Date of Award

Spring 5-2020

Document Type

Thesis

College

College of Arts and Sciences

Degree Program

Biology

Degree Name

Master of Science

Thesis Advisor

Dr. Eric Birgbauer

Committee Member

Dr. Matt Stern

Committee Member

Dr. Kathryn Kohl

Keywords

Lysophosphatidic Acid, LPA, LPA Receptors, LPA4, Chicken, Growth Cones

Abstract

Growth cones direct axon pathfinding during neural development by detecting environmental stimuli, known as axon guidance molecules. In vitro studies have shown inhibitory axon guidance molecules to cause growth cones to change morphology to what is called a collapsed growth cone. Lysophosphatidic acid (LPA) has been demonstrated to collapse growth cones in culture, and thus it may act as an inhibitory molecule. However, identification of what LPA receptor is responsible for this physiological response is unknown. To investigate which receptor elicits collapse, we focused on LPA receptor-4 (LPA4) by designing a CRISPR construct to mutate Lpar4 in chick retinal ganglion cells (RGCs). Through retinal injections, we will introduce our CRISPR construct and isolate mutated retinal tissue to observe whether mutating Lpar4 abolishes RGC growth cone collapse. Preliminary data suggests our injection delivery system can successfully target RGCs. Moreover, we have successfully cloned five guide RNAs (gRNA) and obtained sequence results from gRNAs1-3 that show gRNA 1 and 2 have a high mutation rate in a DF1-Cas9 cell line.

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