Fig. 1. Hand genes are expressed and developmentally regulated in the gut. (A) Transcripts encoding Hand2 (red), Hand1 (green), Phox2b (blue) and Mash1 (purple) quantified relative to ß-actin and plotted as a function of age. (B,C) ß-galactosidase expressed under the control of the Hand1 promoter in transgenic mice to locate sites of Hand1 expression. ß-galactosidase immunoreactivity visualized by confocal microscopy (green). Hu (B; red) or Kit (C; red) immunoreactivities co-localized with that of ß-galactosidase to identify, respectively, neurons and interstitial cells of Cajal. (D) In situ hybridization reveals Hand1 transcripts in muscle layers, but not ganglia, of adult mouse intestine, whereas transcripts encoding Hand2 are located in neurons within ganglia, but not smooth muscle. (E) Transcripts encoding Hand1, but not those encoding p75^NTR, are detected, by reverse transcriptase (RT)-PCR, in a preparation from which crest-derived (CD) cells have been removed by immunoselection (nCD). Transcripts encoding Hand1 cannot be detected in a p75^NTR-rich immunoselected preparation of crest-derived cells. (F) Transcripts encoding Hand2 are detected by RT-PCR in mucosa-free preparations of bowel wall (ext wall) but not in isolated mucosa from adult mouse duodenum (d), ileum-jejunum (i) and colon (c). (G,H) Transcripts encoding Hand2 were found in Hu+ (G; green) and B-FABP+ (H; red) cells. Arrows in G show Hand2 transcripts in cells that are not Hu⁺. d, duodenum; c, colon; CD, crest-derived; CM, circular muscle; G, ganglion; i, ileum-jejunum; LM, longitudinal muscle; M, size markers; nCD, non-crest-derived; pl, plasmid carrying DNA encoding Hand2 (positive control). Scale bars: 50 µm.