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The fibroblast growth factor (FGF)/MAPK pathway plays an important role in early Xenopus developmental processes, including mesoderm patterning. The activation of the MAPK pathway leads to induction of Xenopus Brachyury (Xbra), which regulates the transcription of downstream mesoderm-specific genes in mesoderm patterning. However, the link between the FGF/MAPK pathway and the induction of Xbra has not been fully understood. Here we present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posteriormesoderm during early embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the formation of the trunk and tail structures. Overexpression of Ets-2 resulted in the induction of Xbra, and expression of the dominant-negative Ets-2 inhibited FGF- or constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in vitro. These results taken together indicate that Xenopus Ets-2 plays an essential role in mesoderm patterning, lying between the FGF/MAPK pathway and the Xbra transcription.
Figure 1
Overexpression of Ets-2 induces posteriorized embryos. A, tadpole stage (stage 35) embryos and sibling embryos that have been injected with Ets-2 mRNA into dorsal marginal zones (DMZ) or ventral marginal zones (VMZ) at four-cell stage at indicated doses. B, expression of marker genes in whole embryos that were injected with 1 ng of Ets-2 mRNA into the dorsal marginal zones. Injected embryos were cultured until sibling embryos reached stage 26 or 37. Expression of indicated marker genes was analyzed by RT-PCR. EF1-α served as a loading control. RNA from whole embryo (indicated as embryo) provides a positive control. No signal was observed in the absence of reverse transcription (−RT).NCAM, neural cell adhesion molecule.
Figure 2
Ets-2 rescues the defects by XFD. Embryos were injected into the dorsal marginal zones at four-cell stage and cultured until stage 35. 0.5 ng of XFD mRNA was injected with or without 1.5 ng of Ets-2 mRNA.
Figure 3
Ets-2 induces expressions of mesodermal markers in animal cap explants. As shown in A, Ets-2 mRNA was injected into animal poles of two-cell stage embryos at the indicated doses. Animal caps were dissected at blastula stage and cultured until sibling embryos reached stage 11. Expression of indicated marker genes was analyzed by RT-PCR. −RT, absence of reverse transcription. As shown in B, Ets-2 mRNA was injected as in panel A. Animal caps were dissected at blastula stage and cultured until sibling embryos reached stage 26. Indicated markers were analyzed by RT-PCR.
Figure 4
Inhibition of Ets-2 causes defects in mesodermal patterning. A, a schematic diagram of Ets-2 construct fused with an En-R. B, tadpole stage (stage 35) embryos that have been injected with 1 ng of mRNA encoding EtsδN En-R into dorsal or ventral marginal zones at the four-cell stage.DMZ, dorsal marginal zone; VMZ, ventral marginal zone. C, an inhibitory effect of EtsδN En-R on FGF- or MAPKK SESE-induced Xbra expression in isolated animal caps. Animal caps were dissected at blastula stage from embryos that had been injected with EtsδN En-R mRNA (1 ng) together with MAPKK SESE mRNA (0.1 ng) at the two-cell stage and were cultured until sibling embryos reached stage 11. On FGF treatment, animal caps were cultured in the medium including 50 ng/ml bFGF. Expression of Xbra was analyzed by RT-PCR. D, wild type Ets-2 rescued the reduction of Xbra expression caused by EtsδN En-R. EtsδN En-R mRNA (50 pg) was injected together with wild type Ets-2 mRNA (1 ng) into marginal zones of the two-cell embryo. Injected embryos were cultured until sibling embryos reached stage 11. Expression of Xbra was analyzed by RT-PCR with injected whole embryos.
Figure 5
Ets-2 is necessary for patterning of posteriormesoderm. Embryos were injected with Ets-2 MO (50 ng) or control MO (50 ng) into dorsal marginal zones at the two-cell stage and cultured until stage 33. For rescue of Ets-2 depletion, Ets-2 mRNA (1.5 ng) was co-injected with Ets-2 MO.
Figure 6
Ets-2 is essential for Xbra transcription. A, Ets-2-regulated expression of reporter gene construct containing a 1.5-kb fragment of the Xbra promoter region. The Xbra promoter construct was co-injected with Ets-2 mRNA or EtsδN En-R mRNA into animal poles of two-cell stage embryos. Animal caps were dissected at stage 8 and cultured with or without FGF (50 ng/ml). Cultured animal caps were assayed for luciferase activity at stage 11. B, the sequence of the wild type and the mutant Ets binding sites. C, Ets-2 bound to the −310/−271 region of the Xbra promoter. The gel mobility shift assay was performed using radiolabeled double-strand oligonucleotide probe of the Ets-binding site. After incubation of the radiolabeled probe with protein extracts, DNA-protein complex was analyzed by autoradiography following electrophoresis of binding reactions on 4% polyacrylamide gels. The upper arrow indicates the position of DNA-protein complex. The recombinant GST or GST-Ets-2 fusion protein was incubated with radiolabeled oligonucleotide probe containing the Ets-binding site. For competition assay, binding reactions were preincubated with a 200-fold molar excess of unlabeled oligonucleotide probe as competitor, and then binding reactions with GST-Ets-2 and labeled probe were incubated as described above. The same competition assay was performed with unlabeled mutant oligonucleotide.
