Figure 3

En2 KO GNPs exhibit normal survival but diminished response to neuritogenic signals compared to WT GNPs. KO and WT GNPs were cultured in defined media without and with PACAP (10 nM), IGF1 (10 ng/ml) or both, and the percent of living, neurite-bearing cells was assessed under phase microscopy. (A) KO and WT GNP morphology was qualitatively similar in response to PACAP and IGF1, though numbers of neurite-bearing cells differed, as quantified in B-C. Arrows denote neurites extending many cell bodies from neuronal somas. Arrowheads identify cells without neurites >2 cell somas. (B) PACAP and IGF1 each induced fewer neurite-bearing KO GNPs compared to WT GNPs. However, in combination IGF1 and PACAP stimulated KO GNP neuritogenesis equivalent to WT. For each group, 2–3 dishes were assessed in each experiment for each genotype, derived from three experiments, yielding N = 6-9 dishes/group/genotype. (C) Anti-mitogenic growth factors failed to overcome differentiation deficits observed in the absence of En2. FGF2 reduced neurite outgrowth in both genotypes, whereas IGF1 again stimulated neuritogenesis in WT cells only. In combined factor treatment, WT cells exhibited outgrowth equivalent to control media, but KO cells displayed outgrowth inhibition similar to FGF2 treatment alone. These data suggest cell cycle exit and differentiation are mediated through different pathways in GNPs, and the absence of En2 reduces IGF1 neuritogenic effects. N = 6-14 dishes/group/genotype. (D) Cell survival is similar in 24-h culture in WT and KO GNPs in response to trophic factors (compared to counts at 2 h; 200–400 cells counted/dish at 24 h), suggesting differences in DNA synthesis and neuritogenesis are unlikely to reflect differences in survival or death. N = 6-11 dishes/group/genotype. t-test *, compared between genotypes, p ≤ 0.05; t-test #, compared to genotype control, p ≤ 0.05; ## p ≤ 0.01; ### p ≤ 0.001; t-test %, compared within genotype, p ≤ 0.0001.