Bmp signaling is necessary and sufficient for ventrolateral endoderm specification in Xenopus - PubMed (original) (raw)

Bmp signaling is necessary and sufficient for ventrolateral endoderm specification in Xenopus

Andrea Wills et al. Dev Dyn. 2008 Aug.

Abstract

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.

Copyright (c) 2008 Wiley-Liss, Inc.

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Figures

Figure 1

Figure 1. Bmp4 and Alk3Q233D induce endodermal markers

(A) Embryos were injected with doses of Bmp4 mRNA ranging from 0.1 ng to 1.0 ng. Ectoderm was explanted from these embryos, harvested at stage 10.5 and analyzed by RTPCR for the expression of _Sox17_β and Xbra. (B) Embryos were injected with 0.5 or 1.0 ng Bmp4. Ectoderm was explanted and harvested at stage 10.5. RT-PCR was performed using primers for Sox17β, VegT, Mixer, Mix.1, Cerberus (Cer). (C) Embryos were injected with 0.5ng of Bmp4 and explants were performed as in (A), but with primers for Xhex or Xhox3, Msx1, and _Sox17_β. (D) Embryos were injected with 0.5 ng Alk3 Q233D and explants were performed as in (A). ODC was used as a loading control for all experiments (A – D). WE, whole embryo; -RT, minus reverse transcriptase.

Figure 2

Figure 2. Bmp2, Bmp4, Bmp7 and Alk3 are expressed in vegetal cells

Animal and vegetal hemispheres were explanted at stage 8.0, cultured and harvested at stage 10.5. RT-PCR analysis was performed using primers for _Bmp2, Bmp4, Bmp7, Alk3, Sox17_β and Xbra. cDNA amounts were normalized. ODC was used as a loading control. WE, whole embryo; -RT, minus reverse transcriptase.

Figure 3

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), and (D) Noggin (0.5 ng) and assayed by in situ hybridization with a probe for _Sox17_β at stage 10.5. (A) is a control injected with water. In F-J, 2-cell stage embryos were injected vegetally with 200pg 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_β expression.

Figure 4

Figure 4. Bmp signaling is essential for endoderm formation in Xenopus

X. tropicalis embryos were injected at the two-cell stage with 10ng per blastomere of each MO indicated, then analyzed for expression of _Sox17_α (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 ventral blastomere at the 4-cell stage with 0.5ng of mouse Bmp4. Nuclear β-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

Figure 5. Combination of Wnt and Bmp signaling is required for endoderm formation

X. tropicalis embryos were injected with 10ng 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_α and Mixer, or the dorsoanterior endoderm markers Cerberus and Xhex.

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