of Tgif incorporated into Smad protein complexes can regulates the level of downstream target gene expression (Figure 1.6) (Bertolino et al. 1995; Wotton et al. 1999a). Given the role of Tgifin regulating TGF-P signalling, I hypothesized that Tg/J-dependent regulation of downstream genes is responsible for enabling TGF-P signalling to inhibit proliferation. In that case, mutant MEFs lacking Tgif would be more sensitive to the anti-proliferative effect of TGF-p. To investigate this hypothesis, the rate of [ H]-thymidine 3  80  incorporation into MEFs was assessed after treatment with TGF-p\ Tgif* * MEFs 1  exhibited a typical inhibition of proliferation in response to TGF-P treatment: growth inhibition was detected at TGF-P concentrations as low as 0.08 ng/mL, or 3 pM, and reached a maximum 2-fold inhibition at 0.6 ng/mL, or 25 pM (Figure 4.6A) (Sirard et al. 2000). Tgif ' MEFs proliferated more slowly compared with Tgif* * MEFs in 1  1  response to increasing concentrations of TGF-p. Since Tgif ' cells normally 1  proliferated at a lower rate, the level of [ H]-thymidine uptake was normalized to the 3  maximum incorporation level. Through this transformation it became apparent that the degree of growth inhibition between Tgif* * and Tgif ' MEFs was similar at similar 1  1  concentrations of TGF-fi (Figure 4.6B). This result indicates Tgif does not regulate proliferation by modulating TGF-P response genes in MEFs.  4.5.2.  Proliferation in response to retinoic acid signaling  Similar to TGF-P, RA also functions to arrest proliferation (Goyette et al. 2000). Additionally, TGIF was reported to compete for DNA binding with the RXRa retinoic acid receptor (Bertolino et al. 1995). Therefore, I hypothesized the expression of Tgif may play a role in regulating RA-mediated transcription of downstream cell cycle genes. It would then be predicted that the rate of proliferation in MEFs lacking Tgif would mimic exposure to increased RA. To investigate this possibility, growth arrest in response to treatment with 9-cis RA and aW-trans-KA in MEFs was assessed. Similar to keratinocytes, treatment of Tgif* * MEFs with 10" M 9-cis-RA caused a reduction 1  12  in DNA synthesis, with a maximal effect seen at 10" M , which caused an 80% 6  reduction in proliferation (Goyette et al. 2000). A\l-trans-RA  was also effective in  inhibiting proliferation with a maximal effect at 10" M , that resulted in a 70%> 6  reduction in proliferation. When normalized, these dose-response experiments again 81  -wildtype -mutant  TGFP (na/mL) TG1 P (pM) ::  -wildtype - mutant  TGFp (ng/mL) TGFP (pM)  -o- wildtype -B-mutant  -12 -11 -10 -9 -8 Log cis-RA (M)  -7  1.2  s  o  ft. >• 03  1.0 0.8 -  -wildtype -mutant  0.6 0.4 0.2 0.0 -  -12 -11 -10 -9 -8 -7 Log all-trans RA (M)  1.0  u  0.8  KJ i  0.6  -wildtype - mutant  0.4 0.2 0.0 0.01  0.1  1  U0126 (uM) 82  10  100  Figure 4.6 Analysis of MEF proliferation in response to TGF(3, 9-cis RA, all-rrara-RA and U0126. (A) Primary Tgif* and Tgif ' MEF cells were grown in the presence of 0.6 1  pg/mL-10 ng/mL TGF-P for 24 hours and DNA synthesis was measured by [ H]thymidine incorporation as described. Mutant MEFs displayed lower proliferation at all concentrations of TGF-P relative to wild-type MEFs. (B) Results from (A) were normalized to the maximum value of each genotype since mutant MEFs consistently showed reduced proliferation. The proliferation inhibitory response was similar between wild-type and mutant MEFs at all concentrations of TGFp. (C-D) Fresh 9-cisRA (C) and al\-trans-RA (D) were added to MEFs on a daily basis for 5 days and [ H]thymidine incorporation was performed on day 6. Results were normalized as in 3  (B). The proliferative inhibitory response to 9-cis-RA and all-/rara'-RA was similar between wild-type and mutant MEFs. (E) 0.01-100 uM U0126 was added to MEFs and [ H]thymidine incorporation was performed after 24 hours of culture. The proliferative inhibitory response to U0126 was similar between wild-type and mutant MEFs. A l l results are from representative experiments. For each, three experiments were performed in triplicate using cells derived from independent Tgif* and Tgif embryos. Error bars represent standard deviation of the mean. Cpm, counts per minute.  83  showed similar growth arrest responses between Tgif* * and Tgif ' MEFs (Figure 1  1  4.6C,D). Therefore, and contrary to expectation, Tgif does not appear to regulate proliferation in MEFs by modulating the transcriptional levels of cell cycle genes downstream of the RA pathway.  4.5.3. Proliferation in response to R a s / M A P K inhibitor, U0126 The Ras/MAP Kinase pathway is activated downstream of many mitogenic factors, such as the Fgf (fibroblast growth factor) family (Lodish et al. 2000). TGIF protein can be stabilized by phosphorylation of two sites near its C-terminus via the Ras/MAP kinase pathway (Lo et al. 2001). It is possible that the Ras/MAP Kinase pathway may modulate Tgif s stability, and hence its activity in regulating proliferation. To address this possibility, U0126, an inhibitor of the kinase activity of M E K in the Ras/MAP Kinase pathway, was used to inhibit the phosphorylation and stability of Tgif (Lo et al. 2001). The reduction of Tgif activity by U0126 would be predicted to reduce proliferation in wild-type cells, and therefore this treatment would mimic mutant MEFs lacking Tgif. U0126 inhibited cell proliferation at 0.01 uM and almost completely inhibited cell cycling at 100 uM. However, the proliferative response to U0126 treatment or Ras/MAP kinase inhibition in Tgif* * and Tgif ' MEFs 1  1  did not differ significantly (Figure 4.6E). Therefore, TGIF protein phosphorylation and stability, as regulated by the Ras/MAP kinase pathway, did not have a significant function in regulating proliferation in MEFs. Taken together, the pathways known to be modulated by Tgif did not show an increased sensitivity to ligand in Tgif'' cells, and the mutant MEFs displayed growth inhibition responses that were unaltered relative to Tgif*'* cells.  84  4.7  SUMMARY M E F s derived from Tgif ' cells consistently proliferated slower and 1  accumulated to a lower density than did wild-type cells. This decreased proliferation was shown to result from reduced Gi/S progression of the cell cycle. Additionally, mutant embryos exhibited a developmental delay consistent with the observation that 19% of mutant adults were growth retarded. However, in vivo proliferation within the developing neuroectoderm was normal in the mutants at E8. Re-expression of wild-type TGIF in M E F s rescued this proliferative defect, confirming that the loss of Tgif'was critical in regulating progression through the cell cycle. However, TGIF proteins containing mutations in the homeodomain were unable to rescue this phenotype, indicating the homeodomain is necessary for Tgif to function during cell cycle regulation. Finally, the slow proliferation of mutant M E F s in standard culture conditions was not the result of enhanced sensitivity to TGF-P or R A , or reduced responsiveness to M A P K signalling.  85  5.  C H A P T E R 5 DISCUSSION  5.1.  M U T A N T M I C E DID N O T E X H I B I T H P E , B U T DID E X H I B I T H P E RELATED DEFECTS  Medical genetics has provided abundant and significant insights into normal development and disease progression (Epstein et al. 2003). One recent and notable study associated the SLITRK1 gene with Tourette's Syndrome. This study is providing insights into and understanding of neuronal growth, guidance and branching, as well as generating hypotheses for the etiology of this disease (Abelson et al. 2005). Similarly, this study of Tgif, a gene mutated in some HPE patients, has provided important insights and generated hypotheses for future studies of forebrain development and the etiology of HPE. Deletion, nonsense and missense mutations in TGIF have been identified in a subset of HPE patients (Overhauser et al. 1995; Gripp et al. 2000; Chen et al. 2002; Aguilella et al. 2003; Bendavid et al. 2005a; Bendavid et al. 2005b). Significantly, HPE can develop in patients carrying heterozygous mutations in TGIF, suggesting the processes being disrupted are exquisitely sensitive to the level of TGIF. Paradoxically, most carriers do not develop HPE, indicating a low level of penetrance for this disorder, and that the manifestation of the phenotype may be dependent on genetic modifiers (Nanni et al. 1999; Aguilella et al. 2003). Here, I showed that mice lacking Tgif were viable and fertile without overt evidence of an HPE phenotype. However, some mutant mice did develop multiple defects including laterality and eye defects, growth retardation and kinked tails, suggesting that Tgif 'may be involved in many processes during development. As of May 2006, the characterization of Tgif mutant mice has been reported independently by three other groups, and all indicating a lack of a HPE phenotype in the mutant mice 86  (Shen and Walsh 2005; Bartholin et al. 2006; Jin et al. 2006). Furthermore, those groups did not report microcephaly, eye defects, kinked tails, or laterality defects with mutant mice on C57BL/6 and 129 genetic backgrounds.  5.1.1. Lack of HPE phenotype Mice generated through loss-of-function experiments often do not develop the expected phenotypes, and there are many explanations. One unexplained phenomenon in mammalian genetics is that the corresponding mutation in mice often results in a much stronger phenotype in humans (Bier and McGinnis 2003). A classic example is that heterozygous mutations in SHH result in HPE in humans, but comparable abnormalities are only observed in homozygous Shh mice (Chiang et al. 1996). Additional examples include Ptch and FoxHl (Goodrich et al. 1997; Hoodless et al. 2001). Likewise, heterozygous mutations in TGIF are found in patients, but homozygous mutant mice described here developed only microforms, or associated defects, of HPE. The basis for these differences in mouse and human CNS development are beginning to be explored (Li et al. 2005). The loss of Tgif cm potentially be compensated by rgz/paralogues. In mice, three rgz/paralogues have been identified: Tgif2, Tex, and Tgiflx/y (Melhuish et al. 2001; Bianco-Arias et al. 2002; Lai et al. 2002). Tgif has been shown to possess many similar biochemical properties as Tgif, and its expression has also been detected in the developing nervous system (Jin et al. 2005). Tex and Tgiflx/y are also predicted to contain many of the same functional domains as Tgif. In order to address the potential roles of these paralogues, loss-of-function mice for these genes could be generated and bred with Tgif " mice to evaluate the potential redundancy amongst these genes. 1  87  As discussed earlier, the penetrance for heterozygous TGIF mutations for HPE is only 10% in humans; the penetrance of mutations in the other HPE genes is similarly incomplete (Dubourg et al. 2004). In this study I examined 80 adult mice: 25 were Tgif + / +  (31%), 36 were Tgif* ' (45%), and 19 were Tgif ' (24%). It is possible that some 1  1  Tgif mutants developed HPE at low penetrance and died undetected as dams are less likely to nurture their abnormal neonates than are humans. Consistent with this possibility, a significant number of neonates can be lost (greater than 50%, or 15 mutant mice in this example) and not be detected statistically at a significance level of 95%). In order to detect a decrease in viability perinatally, pups could be examined for HPE before they are born. Another possibility is that the dissection methods used in this study were insufficiently sensitive to detect mild HPE cases since forebrain defects in many human HPE patients can be subtle and only be detected by MRI (Takahashi et al. 2004). A detailed histological study could address this possibility. Detailed histology was conducted by another group, and while HPE was not detected, the mice examined in that study were on the C57BL/6J genetic background (Shen and Walsh 2005). background  The most likely explanation for a lack of HPE defect in mice is the influence of genetic background. The degree to which the function of other genes and alleles contribute to a given phenotype is complex (Nadeau 2003). For example, one variant of TGF-fil in patients caused severe impairment and lung disease relative to patients with different alleles that exhibited only mild impairment (Drumrn et al. 2005). Likewise, Tgif function in humans is possibly influenced by genetic background as evidenced by HPE mutations that exhibit variable expressivity and a low degree of 88  correlation between genotype and phenotype. Furthermore, sometimes carriers of the same mutation in the same family develop a wide spectrum of defects, ranging from no defect to severe HPE (Aguilella et al. 2003). However, twin studies that can better separate the effects of genetic and environmental influences have not yet been performed. Multigenic mutation hypotheses have been proposed for HPE and other multifactorial diseases. It is thought that forebrain development and other biological processes have evolved to be regulated by redundant mechanisms and that disruptions to more than one gene or pathways would disturb the developmental program sufficiently to cause abnormalities. This hypothesis was proposed when multiple HPE patients including two patients from different families that carry mutations in both Shh and TGIF were identified (Ming and Muenke 2002). Interestingly, TGIF is located only 5 Mb from TWISTED (TSG),  GASTRULATION  a member of the BMP pathway; BMP plays a role in regulating dorsal-ventral  patterning of the forebrain (Petryk et al. 2005). Recently, many patients were identified with sub-karyotypic microdeletions that encompass TGIF (Bendavid et al. 2005a; Bendavid et al. 2005b). Patients with microdeletions or large rearrangements over the HPE4 region that affected both TGIF and TSG function may develop more severe symptoms compared to patients with smaller rearrangements that only affected TGIF. Studying the correlation between the severity of HPE symptoms and the extent of chromosomal rearrangements in patients may shed light on the effect of disrupting one or both genes. The influence of genetic background in mice has long been recognized, but the prohibitive costs of mouse genetics research likely precluded further investigations (Bonyadi et al. 1997; LeCouter et al. 1998; Nadeau 2003). Both amino acid 89  polymorphisms and regulatory variations are known to cause variations in the temporal or spatial patterns, and absolute level of expression in different inbred mouse strains (Cowles et al. 2002). The loss of Cdo, a co-receptor for Shh, caused HPE only on selected genetic backgrounds exemplifies this phenomenon (Zhang et al. 2006). Tgif ' mice generated from this study displayed a variety of low penetrant 1  defects. In contrast, three other groups did not report these findings. Tgif'is a simple, 3 exon-containing gene encompassed within 7 kb of genomic DNA. The targeting constructs from all four groups were similarly designed to remove the majority of coding sequences with only minor differences. I discovered the loss of Tgif resulted in pleiotropic defects only on the 129/Sv/CDl background, while mutants on the C57BL/6J and 129/Sv backgrounds did not develop any of the defects. The other three groups did not report the phenotypes I observed, but they only analyzed mutant mice on the C57BL/6J and 129/Sv/C57BL/6J genetic backgrounds (Shen and Walsh 2005; Bartholin et al. 2006; Jin et al. 2006). I also examined a large number of mice, over 450, of which 92 were homozygous mutant, and detected phenotypes that occurred with low penetrance. For example, thorough analysis of the weight of 139 mice revealed 19% of mutant mice displayed a growth retardation phenotype relative to littermate controls. Interestingly, this result is in agreement with the decreased, but not statistically significant, weights reported by one study that did examine the weight of mice (Shen and Walsh 2005). This result was likely masked when the weights of 54 mice were averaged. The precise mechanism by which genetic background can influence manifestation of diseases such as HPE can vary. However, haploinsufficient mutations in patients suggest a reduction in the level of Tgif, perhaps below a threshold level, in  90  the developing forebrain is sufficient to cause HPE development in a sensitive genetic background (Nadeau 2001).  5.1.2. HPE-related defects 7_-*ymutant mice developed laterality and eye defects and kinked tails, as well as growth retardation. Laterality defects are often associated with HPE and likely arose from disruptions to a common developmental process (Roessler and Muenke 2001; Cohen 2003). The forebrain is in part patterned by the ventral midline and the prechordal plate, which instructs neural plate cells of their midline and lateral positions. The same midline cells coordinate and instruct the surrounding mesodermal cells of their position relative to the left-right axis. Consistent with this idea, disruptions to the signalling pathways that transduce these cues lead to both laterality defects and HPE (Roessler and Muenke 2001). Additionally, kinked tails are also indicative of midline defects (Purandare et al. 2002). Finally, eye defects and growth retardation are often exhibited by HPE patients (Cohen 2003). An additional line of evidence from studies conducted in chick spinal cord suggests Tgif may play a role during forebrain patterning. Tgif expression was detected in the dorsal medial spinal cord, and a series of overexpression experiments indicated Tgif repressed transcription of dorsal expressing genes that regulate the dorsoventral identity of neurons (Knepper et al. 2006). Consistent with this hypothesis, abnormal patterning of the forebrain dorsoventral axis can cause HPE. One caveat associated with overexpression studies is the possibility that ectopic expression can cause artifacts by competing for binding factors and disrupting protein complexes. For instance, since Tgif normally interacts with a multitude of proteins, such as p300, high levels of Tgif may quench and disrupt such protein complexes. Consequently, it is important to 91  verify that the same genes shown to be regulated by 7g//in overexpression studies are also misregulated in loss-of-function animals, such as the Tgif ' mice. Analysis of 1  several forebrain and ventral markers was conducted in this study, as well as by another group, but differences in the expression pattern of these ventral markers were not detected (Jin et al. 2006).  5.1.3. Future Directions To further address the functions of Tgif during forebrain development I propose three genetic strategies in an attempt to uncover an HPE phenotype in Tgif mutant mice. Genetic strategies are being proposed because Tgif ' mice are viable and fertile and can 1  thus be used strategically. This is a realistic and promising objective, since patients with rgj/mutations develop HPE, and HPE can develop in mice. Developing an additional HPE mouse model would be a valuable reagent that could provide insights into the mechanism of forebrain patterning. My first proposal is to identify an inbred genetic background in which Tgif mutant mice develop HPE. Since mutant mice on the 129/Sv/CDl background developed HPE related defects, and CD1 outbred mice were originally derived from Swiss mice, Swiss inbred mice may be a good candidate inbred strain (Chia et al. 2005). A variety of other inbred strains are also available from the Jackson Lab, such as the widely available and studied Balb/cJ, FBV, CBA, and C3H inbred strains. For example, on the C57BL/6J genetic background, the retinoblastoma pi 30 null mice were viable and fertile, but a plethora of functions were revealed in the Balb/cJ genetic background. If this strategy was successful, markers for progressive steps during forebrain patterning would be used to determine the precise function of Tgif during HPE development. In addition, modifier genes in the affected strain could also be 92  identified using QTL analysis. Identification of modifier genes may also be informative since they may play a role in regulating or interacting with Tgif during forebrain patterning (Neumann et al. 1994; Bonyadi et al. 1997). Finally, penetrance does not have to be 100% to identify such genetic modifiers. The major challenge in this experiment would be the expense of housing multiple lines of mice. However, the advantage of this experiment is the simplicity of the procedure. The second strategy is to generate compound mutants. This strategy is traditionally used by geneticists and has proven successful for many genes. A few notable examples include Smad2 ';Nodat ', Smad2 ~;Smad3 ~', Cerl" '\leftyV ', and gsc +,  /  +l  +l  l  l  '';HNF3B ' double mutant mice (Filosa et al. 1997; Nomura and L i 1998; Perea-Gomez +/  et al. 2002; Dunn et al. 2004). The reason for this success is that multiple genetic lesions can disrupt functional redundancy amongst genes and pathways that would otherwise compensate and mask the effects of single mutations. Consequently, digenic and multigenic hypotheses were proposed for many genetic diseases, including HPE (Ming and Muenke 2002). Candidate mutations to introduce with the Tgif mutation include genes in the Nodal, RA, BMP, Shh, Fgf and Wnt pathways. For example, Shh mutant mice are an excellent candidate since two patients carried mutations in both Shh and Tgif (Ming and Muenke 2002). Alternatively, Zic2 mutant mice is another candidate since Zic2 is an important HPE gene that regulates Nodal signalling. Since many mutants in these pathways are available, this strategy is feasible. The advantages and disadvantages are similar to the last strategy. The third strategy is to identify mutants using an ENU-mutagenesis screen (Cordes 2005). ENU-mutagenesis is a method used to create high rates of point mutations that can saturate the mouse genome. While the second strategy requires the assessment of a good hypothesis and the selection of a likely candidate, a mutagenesis 93  screen identifies candidates based on function. With a clearly defined phenotypic screening strategy, E N U mutagenesis screens have been used to successfully identify novel genes and alleles of known genes that have provided valuable insights into developmental mechanisms (Anderson 2000). If Tgif ' mice are sensitized to 1  developing forebrain defects, the introduction of an additional mutation may be sufficient to further disrupt and allow HPE to develop. Identification of such genes may give insights into Tgif function and forebrain patterning. On this note, many genes that cause H P E are not yet known. Only 20% of non-syndromic, chromosomallynormal H P E patients have known mutations. A screen may identify candidate H P E genes and genes previously not suspected to be involved in forebrain patterning.  5.2.  M U T A N T M I C E EXHIBITED L A T E R A L I T Y D E F E C T S Events determining left-right patterning take place early during embryogenesis  before the onset of organogenesis.  Soon after the breaking of symmetry, expression of  the T G F - P family member Nodal becomes enhanced on the left side of the node during gastrulation (Hamada et al. 2002). The left-sided Nodal signal is relayed to the left L P M , where downstream target genes such as lefty 1, lefty2 and pitx2 are activated. The left-sided signals and pathways are barred from the right side by midline mesodermal cells. Laterality defects were discovered in 13%> of mutant mice, with 2 mice showing complete situs inversus, indicating that Tg/Jplays a role in laterality determination. Expression analysis by in situ hybridization revealed that while changes to Nodal and lefty2 expression were not detected, 1 out of 20 mutant embryos did exhibit bilateral pitx2 expression. Bilateral pitx2 expression suggests the loss of Tg//may disrupt laterality at a molecular level upstream of pitx2. The low penetrance of this defect 94  impeded further studies, but this result does suggest TGIF is a novel candidate gene for laterality disorders in humans. Laterality mutants can be classified based on the nature of the disruption to the laterality pathway and the type of situs defect that results. Situs inversus is caused by disruptions to the early symmetry breaking event. The classic mutant that is disrupted during this process is situs inversus mice. At the molecular level, the defect is likely a failure in generating a nodal flow. This defect leads to the right-sided expression of genes normally expressed on the left side. In contrast, situs ambiguus phenotypes are caused by perturbations to relatively later events when laterality information is transduced inappropriately, causing laterality defects to each organ independently, or else loss-of-midline defects. The consequences of such defects include bilateral, or absence of, expression of genes normally expressed only on the left side. One example of such a defect is mutants in the Nodal signal transduction pathway. The varieties of laterality defects, both situs inversus and situs ambiguus, in Tgif  ~~ ' mice is unusual and intriguing because this suggests Tgif may function during multiple stages of laterality determination. To my knowledge, only one mutant, Zic3, exhibits both situs inversus and situs ambiguus (Purandare et al. 2002). Zic3 regulates  Nodal signalling, consistent with the fact that Zic3 mutants initiate Nodal expression normally but fail to maintain it. Therefore, in Zic3 mutants, NodaVs function is disrupted not only during early symmetry breaking, but also later when the leftness signal is propagated to the organ primordia. It is relevant to note here that mutations in Zic2, a paralogue of Zic3, are found in HPE patients. In addition, situs amgiguus in rgz/mutants may originate from abnormal midline development, an idea that is supported by the kinked tails phenotype (Purandare et al. 2002). An example of a midline mutant is Shh (Meyers and Martin 95  1999). Midline mutants initiate the breaking of symmetry at the node normally, and transfer asymmetric expression from the node to the L P M correctly. However, defects first become apparent when asymmetric expression and differentiation is not maintained when the midline structures are disrupted. Such defects are typically examined using midline markers such as shh, hrachyury, HNF3B and lefty 1. shh was used to examine the midline in Tgif mutant embryos, but expression was not different from control embryos.  5.2.1. Molecular basis of laterality defects  Loss of Tgif'may cause laterality defects through inappropriate modulation of the Nodal and RA signalling pathways. TGIF has been shown to repress the level of TGF-P-activated transcription through interaction with SMAD2 and SMAD3. Since Smad2 and Smad3 mediate Nodal signalling, the loss of Tgif is predicted to increase the sensitivity of cells to the level of Nodal (Wotton et al. 1999a). Therefore, ectopic Nodal response may lead to inappropriate symmetry breaking in the node in Tgif mutants. Later, ectopic Nodal response may also become propagated to both the left and right sides of the embryo. I examined expression of Nodal by in situ hybridization but did not detect differences in its expression pattern, but it is possible that in situ results were not sufficiently sensitive to detect changes in Nodal expression level. A more sensitive method would be to use the Nodal-lacZ transgenic reporter that can detect subtle differences in Nodal levels (Meyers and Martin 1999; Brennan et al. 2001). Alternatively, given the low penetrance of this phenotype it may be necessary to examine a greater number of embryos in order to characterize the molecular defect. Nevertheless, an absence of changes to Nodal expression level is not unexpected, since Nodal expression is carefully regulated by multiple redundant mechanisms (Meno et al. 96  1997; King and Brown 1999; Constam and Robertson 2000b; Constam and Robertson 2000a). A second potential molecular mechanism by which Tgif'may disrupt laterality determination is its ability to modulate RA signalling. Recent reports demonstrated that RA and Shh are involved in breaking of the left-right symmetry (Tanaka et al. 2005; Vermot and Pourquie 2005). Since TGIF has been shown to compete, and thus inhibit, RA signalling and repress RA responsive transcription, the loss of Tgif may disrupt this step of the laterality determination pathway (Bertolino et al. 1995; Bertolino et al. 1996; Bartholin et al. 2006). The nodal flow model predicts the left-ward transport of morphogen proteins such as Shh and RA (Tanaka et al. 2005). It is conceivable that the loss of 7gz/may sensitize cells to low levels of RA on the right side and result in subsequent activation of Nodal expression on both sides. 'Situs amgiguus in Tgif " mice may also originate from abnormal midline 1  development. This possibility is supported by the appearance of kinked tails in these mice (Purandare et al. 2002), and bilateral pitx2 expression. Consistent with this, Shh and RA signalling have been shown to regulate lefty I expression in the left side of prospective floor plate of neural tube (Tsukui et al. 1999). The function of lefty I in the midline is crucial for restricting Nodal to the left L P M (Meno et al. 1998). Tgif 'is expressed in the forebrain midline and may modulate lefty 1 expression. Alternatively, Tgif 'may play a direct role in midline integrity.  5.2.2. Future directions The challenge to further dissecting the mechanism of the laterality defect is the low penetrance of this phenotype. To address this issue, I would apply a similar strategy to what was outlined above. Perhaps the 7g7/mutation will contribute to a 97  higher penetrance of situs defects in other genetic backgrounds, in combination with other laterality mutants, or in combination with novel mutations that can be generated by ENU mutagenesis. If successful, marker analysis can better define the temporalspatial disruption caused by the loss of Tgifs function during both laterality and forebrain development.  5.3.  R O L E OF TGIF DURING C E L L C Y C L E C O N T R O L This study provides the first evidence of a proliferative function for Tgif in  mammalian cells. MEF cell lines lacking Tg/Jproliferated at a slower rate and did not reach the same density as normal controls. Further cell cycle analysis revealed that mutant MEFs were defective in Gi/S progression, and that re-expression of human TGIF rescued this proliferation defect. Consistent with observations made in this study, the yeast protein Tos8, a TALE homeodomain transcription factor related to TGIF, participates in G|/S transition events (Horak et al. 2002). Furthermore, expression of human TGIF (CPR1) restored cell cycle progression by overcoming mating pheromone-induced Gi arrest in yeast (Edwards et al. 1997). Also of relevance, the Drosophila  TGIF orthologues achintya and vismay were shown to activate  downstream target genes whose functions were necessary during spermatogenesis and meiosis (Ayyar et al. 2003; Wang and Mann 2003). This suggests that a role for Tgif in the regulation of the cell cycle may be conserved in eukaryotes from yeast to mammals. Accordingly, the expression patterns of Tgif support its role in proliferation. In the mouse embryo 7g//expression was detected in proliferating cell populations, including those within the forebrain, branchial arches, lung, limb buds, and tongue (Bertolino et al. 1996). Tgif expression is also widespread in the developing central nervous system undergoing dramatic growth and expansion (Bertolino et al. 1996; Shen 98  and Walsh 2005; Jin et al. 2006). Furthermore, Tgif may regulate the generation of neuronal precursors in the neural tube (Knepper et al. 2006). Altogether, the expression analyses currently available indicate Tgi/potentially plays a role in the proliferation of multiple neuronal populations, as well as other tissues. Consistent with this idea, some Tgif'' mice were growth retarded and mutant embryos were often developmentally delayed as early as E8 when somite numbers were used to assess developmental progress. To determine whether embryonic cells in vivo also showed a reduced proportion of cells in S phase, in vivo BrdU incorporation experiments were conducted. However, assays of in vivo proliferation did not demonstrate a significant difference in the number of proliferating cells in the developing forebrains of wild-type and mutant embryos at E8. While in vivo BrdU incorporation was useful in mutant mice that displayed dramatic proliferation defects, such as Pox6 (Warren et al. 1999), it may not be sufficiently sensitive to detect small proliferation changes. Furthermore, only when global proliferation is perturbed will a dramatic reduction in proliferation be detected; isolated effects in specific progenitors are unlikely to be detected. Alternatively, in vivo proliferation may not be altered by the loss of Tgif at the stage examined. Cell cycle is tightly regulated in vivo during development, and other studies have failed to detect in vivo proliferation defects after initially observing such defects in vitro. For example, in vivo proliferation defects could not be demonstrated in mice lacking the Dtype cyclin-dependent kinases Cdk4 and Cdk6, and in mice lacking the D-cyclins, cyclin DI, D2 and D3 (Kozar et al. 2004; Malumbres et al. 2004). TGIF possesses many well characterized properties that link its functions to many signalling pathways. Tgif can suppress TGF-P responsive transcription (Wotton et al. 1999a). Similarly, Tgif competed with RA during transcription activation of RXR 99  responsive elements, such as the cellular retinol-binding protein II promoter, through direct competition for DNA binding elements (Bertolino et al. 1995). More recently, Tgif was shown to function as a competitive antagonist independent of direct DNA binding; Tgif directly bound to the ligand binding domain of the RXR family of receptors and repressed downstream transcription through recruitment of CtBP complexes (Bartholin et al. 2006). TGIF protein stability can be modulated by the Ras/MAP kinase pathway (Lo et al. 2001). However, while it was expected that the loss of Tgif in mutant MEFs may cause hypersensitivity to TGF-P or RA, an increased growth arrest in these cells was not observed. Further studies utilizing TGIF mutations identified in HPE patients addressed the mechanism of TGIF in regulating proliferation. These experiments indicated mutations located in the homeodomain were not able to rescue the proliferative defect. These results suggest a novel mechanism by which Tgif may regulate cell cycle genes or downstream targets through DNA binding or protein-protein interactions through the homeodomain. Given that only transcriptional repressive activity has been demonstrated for TGIF, its target genes may be inhibitors of Gi/S progression.  5.3.1.  PROLIFERATION DURING NEURAL  DEVELOPMENT  Proliferation plays an important and decisive role during development of a multicellular organism; morphogenesis coordinates differentiation, controlled proliferation and other processes. For example, prior to establishment of the primary axis, the mouse embryo is radially symmetric. Nodal signalling and the localization of its antagonists establish the anteroposterior axis through the appropriate regional proliferation of visceral endoderm cells (Yamamoto et al. 2004). Thus, the  100  proliferation of distal visceral endoderm determines the location of the future anterior side. Proliferation defects are implicated in forebrain patterning and HPE etiology (Xuan et al. 1995; Lukaszewicz et al. 2005). MRI studies of HPE patients demonstrated a correlation between reduced size of the developing telencephalon and the severity of HPE (Takahashi et al. 2004). Furthermore, proliferation function is associated with most HPE genes. Shh is mutated most frequently in HPE patients (Roessler et al. 1996); reduction in Shh signalling leads to HPE in mice and men. Shh signalling patterns the dorsoventral axis of the forebrain and spinal cord, through the coordination of three interdependent mechanisms: (1) region-specific expression of multiple transcription factors along the axis (Briscoe et al. 2001; Xu et al. 2005); (2) proliferation of neuronal progenitors (Britto et al. 2002); (3) sorting and partitioning of similar neuronal progenitors (Wijgerde et al. 2002). Six3 is a second HPE gene (Wallis et al. 1999). Recent reports showed Six3 promotes proliferation of neuronal precursors by competing for binding to Geminin (Del Bene et al. 2004). Geminin normally suppresses cell cycling by binding to and inactivating Cdtl, an important component of the replication complex that mediates proliferation. Accordingly, over-expression of Geminin, or the loss of Six3, led to inactivation of Cdtl activity and reduced proliferation, and subsequently the development of cyclopia, a severe form of HPE, the loss of forebrain, as well as premature differentiation of neurons. The third HPE gene is Zic2. While the molecular functions of Zic2 are poorly understood, the activity of Zicl, a paralogue of Zic2, is associated with the expansion and differentiation of neuronal progenitors (Aruga et al. 2002). Interesting, Zic3, another paralogue of Zic2, is involved in laterality determination and has been shown to 101  modulate Nodal signalling. Nodal signalling has been shown to regulate morphogenesis in part through regulating proliferation (Yamamoto et al. 2004). In fact, intriguing parallels can be drawn between Zic2 and Tgif. Reduction in the activity of both genes can sensitize cells to Nodal signalling. Additionally, both transcription factors have been shown to directly bind to the promoter of D1A dopamine receptor and regulate its expression (Yang et al. 2000a; Yang et al. 2000b). Finally, other pathways associated with HPE, such as megalin and the BMP4 pathway were shown to directly regulate proliferation and apoptosis in the developing neural tube (Ohkubo et al. 2002; Spoelgen et al. 2005). In addition, the mouse mutant flat-top  exhibited a telencephalon regionalization defect caused by a proliferation defect  specific to the forebrain (Hentges et al. 1999). It is interesting to note they also exhibited kinked neural tube and laterality defects. Unfortunately the function offlattop is not currently known. Therefore, based on the proliferative defect observed in rgz/mutants, it is tempting to speculate that a reduction in Tg-z/activity may reduce the rate of proliferation and cause HPE in patients. Since Nodal has been shown to promote proliferation, Tgif may be mediating this function through the Nodal pathway. Consistent with this hypothesis, Smadl and Smad4 mutants exhibited reduced proliferation (Sirard et al. 1998). Alternatively, ectopic RA is associated with HPE and reduced proliferation (Schneider et al. 2001). Interestingly, exencephaly incidence was higher in embryos born from pregnant mutant mice exposed to teratogenic levels of RA (Bartholin et al. 2006). Exencephaly is not a forebrain related birth defect, but results from neural tube closure defects instead, and can result from disruptions to cell cycle genes (Juriloff and Harris 2000).  102  However, in vitro studies with MEFs indicate that the proliferation defect was not mediated by the nodal, RA, or M A P K signalling pathways. Whether these signalling pathways may be modulated by Tgifin specific neuronal populations remains to be addressed. Regardless, Tgif'may function downstream of multiple signalling pathways that regulate and coordinate cell cycle with other morphogenetic events. A n important example is string, a cell cycle regulator in Drosophila that coordinates cell cycle regulation and morphogenesis through integrating inputs from numerous signalling pathways (Skaer 1998).  5.3.2. Future directions To address whether Tgif regulates neurogenesis in the forebrain and whether proliferation is an important mechanism in achieving this, I propose three approaches. A chick overexpression study indicated Tgif'may regulate the identity of dorsal neurons of the spinal cord (Knepper et al. 2006), suggesting neurons in the Tgif ' mice may not 1  have properly differentiated. To test this hypothesis, expression of dorsal expressing genes in Tgif ' forebrains can be assessed to determine whether dorsal progenitors are 1  properly specified. If this hypothesis is correct, mutant forebrains would exhibit reduced expression of dorsal genes and possibly a concomitant dorsal shift of ventral markers. A second approach is to direct ES cells to differentiate into telencephalic progenitors. ES cells would have to be generated from wild-type and mutant blastocysts (Hoodless et al. 2001). ES cells can be cultured to generate motor neurons, telencephalic neurons, haematopoietic lineages, and many other lineages of interest (Kennedy et al. 1997; Wichterle et al. 2002; Watanabe et al. 2005). Mutant ES cells could be used to differentiate into dorsal, ventral and other intermediate telencephalic 103  neuronal progenitor populations in vitro to address whether Tgif'plays a role during differentiation. In vitro culturing methods are sometimes more sensitive than in vivo conditions where defects may be masked by compensatory mechanisms. If mutant ES cells exhibited abnormal neurogenesis, a variety of questions could be posed. For example, is the differentiation of a specific neuronal progenitor regulated by Tgif? Do the TGF-R, Nodal or M A P K signalling pathways modulate the activity of Tgif! What is the relationship (epistatic, synergistic, parallel, sequential) of other signalling pathways, such as Shh, BMP, and Wnt, with respect to regulating neurogeneis? Is the effect achieved through proliferation? Do patient mutations disrupt these functions and can give insights into HPE etiology? ES cells can form embryoid bodies when cultured in suspension (Kennedy et al. 1997). I have previously created ES cell lines overexpressing 7g//and have compared the embryoid bodies generated from wild-type ES cells and ES cells overexpressing Tgif Embryoid bodies overexpressing Tgif consistently larger than control embryoid bodies based on their size measurements (data not shown), support the proliferation function of Tgi/discussed here, and further suggest Tgif activity may promote neural progenitor cells. The in vivo proliferation rate of wild-type and mutant telencephalons was examined in this study, but a difference was not detected. The developing telencephalon likely comprises a large variety of neuronal progenitor cells that are not yet well characterized. Unless global proliferation is perturbed by the loss of Tgif, a reduction in proliferation in specific lineages is unlikely to be detected. In contrast, retinal neurogenesis is better characterized than in any other region of the CNS (Dyer and Cepko 2001). Therefore, my final proposal is to further characterize cell cycle defects in mutant retina. Defects would be expected in the retina since it is a forebrainderived neural tissue. Though not all mutant mice developed eye defects, many mice 104  may have subtle defects not revealed by this study. Consistent with this hypothesis, HPE patients often developed eye defects. Initial experiments should include an expression study of Tgif'in the developing retina to identify retinal populations most likely to be disturbed in the mutants. Retinal progenitors are well characterized, as is the process of their differentiation into postmitotic neurons and glia (Dyer and Cepko 2000). The expectation is mutant retina would have reduced numbers of retinal progenitors due to defects in Gj/S progression, and later-born neurons may even fail to be born.  5.4.  FINAL COMMENTS At the time this thesis project started relatively little was known about the  genetic program involved in the patterning, growth and differentiation of the forebrain. Few molecular markers expressed in regionally restricted patterns were available for analysis, even though many of the major signalling pathways were known to be involved. However, the same signalling pathways play earlier roles during development and prevented detailed analysis of forebrain defects in mutants, and interpretations of observations were further hampered by overlapping expression and functional redundancy of related family members. This thesis project set out to determine whether the loss of Tgif'in mice would lead to HPE development and allow the characterization of the underlying molecular mechanisms. The experimental system used provided the answer that HPE does not develop in mice lacking one or both alleles of Tgif, unlike in humans. However, a number of related defects were observed lending credence to Tgifs function in forebrain development. A significant contribution from this project is the discovery of Tgifs function during cell cycle, and to provide the novel hypothesis that Tgifs 105  function during cell cycle regulation plays a role in the etiology of HPE. Consistent with this hypothesis, multiple studies in this field indicate proliferation is integral to forebrain development. Preliminary evidence shown here indicates reduced proliferation is a possible mechanism for HPE development, but this mechanism will require further investigations. The last five years have witnessed the identification of many patterning genes and subregionally expressed genes that have provided a better understanding of the genetic pathways during the patterning process. Sophisticated conditional strategies have become useful for manipulating spatio-temporal gene expression and allow better dissection of the underlying developmental mechanisms. This knowledge has culminated in the ability to induce ES cells to differentiate into neurons that have therapeutic potential. The task of identifying and detailing the molecular mechanisms that pattern and generate the entire repertoire of neurons of our brain is still a formidable task but is now closer to reality. 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