The Role of Homeobox Transcription Factors Six3 and Six6 in Retinal Development
Diacou, Raven Gwendolyn
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The potential of stem cell-based therapeutics has reignited interest and attention to the development of organs and cell fate specification. In patients, irreversible vision loss occurs due to neurodegenerative eye disease. A major goal in the field is identifying the mechanisms that are required to direct groups of stem cells towards the heterogenous cell types that comprise the retina. The retina serves as an easily accessible model of neural development as diverse neuronal cells are generated from a pool of multipotent progenitors. The genetic mechanisms regulating multipotent retinal progenitors and their differentiation are partially understood. Six3 and Six6 are homeobox transcription factors expressed in multipotent retinal progenitor cells. Although Six3 is required for eye specification, the role of Six3 and Six6 in retinal progenitor cells is largely undetermined as Six3-null mice fail to initiate eye development and until now Six3 was not studied in the context of retinal development after eye specification. The Six3;Six6 compound mutant retinas displayed novel retinal phenotypes (reduced proliferation, aberrant differentiation, and perturbed expression of several important transcription factors), which were not observed in either Six3 or Six6 null retinas. Although Six3 and Six6 regulate several known genes, the full gene regulatory network controlled by Six3 and Six6 has not been determined. Therefore, I investigated the genetic network regulated by Six3 and Six6 in retinal progenitor cells by 1) transcriptome profiling and 2) functional assessments of two molecular mechanisms downstream of Six3 and Six6 joint functions. In chapter 1, I review the process of eye development, followed by characterization of the genes required to maintain multipotent retinal progenitor cells and describe the histological and molecular phenotype observed. I describe two major retina phenotypes of the Six3;Six6 compound null retinal phenotype; expansion of ciliary margin fate and reduction of retinogenic genes in retinal progenitors resulting in reduced eye size. In chapter 2, I describe the mouse mating strategy used to generate Six3;Six6 compound null retinas and force expression of Sox2 in Six3;Six6 compound null retinas, detail methods to profile embryonic mouse retinas by RNA-sequencing and a method to culture embryonic mouse eyecups in soluble recombinant proteins. In chapter 3, I use RNA-sequencing to identify the genes dysregulated in the Six3;Six6 compound null retinas. From this analysis I identified 800 differentially expressed genes and confirmed the upregulation of ciliary margin fate and Wnt/Bcatenin pathway in the Six3;Six6 compound null retinas. Furthermore, Wnt3a, Wnt16, and receptor Fzd1 were identified from the RNAseq as a putative Wnt components regulating the CM expansion phenotype observed in Six3;Six6 compound null retinas. In chapter 4, I tested the effects of exogenous Wnt signaling on retinas and demonstrated Wnt3a was sufficient to promote ciliary margin fate. In chapter 5, I tested a candidate mediator of Six3 and Six6, transcription factor Sox2, by driving expression of Sox2 in Six3;Six6 compound null retinas through a genetic mouse mating strategy which partially restored aspects of the retinal phenotype observed in Six3;Six6 compound null retinas. This thesis provides a groundwork for identifying genes directly regulated by Six3 and Six6 during retinal development and may aid in the identification of novel biomarkers for patients with developmental eye disease and diseases of the anterior segment and inform genetic instruction of eye development in stem cell regeneration efforts. I propose Six3 and Six6 maintain neuroretinal progenitors by regulating several retinogenic programs, while antagonizing Wnt/B-catenin to suppress ciliary margin fate.