Date of Award

January 2018

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Diane C. Darland

Abstract

Progressive restriction of neural stem and progenitor cell fates in the cortex has been linked to a clearly defined sequence of transcription factors that regulate neurogenesis and gliogenesis in the developing cortex. In recent years, increasing attention has been paid to the epigenetic regulatory mechanisms contributing to cell fate choice, largely focusing on intrinsic properties of the neural stem cells (NSCs), themselves, rather than microenvironmental influences such as vascular investment. To investigate the influence of vascular cells on cortical NSC fate choice, we developed a novel coculture system that utilizes Transwell® membranes and primary cultures that include an enriched NSC population, brain-derived fibroblast/perivascular cells, and brain-derived endothelial cells. This porous membrane system allows for investigation of juxtacrine, paracrine, and autocrine interactions that influence gene expression to direct cell fate. We conducted a transcriptome-wide analysis of altered gene expression in the NSCs in response to vascular coculture. Ingenuity pathways analysis (IPA®) identified several key pathways that were enriched in the dataset including those linked to Leukemia inhibitory factor (Lif) signaling. We observed an upregulation of Glial fibrillary acidic protein, Gfap, in NSCs cultured in vascular coculture conditions. We used an inhibitor of the Polycomb-repressive complex 2 (PRC2) subunit, Ezh2, and quantified differences in Gfap expression in NSC derived from E11.5 and E13.5 NSC. These time points bracket a window of early forebrain angiogenesis that accompanies rapid proliferation of NSC and the expansion of the neuroepithelium. The E11.5 NSC increased Gfap expression in response to vascular cell conditioned medium (CM) and Gfap levels further increased with Ezh2 inhibition. In contrast, the Ezh2 inhibitor had no effect on the response of the E13.5 NSC to vascular cell CM. Our findings indicate that vascular investment in the developing cortex can contribute to NSC fate decisions via soluble and contact-mediated cell-cell interactions. Further, vascular cells may help direct the shift from neurogenesis to gliogenesis during a window of NSC sensitivity in early cortex formation.

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