DOI
https://doi.org/10.25772/V2E4-T067
Defense Date
2017
Document Type
Thesis
Degree Name
Master of Science
Department
Biology
First Advisor
Robert Tombes, PhD
Abstract
Ca2+/Calmodulin-dependent protein kinase type II (CaMK-II) is a Serine/Threonine protein kinase that is activated by Ca2+ and Calmodulin to phosphorylate substrates involved in myriad developmental processes. This project implicates CaMK-II in specification of HSCs, and zebrafish provide an ideal embryonic model to study hematopoiesis. Zebrafish genetic manipulation was achieved through: incubation in chemical inhibitors; injection of notochord-targed WT and DN CaMK-II constructs with Transposase; and injection of camk2g1 translation-blocking morpholino antisense oligonucleotide (MO). Whole-mount in situ hybridization (WISH) and immunolocalization on zebrafish embryos allowed visualization of key HSC markers and pathway components that implicated CaMK-II in the specification of HSCs. CaMK-II is a negative regulator of shh expression during HSC specification, but CaMK-II does not influence Shh during its well-documented role in vasculogenesis. CaMK-II appears to affect the spatial distribution of Shh protein, which accumulates near the notochord source and differentially affects expression of Shh target genes based on their distance from the notochord. This project also identifies the specific timing requirement for CaMK-II during HSC specification, as inhibition of CaMK-II consistently reduces HSC specification, but only if administered before 18hpf. CaMK-II also downregulates ezh2 in the DA during the time of HSC specification, and the Ezh2 inhibition rescues the loss of HSCs, suggesting that CaMK-II regulates the secretion of Shh from the notochord to epigenetically regulate expression of key HSC specification genes in the DA through EZH2 methyltransferase.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-15-2017