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BACKGROUND: In Xenopus, the endoderm germ layer is derived from the vegetal blastomeres of cleavage-stage embryos. Cell transplantation experiments have revealed that the endodermal fate becomes gradually fixed during the late blastula stages. Sox17alpha, Mix.1, Mixer and GATA-4 encode vegetal zygotic transcription factors with endoderm-inducing activity. The accumulation of their transcripts during the late blastula stages may cause determination of the endodermal fate. VegT, a T-box transcription factor, the maternal transcripts of which are vegetally localised, is also required for endoderm formation.
RESULTS: We analysed the events leading to the progressive accumulation of the transcripts for Sox17alpha, Mix.1, Mixer and GATA-4. Two phases could be distinguished in the endodermal programme. In phase 1, Sox17alpha, Mix.1, and the genes encoding transforming growth factor beta-related signalling molecules Xnr1, Xnr2 and Derrière were activated cell-autonomously at around the mid-blastula transition (MBT) by maternal determinants. In phase 2, TGFbeta signalling, possibly involving Xnr1, Xnr2 and Derrière, led to the activation of Mixer and GATA-4 in late blastula stages and to the reinforcement of the expression of Sox17alpha and Mix.1. Overexpression of VegT in animal caps triggered a developmental programme qualitatively similar to that observed in vegetal blastomeres, except that Xnr1 and GATA-4 were not activated by the early gastrula stage.
CONCLUSIONS: Our results support a two-step model for endoderm determination between fertilisation and the onset of gastrulation. The initial cell-autonomous activation of early endodermal genes by maternal determinants including, but not limited to, VegT is relayed by the action of zygotic TGFbetas such as Xnr1, Xnr2 and Derrière.
Figure 1.
Expression profile of early endodermal genes. The temporal gene-expression profiles of Sox17α, Mix.1, Derrière, Xnr1, Xnr2, Mixer, GATA-4 and the fibroblast growth factor (FGF) receptor gene (FGFR) were analysed by RT–PCR. FGFR is ubiquitously expressed and served as a loading control. Expression of Sox17α starts at stage 8; that of Mix.1 and Derrière starts at stage 8.5; expression of Mixer and GATA-4 is first detected at stage 9.5; transcripts for Xnr1 and Xnr2 are detected from stage 9. The timing of restriction and gradual determination of the fate of presumptive endodermal blastomeres is shown at the top.
Figure 2.
Cellular communication after the MBT is required for the activation or full expression of early endodermal genes. Embryos were dissociated (dis) at the stages indicated in (a,c) and, in some cases, reaggregated at later stages as shown. (b,d) The indicated transcripts were then detected by RTâPCR at (b) stage 10 or (d) stages 9, 9.5 and 10. As a positive control, intact embryos of the appropriate stage were analysed in parallel.
Figure 3.
Post-MBT cellular communication occurs during the late blastula stage. Embryos were dissociated (dis) and reaggregated at the stages indicated in (a); dotted lines show the period of dissociation. (b) The indicated transcripts were then detected by RTâPCR at stage 10, with intact stage 10 embryos as a positive control.
Figure 4.
Signalling molecules in vegetal pole explants are more active after the MBT. (a) Stage 9 (St 9) animal caps (AC) were cultured in contact with stage 7 or stage 9 vegetal pole explants (VPE) for 1 h. Animal caps were then removed and cultured alone until the equivalent of stage 10. (b) The indicated transcripts were then detected by RTâPCR, with whole stage 10 embryos (WE) as a positive control and unconjugated animal caps as a negative control.
Figure 5.
TGFβ like signals are involved in the post-MBT cellular communication. (a) Dominant-negative ActIIR mRNA (DnActIIR, 4 ng RNA per blastomere) was injected vegetally into four-cell embroys. At stage 10 (St 10), vegetal pole explants (VPE) from injected or control, uninjected embryos were removed and analysed. (b) The indicated transcripts were then detected by RTâPCR, with whole stage 10 embryos (WE) as a positive control. Although, in the experiment shown here, expression of Sox17α is not affected in the DnActIIR-injected vegetal pole explants, this gene was also partially downregulated in other repetitions of this experiment (data not shown). (c) Constitutively active Alk4 RNA (CA-Alk4, 100 or 400 pg per embryo) was injected vegetally into four-cell embryos, which were immediately dissociated and cultured until the equivalent of stage 10. (d) The indicated transcripts were then detected by RTâPCR, with intact stage 10 embryos as a positive control and uninjected dissociated embryos as a negative control.
Figure 6.
Sox17α and Mix. 1 are direct targets of maternal factors, whereas expression of Mixer requires zygotic proteins. (a) Embryos were treated continuously with CHX from stage 7 onwards and analysed at stages 9, 9.5 and 10 in parallel with control, untreated embryos. The dotted line shows the period of treatment with CHX. (b) The indicated transcripts were then detected by RTâPCR of untreated and CHX-treated embryos.
Figure 7.
Overexpression of Xnr1, Xnr2 or Derrière induces prospective ectoderm to express endodermal genes. (a)Derrière, Xnr1 or Xnr2 mRNA was injected into the animal part of both cells of two-cell embryos, which were cultured until stage 8 (St 8). Animal caps were then dissected, cultured until the equivalent of stage 10 and analysed. (b,c) The indicated transcripts were then detected by RT–PCR from embryos injected with the indicated amounts of (b) Xnr1 and Xnr2 mRNA and (c) Derrière mRNA. Whole stage 10 embryos (WE) acted as a positive control and uninjected animal caps as a negative control.
Figure 8.
VegT triggers a two-step partial endodermal program in animal caps. (a)VegT mRNA was injected into the animal part of both cells of two-cell embryos, which were cultured until stage 8 (St 8). Animal caps were then dissected, cultured until the equivalent of stage 10 and analysed. (b) The indicated transcripts were then detected by RTâPCR from embryos injected with the indicated amounts of VegT mRNA. A very weak induction of Xnr1 in the VegT-injected animal caps at stage 10 was only observed in two out of four repetitions of this experiment. (c)VegT mRNA was injected into two-cell embryos as in (a). At stage 8, animal caps were then dissected and either dissociated or left intact, cultured until the equivalent of stage 9 or stage 10 and analysed. (d) The indicated transcripts were then detected by RTâPCR. Whole stage 10 embryos (WE) acted as a positive control and uninjected animal caps as a negative control.
Figure 9.
A model for the cell-fate determination of vegetal pole blastomeres to endoderm. For simplicity, only molecules that have been analysed in this study are considered in this model, which is therefore necessarily incomplete. X represents an additional material endoderm determinant acting upstream of Xnr1 and GATA-4. Dotted lines indicate the upper boundary of the presumptive endodermal region. Levels of gene expression are represented by the size of the lettering. See text for details.
