Title

Combined in vivo Fate Mapping of the Immediate Progeny and Complete Lineage of Radial Glia Reveals Progenitor Diversity in Embryonic Neocortex

Date of Completion

January 2010

Keywords

Biology, Neurobiology|Health Sciences, Human Development

Degree

Ph.D.

Abstract

Radial glia cells (RGCs) are a class of neural progenitor cells that generate neurons and subsequently astrocytes in the cerebral cortex. The degree to which RGCs are heterogeneous in terms of their capacity to produce neurons versus astrocytes at different developmental periods remains largely unknown. Studies using techniques such as transgenic mouse models and retrovirus have advanced our understanding of RGC lineage in the CNS, however further work is required to determine if subpopulations of RGCs exist in the dorsal telencephalic VZ, and if these subpopulations differentially contribute to neurons and astrocytes of the mature brain. ^ The overall objective of this thesis is to study diversity across the embryonic RGC population and map the fates of their progenies. To this end, I utilized in utero electroporation (IUE) to transfect subsets of RGCs in vivo by two complementary genetic strategies amenable to IUE, the Cre/loxP recombination system and the piggyBac transposition system. The episomal Cre/loxP plasmids allowed us to label the terminally differentiated 'immediate progeny' of RGCs from mid- and late neurogenesis. Four promoters, Nestin, Talpha1, GLAST and GFAP were used to drive Cre recombination of floxed eGFP. We found that the immediate progeny of mid-neurogenic RGCs were neurons of similar identity across promoter conditions. In contrast, the immediate progeny generated from late stage RGCs differed: while Nestin+ and Talpha1+ RGCs gave rise to mostly neurons, GLAST+ and GFAP+ RGCs generated a greater ratio of cortical astrocytes to neurons. This finding suggests that progressive diversification exists among subsets of RGCs from mid-to late embryogenesis. To determine if RGCs were restricted in their fates to generate neurons versus astrocytes throughout neural development, I traced the complete cell lineage of RGCs. PiggyBac transposition driven by previously mentioned four promoters was used to insert eGFP into the genome of progenitor cells. While lineage tracing of Nestin+ and GLAST+ RGCs from early neurogenesis showed primarily neuronal progeny, lineage tracing from mid-neurogenesis revealed that Nestin+ progenitors mostly generated neurons while GLAST+ progenitors generated primarily astrocytic lineages. Embryonic analysis of the overlap between Nestin+ and GLAST+ progenitor populations revealed a distinct GLAST+/Nestin- progenitor subset. Our results suggest a dynamic GLAST+ progenitor pool that diverges at mid-neurogenesis from the consistently neuronal Nestin+ population, switching from generating mostly neurons to mostly astrocytes. Taken together, my thesis provides evidence for progressive diversification among RGCs that contributes differentially towards neuronal and astrocytic production. In vivo evidence for the presence of neuronally restricted and progressively changing progenitor pools is critical to our understanding of normal brain development and use of therapeutic approaches involving neural stem cells. ^