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Fig. 1. Repression of β-catenin signaling in the endoderm is necessary and sufficient for liver and pancreas development. (A) 32-cell stage Xenopus embryos were injected with either a pCSKA-Wnt8 plasmid (250 pg) or stabilized pt-β-catenin RNA (250 pg) in the D1 anterior endoderm cells. Other embryos were injected with RNA encoding Dkk1 (500 pg) or Gsk3β (500 pg) into D4 posterior endoderm cells to repress Wnt signaling. (B) In situ hybridization at stage 35 with the liver marker for1, or with a combination of pancreas/duodenum marker pdx1/xlhbox8 and the lung marker nkx2.1, or with the intestinal marker endocut. Some embryos were hybridized with just pdx1. Arrowheads indicate ectopic or repressed gene expression. The solid red line indicates the relative size of the foregut domain. Gut tubes were isolated at stage 42 to visualize organ bud morphology. The dashed red line outlines the liver bud. L, liver; P, pancreas; Lu, lungs. (C) In situ hybridization to Gsk3β-injected guts with liver markers for1, ambp, the early pancreas marker ptf1a and the exocrine pancreas marker elastase. (D) A sectioned embryo co-injected with Gsk3β and β-gal RNA shows β-gal-staining nuclei (blue) and for1 expression (brown) localized to the endoderm. |
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Fig. 2. Temporal regulation of β-catenin/Tcf activity during endoderm pattering. (A) At the 32-cell stage, Xenopus embryos were injected in the anterior D1 cells with RNA encoding the fusion protein GR-LEFδN-βCTA (800 pg), which constitutively activatesβ -catenin target genes in the presence of dexamethasone (Dex). Dex (1μ M) was added to the media of injected embryos at the indicated stages and embryos were assayed by for1, pdx1 and endocut in situ hybridization at stage 35. (B) Addition of Dex to GR-LEFδN-βCTA-injected embryos from stage 30 to 42, followed by hhex in situ, revealed enlarged liver buds. (C) 32-cell stage embryos were injected in posterior D4 cells with RNA encoding GR-δNTcf3 (800 pg), which represses β-catenin/Tcf target genes when activated. Dex (1 μM) was added to the media of injected embryos at the indicated stages and embryos were assayed by for1, pdx1 and endocut in situ hybridization at stage 35. (D) GR-δNTcf3 was injected into D1 cells at the 32-cell stage, and when Dex was added from stages 30 to 42 some embryos exhibited smaller liver buds based on for1 in situ hybridization. No effect was observed in uninjected embryos treated with Dex. |
