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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.
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 (AâC), Mxr (DâF), and
Xbra (GâI). 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
Studentâs 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 (AâD) and Mxr (EâH) 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.
