Figure 6
Loss of Gpc1 results in weakened fibroblast growth factor (FGF) signaling and premature neuronal differentiation in brain. (A-D) In situ hybridization for Pyst1. (E-H) In situ hybridization for Sprouty 2 (Spry2). Both are markers of FGF signaling and only for Spry2 was a noticeable change observed between genotypes, with apparent reduced expression in Gpc1-/- embryos at both embryonic day (E)8.5 and E9.5. (I) Real-time quantitative RT-PCR of Pyst1, Spry1 (a third FGF-target gene) and Spry2 from RNA isolated from E9.5 wild-type and Gpc1-/- forebrains. The data imply a significant reduction (32.5%) in Spry2 message levels in the Gpc1 mutant (*P < 0.01), and a possible reduction in Spry1. (J) Erk enzymatic activity was measured by in vitro phosphorylation in E9.5 dorsal forebrain explants from Gpc1+/+ and Gpc1-/- embryos. Such explants display a high level of basal (unstimulated) Erk activity, presumably due to the actions of endogenous growth factors. Erk activity was found to be 41% lower in mutant tissue than in wild type (*P < 0.005; units are cpm × 104). (K, L) Sagittal sections of E9.5 Gpc1+/+ (K) and Gpc1-/- (L) embryos were immunostained with Tuj1 (red), a marker for postmitotic neurons, and counterstained with the nuclear marker bizbenzamide (blue). (K', L') Enlarged images from white boxes in (K) and (L), corresponding to similar locations in the anterior midbrain. Note the greater density of Tuj1-positive cells in the mutant midbrain. White arrowheads in (K, L) point to the ventral forebrain, where virtually no Tuj1-positive cells are found in the wild-type, but several are present in the Gpc1-/- brain. (M, N) Numbers of Tuj1-positive cells within the forebrain (FB) and midbrain (MB) were totaled over serial sections of whole embryos at E8.5 (M) and E9.5 (N). Note the >2-fold increase in Tuj1-positive cells in Gpc1-/- brains at E9.5. In contrast, no significant difference is observed at E8.5. At E9.5, N = 7 for +/+ and N = 4 for -/-; at E8.5, N = 4 for each genotype. *P < 0.005.