Figure 2.
HGF promoted neurogenesis and neurite growth in cultured E12.5 ENS precursor cells. A–C, E12.5 ENS precursors immunoselected with p75NTR antibody were maintained in culture for 48 h in the presence of HGF plus 1 pg/ml GDNF before TuJ1 and/or MET immunohistochemistry and DAPI nuclear staining. All TuJ1+ enteric neurons were MET-IR. D–F, M, N, HGF caused a dose-dependent increase in TuJ1-IR neuron number and neurite length. *p < 0.05, ANOVA with Dunn's multiple-comparison test. F, M, N, MET blocking antibody (Aby) reduced TuJ1+ neuron number and neurite length in surviving cells. Control (Ctrl) is 50 ng/ml HGF plus 1 pg/ml GDNF. **p < 0.01, Student's t test. G–L, O, P, When ENS precursors were grown in GDNF alone, the MEK inhibitor PD98059 (PD) had no effect on neuron number or neurite length, but the PI-3K inhibitor LY294002 (LY) reduced neuron number and neurite length. In contrast, in HGF (50 ng/ml) plus GDNF (1 pg/ml)-treated cells, both MEK and PI-3K inhibition reduced neurite length (P), whereas only PI-3K inhibition reduced neuron number (O). *p < 0.01, ANOVA with Dunn's multiple-comparison test. Scale bar in C applies to A–F. Scale bar in L applies to G–L. (N ≥ 3 biological replicates/group; 12 individual wells/group).