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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. |
