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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. |
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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. 5. Regulation and function of Xenopus hhex. (A) Analysis of hhex expression by in situ hybridization to bisected stage-18 embryos (anterior left). (a) Schematic of a stage-18 bisected embryo showing the presumptive foregut (fg, green) and hindgut domain (hg). (b) Injection of GR-LEFδN-βCTA RNA (800 pg) into the D1 anterior endoderm cell has no effect without Dex. (c) Addition of Dex (1 μM) at the midgastrula repressed hhex expression as does (d) D1 injection of stabilized pt-β-catenin RNA (250 pg). (e) Uninjected control embryo. (f) Injection of δNTcf3 RNA (800 pg) or (g) Gsk3β RNA (500 pg) in posterior D4 cells results in ectopic hhex expression (arrowhead). (h) Co-injection of Gsk3β and β-gal RNA reveals that the blue β-gal stain co-localizes with ectopic hhex in the endoderm. (B) Hhex is required for liver and pancreas development. 32-cell stage embryos were injected with either an antisense hhex morpholino oligo (HexMO, 80 ng) in the D1 cells or with Gsk3β or Gsk3β plus HexMO in D4 cells. At stage 35, embryos were assayed by in situ hybridization with liver (for1) or pancreas/duodenum (pdx1) probes. |
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Fig. 7. Vent2 mediates β-catenin function. (A) Xenopus embryos were injected with the indicated hhex:luciferase constructs with or without Vent2 RNA (500 pg) in D1 anterior or D4 posterior cells at the 32-cell stage. The bar chart shows the normalized relative luciferase activity at gastrula stage, indicating that Vent2 represses the hhex promoter. (B-D) In situ hybridization of bisected stage-18 embryos with the probes indicated. (E) Injection of Gsk3β RNA (500 pg) in the posterior endoderm repressed vent2 expression. (F) Embryos were injected at the 32-cell stage with Vent2 RNA in anterior D1 cells or in posterior D4 cells with either Gsk3β or Gsk3β plus Vent2, followed by in situ hybridization at stage 18 with hhex, and stage 35 with for1 or pdx1 probes. (G) These data suggest a molecular pathway in which Wnt/β-catenin signaling promotes vent2 expression and Vent2 represses hhex transcription. |
