Fig. 2. Hhip (Hip1 in figure) function is required for inhibition of Hh signaling and proper pancreas morphogenesis. Staining for Hhip promoter regulated ß-galactosidase activity in heterozygous and homozygous embryos (A,B; E12.5). ß-galactosidase activity in stomach (arrowhead) and duodenum (arrows) is significantly increased in Hhip^–/– embryos. `Real time' PCR analysis of Gli expression in E17.5 pancreas (C). Gli expression levels are shown relative to the level of actin mRNA. To facilitate comparison, expression in wild-type pancreas (white bar) has been adjusted to `1'. Hhip^+/– (2.7±1.4; light-gray bar) and Hhip^–/– mutant pancreas (7.8±0.6; dark-gray bar). Error bars shown are ±s.d. Pancreas and spleen (arrows) are deformed in Hhip^–/– embryo at E18.5 (F,G) compared with wild type (D,E). Higher magnification reveals that the connection between ventral pancreas and duodenum is confined to the dorsal region of the duodenum (E, broken line) in wild-type embryos but extends laterally in Hhip^–/– embryos (G, broken red line). (H-K) In some cases, ectopic pieces of pancreas are integrated within the duodenum (red arrows), as shown by Feulgen staining (I) and staining for amylase (K), a marker of pancreatic exocrine cells. d, duodenum; dp, dorsal pancreas; li, liver; s, stomach; sp, spleen; vp, ventral pancreas.