DOI
https://doi.org/10.25772/J941-CH59
Defense Date
2020
Document Type
Thesis
Degree Name
Master of Science
Department
Biology
First Advisor
Robert Tombes
Second Advisor
Sarah Rothschild
Abstract
Ca2+/calmodulin dependent protein kinase II (CaMKII) is an important signaling molecule involved in many developmental processes. This project aims to understand the role of CaMKII in specification of hematopoietic stem cells (HSCs) in zebrafish. HSCs undergo hematopoiesis to specify all cells that constitute blood. The process of hematopoiesis along with the specification of hematopoietic stem cells is conserved in vertebrates. CaMKII is expressed in tissues important for HSC specification, including the somites, notochord, and dorsal aorta. The dorsal aorta is the site of HSC specification in zebrafish. Injection of camk2g1 translational blocking antisense oligonucleotides (MO) or Cas9 guide RNAs targeting camk2g1 leads to a loss of HSCs. Using a pharmacological inhibitor of CaMKII, KN93, it is demonstrated that CaMKII is required for HSC specification in a time-dependent manner. Inhibition of CaMKII after 16 hours post fertilization (hpf) in zebrafish embryo does not result in HSC loss. Additionally, HSC specification can be recovered by washing out KN93 at 48 hpf but not at 72 hpf. Using whole mount in-situ hybridization (WISH) and immunolocalization, loss of CaMKII results in increased expression of the epigenetic regulator, enhancer of zeste 2 (ezh2), which trimethylates lysine 27 on histone 3 (H3K27me3). Altered methylation could lead to decreased expression of Notch ligand, Jag1a, inhibiting HSC specification. Understanding the signals that guide HSC specification is important for differentiation of HSCs from induced pluripotent stem cells in order to remedy primary immunological disorders and hematological malignancies.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-28-2020