Date on Honors Thesis

Spring 4-2023

Major

Chemistry/pre-medicine and Spanish

Minor

Cell Biology

Examining Committee Member

Dr. Gary ZeRuth, Associate Professor, Thesis Advisor

Examining Committee Member

Dr. Dena Weinberger Assistant Professor, Committee Member

Examining Committee Member

Dr. Christopher Trzepacz, Assistant Professor, Committee Member

Abstract/Description

The Krüppel-like transcription factor, Gli-similar 3 (Glis3) has been implicated in several human pathologies including neonatal diabetes, congenital hypothyroidism, and polycystic kidney disease. Numerous genome-wide association studies (GWAS) have additionally identified Glis3 as a risk locus for the development of both type 1 and type 2 diabetes mellitus. Our previous data suggest possible roles for Glis3 in endocrine pancreas specification in mice, but despite its clinical significance, much remains unknown about the role(s) Glis3 plays during development. To elucidate Glis3 gene function, a CRISPR-mediated knockout line of zebrafish was developed by deleting a segment of the Glis3 coding region resulting in a non-functional protein product. Founder fish were subsequently outcrossed to give rise to offspring that are heterozygous for the knockout mutation. In this study, fish heterozygous for the Glis3 deletion were crossed to produce zebrafish of varying genotypes, including those lacking a functional copy of the Glis3 gene. Genotypes were determined by PCR using tail biopsies and primers that flank the deleted region of the Glis3 locus. At select time points in development, the resulting embryos were analyzed via fluorescence imaging to determine the effect of the Glis3 knockout on embryonic pancreatic development. Analysis of the knockout embryos demonstrated that development of the principal islet remained largely unaffected by the absence of Glis3. In addition to fluorescence microscopy, immunohistochemistry was employed to further characterize the development of the secondary islets. In a similar fashion to mice, the knockout zebrafish displayed a phenotype of polycystic kidney disease that was visible by 30 dpf.

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