Date of Degree

2-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Mark Emerson

Committee Members

Christine Li

Andreas Kottmann

Christine Rushlow

Claude Desplan

Subject Categories

Bioinformatics | Biology | Developmental Biology | Developmental Neuroscience | Molecular and Cellular Neuroscience

Keywords

retina, photoreceptors, cones, chick, progenitors

Abstract

There are two types of photosensitive cells of the retina that contribute to image formation: Cone photoreceptors that mediate color discrimination and rods that provide photosensitivity in low-light conditions. Given the importance of cones in high acuity and color vision, deficiencies in this cell type that result from ailments such as retinitis pigmentosa and macular degeneration can lead to a debilitating loss of vision. Currently, one of the most pressing goals in the field of retinal development is the elucidation of the gene regulatory networks (GRN) involved in inducing an undifferentiated cell into becoming a functional cone photoreceptor.

Recently, an enhancer element that drives transcription of the THRB gene in the developing retina, THRBCRM1, was found to be active in a specific lineage of progenitor cells that are restricted to produce only cones and horizontal cells5. Further exploration revealed that the transcription factors OC1 and OTX2 are required simultaneously for activation of THRBCRM1 and both are present in cells positive for THRB mRNA. This work largely focuses on a closer exploration of the transcriptional changes that underlie the emergence of this class of restricted progenitor cells from multipotent progenitors and the production of cone photoreceptors.

First, collecting and sequencing the mRNA of the THRBCRM1 restricted progenitors allows for a snapshot of the gene expression levels in this cell population. Thorough analysis of this data, I determined some of the changes necessary for the transition of these progenitors to a restricted state, namely the downregulations of a class of proteins known to be involved in the multipotent state.

Second, using an artificial version of OC1 designed to exclusively repress its targets, a new sequencing dataset was produced and, in combination with our THRBCRM1 data, candidate genes downstream of OC1 were screened for importance in the establishment of cone fate. A novel cone-related gene involved in the regulation of rod fate in early retinal development was identified.

Lastly, the initial dataset was combed for enriched cone genes that might serve as a novel marker for early cones. With the use of our genetic toolset in chicken retinas and different strains of transgenic mice, the expression pattern and putative marker specificity of a novel cone gene is described. Additionally, a BAC-GFP transgenic line is shown to be a faithful reporter of early cones. This tool was used for the collection of cone precursors at an early developmental time point (E14.5) for single-cell RNA sequencing, providing a dataset enriched in early cone precursors.

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