Engleka MJ et al. (2001), VegT activation of Sox17 at the midblastula tra...

XB-ART-8516

Dev Biol 2001 Sep 01;2371:159-72. doi: 10.1006/dbio.2001.0366.

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VegT activation of Sox17 at the midblastula transition alters the response to nodal signals in the vegetal endoderm domain.

Engleka MJ , Craig EJ , Kessler DS .

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In Xenopus, the prospective endoderm and mesoderm are localized to discrete, adjacent domains at the beginning of gastrulation, and this is made evident by the expression of Sox17 in vegetal blastomeres and Brachyury (Xbra) in marginal blastomeres. Here, we examine the regulation of Sox17alpha expression and the role of Sox17alpha in establishing the vegetal endodermal gene expression domain. Injection of specific inhibitors of VegT or Nodal resulted in a loss of Sox17alpha expression in the gastrula. However, the onset of Sox17alpha expression at the midblastula transition was dependent on VegT, but not on Nodal function, indicating that Sox17alpha expression is initiated by VegT and then maintained by Nodal signals. Consistent with these results, VegT, but not Xenopus Nodal-related-1 (Xnr1), can activate Sox17alpha expression at the midblastula stage in animal explants. In addition, VegT activates Sox17alpha in the presence of cycloheximide or a Nodal antagonist, suggesting that Sox17alpha is an immediate-early target of VegT in vegetal blastomeres. Given that Nodal signals are necessary and sufficient for both mesodermal and endodermal gene expression, we propose that VegT activation of Sox17alpha at the midblastula transition prevents mesodermal gene expression in response to Nodal signals, thus establishing the vegetal endodermal gene expression domain. Supporting this idea, Sox17alpha misexpression in the marginal zone inhibits the expression of multiple mesodermal genes. Furthermore, in animal explants, Sox17alpha prevents the induction of Xbra and MyoD, but not Sox17beta or Mixer, in response to Xnr1. Therefore, VegT activation of Sox17alpha plays an important role in establishing a region of endoderm-specific gene expression in vegetal blastomeres.

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Species referenced: Xenopus
Genes referenced: cer1 eef1a2 eng fgfr1 gsc mixer myod1 nodal nodal1 odc1 sox17a sox17b sox17b.2 tbx2 tbxt vegt wnt8a

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	FIG. 1. VegT and Nodal function are required for endodermal gene expression. At the four-cell stage, each blastomere was injected vegetally with 300 pg of Eng-VegT mRNA or 100 pg of Cer-S mRNA. Uninjected (A, D, G), Eng-VegT-injected (B, E, H), and Cer-S-injected (C, F, I) embryos were fixed at the early gastrula stage (stage 10.25) and sagittal sections were analyzed by in situ hybridization for the expression of Sox17a (A–C), Mxr (D–F), and Xbra (G–I). Arrowheads indicate the dorsal blastopore lip. Scale bar, 0.25 mm.
	FIG. 2. Sox17a and Mxr differ in the onset of expression. (A) Sox17a is expressed at the midblastula transition prior to the expression of Mxr and Xbra. Intact embryos were harvested at stages 8.5, 9.0, 9.5, and 10.25 for RT-PCR analysis of Sox17a, Mxr, and Xbra expression. (B) Sox17a and Mxr differ in the onset of expression in response to VegT and Xnr1. At the two-cell stage, the animal pole was injected with 300 pg of VegT or 500 pg of Xnr1 mRNA. Animal explants were prepared at stage 8 from uninjected (Control) or injected embryos and both explants and whole embryos (WE) were harvested at stages 9.0, 9.5, and 10.25 for RT-PCR analysis of Sox17a, Mxr, and Xbra expression. Ornithine decarboxylase (ODC) served as a control for RNA recovery and loading. Stage 10.25 whole embryo mRNA was used in a cDNA synthesis reaction without the addition of reverse transcriptase to control for PCR contamination (10.25-RT).
	FIG. 3. VegT activation of Sox17a is not dependent on Nodal signals or protein synthesis. (A) At the one-cell stage, the animal pole was injected with 1 ng of Cer-S mRNA and, at the two-cell stage, 300 pg of VegT or 500 pg of Xnr1 mRNA was injected. Animal explants prepared at the blastula stage were harvested at the gastrula stage for RT-PCR analysis of Sox17a, Mxr, and Xbra expression. (B) At the two-cell stage, the animal pole was injected with 300 pg of VegT mRNA. Animal explants prepared at stage 7 were cultured with or without cycloheximide (Chx, 5 mg/ml) and were harvested at stage 10.25 for RT-PCR analysis. EF1a served as a control for RNA recovery and loading. Whole embryos served as positive control (WE) and an identical reaction without reverse transcriptase controlled for PCR contamination (WE-RT).
	FIG. 4. Initiation of Sox17a expression at the midblastula transition requires VegT function, but not Nodal. At the four-cell stage, each blastomere was injected vegetally with 300 pg of Eng-VegT mRNA or 100 pg of Cer-S mRNA. Uninjected (A), Cer-S-injected (B), and Eng-VegT-injected (C) embryos were harvested at stage 8.5 and analyzed for Sox17a expression by in situ hybridization. Sox17a expression was unaffected by Cer-S (B), but greatly reduced by Eng-VegT (C). Vegetal views with arrows indicating the perinuclear staining of Sox17a-positive cells are shown. Scale bar, 0.25 mm. (D) Quantitation of Sox17a-positive cells. The mean and standard error of the number of Sox17a-positive cells per embryo (n 5 36) are shown. Statistical significance was assessed using the Student’s t test (*, P , 0.001).
	FIG. 5. Sox17a inhibits mesodermal gene expression in the marginal zone. At the four-cell stage, a single blastomere was injected in the marginal zone with 500 pg of Sox17a mRNA (B, D, F, H). Uninjected (Control) and injected embryos were collected at stage 10.25 for in situ hybridization analysis of Goosecoid (Gsc; A, B), MyoD (C, D), Xwnt8 (E, F), and Xbra (G, H) expression (vegetal views, dorsal up). Sox17a misexpression resulted in reduction of Goosecoid (B) expression and a gap in the expression domains of MyoD (D), Xwnt8 (F), and Xbra (H). Arrowheads indicate the regions of reduced gene expression. Scale bar, 0.25 mm.
	FIG. 6. Cell autonomous inhibition of mesodermal gene expression by Sox17a without ectopic endodermal gene expression. At the four-cell stage, a single blastomere was injected in the marginal zone with the fluorescent lineage marker, Oregon Green-dextran (OGD), alone (A, B, E, F), or together with 500 pg of Sox17a mRNA (C, D, G, H). Injected embryos were collected at stage 10.25 for in situ hybridization analysis of Xbra (A–D) and Mxr (E–H) expression. Vegetal views (dorsal up) of the in situ staining pattern (B, D, F, H) or merged images of OGD-positive cells and the in situ stain (A, C, E, G) are shown. The position of OGD-positive, Sox17a-expressing cells corresponds precisely to the gap in Xbra expression, but no ectopic Mxr expression was observed. Arrowheads indicate the position of OGD-positive cells. Scale bar, 0.25 mm.
	FIG. 7. Sox17a alters the response to Xnr1. At the one-cell stage, the animal pole was injected with 250 pg of Sox17a mRNA and at the two-cell stage, 30 pg of Xnr1 mRNA was injected. Animal explants were prepared at the blastula stage and harvested at the gastrula stage for RT-PCR analysis of Xbra, MyoD, Mxr, and Sox17b expression. While Xnr1 induced both mesodermal and endodermal genes, Sox17a coexpression prevented the induction of the mesodermal genes without affecting the response of the endodermal genes. ODC served as a control for RNA recovery and loading. Whole embryos served as a positive control (WE) and an identical reaction without reverse transcriptase controlled for PCR contamination (WE-RT).
	FIG. 8. Direct activation of Sox17a by VegT establishes the vegetal endodermal domain by altering the response of vegetal cells to Nodal signals. (A) At the midblastula stage, maternal VegT directly activates Sox17a expression independent of Nodal signals. VegT also induces the vegetal expression of Nodal-related genes at this stage (Clements et al., 1999; Kofron et al., 1999) (B) At the late blastula and early gastrula stages, Nodal signals maintain the expression of Sox17a and induce the expression of other endodermal genes (Mxr). The presence of Sox17a in vegetal cells prevents the vegetal induction of mesodermal genes (Xbra) in response to Nodal signals. In marginal cells that lack Sox17a, Nodal signals induce mesodermal gene expression. This model accounts for the establishment of the vegetal endoderm domain distinct from the mesoderm domain in the early Xenopus embryo. In addition, this model provides a mechanism for the involvement of Nodal signals in the establishment of both the endodermal and mesodermal germ layers, tissues that are spatially and functional distinct.