Murray State Theses and Dissertations

Abstract

Maintenance of proper blood glucose homeostasis is governed by the endocrine cells of the pancreas. Dysregulation of blood glucose is characteristic of the disease, diabetes mellitus. Relatively little is known about the molecular mechanisms that underlie diabetes. The genes associated with more common forms of diabetes remain more enigmatic and likely have less direct roles in blood glucose homeostasis such as glucose sensing, insulin secretion, and β cell maintenance. GLIS3, a transcription factor involved in pancreatic development as well as the transcriptional regulation of insulin has been identified as a risk locus for type 1 and type 2 diabetes by numerous GWAS. Moreover, homozygous mutation of GLIS3 results in a human multisystem syndrome that includes neonatal diabetes and polycystic kidneys. The specific roles of GLIS3 in the developmental specification of endocrine cells and its later roles in regulating insulin production in mature β cells is not clearly defined. Uncovering these mechanisms could lead to novel therapies for the treatment and prevention of diabetes.

To elucidate the role of glis3 in pancreas development, we utilized the model organism Danio rerio. We characterized the spatial expression pattern of glis3 in zebrafish throughout development. glis3 is expressed in brain, thyroid, and kidney tubules in the zebrafish during early development. At early time points, glis3 expression was observed broadly in the endoderm while pancreas specific expression was not evident until after 72 hpf. Once turned on in the pancreas, expression remains through the end of pancreas development and subsequent β cell mass expansion. Morpholino-mediated knock down of glis3 expression in early development showed significant reduction in expression of pdx1 and ptf1a, important pancreas identity markers as well as reductions in the expression of pancreatic hormones, insulin, glucagon, and somatostatin. Given that morpholino-mediated knockdown only lasts for 2-5 days until the morpholinos become diluted, CRISPR/Cas9 technology was used to generate a line of zebrafish lacking glis3 expression. Once a glis3 CRISPR knockout line is fully established, further manipulation will be performed to elucidate the role of glis3 in the development of the pancreas. The generated mutant fish will be assessed for their ability to respond to nutrient overstimulation as well as their ability to regenerate β cells when compared to wild types.

Year manuscript completed

2022

Year degree awarded

2022

Thesis Advisor

Gary ZeRuth

Committee Chair

Gary ZeRuth

Committee Member

Dena Weinberger

Committee Member

Chris Trzepacz

Committee Member

Christopher Lennon

Committee Member

Bikram Subedi

Document Type

Thesis

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