Fig. 1. Foxf mutants have aganglionic megacolon with smooth muscle hypoplasia and occasional anal atresia. (A,B) Lower gastrointestinal tract of E18.5 wild-type (A) fetus and Foxf1^-/+; Foxf2^-/+ litter mate (B). Arrowheads indicate the colon, which is thin-walled and distended in the mutant. (C) Vibratome section of the intestine of a wild-type E12.5 embryo, whole-mount in situ hybridized with a Foxf2 probe, showing Foxf2 expression in the mesenchyme next to the endodermal epithelium. (D,E) Higher magnification of the colon in A and B. (F) Anal atresia and megacolon in E18.5 Foxf2^-/- mutant. (G-I) Hematoxylin and Eosin stained sagittal sections through the rectum and lower colon of E18.5 wild-type (G), Foxf1^-/+; Foxf2^-/+ (H) and Foxf2^-/- (I); insets show higher magnifications of the distal colon wall, which is thin and flat in the mutants. (J,K) Thin (1 µm) section of wild-type (J) E18.5 distal colon showing the stratification with epithelium (ep), mesenchyme (mc), circular (cm) and longitudinal (lm) musculature, and mesothelium (mt). The mesodermal layer in the Foxf1^-/+; Foxf2^-/+ (K) is hypoplastic and consists of loosely associated, poorly differentiated SMCs. (L,M) Immunostaining with antiserum against the neuronal marker neurofilament shows the plexus of enteric nerves in the mesodermal layer of E18.5 wild-type distal colon (L), whereas no neurons are detected in the mutant (M; Foxf1^-/+; Foxf2^-/+). Insets show the distal colon wall at higher magnification and arrowheads indicate the enteric plexus. Staining in the gut lumen is due to the non-specific stickiness of the meconium and goblet cell mucins. (N,O) Immunostaining with anti-SMA reveals SMC hypoplasia in Foxf2^-/- distal colon (arrowheads), but normal SMC investment of arteries (Ar).