Fig. 4. xTGIF2 is required in vivo for establishing the pancreatic region within the endoderm. (A) Both dorsal vegetal (VD) blastomeres of eight-cell stage embryos were injected with a combination (5 ng each) of two antisense morpholino oligonucleotides (TGIF2-Mo) targeting both Xenopus laevis TGIF2 pseudoalleles (see Fig. S3 in the supplementary material). TGIF2-Mo-injected VD, uninjected VD and ventral vegetal (VV) explants were dissected at stage 10 and assayed for expression of the indicated markers by RT-PCR analysis. (B) Whole-mount in situ hybridization using Pdx1 probe. Embryos injected with TGIF2-Mo showed reduction of Pdx1 expression domain (80%; n=40). Pdx1 staining was rescued in embryos injected with TGIF2-Mo and mouse Tgif2 (mTgif2; 500 pg) mRNA (70%; n=25). Embryos left untreated show normal Pdx1 expression domain in the pancreatic/duodenum endoderm, as indicated by the white bracket. (C) Whole-mount in situ hybridization with Hex. Embryos injected with TGIF2-Mo or left untreated were stained at stage 34. Hex expression domain in the hepatic endoderm is indicated by the yellow bracket. (D) Analysis of the expression of the pancreatic differentiation markers, insulin and amylase, by whole-mount in situ hybridization. Arrowheads indicate normal domains of expression of insulin and amylase in the pancreatic bud of tadpole gut tubes.