Dissertations and Theses

Date of Award

2024

Document Type

Thesis

Department

Biology

First Advisor

Mark Emerson

Second Advisor

Osceola Whitney

Third Advisor

Christine Li

Keywords

Retina, Little Skate, Single Cell RNA Sequencing, Alternative Splicing, Immunohistochemistry, Marine Biological Laboratory

Abstract

The retinal development of Elasmobranchs (the superclass comprising sharks, skates and rays) is a poorly understood phenomenon, but it represents a critical component of the broader picture of vertebrate visual development. Evolutionary divergence between these organisms and the mammals is vast, with the last common ancestor of the two clades living roughly 420 million years ago. Remarkable conservation of retinal cell type and arrangement is exhibited across almost all vertebrates, elasmobranchs included, but there are notable differences among some members of this clade that demand explanation, and which may elucidate shared underlying principles of vertebrate visual development. The Little Skate (Leucoraja Erinacea) is a fascinating model for elasmobranch-specific retinal innovation, and this is underscored by two significant morphological oddities in their retinal design. The best studied of these is their “simplex” photoreceptor layer, composed of only one type of photoreceptor: a rod cell capable of single photon detection in the dark and bright light detection in bright backgrounds, with each distinct capability achieved after a process of adaptation to the background luminance. The other morphological oddity, which is until this study not well characterized, is the presence of a dense packing of cells in the inner nuclear layer (INL), present at the hatchling stage and disappearing in adulthood, that are apparently neither one of the mature canonical cell types, nor a known progenitor. This study reveals these cells to be largely post-mitotic photoreceptor precursor cells which intercalate into the photoreceptor layer at some point between hatching and adulthood, although the timeline is unclear. Single cell analysis reveals that these cells express canonical markers of both the cone and rod pathway, suggesting the precursors utilize elements of the cone and rod program to generate their apparent hybrid photoreceptor structures. Analysis of little skate onecut1, a transcription factor gene critical to the development of cone photoreceptors in studied species, was found to be alternatively spliced in a manner not characterized in retinal development, and which may have implications on the little skate’s ability to generate cone-like rods. Bulk RNA-seq analysis shows the two distinct splice isoforms are coexpressed in the retina at all studied stages (embryo, hatchling, adult), with the ratios of the transcript types, as well as the bulk expression of the gene itself, varying across developmental stages. The implications of this finding are not yet entirely clear, but there is precedence for the discovery of alternative splicing of this gene into two isoforms, as the report on the initial discovery of this gene found two onecut1 splice isoforms in the rat liver. How the gene regulatory activity of onecut1 is affected by the coexpression of two isoforms in the retina is a significant outstanding question, with the results presented here and those in the original onecut1 discovery study (Lannoy et. al, 1998) providing context and structural information that will be useful in this investigation. Finally, to attempt overexpression experiments in the little skate at critical timepoints of developmental processes, it is important to have cell cycle dynamic information in the retina of the developing skate. An analysis of the G2 phase of the cell cycle was carried out in the embryonic little skate retina, revealing the length of this phase to be 4-8 hours. This information is useful for the proper implementation of cell-culture time windows in electroporation experiments.

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