Degree Type

Dissertation

Date of Award

2017

Degree Name

Doctor of Philosophy

Department

Genetics, Development and Cell Biology

Major

Molecular, Cellular and Developmental Biology

First Advisor

Jeffrey M. Trimarchi

Abstract

Deciphering the mechanisms of development of retinal neurons is not only of immense interest to developmental biologists, but is also vital for regenerative therapeutic applications. To attain this goal, it is critical to understand how specific intrinsic factors control cell fate decisions and neuronal maturation processes. In the retina, Atoh7 is a highly conserved transcription factor that is essential for retinal ganglion cell development in the developing mouse and zebrafish. Atoh7 labels a subset of cells in the developing retina that are progressing from a progenitor to a differentiated state. To capture cells during the window when the cell fate decision is made, we performed transcriptome profiling of Atoh7+ individual cells isolated from mouse retina, thereby obtaining a wealth of information about how distinct types of retinal neurons are produced. Our efforts also led us to uncover several genes whose expression closely tracked with that of atoh7. These genes we believe will play important roles in retinal cell fate choice or early neuronal maturation. We further assayed the expression of these genes and found them to be expressed in the developing mouse and zebrafish retina by in situ hybridization. One of these genes was Trim9, an E3 ubiquitin ligase, that plays a highly significant role in axon branching and guidance in the mouse brain. Given the correlation of expression of Trim9 with Atoh7, we hypothesized that this gene may play roles in driving cell fate acquisition and neuronal differentiation in the vertebrate retina. However, examination of mice and zebrafish harboring mutations in Trim9 failed to reveal a retinal phenotype. We have also used CRISPR-Cas9 mediated genome editing in zebrafish to mutate several additional genes chosen from our single cell transcriptomic data. From our experience, single cell transcriptomics combined with CRISPR-Cas9 mediated genome editing, despite some potential hurdles, is a powerful approach for the study of gene networks governing cell fate decisions.

Copyright Owner

Rebecca Chowdhury

Language

en

File Format

application/pdf

File Size

194 pages

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