Publication Date

2009-12-23

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Neuroscience (Medicine)

Date of Defense

2009-11-13

First Committee Member

John Bixby - Committee Chair

Second Committee Member

nirupa Chaudhari - Committee Member

Third Committee Member

mary lou king - Committee Member

Fourth Committee Member

gerhard dahl - Committee Member

Fifth Committee Member

Pantelis Tsoulfas - Mentor

Abstract

Identifying the mechanisms that regulate neural precursor cell (NPC) proliferation and differentiation is important for understanding CNS development among different vertebrates. My work has focused specifically on understanding how mitogenic factors, like basic fibroblast growth factor (FGF2), regulate the NPC cell cycle. Mitogenic factors and serum are thought to drive cell cycle and therefore proliferation mainly by activating G1-type cyclin-dependent kinases (CDKs). The general hypothesis being addressed here is that FGF2 also promotes cell cycle progression of NPCs through the degradation of the cell cycle inhibitor p27Kip1. I show that, in the presence of FGF2 in vitro, embryonic rat cortical NPCs express high protein levels of the CDC28 protein kinase regulatory subunit 1b (Cks1b), a component of the SCFSkp2 E3 ubiquitin ligase complex that targets p27Kip1 for proteasomal degradation. I also show that NPCs maintained in FGF2 express undetectable levels of p27KIP1, while removal of FGF2 results in increased p27Kip1 protein expression and decreased protein expression of Cks1b. RNA expression data shows that Cks1b mRNA is reduced in non-dividing NPCs but is present in dividing NPCs, suggesting that Cks1b is being regulated at the transcriptional level. Analysis of the putative promoter of Cks1b reveals numerous conserved transcription factor consensus sites that could potentially play a role in regulation of Cks1b transcription, including consensus sites for E2F and the cell cycle-dependent element (CDE) cell cycle genes homology region (CHR) tandem repressor element. I use chromatin immunoprecipitation and luciferase assays to identify which E2Fs occupy and regulate the transcription of Cks1b under different conditions of mitogen stimulation. The data show that E2F4 occupies the promoter of Cks1b in non-dividing NPCs while E2F1 binds exclusively in proliferating NPCs. Mutation of either the E2F or CDE/CHR consensus sites independently de-represses the activity of a Cks1b promoter reporter in NPCs in G0/G1, while mutation of both sites delays induction of promoter activity. Finally, I use in ovo electroporation to determine if p27Kip1 has an additional role in neuronal differentiation during early spinal cord development. I show that ectopic expression of p27Kip1 is insufficient to induce neuronal differentiation in spinal cord progenitors.

Keywords

CNS; P27; Stem Cells; FGF

Share

COinS