DOI
https://doi.org/10.25772/8NFE-WE42
Defense Date
2010
Document Type
Thesis
Degree Name
Master of Science
Department
Anatomy & Neurobiology
First Advisor
Jack Haar
Second Advisor
Joyce Lloyd
Abstract
Krüppel-like factors (KLFs) are a family of 3 Cys2/His2 zinc finger transcription factors with a diverse set of roles in cellular differentiation, cell cycle regulation, tumor suppression, erythropoiesis, angiogenesis, and other processes. During embryonic development, KLF2 has a role in vessel maturation. Adult conditional KLF4 knockout mouse embryos have thickened arterial intima follow vascular injury. Breeding KLF2+/- and KLF4+/- mice resulted in the generation of KLF2/KLF4 double knockout (DKO) embryos. KLF2/KLF4 DKO embryos died by E10.5 with cranial bleeding. Using immunohistochemistry, embryo whole-mounts were examined for differences in gross vascularization between wild-type (WT), KLF2-/- and KLF2/KLF4 (DKO embryonic day 9.5 (E9.5) embryos. No obvious gross capillary abnormalities were noted in E9.5 KLF2/KLF4 DKOs, although the posterior cardinal vein appeared to narrow rostral to caudal in KLF2-/- and KLF2/KLF4 DKO embryos. Light and electronic microscopy were employed to investigate potential structural and ultrastructural phenotypes in KLF2/KLF4 DKO embryos. Microscopy confirmed hemorrhaging near and endothelial breaks in the primary head vein (PHV) in E9.5 KLF2/KLF4 DKOs (n=8) and E10.5 KLF2-/-KLF4+/- embryos (n=1). Electron micrographs illustrated a disrupted endothelium in KLF2/KLF4 DKOs with endothelial cells having filopodia-like projections. Surprisingly, KLF2-/- embryos had the presence of wider medial PHV endothelial gaps compared to WT at the electron micrograph level. Density counts revealed a 15% reduction in midline cranial mesenchyme at the level of hemorrhaging in KLF2/KLF4 DKOs compared to KLF2-/- (n=3). An in-situ hybridization localized KLF2 RNA expression to the endothelium of the PHV. A quantitative reverse transcriptase polymerase chain reaction assay revealed that the eNOS expression is synergistically regulated by KLF2 and KLF4, as a shared downstream target. It is proposed that KLF2 and KLF4 share in the regulation of multiple gene targets, leading to early death by E10.5.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
August 2010