Figure 2
IGF1 stimulates greater DNA synthesis in En2 KO GNPs in culture and in vivo . (A) Mitogenic stimulation of KO and WT GNPs was measured in 24 h cultures using 3H-dT incorporation. IGF1 (10 ng/ml) and high-dose insulin (10 μg/ml) differentially increased KO DNA synthesis compared to WT, while Shh (3 μg/ml) elicited similar increases in both genotypes. Co-administration of IGF1 and Shh synergistically increased DNA synthesis, though differential genotype responses to IGF1 remained. Data presented as percent control 3H-dT incorporation ± SEM; control: 350–550 cpm/well; growth factors: 800–2,200 cpm/well; 4–6 mice/genotype/experiment; 3–6 wells/treatment/experiment, derived from three experiments. (B) IGF1 elicited differential genotype responses at doses above 1 ng/ml (Note: IGF1 elicited 3-4-fold increased KO DNA synthesis in Figure 2A-B; WT responses were less consistent across experiments.) N = 6-9 wells/group, from 3 experiments. (C) Anti-mitogens reduced DNA synthesis similarly across genotypes. FGF2 (10 ng/ml) and PACAP (10 nM) attenuated, but did not abolish, differential genotype responses to IGF1. N = 6-9 wells/group from three experiments. (D) In vivo, IGF1 (10μg/gbw) also differentially increased cerebellar 3H-dT incorporation (see Methods). 3H-dT incorporation after saline injection was not different between genotypes (WT = 73% + 1.6; KO = 66% + 3.5; p = 0.08). IGF1 significantly increased DNA synthesis over saline injection in both genotypes, however WT DNA synthesis increased 9.7% while KO DNA synthesis increased 28%. Two-way ANOVA yielded significant genotype × IGF1 interaction. KO: N = 9 saline, N = 6 IGF1; WT: N = 12 saline, N = 6 IGF1; N = 6 experiments, a minimum of one pup per genotype was injected with saline and IGF1 in each experiment. Con: Control; F: FGF2; P: PACAP: t-test *, compared between genotypes, p < 0.05; **p < 0.01; ***p < 0.001; t-test #, compared to genotype control, p < 0.05; ##p < 0.01; ###p < 0.001; %, compared within genotype, p < 0.05; $: two-way ANOVA, IGF1 F(1,29) = 28.5, p < 0.0001, genotype F(1,29) = 0.11, p = 0.74, genotype × IGF1 interaction F(1,29) = 4.88, p = 0.035.