Fig. 4.

Shh overexpression inhibits ENCC proliferation and promotes neuronal differentiation. Chick E5 hindgut was cultured for 3 days with or without Shh protein or cyclopamine. The rate of ENCC proliferation without additive (A, example of BrdU⁺ p75⁺ ENCC shown in inset) was 29%, and was markedly reduced by Shh protein (B) and increased by cyclopamine (C), as shown quantitatively (D). Cultured guts were stained for activated caspase 3 and Tuj1, and no apoptotic enteric neurons were observed in control (E) or Shh-treated (F) intestine. Apoptosis was seen in an E8 dorsal root ganglion (DRG) used as a positive control (G). Tuj1 and neurofilament, which are markers of early and late neuronal differentiation, respectively, show that Tuj1⁺ neurons are present in all treatment groups (H-J′), while neurofilament is increased following Shh treatment (H″-J″). *P<0.002, **P<0.0004.