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.

Williams-Beuren Syndrome (WBS) is an autosomal dominant neurodevelopmental disorder caused by hemizygous deletion of a 1.5 Mb region on chromosome 7q11.23. Symptoms are numerous and include behavioural and cognitive components. One of the deleted genes, GTF2IRD1, a putative transcription factor, has been implicated in the neurological features of WBS by studying patients with atypical deletions of 7q11.23. Gtf2ird1-targeted mice have features consistent with the WBS phenotype, namely reduced innate fear and increased sociability. To identify neural targets of GTF2IRD1, microarray analyses were performed comparing gene expression in whole brains of Gtf2ird1-/- and wildtype (WT) mice at embryonic day 15.5 and at birth. Overall, the changes in gene expression in the mutant mice were not striking, with most falling in the range of 0.3 to 2 fold. qRT-PCR was used to verify the expression levels of candidate genes and examination of verified genes revealed that most were located on chromosome 5, within 50 Mb of Gtf2ird1. Expression of these candidate genes in Gtf2ird1-/- mice was found to be the same as in WT 129S1/SvImJ mice, indicating the differences were the result of flanking chromosomal material from the, 129-derived, R1 ES cells from which the Gtf2ird1-/- mice were generated, and that expression differences were unrelated to Gtf2ird1 dosage. Further analysis found that while many genes showed decreased expression using primers targeting the 3’ UTR, expression of upstream exons was not affected. Transcripts using alternative polyadenylation sites were identified using 3’ RACE, and qRT-PCR showed that expression of different 3’ UTR isoforms can occur in a strain specific manner. Expression analysis of previously identified GTF2IRD1 targets also failed to demonstrate an in vivo effect. In summary, I was unable to find any in vivo neuronal targets of this putative transcription factor, despite its robust expression in the developing rodent brain.

Epigenetic studies hold the promise of addressing some of the fundamental questions of human biology including development, cell differentiation, and the aetiological mechanisms of complex disease. Over the last years, several new large scale high throughput technologies have been developed to allow genome wide profiling of epigenetic signals such as DNA methylation and histone modifications. Two of such technologies were developed in our laboratory enabling a genome wide microarray based profiling of DNA methylation signatures and a high throughput method for the site specific interrogation of the density of methylated cytosine. Using these techniques, we identified a DNA methylation difference in the 3’UTR of the DLX1 gene with potentially functional implications to discordance in risk taking behavior in a single pair of MZ twins. We modeled a power analysis on the effect size of the detected difference and determined that approximately 6~25 discordant twin pairs will be adequate to yield 80% power across the entire 12 K CpG island microarray platform using our epigenomic microarray profiling technique. We performed a DNA methylome analysis of MZ twins in white blood cells (WBC), buccal epithelial cells, and gut (rectum) biopsies (N=57 pairs in total) using 12K CpG island microarrays providing the basis for the first annotation of epigenetic metastability of ~6,000 unique genomic regions in MZ twins. We performed a classical twin study on DNA methylation differences in WBC and buccal epithelial cells from 39 pairs of MZ twins to 40 pairs of DZ twins. DZ co-twins exhibited significantly higher epigenetic difference compared to the MZ co-twins in buccal cells (p=1.2x10-294). While such higher epigenetic discordance in DZ twins can result from DNA sequence differences, our in silico SNP analyses and comparison of methylomes in inbred vs. outbred mice favour the hypothesis that this is due to epigenomic differences in the zygotes. This study suggests that molecular mechanisms of heritability may not be limited to DNA sequence differences.

Behaviour genetics deals with complex phenotypes which respond flexibly to environments animals experience. Change of phenotype in response to environment is phenotypic plasticity. A central question is how genes influence plasticity. I study plasticity and gene by environment interactions (GEI) relating to behaviours, metabolic, and genomic phenotypes of adults of the fruit fly Drosophila melanogaster. Chapters 1-3 study cuticular hydrocarbon (CH) levels of male flies. Chapter 1 shows male CH levels respond to time of day and light. Methods are developed to reduce high variability of CH. I show variation in CH parallels activity of two classes of CH synthesis hormones. Analysis of rate of variation gives estimates of turnover rates of CH and the metabolic cost of signaling. Chapter 2 studies mixed groups of genetically different flies, “hosts” and “visitors”. GEI of CH are found with both abiotic factors and with social mix. Social mix results in GEI as strong as abiotic factors. Indirect Genetic Effects (IGE) theory is used to show frequency-dependent IGE interactions. Chapter 3 shows that males in mixed social environments reduce expression of clock and CH synthesis genes, resulting in different signals. Females mate more often with males in a mixed group than with single-genotype males. Plasticity in male gene expression in response to social environment leads to different signals, mating levels, and potentially different fitness. Chapter 4 deals with behaviour, metabolite, and genomic phenotypes in flies differing in foraging gene alleles, as the food environment is changed. Strong GEI is found, structured by food type, chemical class of metabolite, and gene metabolic roles. A concept called “relative nutrient sensitivity” suggests an interaction between foraging and the insulin signaling pathway. I demonstrate epistasis between for and insulin with quantitative genetic methods and bioinformatics. These results lead to the conclusion that GEI are common in many fly phenotypes in response to well studied environments such as food and less studied ones such as social group. Some implications of this for maintenance of genetic variance are discussed.

Development of genome-wide reagents has allowed systematic analysis of gene function. The experimental accessibility of budding yeast makes it a test-bed for technology development and application of new functional genomic tools and resources that pave the way for comparable efforts in higher eukaryotes. In this Thesis, I describe a two-color GFP-RFP reporter system I developed to assess the consequences of genetic perturbations on a promoter of interest. The dual-reporter system is compatible with the synthetic genetic array methodology, an approach that enables marked genetic elements to be introduced into arrays of yeast mutants via an automated procedure. I use this approach to probe cell cycle-regulation of histone gene transcription by introducing an HTA1 promoter-GFP reporter gene construct into an ordered array of ~4500 yeast deletion mutants. I scored defects in reporter gene expression for each mutant, generating a quantitative analysis of histone promoter activity. The results of my screen motivated a number of follow-up experiments, including chromatin immunoprecipitation, transcript profiling and genome-wide analysis of nucleosome positions, which revealed a previously unappreciated pathway that specifies regions of repressed chromatin in a cell cycle-sensitive manner. A novel aspect of this pathway is that it involves histone chaperones and a chromatin boundary element. Specifically, we discovered that the histone chaperone Rtt106 works with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at histone promoters which is bound by the protein Yta7. It was clear from previous work that Asf1 and HIR repress transcription at HTA1 and that HIR localizes to and functions through a specific element in histone promoters. However, there was no previous data demonstrating a role for Rtt106 in cell cycle-dependent gene transcription. In sum, I describe a new genomic screen that I used to discover a novel pathway regulating cell cycle-dependent transcription. While I examined histone gene expression as proof-of-principle, my screening system could be applied to virtually any pathway for which a suitable reporter can be devised. I anticipate this methodology will enable yeast researchers to collect quantitative data on hundreds of gene expression pathways.

The vertebrate hindbrain is divided early in embryogenesis along its anterior-posterior axis into eight segments known as rhombomeres. This provides an excellent model for studying early segmentation and region-specific transcriptional domains. MafB, a basic domain leucine zipper transcription factor, is the first gene known to be expressed in the presumptive rhombomere 5 and 6 domain (r5-r6). MafB expression is directly activated by the homeodomain protein vHnf1. vHnf1 and MafB share an anterior expression limit at the r4/r5 boundary but vHnf1 expression extends beyond the posterior limit of MafB and, therefore, cannot establish the posterior expression boundary of MafB. Through the use of in situ hybridization, immunofluorescence, and chromatin immunoprecipitation analyses, I have determined that the caudal-related homeodomain protein Cdx1 establishes the posterior boundary of MafB by directly inhibiting expression beyond the r6/r7 boundary. My results indicate that MafB is one of the earliest direct targets of Cdx1.

Paediatric undifferentiated soft tissue sarcomas (USTSs) are a diagnostically challenging group of neoplasms. We hypothesized that USTSs contain distinct subgroups that can be identified based on their morphology, genomic aberrations and expression profiles. We sought to characterize genomic aberrations within primitive round cell (PRC) sarcomas which may underlie aberrant expression patterns. Using molecular and cytogenetic analyses, we identified 5 of 18 CIC-DUX4-positive PRC sarcomas. The consistent involvement of the CIC-DUX4 fusion in a subset of PRC sarcomas suggests a central role for the fusion transcript in such tumours. These analyses also identified a cohort of CIC-DUX4-negative USTSs with no established genetic markers. We performed integrative copy number and expression profiling, and identified significant genomic and transcriptomic changes. We propose that these genes are involved in biological pathways that are important to the initiation and progression of undifferentiated sarcoma, and may provide novel insights into the biological events responsible for sarcomagenesis.

This study examined the differential gene expression patterns between endothelial cells (EC) from 2D monolayer and EC from angiogenic capillary-like network in 3D matrices. Our microarray analysis comparing 3D to 2D EC cultures detected upregulation of 854 protein-coding genes and downregulation of 863 genes. We show that Notch signaling pathway is highly regulated in angiogenesis, induced by change in ECM dimension. Notch target genes Hey1, HeyL, Hes1 and Hes4 transcription factors were upregulated in 3D angiogenic EC, which were confirmed with qRT-PCR. Moreover, we are the first to report enrichment of FoxS1 transcription factor mRNA during angiogenesis in 3D ECM. Next, we asked whether epigenetic mechanisms partly mediate cis-trans response in angiogenesis. Our sodium bisulfite sequencing analyses did not indicate a role for DNA methylation in the expression of key Notch signaling components. However, our pilot studies indicate a potential role for lncRNAs in controlling EC phenotype in angiogenic response.

The vertebrate hindbrain is divided early in embryogenesis along its anterior-posterior axis into eight segments known as rhombomeres. This provides an excellent model for studying early segmentation and region-specific transcriptional domains. MafB, a basic domain leucine zipper transcription factor, is the first gene known to be expressed in the presumptive rhombomere 5 and 6 domain (r5-r6). MafB expression is directly activated by the homeodomain protein vHnf1. vHnf1 and MafB share an anterior expression limit at the r4/r5 boundary but vHnf1 expression extends beyond the posterior limit of MafB and, therefore, cannot establish the posterior expression boundary of MafB. Through the use of in situ hybridization, immunofluorescence, and chromatin immunoprecipitation analyses, I have determined that the caudal-related homeodomain protein Cdx1 establishes the posterior boundary of MafB by directly inhibiting expression beyond the r6/r7 boundary. My results indicate that MafB is one of the earliest direct targets of Cdx1.

Paediatric undifferentiated soft tissue sarcomas (USTSs) are a diagnostically challenging group of neoplasms. We hypothesized that USTSs contain distinct subgroups that can be identified based on their morphology, genomic aberrations and expression profiles. We sought to characterize genomic aberrations within primitive round cell (PRC) sarcomas which may underlie aberrant expression patterns. Using molecular and cytogenetic analyses, we identified 5 of 18 CIC-DUX4-positive PRC sarcomas. The consistent involvement of the CIC-DUX4 fusion in a subset of PRC sarcomas suggests a central role for the fusion transcript in such tumours. These analyses also identified a cohort of CIC-DUX4-negative USTSs with no established genetic markers. We performed integrative copy number and expression profiling, and identified significant genomic and transcriptomic changes. We propose that these genes are involved in biological pathways that are important to the initiation and progression of undifferentiated sarcoma, and may provide novel insights into the biological events responsible for sarcomagenesis.