UNC-6/Netrin guides circumferential migrations along the dorso-ventral axis in C.elegans. Its receptors, UNC-40/DCC and UNC-5, mediate both attraction and repulsion of migrating cells or growth cones from sources of UNC-6. seu-2(ev523)and seu-3(ev555) (suppressor of ectopic unc-5) were identified as suppressors of ectopic UNC-5 in the touch receptor neurons(Colavita and Culotii, 1998). Like other components of UNC-6 signaling, seu-2 and seu-3 have roles not just in the migration of axon growth cones, but also in the repulsive migration of other cell types, specifically the distal tip cells (DTCs). Similar to observations in the touch receptor neurons, both seu-2 and seu-3 are able to suppress ectopic expression of the UNC-5 receptor in the DTCs. Genetic analysis of seu-2; seu-3 double mutants reveals that these genes function within the same signaling pathway for repulsive unc-6 guidance. seu-2 also appears to act in attractive unc-6 guidance. Mutations in seu-2 result in ventral to dorsal axon guidance defects in the HSN and ray 1 neurons. Double mutant analyses of seu-2 with either unc-40 or unc-6 null mutations exhibited HSN and ray 1 axon guidance defects at similar penetrance to either single mutant. These results suggest that seu-2 functions in the attractive unc-6-unc-40 dependent signaling pathway for HSN and ray 1 axon guidance. seu-2 was found to encode a G protein coupled receptor. Whole genome sequencing was used to identiy that seu-3 encodes the novel protein K09C6.9. K09C6.9 is predicted secreted protein that is expressed throughout development. Taken together, the phenotypes, method of isolation and genetic interactions of seu-2 and seu-3 make them interesting candidate mediators of UNC-6 signaling. I utilized genes, such as seu-2 and seu-3, to further elucidate other signaling components governing cell migration and axon guidance.

Cilia are microtubule-based organelles that project into the extracellular space and have various functions including transducing sensory information, regulating developmental signalling pathways, and generating directed fluid flow, making them important regulators of vertebrate development and homeostasis. Despite their importance, there are many aspects of cilia formation and function that remain poorly understood. The planar cell polarity (PCP) pathway is a branch of Wnt signalling that provides positional information to cells and is required for polarized morphogenic cell movements. Previous studies of PCP effector proteins suggested that PCP signalling was required for cilia formation. However, these proteins are not specific to the PCP pathway and are shared with other branches of Wnt signalling. To determine the role of a core and specific PCP regulator on ciliogenesis, I examined maternal-zygotic (MZ) vangl2 zebrafish mutants using an in vivo marker of cilia, Arl13b-GFP. Analysis of MZvangl2 mutants revealed that PCP is not required for cilia formation but is required for the posterior tilting and posterior positioning of motile cilia, essential for directed fluid flow. A parallel branch of studies suggested that cilia are actually required to regulate PCP signalling because defects in PCP-mediated morphogenic movements were observed with the knockdown of certain proteins that localize at or near cilia or basal bodies. To determine whether cilia were required to establish PCP, I generated MZ-intraflagellar transport-88 (IFT88) mutants, where ciliogenesis is completely abolished. Analysis of MZift88 mutants revealed that cilia are not directly required for PCP-mediated morphogenic movements. However, I observed that MZift88 mutants had defects in oriented cell divisions (OCD) occurring during gastrulation. Remarkably, these divisions occur prior to cilia formation, suggesting a cilia-independent role for IFT proteins in cell divisions, which may have important consequences on the interpretation of the role of cilia in disease.

Biological outputs of insulin/IGF signaling are regulated through essential mediators, such as IRSs, PI3-kinase, and PKB/Akt. These mediators serve critical roles in signal propagation, feedback, and as junctions for crosstalk with other pathways. Abnormal insulin/IGF signaling results in disease, such as obesity, diabetes, and cancer. Given the vital role of this signaling pathway to human health, unraveling its regulatory mechanisms is crucial. Components of this pathway are highly conserved throughout evolution. PTEN, one of the well-defined regulators of this pathway, functions as a lipid phosphatase that negatively regulates insulin/IGF-1 signaling at the PIP3 level, a phosphoinositol that is upregulated by activated PI3-kinase in both Drosophila and mammals. To discover genetic modulators of PTEN in Drosophila, we performed a loss-of-function genetic screen to identify molecules that modify the phenotype elicited by PTEN overexpression in the Drosophila eye. From this screen, we identified a member of the Dbl-family, the guanine nucleotide exchange factor DRhoGEF2, which suppresses the PTEN-overexpression eye phenotype via its effects on dPKB/dAkt activation. By conducting a genetic rescue, we established that PDZ-RhoGEF, a member of the regulator of G-protein signal (RGS)-like domain containing Rho GEFs (RGS-RhoGEFs) subfamily of Dbl-family GEFs, is the mammalian counterpart of DRhoGEF2. PDZ-RhoGEF is essential for cell proliferation and survival through ROCK-dependent activation of IRS/PI3-kinase signaling cascade, which has a major impact on adipose tissue homeostasis. Through an integrative approach, we have demonstrated that DRhoGEF2/PDZ-RhoGEF-dependent signaling has tissue-specific effects on insulin/IGF-signaling throughput in both Drosophila and mammals. Particularly, we have demonstrated the role played by PDZ-RhoGEF in diet related pathology, provides an alternative therapeutic opportunity in disease intervention.

The scaffolding protein anillin plays a crucial role during cytokinesis – the physical separation of daughter cells following chromosome segregation. Anillin binds filamentous F-actin, non-muscle myosin II and septins, and in cell culture models has been shown to restrict actomyosin contractility to the cleavage furrow. Whether anillin also serves this function during the incomplete cytokinesis that occurs in developing germ cells has remained unclear. Localization of anillin to several actin-rich structures in developing male germ cells also suggests potential roles for anillin outside of cytokinesis. In this study, I demonstrate that anillin is required for cytokinesis in dividing Drosophila spermatocytes. In addition, spermatid individualization is defective in anillin-depleted cells, although similarities to another cytokinesis mutant, four wheel drive, suggest this may be a secondary effect of failed cytokinesis. Anillin, septins and myosin II stably associate with the cleavage furrow in wild-type dividing spermatocytes. Anillin is necessary for recruitment of septins to the cleavage furrow, and for maintenance of Rho, F-actin and myosin II at the equator in late stages of cytokinesis. Membrane trafficking appears unaffected in anillin-depleted cells, although, unexpectedly, ectopic expression of one membrane trafficking marker, DE-cadherin-GFP, suppresses the cytokinesis defect. DE-cadherin-GFP recruits β-catenin (armadillo) and α-catenin to the cleavage furrow and stabilizes F-actin at the equator. Taken together, my results suggest that the anillin-septin and cadherin-catenin complexes can serve as alternative means to promote tight physical coupling of F-actin and myosin II to the cleavage furrow and successful completion of cytokinesis.

Genes are typically expressed in equal amounts from both parentally inherited chromosomes. However, recent studies have demonstrated that genes can be preferentially transcribed from a locus. Non-random preferential expression of alleles can occur in a parent-of-origin pattern, known as imprinting, where epigenetic factors regulate their transcription. Alternatively, it can occur in a haplotype-specific pattern, where cis-acting polymorphisms in regulatory regions are thought to underlie the phenomenon. Both forms of unequal allelic expression have been associated with human disease. Consequently, it is important to identify genes subject to unequal allelic expression and characterize mechanisms that regulate differential transcription. This thesis presents the results of a screen for unequal allelic expression where approximately 50 murine transcripts homologous to genes on human chromosome 7 were analyzed. Human chromosome 7 was selected due to its association with several human disorders that show parent-of-origin effects. The screen identified non-imprinted preferential allelic expression in numerous transcripts and demonstrated that such patterns can occur in tissue specific patterns. Paraoxonase-1 (Pon1), a gene implicated in arthrosclerosis, was identified as having a dynamic pattern of allelic expression which varies throughout embryonic development. This finding represents the first report of a developmentally regulated pattern of allelic variance. Carboxypeptidase-A4 (Cpa4) was identified as having a tissue-specific imprinted pattern of expression, where the maternal allele was preferentially expressed in all embryonic tissues, with the exception of the brain. The Krüppel-like factor 14 gene (Klf14), a novel imprinted transcript, was found to have ubiquitous maternal expression in all human and murine tissues analyzed. A differentially methylated region, generally associated with imprinted transcripts, was not found in the gene’s CpG island, nor was a differential pattern of histone modifications identified. However, it was determined that maternal methylation regulates the transcript. The data in this thesis contribute to our understanding of the numerous patterns of allelic expression that exist in nature and the diverse mechanisms that regulate them. Ultimately, quantitative analyses of allelic expression patterns and the identification of their underlying genomic DNA sequences will become standard protocol in all biomedical studies.

Highly conserved genes that are abundantly and specifically expressed in the retina are likely to be important for retinal physiology or development. Prdm8 is expressed abundantly in retina and belongs to the PRDM (PR domain containing) protein family, conserved transcription factors important in the regulation of development and cancer. To begin to understand the role of Prdm8 in the retina, I cloned the cDNA and characterized Prdm8 expression in the mouse. Prdm8 encodes a PR domain and three zinc finger domains and is expressed predominantly in the developing and mature nervous system, including the retina, hippocampus and cerebellum. In the developing retina, Prdm8 mRNA is enriched in differentiating neurons. Together, these findings suggested that Prdm8 might be important for both neural development and maintenance. To determine whether Prdm8 is necessary for neural development, I generated Prdm8 null mice. Prdm8eGFP/eGFP mice are born at expected Mendelian ratios and viable, but have a hypocellular retina, develop prominent ventral skin lesions and display defects in hindlimb coordination, impaired spatial learning and memory, and changes in the volume of brain substructures. To better understand the role of Prdm8 in development, I focused on the Prdm8eGFP/eGFP retinal phenotype. I determined that Prdm8 is expressed in subsets of bipolar, amacrine and ganglion cells, and at a low level in photoreceptors. In the Prdm8eGFP/eGFP retina, cell loss was restricted to the inner nuclear layer, due to a lack of rod bipolar and cone bipolar subtypes. I found that bipolar cells were specified, but lost due to a failure in maturation, including a decrease in Vsx1 and Bhlhb4 expression, two transcription factors necessary for bipolar cell development. Consistent with this defect, Prdm8eGFP/eGFP mice exhibited impaired electroretinogram b-wave responses. Given the abundant expression of Prdm8 in the developing and mature nervous system and the array of neurobehavioural phenotypes identified, Prdm8 is likely to be necessary for development in other regions of the brain. My work provides novel insight into the genetic control of visual disorders and a useful framework for understanding the role of Prdm8 and this class of transcription factors in the development of other parts of the nervous system.

The putative transcription factor Prdm8 is a member of the PR domain-containing protein family, which are regulators of cell proliferation and differentiation. Prdm8 is expressed in the developing and adult retina, most abundantly in the inner nuclear layer, where retinal bipolar interneurons reside. Adult Prdm8eGFP/eGFP retinas have a near complete absence of rod bipolar and Type 2 OFF cone bipolar cells. Prdm8 is required for late bipolar cell differentiation. To begin to identify downstream targets of Prdm8, I compared gene expression of wild-type and Prdm8eGFP/eGFP retinas at P6 by microarray analysis. I identified 75 differentially expressed genes, many of which are involved in neurogenesis and neurite development. Differentially expressed genes include transcription factors such as Sox6 (2.4-fold up in mutant), Lhx4 (1.6-fold up), and Eomes (1.4-fold down). Differentially expressed genes of the Prdm8eGFP/eGFP retina are strong candidates to be controlled by Prdm8, suggesting Prdm8 may function in a transcription factor cascade that regulates late bipolar cell differentiation.

During development of the vertebrate embryo, a highly conserved tissue called the organizer forms during gastrulation, and is required for establishment of the basic body plan. In mouse, the organizer gives rise to the node and notochord, which are both transient signaling centres involved in patterning the body axes. The genetic regulation and morphogenesis of these tissues, particularly in the mouse, is not well understood. To follow the formation of these tissues we used time-lapse live imaging together with conventional cell lineage tracking. This showed that the notochord has distinct morphogenetic origins along the anterior-posterior axis: anterior head process forms by condensation of dispersed midline organizer cells; trunk forms by convergent extension of node cells; tail forms from posteriorly migrating node cells—this challenges the previously accepted model that tail notochord forms by node regression. We have also found there are distinct genetic requirements within these different regions. Previous mouse mutant analysis showed that conserved transcription factors Foxa2 and Noto are required for either all notochord regions or just tail notochord, respectively. We found a novel genetic interaction between the two demonstrated Foxa2 compensates for Noto specifically in the trunk notochord. Furthermore, we found Noto has a conserved role in regulating axial (notochord) versus paraxial (somite) cell fate. Therefore, we proposed there are three distinct regions within the mouse notochord, each with its own unique morphogenetic origins and genetic control. We have also conducted two microarray-based screens to identify novel gene expression patterns in the node and notochord. First, we compared Foxa2 mutant and wild type gastrula embryos. Second, we isolated notochord progenitors from early somite stage embryos. Extensive in situ hybridization screening based on both data sets revealed over 50 node and notochord expression patterns. Lastly, we screened Foxa2-bound chromatin regions near these notochord-specific genes using a transient zebrafish expression assay, and identified two novel notochord cis-regulatory modules. Together, we found a combination of classical genetics, embryology, and novel imaging techniques, has given us a better understanding of the morphogenesis and genetic regulation of pattern formation in the developing mouse embryo.

p53 functions as an important tumour suppressor through its ability to regulate a number of important cellular processes such as cell cycle arrest, apoptosis, DNA repair, senescence, and angiogenesis. An in vivo genetic modifier screen performed using Drosophila melanogaster resulted in the identification of D. melanogaster brain tumour (brat) as a putative modifier of of the p53 small eye phenotype. Mammalian homologs of brat are members of the tripartite motif family that contain a c-terminal NHL domain. We focus on elucidating the in vivo role of one such homolog, BERP, through the generation and characterization of a classical gene-deletion mouse mutant. We report that BERP-deficient mice exhibit enhanced learning/memory, increased fear, impaired motor coordination, and increased resistance to PTZ -induced seizures. Electrophysiological and biochemical studies show a decrease in mIPSC amplitude along with a decrease in cell surface expression of gamma2 subunit-containing GABA A receptors in the brains of BERP-deficient mice. In addition, no effect of genotype is apparent when examining BERP mRNA levels in the brain. This suggests that the decreased cell surface expression of gamma2 subunit-containing GABA A receptors is likely a posttranscriptional phenomenon and supports the possibility that BERP may be involved in the intracellular trafficking of GABA A receptors. In investigating the possible relationship between BERP and p53, we identify the presence of a transcriptionally competent p53 response element within the first intron of the human BERP genomic locus and demonstrate that the BERP expression is up regulated in a p53-dependent manner both in vitro and in vivo. These results support the interpretation that BERP is a novel p53-regulated gene and suggest a new role for p53 in the regulation of GABA A receptor trafficking and epileptogenesis.

The GATA transcription factors consist of six family members that bind the consensus DNA binding element W-GATA-R, and are poorly characterized in the central nervous system (CNS). In this thesis we identify GATA4 to be expressed in the neurons and glia of normal murine and human embryonic and adult CNS with significant loss in Glioblastoma Multiforme (GBM). GBM is the most common and lethal primary brain tumour and exhibits multiple molecular aberrations. Here we report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumour suppressor gene. Although GATA4 was expressed in normal brain, loss of GATA4 was observed in GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor like phenotype, formation of neurospheres and the ability to differentiate into astrocytes, neurons and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures and brain tumour initiating cells suppressed tumour growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21CIP1, independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT, the DNA repair enzyme often implicated in temozolomide resistance. Instead GATA4 reduced expression of APNG, a DNA repair enzyme poorly characterized in GBM mediated temozolomide resistance. Identification and validation of GATA4 as a tumour suppressor gene and its downstream targets in GBM may yield promising novel therapeutic strategies.