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The Iroquois genes code for homeodomain proteins that have been implicated in the neural development of Drosophila and vertebrates. We show here for the first time that Xiro-1, one of the Xenopus Iroquois genes, is expressed in the Spemann organizer from the start of gastrulation and that its overexpression induces a secondary axis as well as the ectopic expression of several organizer genes, such as chordin, goosecoid, and Xlim-1. Our results also indicate that Xiro-1 normally functions as a transcriptional repressor in the mesoderm. Overexpression of Xiro-1 or a chimeric form fused to the repressor domain of Engrailed cause similar phenotypes while overexpression of functional derivatives of Xiro-1 fused with transactivation domains (VP16 or E1A) produce the opposite effects. Finally, we show that Xiro-1 works as a repressor of bmp-4 transcription and that its effect on organizer development is dependent on BMP-4 activity. We propose that the previously observed down regulation of bmp-4 in the dorsal mesoderm during gastrulation can be explained by the repressor activity of Xiro-1 described here. Thus, Xiro-1 seems to have at least two different functions: control of neural plate and organizer development, both of which could be mediated by repression of bmp-4 transcription.
Figure 1. Xiro-1 expression. A: Saggital sections of a stage 10+ embryos were analyzed by in situ hybridization for the expression of Xiro-1 (arrow). Arrowhead: blastopore; asterisk: ectodermal expression. B-E: Embryos were fixed at stage 10+ and after in situ hybridization, were sectioned into halves to examine internal gene expression. The border of the blastocoel cavity is indicated by a black line (top); the arrowhead indicates the dorsal blastopore lip (bottom). B: Xiro-1 expression in the involuting marginal zone is indicated by a white line; asterisk: ectodermal expression. C: Chd expression; D: Gsc expression; E: Cer expression. The expression of Xiro-1 is indicated in all three cases (C-E) to show its overlapping expression with Chd, Gsc, and Cer. F: After gastrulation, Xiro-1 expression is detected in the ectoderm (arrow), notochord (n, arrowhead), (saggital section stage 17). G: At stage 25, Xiro-1 is located in the neural tube and can easily be seen in the somites (arrow). H: A section from a stage-25 embryo following in situ hybridization for Xiro-1. Expression is observed in the notochord (arrowhead), somites and neural tube (n: notochord; NT: neural tube).
Figure 3. Xiro-1 regulates the expression of mesodermal genes by repression. When injected into one blastomere of 2-cell stage embryos, in the presumptive ventro-lateral mesodermal region, 2 ng of either Xiro-1 (A,C,E,G) or EnR-Xiro (B,D,F,H) produced the ectopic expression of chordin (A and B: arrowheads, A: 37% n = 104 and B: 38% n = 132) and goosecoid (C and D: arrowheads, C: 34% n = 95 and D: 54% n = 131). Ventral genes were inhibited by these treatments. Inhibition of Xvent-1 (E) and Xwnt-8 (G) by Xiro-1 was observed in 89 and 91% of cases, respectively (E and G: arrowheads, E: n = 162 and G: n = 172) while EnR-Xiro caused an inhibitory effect in 92% (F: arrowhead, n = 158) and 97% (H: arrowhead, n = 129) of cases, respectively. All figures shown are vegetal views of stage 10.25 embryos with the dorsal region at the top. Dexametasone treatment was started at stage 5 until the embryos were fixed.
Figure 6. Xiro-1 represses bmp-4 expression. Embryos were analyzed for bmp-4 expression (A,B: vegetal view; C,D: animal view). Overexpression of 2 ng of Xiro-1 or EnR-Xiro repressed the expression of bmp-4 when injected into one blastomere of 4-cell stage embryos. Xiro-1 inhibited bmp-4 expression in the ventro-lateralmesoderm and ectoderm (A and C: arrowheads, 95% n = 41). EnR-Xiro behaved in the same manner, repressing bmp-4 expression in 98% of cases (B and D: arrowheads, n = 53).
