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Vascular endothelial growth factor (VEGF) regulation of early forebrain neuralepithelium development.

Purpose: We investigated the role of VEGF in regulating neural development in early mouse forebrain. VEGF exists in the mouse primarily as three isoforms, VEGF 120, 164, and 188, that differ in their ability to bind heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Our hypothesis is that the bioavailability of VEGF mediates vascular cell-radial glia interactions, including radial glia proliferation and differentiation during developmental neurogenesis and angiogenesis. Therefore, we investigated proliferation of neural stem cells and differentiation of the intermediate progenitor cells in primitive cortex in a transgenic mouse model of altered VEGF isoform expression. We examined an embryonic (E) time point associated with extensive neuralepithelial expansion and vascular investment preceding cortical layer formation.

Methods: E11.5 mouse embryos expressing all or only single VEGF isoforms were fixed in 3.7% buffered paraformaldehyde, equilibrated in 30% sucrose and cryosectioned at 30 [micro]m. Sections were permeabilized in 3% donkey serum, 1% goat serum, 1% bovine serum albumin, and 0.1% Triton-X 100 in phosphate-buffered saline overnight. Primary antibody (Ab) incubation was overnight with either Phospho-Histone H3 (PHH3, 1:200) to label mitotic cells or T-box related protein 2/eomes (Tbr2, 1:400) to identify the intermediate progenitor cells. A Horseradish peroxidase-conjugated secondary Ab was used to visualize bound primary Ab and developed using Vectastain ABC kit and diaminobenzidine as a substrate. Sections were counterstained with methyl green and quantified for the number of PHH3- and Tbr2-positive cells and total nuclei in the forebrain neuralepithelium using design-based stereology with Stereo-Investigator software (MicroBrightfield, V.9). An optical fractionator optical dissector probe (75 [micro][m.sup.2]) was used with a randomly placed grid of 150 [micro]m X 200 [micro]m and a 10-section counting interval.

Results: Quantification of proliferating cells showed that the VEGF 120 mice have significantly reduced numbers of PHH3-positive cells compared to wildtype (Figure 1A; p=0.015, t test). This decrease in proliferating cells corresponded to a decrease in total cell number (Figure 1B; p=0.02, t test) and neuralepithelial volume (Data Not Shown, p=0.007, t test) in the VEGF 120 forebrain. Quantification of differentiating cells showed that there was no change in Tbr2+ positive cells (Figure 1C; p=0.87, t test). The ratio of intermediate progenitor cells relative to proliferating cells was significantly higher in VEGF 120 forebrain (Figure 1D; p=0.0001, t test) reflecting a shift in proliferating versus differentiating precursor populations.

Conclusions: Loss of the localized VEGF isoforms (VEGF 164 and VEGF 188) in the VEGF 120 mice reduces proliferation of neural stem cells at the ventricular surface in the developing forebrain leading to a decrease in overall neuralepithelial cell number and volume. However, the VEGF120 mice have comparable numbers of Tbr2-positive cells indicating that the diffusible VEGF 120 isoform is sufficient to allow specification and survival of the intermediate progenitor population. These data support a critical role for VEGF and its bioavailability during early cortical development.


(1) Cain, J.T. *, (1) Berosik, M.A., (1) Frisch, S., (1) Odens, EW., (1) Urquhart, S. (1) Dvorak, S., and (1) Darland, D.C.

(1) Biology Department, University of North Dakota, Grand Forks, North Dakota, United States of America
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Title Annotation:Graduate communications in the A. Roger Denison Competition
Author:Cain, J.T.; Berosik, M.A.; Frisch, S.; Odens, P.W.; Urquhart, S.; Dvorak, S.; Darland, D.C.
Publication:Proceedings of the North Dakota Academy of Science
Date:Apr 1, 2011
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