Genetic and epigenetic regulation of ocular development and homeostasis
Vertebrate retinogenesis is a complex developmental process that requires the dynamic orchestration of multiple tissues and cell types, morphological changes, and intrinsic and extrinsic cellular signaling. Intertwined with these processes are coordinated genetic and epigenetic mechanisms that function to regulate cellular state, proliferation, differentiation, and maintenance of retinal progenitor cells and neurons. Disruption of the DNA methylome or micro-RNA pathways, the regulatory pathways examined in this study, results in aberrant cellular proliferation, differentiation, and retinal homeostasis. Additionally, defects in these pathways are known to result in tumorigenesis, embryonic lethality, and neurodegenerative disease. Data presented in this thesis address the role of maintenance and de novo DNA methylation in the retina. Utilizing the zebrafish as a model for vertebrate retinal stem cell (RSC) maintenance and development, I demonstrated a requirement for dnmt1, the maintenance DNA methyltransferase, in RSC maintenance through the regulation of cell cycle genes, cell survival, and dysregulation of retroviral elements (RE). Additionally, I generated combinatorial mutants for the six de novo DNA methylation genes using TALEN and CRISPR/Cas9 mutagenesis to assess the function of these enzymes during retinogenesis. The tested combinations of mutant alleles lack overt phenotypes, however further analyses will be critical for determining their gene-specific and likely overlapping roles within the retina. Finally, I aimed to characterize the role of the dual-specificity phosphatase enzyme, Dusp11, within the retina. Utilizing a Dusp11, splice-mutant mouse line, I lay the groundwork for characterizing this enzyme in vivo and assessing its roles in retinal maintenance and potential contribution to retinal degeneration diseases. Through the use of Spectral Domain Optical Coherence Tomography (SD-OCT), I performed longitudinal experiments to assess retinal lamination and structure. Additionally, preliminary immunohistochemistry unveiled potential cell type-specific localization of Dusp11. These findings require further validation, but provide a first glimpse into ocular requirements for Dusp11 function and potentially revealing a unique genetic regulator that contributes to retinal degenerative diseases.