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Here we show that Bmp signaling is necessary and sufficient for the specification of ventral endoderm in Xenopus embryos. Overexpression of Bmp4 in ectoderm induces markers of endoderm, including Sox17beta, Mixer, and VegT, but cannot induce the expression of the dorsoanterior markers, Xhex and Cerberus. Furthermore, knockdown approaches using overexpression of Bmp antagonists and morpholinos designed against Bmp4, Bmp2, and Bmp7 demonstrate that Bmp signaling is critical for ventral, but not dorsoanterior endoderm formation. This activity is not simply a result of embryonic dorsalization as markers for dorsal endoderm are not expanded. We further show that endodermal cells of either ventral or dorsal character do not form when both Wnt and Bmp signals are abolished. Overall, this report strongly suggests that Bmp plays an essential role in ventral endoderm specification.
Figure 3. Bmp antagonists disrupt early endoderm formation. One-cell embryos were injected vegetally with mRNA encoding the following Bmp Antagonists. B: DN-Alk3 (0.5 ng). C: Chordin (0.5 ng). D: Noggin (0.5 ng). The embryos were assayed by in situ hybridization with a probe for Sox17 beta at stage 10.5. A: A control injected with water. F-J: Two-cell-stage embryos were injected vegetally with 200 pg of lacZ mRNA, and mRNA for Chordin or Noggin as indicated. Embryos were fixed at stage 11, stained for lacZ using red gal substrate, and analyzed for Sox17 beta expression.
Figure 4. Bmp signaling is essential for endoderm formation in Xenopus. X. tropicalis embryos were injected at the two-cell stage with 10 ng per blastomere of each MO indicated, then analyzed for expression of Sox17 alpha (A, B) or Mixer (B) at stage 10.5. Miniruby fluorescent dextran was used as a tracer for MO injection. In B, a subset of MO-injected embryos were re-injected in one ventralblastomere at the 4-cell stage with 0.5 ng of mouse Bmp4. Nuclear beta -galactosidase (red) was used as a tracer. C: Triple Bmp morphant embryos were also analyzed for expression of Xbra, Derriere, Myf5, and Vent2.
Figure 5. Combination of Wnt and Bmp signaling is required for endoderm formation. X. tropicalis embryos were injected with 10 ng per blastomere of each MO indicated at the 2-cell stage, then analyzed at stage 10.5 for expression of the general endoderm markers Sox17 alpha and Mixer, or the dorsoanterior endoderm markers Cerberus and Xhex.
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