Date Approved

2022

Degree Type

Open Access Thesis

Degree Name

Master of Science (MS)

Department or School

Biology

Committee Member

Robert Winning, PhD

Committee Member

Hannah Seidel, PhD

Committee Member

David Kass, PhD

Abstract

EphA4 is a receptor tyrosine kinase that is responsible for cellular adhesion and locomotion using repulsion signaling in early development of Xenopus laevis. EphA4 regulates cellular locomotion by controlling proteins within its signal transduction pathway, which may include the protein cofilin. Cofilin actively severs and removes actin monomers, thus altering the actin cytoskeleton, leading to the cessation of cellular crawl. During the gastrulation phase of embryonic development, individual cells are relocating to create the three primary germ layers of the organism. Failure of this process to occur results in attrition of the embryos, frequently by way of embryonic exogastrulation. In order to characterize the role of the cofilin gene in embryonic development, we generated mutations of this gene using CRISPR-Cas9 technology. We demonstrated that mutation of the cofilin gene in X. laevis embryos resulted in exogastrulation among the majority of embryos analyzed. Our finding coincides with results observed for EphA4 knockdown in Xenopus in previous studies. It was also discovered that there is maternal cofilin mRNA remaining in all embryos beyond gastrula stage. We also attempted a rescue experiment in which cofilin mutation using CRISPR-Cas9 was supplemented by injection of various concentrations of cofilin mRNA. Rescue was unsuccessful in every attempt, and when cofilin mRNA was injected alone, exogastrulation rates persisted at high levels, suggesting that cofilin activity levels need to be tightly regulated and within a specific range.

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