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Abstract

Albinism has long been known for its unusual combination: poor vision that results from a mutation in a gene involved in the production of melanin or the maintenance of melanosomes. That mutations to these pigmentation genes cause foveal hypoplasia, the underdevelopment of the pit-like fovea in the posterior of retina, has been known for over 100 years, but how these genes cause this change is unknown. A significant reason for this is the absence of a foveated developmental model. While a number of animals including some primates, birds, fish and lizards have foveated retinas, many are limited by a lack of biological tools—including gene editing. In this dissertation, I provide a detailed protocol on gene editing in brown anole lizards through a surgical procedure where the developing oocytes of adult females are injected with CRISPR Cas9. The establishment of this tool allowed us to mutate tyrosinase (tyr), the gene most commonly mutated in albinism, and tfec, a gene suspected to cause piebaldism when mutated, to see changes in skin pigmentation. Both showed loss of skin pigmentation, tyr-/- lost melanin in melanophores and tfec-/- lost guanine crystals in iridophores, a structural chromatophore. After observing pigmentation changes, we assessed both tyr-/- and oca2 -/- albino lizards for ocular changes. We showed both albino mutants exhibited foveal hypoplasia, a phenotype in humans with albinism that impacts visual acuity. Foveal hypoplasia and loss of melanin pigmentation encourage continued work in the lizard as a promising model for understanding foveal hypoplasia in the context of albinism. Lastly, we saw tfec-/- mutants have a significant reduction in the conus papillaris, a reptilian vascular structure similar to the avian pecten suspected to be involved in retinal health through nutrient delivery. However, our lizards survive without this structure, suggesting this structure may be non-essential. Collectively, these three knockouts show the power of gene editing and the importance of genes known for pigmentation in ocular development.

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