Figure 4
The Gpc1 mutant brain phenotype emerges early in neural development. (A) Lateral images of embryonic day (E)8.5, E9.5 and E11.5 wild-type (+/+) and Gpc1-/- (-/-) embryos. Because the developmental stage of embryos is highly variable at E8.5 to E9.5, not every image is fully representative of the average for that genotype (for quantitative data, see (B, C) below). (B) Gpc1-/- embryos display smaller brains starting at E9.5. Brain size was estimated from photographic images, as described in Additional file 2. Averages of estimated cranial area (at E8.5 and E9.5) and cranial volume (E11.5) were obtained for wild-type and homozygous mutant embryos in multiple litters (N = 23 for +/+ and N = 15 for -/- at E8.5; N = 25 for +/+ and N = 22 for -/- at E9.5; N = 10 for +/+ and N = 18 for -/- at E11.5). Units are pixels2 (× 104) and pixels3 (× 106) for area and volume measurements, respectively. Note the 14.1% decrease in area (* and **P < 0.05) that emerges at E9.5 (consistent with an approximately 20.4% decrease in volume). (C) Comparison of cranial area with somite number in E8.5 and E9.5 wild-type (+/+) and Gpc1-/- (-/-) embryos. Linear regression lines demonstrate that, even when variation in somite number is controlled for, Gpc1-/- brains are of normal size at E8.5, and abnormally small at E9.5. (D-F) Quantification of cell proliferation in the embryonic brain. Sagittal sections of E9.5 embryos were immunostained for phosphohistone H3 (PHH3) (D), counterstained with bizbenzamide (E) and the two images merged (F). Note the concentration of labeled cells along the pial and ventricular margins of the neuroepithelium (outlined). (G, H) Sets of serial sections were stained in this manner from multiple wild-type and Gpc1-/- embryos at E8.5 (N = 5 for both +/+ and -/-), E9.5 (N = 8 for +/+ and N = 7 for -/-), and E11.5 (N = 7 for +/+ and N = 6 for -/-), and numbers of PHH3-labeled cells within the neuroepithelium were counted and normalized to neuroepithelial area for either the entire embryo (G) or the forebrain alone (H). The data show a statistically significant (*P < 0.001) decrease in PHH3-labeling index at E9.5, but no change at E11.5.