ABSTRACT
Regulation of neural stem cell (NSC) fate decisions is critical during the transition from a multicellular mammalian forebrain neuroepithelium to the multilayered neocortex. Forebrain development requires coordinated vascular investment alongside NSC differentiation. Vascular endothelial growth factor A (Vegf) has proven to be a pleiotrophic gene whose multiple protein isoforms regulate a broad range of effects in neurovascular systems. To test the hypothesis that the Vegf isoforms (120, 164, and 188) are required for normal forebrain development, we analyzed the forebrain transcriptome of mice expressing specific Vegf isoforms, Vegf120, VegfF188, or a combination of Vegf120/188. Transcriptome analysis identified differentially expressed genes in embryonic day (E) 9.5 forebrain, a time point preceding dramatic neuroepithelial expansion and vascular investment in the telencephalon. Meta-analysis identified gene pathways linked to chromosome-level modifications, cell fate regulation, and neurogenesis that were altered in Vegf isoform mice. Based on these gene network shifts, we predicted that NSC populations would be affected in later stages of forebrain development. In the E11.5 telencephalon, we quantified mitotic cells [Phospho-Histone H3 (pHH3)-positive] and intermediate progenitor cells (Tbr2/Eomes-positive), observing quantitative and qualitative shifts in these populations. We observed qualitative shifts in cortical layering at P0, particularly with Ctip2-positive cells in layer V. The results identify a suite of genes and functional gene networks that can be used to further dissect the role of Vegf in regulating NSC differentiation and downstream consequences for NSC fate decisions.
Subject(s)
Cell Differentiation/physiology , Cell Proliferation , Neural Stem Cells/physiology , Prosencephalon/physiology , Transcriptome/genetics , Vascular Endothelial Growth Factor A/genetics , Animals , Blotting, Western , Central Nervous System/blood supply , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Epithelium/physiology , Female , Gene Expression , Gene Expression Profiling , Genotype , Immunohistochemistry , Mice , Mice, Transgenic , Microarray Analysis , Mitosis/genetics , Pregnancy , Prosencephalon/cytology , Real-Time Polymerase Chain Reaction , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factor A/metabolismABSTRACT
This work was designed to determine the role of the vascular endothelial growth factor A (VEGF) isoforms during early neuroepithelial development in the mammalian central nervous system (CNS), specifically in the forebrain. An emerging model of interdependence between neural and vascular systems includes VEGF, with its dual roles as a potent angiogenesis factor and neural regulator. Although a number of studies have implicated VEGF in CNS development, little is known about the role that the different VEGF isoforms play in early neurogenesis. We used a mouse model of disrupted VEGF isoform expression that eliminates the predominant brain isoform, VEGF164, and expresses only the diffusible form, VEGF120. We tested the hypothesis that VEGF164 plays a key role in controlling neural precursor populations in developing cortex. We used microarray analysis to compare gene expression differences between wild type and VEGF120 mice at E9.5, the primitive stem cell stage of the neuroepithelium. We quantified changes in PHH3-positive nuclei, neural stem cell markers (Pax6 and nestin) and the Tbr2-positive intermediate progenitors at E11.5 when the neural precursor population is expanding rapidly. Absence of VEGF164 (and VEGF188) leads to reduced proliferation without an apparent effect on the number of Tbr2-positive cells. There is a corresponding reduction in the number of mitotic spindles that are oriented parallel to the ventricular surface relative to those with a vertical or oblique angle. These results support a role for the VEGF isoforms in supporting the neural precursor population of the early neuroepithelium.
