Fig. 3. Development of neurons, but not glia, in explants of fetal gut is Hand2-dependent. (A) Transcripts encoding Phox2b detected by reverse transcriptase (RT)-PCR in fetal mouse gut at E9.5 in wild-type (+/+), Hand2^+/- and Hand2^-/- mice. (B) Sox10+, Phox2b+ and Ret+ cells are detected at E9.5 in the outer mesenchyme of wild-type (H2+/+) and Hand2^-/- (H2-/-) gut. (C) p75^NTR+ cells are found in longitudinal sections cut through the foregut of wild-type and Hand2^-/- mice. (D) Hu+, PGP9.5+ and NSE+ neurons develop in cultures of wild-type gut explanted at E9.5. (E) B-FABP+ (red), as well as Hu+ (green), neurons develop in cultures of wild-type gut explanted at E9.5. B-FABP+ glia, but no Hu+ neurons, develop in cultures of Hand2^-/- gut explanted at E9.5. (F-H) Sox10 (F), Phox2b (G) and Ret (H) immunoreactivities are found in cultured explants of E9.5 gut from Hand2^-/- and wild-type mice. (I) p75^NTR+ precursors (red) are present in explants of E9.5 gut from wild-type and Hand2^-/- mice; however, Hu+ neurons (green) develop in vitro only in the explants of wild-type, and not Hand2^-/-, gut. (J) Co-culture of E9.5 Hand2^-/- gut with somites containing primordial dorsal root ganglia. Hu+ neurons (green) develop. (K) Differentiation of neurons occurs when Hand2^-/- explants are rescued by transfection with Hand2; neurons co-express Hu (green) and Hand2 (red) immunoreactivity. Ctrl, control. Scale bars: 50 µm in B-J; 10 µm in K.