Development and regeneration of the vertebrate eye are the result of complex interactions of regulatory networks and spatiotemporally controlled gene expression events. During embryonic retinal development, the coordination of cell signaling and transcriptional regulation allows for a relatively homogenous sheet of neuroepithelial cells to proliferate and differentiate in-to a multilayered, light sensitive retinal tissue. Following injury, the retinas of many cold-blooded vertebrates, such as the zebrafish, undergo a proliferative response that results not only in new retinal cells of the correct type in the correct location, but also functional integration of these cells and restoration of vision. In order for embryonic retinal neurogenesis to proceed correctly, systems must be in place that restrict subsets of progenitor cells from differentiation. Pools of actively proliferating retinal progenitor cells are maintained to fill the needs of developmental processes and normal growth of the retina. In addition, subsets of radial glia in the retina retain the ability to de-differentiate into proliferating progenitor cells to meet the demands of the regenerating retina. All of these processes rely on the tight coordination of extrinsic and intrinsic cues, as well as regulation of gene expression by transcription factors. Although a considerable amount of work has been conducted to identify key regulators of retinal development and regeneration, many gene regulatory networks which include both master signaling pathways as well as individual transcription factors remain poorly characterized. Some of these factors implicated in retinal development and regeneration are members of the Hairy/Enhancer of Split (Hes) superfamily of genes, including the Hairy-related (Her) factors Her4 and Her9. Her transcription factors are basic-helix-loop-helix-orange (bHLH-O) transcription factors that bind to palindromic E- and N-box canonical sequences in the promoters of target genes. Her factors have been previously shown to play roles in a diverse array of developmental and neurogenic processes, including neural tube closure, floor plate development, somitogenesis, and development of various components of the central nervous system as well as the cranial sensory placodes. The roles of her4 and her9 in retinogenesis, however, remain undefined. To determine the possible roles of her4 and her9 factors in the retina, I characterized the expression patterns of these factors during developmental retinal neurogenesis and/or regeneration, examined loss of function phenotypes, and identified signaling pathways that modulate expression of these factors. Chapter 1 of this dissertation provides an overview of vertebrate retina and retinal development, the known functions of her4 in other tissues, and the Notch-Delta signaling pathway. Chapter 2 provides evidence that her4 is a primary effector of the Notch pathway during retinal development, and examines the role of her4 expressing cells during regeneration of the mature zebrafish retina within the context of both chronic and acute photoreceptor damage paradigms. In addition, generation and validation of the transgenic her4:Kaede zebrafish which was used to identify the lineage of her4-expressing cells is described. Characterization of her9 during retinal development, identification of the retinoic acid signaling pathway as a regulator of her9 expression in the retina, and the role her9 plays during retinal vasculogenesis are discussed in Chapter 3. Chapter 4 discusses the generation of her9 knock-out zebrafish lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterization of mutant phenotypes in mosaic her9 mutant F0 fish. In addition, in Chapter 4 I also discuss the screening processes used to identify and characterize genetic lesions in the her9 allele and establish various lines that stably transmit deleterious her9 alleles in the germline, and provide preliminary data of the her9 mutant phenotype. Finally, in Chapter 5 I discuss conclusions from the data generated from this dissertation, additional studies that would expand upon this work, and the implications of these results on the broader understanding of retinal development and regeneration. My dissertation incorporates reverse genetic analysis in zebrafish, biochemical analysis, transgenesis, and various molecular approaches to help better understand the roles of her4 and her9 during retinal neurogenesis. Moreover, these studies may also contribute to a better understanding of retinal development, and disease pathogenesis. It is my hope that this work could also ultimately contribute, even if in some small way, to the goal of enabling human patients who have suffered from vision loss a means by which a damaged retina could be regenerated and functional vision restored.

Delta-like ligand 4 (DLL4) is a ligand for the Notch family of receptors. DLL4 is an important regulator of angiogenesis and DLL4 blockade promotes non-productive angiogenesis and delays tumour growth. The aim of this thesis was to investigate the effects of anti-DLL4 therapy in solid tumours in combination with a clinically relevant dose of ionising radiation (5 Gy; IR) and to analyse alterations in the Notch pathway induced by the treatments. Combining both treatments resulted in a greater than additive tumour growth delay in LS174T tumours, compared to either treatment alone. DLL4 blockade dysregulated vasculature and increased necrosis in LS174T and HCT-15 (DLL4-expressing and negative cell lines respectively) tumours within 3 days after treatment, but no changes were observed with IR alone. Additionally, combined IR and anti-DLL4 treatment of FaDu tumours (another DLL4-negative cell line) by our colleagues, also resulted in a supra-additive growth delay. These results show that combining IR with DLL4 blockade is an effective strategy for prolonging tumour growth delay and suggest that the stroma/vasculature provide the main therapeutic target for the anti-DLL4 therapy. Analysis of Notch pathway shows that IR upregulated Jag1 in tumour cells, and may inhibit Notch and downregulate DLL4 in the stroma. These changes may potentially affect tumour vessels and response to anti-DLL4 therapy. In vitro, anti-DLL4 therapy induced proliferation in quiescent contact-inhibited endothelial cells and also appeared to abrogate IR-induced inhibition of migration. These results suggest that DLL4 may be important in maintaining vessel quiescence and that IR may in part decrease migration through Notch signalling. Combining IR and DLL4 blockade to target tumour growth is an effective and well tolerated strategy and warrants further validation and refinement to be translated into clinical practice.