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BMP-4 is believed to play a central role in the patterning of the mesoderm by providing a strong ventral signal. As part of this ventral patterning signal, BMP-4 has to activate a number of transcription factors to fulfill this role. Among the transcription factors regulated by BMP-4 are the Xvent and the GATA genes. A novel homeobox gene has been isolated termed Xvex-1 which represents a new class of homeobox genes. Transcription of Xvex-1 initiates soon after the midblastula transition. Xvex-1 transcripts undergo spatial restriction from the onset of gastrulation to the ventral marginal zone, and the transcripts will remain in this localization including at the tailbud stage in the proctodeum. Expression of Xvex-1 during gastrula stages requires normal BMP-4 activity as evidenced from the injection of BMP-4, Smad1, Smad5 and Smad6 mRNA and antisense BMP-4 RNA. Xvex-1 overexpression ventralizes the Xenopus embryo in a dose dependent manner. Partial loss of Xvex-1 activity induced by antisense RNA injection results in the dorsalization of embryos and the induction of secondary axis formation. Xvex-1 can rescue the effects of overexpressing the dominant negative BMP receptor. These results place Xvex-1 downstream of BMP-4 during gastrulation and suggest that it represents a novel homeobox family in Xenopus which is part of the ventral signaling pathway.
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10446269
???displayArticle.link???Mech Dev
Fig. 3. Xvex-1 is expressed as a ventral gene. Whole mount in situ hybridization analysis was performed to determine the spatial pattern of Xvex-1 expression during early embryogenesis. (A) At stage 10 Xvex-1 transcripts can be observed throughout the embryo (animal view). (B) At late stage 10.5 a wave of repression can be observed spreading from dorsal to ventral on the vegetal side including the marginal zone and the yolk-filled endodermal cells. (C) By stage 11.5 the Xvex-1 transcripts are localized to the ventral marginal zone. (D) The ventral localization of the Xvex-1 continues at stage 12.5 but the gap between the expression domain and the blastopore disappears. (E) The onset of neurulation (stage 13) marks a continued lateral restriction in the Xvex-1 pattern of expression. (F) At stage 14 the process of restriction in the lateral extent of Xvex-1 continues. (G) By stage 16 the Xvex-1 expression domain acquires its minimal size on the ventral-most region of the closed blastopore. By stage 26/27 expression of Xvex-1 remains in the same position on the now ventrally localized proctodeum, (H) lateral view, (I) ventral views. Panels B and C vegetal view, D–G posterior view. Panels (A–H), dorsal is to the top.
Fig. 5. Xvex-1 ventralizes Xenopus embryos. Embryos injected with increasing amounts of Xvex-1 mRNA exhibit dose dependent ventralization. At the four-cell stage, embryos were injected in all four blastomeres with the appropriate RNA. (A) Control, LacZ mRNA injected embryo. (B) Embryo injected with 0.4 ng of Xvex-1 mRNA. (C) Injection of 0.8 ng of sense Xvex-1 RNA result in further ventralization. (D) Embryos injected with 1.6 ng of Xvex-1 mRNA exhibit the strongest degree of ventralization.
Fig. 6. Partial loss-of-function of Xvex-1 has dorsalizing effects. Embryos were injected with antisense Xvex-1 RNA in all four blastomeres at the four-cell stage. Embryos were processed for phenotypic analysis (A,B) or for the analysis of the effects on gene expression (C,D). Phenotypic analysis was performed at stage 37/38 (A) control embryos, (B) antisense Xvex-1 RNA injected embryos exhibiting dorsalization and secondary axis induction (red arrowheads). The gsc gene was used as a marker to study the effect of partial loss of Xvex-1 function, (C) control embryos exhibiting the normal gsc expression at stage 10.5, (D) expanded gsc expression in embryos injected with antisense Xvex-1 RNA.
Fig. 7. Regulation of Xvex-1 expression by BMP-4. In order to study the regulatory effect of BMP-4 on Xvex-1 expression RNA encoding different elements of the BMP-4 signaling pathway was injected in all four blastomeres of four-cell embryos. At stage 11 the embryos were processed for whole mount in situ hybridization and probed with the Xvex-1 specific probe. (A) Control embryo exhibiting normal Xvex-1 expression. (B) Embryo injected with BMP-4 mRNA exhibiting expanded expression of Xvex-1. (C) Injection of antisense BMP-4 RNA results in the downregulation of Xvex-1 expression. (D) Embryo injected with the BMP-specific Smad1 mRNA shows expansion in the domain of Xvex-1 expression. (E) Overexpression of Smad5 also results in the upregulation of Xvex-1 expression and its dorsal expansion. (F) Embryo injected with the BMP-specific inhibitory Smad6 results in the downregulation of Xvex-1 expression.
Fig. 8. Xvex-1 can rescue the loss of BMP signaling. Co-injection experiments were performed to determine whether Xvex-1 can rescue the loss of BMP signaling induced by overexpression of the dominant negative BMP receptor. Embryos were injected ventrally with the different mRNAs and subsequently scored for the formation of secondary axes. (A) Embryo injected with 200 pg of mRNA encoding the dominant negative BMPreceptor. (B) Embryo co-injected with 200 pg of dominant negative BMPreceptor RNA together with 120 pg of Xvex-1 mRNA. (C) Embryo injected with 200 pg of dominant negative BMPreceptor mRNA and 200 pg of Xvex-1 sense RNA. The percentage of embryos with secondary axes are shown.
ventx3.2 (VENT homeobox 3, gene 2) gene expression in Xenopus laevis embryos, NF stage 12.5, as assayed by in situ hybridization. vegetal view: anterior/dorsal up.
