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Patterning of the marginal zone in the Xenopus embryo has been attributed to interactions between dorsal genes expressed in the organizer and ventral-specific genes. In this antagonistic interplay of activities, BMP-4, a gene that is not expressed in the organizer, provides a strong ventralizing signal. The Xenopus caudal type homeobox gene, Xcad-2, which is expressed around the blastopore with a gap over the dorsal lip, was analyzed as part of the ventral signal. Xcad-2 was shown to efficiently repress during early gastrula stages the dorsal genes gsc, Xnot-2, Otx-2, XFKH1 and Xlim-1, while it positively regulates the ventral genes, Xvent-1 and Xvent-2, with Xpo exhibiting a strong positive response to Xcad-2 overexpression. Xcad-2 was also capable of inducing BMP-4 expression in the organizer region. Support for a ventralizing role for Xcad-2 was obtained from co-injection experiments with the dominant negative BMP receptor which was used to block BMP-4 signaling. Under lack-of-BMP-signaling conditions Xcad-2 could still regulate dorsal and ventral gene expression and restore normal development, suggesting that it can act downstream of BMP-4 signaling or independently of it. Xcad-2 could also inhibit secondary axis formation and dorsalization induced by the dominant negative BMP receptor. Xcad-2 was also shown to efficiently reverse the dorsalizing effects of LiCl. These results place Xcad-2 as part of the ventralizing gene program which acts during early gastrula stages and can execute its ventralizing function in the absence of BMP signaling.
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Fig. 1. Regulation of ventral genes by Xcad-2. Embryos injected with Xcad-2 mRNA (B,E,H,K) or antisense Xcad-2 RNA (C,F,I,L) or control embryos (A,D,G,J) were analyzed by in situ hybridization with probes specific for the Xpo (A–C), Xvent-1 (D–F), Xvent-2/Vox (G–I) and BMP-4 (J–L) genes. (A–I) Vegetal views. (J–L) Lateral views.
Fig. 2. The temporal pattern of expression of Xcad-2 and BMP-4. RNA samples were collected from 7 h post fertilization until 11.5 h. RT-PCR was performed with Xcad-2, BMP-4 and histone H4 specific primers. The different samples were normalized to the level of histone H4.
Fig. 3. Xcad-2 represses dorsal-specific genes. Embryos injected with sense (B,E,H,K,N) or antisense (C,F,I,L,O) RNA were probed for the expression of gsc (A–C), Xnot-2 (D–F), Otx-2 (G–I), Xlim-1 (J–L) and XFKH1 (M–O) during gastrula stages. Control embryos are also shown (A,D,G,J,M). All panels are vegetal views.
Fig. 4. Regulation of Xcad-2 by dorsal- and ventral-specific genes. Embryos injected diagonally with Xnot-2-capped RNA (B), or radially with BMP-4 mRNA (D) or BMP-4 antisense RNA (E) and probed for Xcad-2 expression are shown. Control embryos are also shown (A,C). The arrows mark the position of the gaps in the Xcad-2 ring of expression
Fig. 5. Xcad-2 can function ventrally in the absence of BMP signaling. Control embryos (A,E,I) or embryos injected with either Xcad-2 mRNA (B,F,J), dominant negative BMP receptor sense RNA (C,G,K) or a combination of both Xcad-2 and dominant negative BMP receptor mRNAs (D,H,L) were probed with the gsc (A–D), Xpo (E–H) or Xvent-1 (I–L) probes.
Fig. 6. Xcad-2 can rescue LiCl-dorsalized embryos. Groups of embryos treated with LiCl and injected ventrally with Xcad-2 sense RNA (D) are rescued to resemble the normal control embryos (A) and not LiCl-treated embryos (B) or embryos treated with LiCl and injected with LacZ mRNA (C). A panel with an enlarged embryo is shown for each treatment.
Fig. 7. Xcad-2 can regulate dorsal and ventral genes in dorsalized embryos. Embryos treated with LiCl were diagonally injected with either LacZ mRNA (A,C) or a combination of LacZ and Xcad-2 sense RNA (B,D). The treated embryos were probed during gastrula stages with the gsc (A,B) or the Xpo (C,D) probes. The gsc radial expression as a result of LiCl dorsalization (A) can be repressed by diagonal Xcad-2 injection (B). In the case of Xpo, LiCl treatment downregulates its expression (C) and Xcad-2 diagonal injection can restore expression of this gene (D). The blue color is the result of the in situ hybridization and the red color is the β-galactosidase lineage tracer. A panel with an enlarged embryo is shown.
Fig. 8. Xcad-2 can reverse the effects of the dominant negative BMP receptor. Xenopus embryos were injected either ventrally at the 4-cell stage with RNA encoding for the dominant negative BMP receptor to induce secondary axis or radially to induce dorsalization. This RNA was co-injected with different mRNAs to test for the inhibition of secondary axis formation. (A,E) Control LacZ-injected embryo. (B) Embryo injected ventrally with a combination of Xcad-2 and LacZ RNAs. (C) Embryo injected ventrally with the dominant negative BMP receptor to induce secondary axes. (D) Embryo co-injected ventrally with Xcad-2 and dominant negative BMP receptor show inhibition of secondary axis formation. (F) Embryo injected radially with Xcad-2 mRNA. (G) Embryo injected radially with the dominant negative BMP receptor. (H) Embryo injected radially with Xcad-2 and dominant negative BMP receptor RNAs. All embryos were lineage traced with LacZ mRNA and stained with Xgal.
