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It is well established that in Xenopus, bone morphogenetic protein (BMP) ventralizes the early embryo through the activation of several target genes encoding homeobox proteins, some of which are known to be necessary and sufficient for ventralization. Here, we used an inhibitory form of Xmsx-1, one of BMP's targets, to examine its role in head formation. Interestingly, ventral overexpression of a dominant Xmsx-1 inhibitor induced an ectopic head with eyes and a cement gland in the ventral side of the embryo, suggesting that Xmsx-1 is normally required to suppress head formation in the ventral side. Supporting this observation, we also found that wild-type Xmsx-1 suppresses head formation through the inhibition of nodal signaling, which is known to induce head organizer genes such as cerberus, Xhex and Xdkk-1. We propose that negative regulation of the BMP/Xmsx-1 signal is involved not only in neural induction but also in head induction and formation. We further suggest that the inhibition of nodal signaling by Xmsx-1 may occur intracellularly, through interaction with Smads, at the level of the transcriptional complex, which activates the activin responsive element.
Fig. 1. Ectopic expression of Xmsx-1 in the anteriorendoderm suppresses head structure formation. Xenopus embryos were either uninjected (A and C, lanes 1, 4, 5) or injected with 500 pg of Xmsx-1 (B and C, lane 2) or 200 pg of BMP-4 (C, lane 3) mRNA in the submarginal zone of the two dorsal blastomeres at the 4-cell stage. (A,B) The embryos were cultured until stage 40 to observe the phenotypes. (C) RT-PCR analysis for the early anterior endomesodermal markers. The dorso-vegetal quarters of the embryos were dissected at stage 10.25 (lanes 1-3) and then subjected to RT-PCR analysis for the indicated markers. Lanes 4 and 5, whole embryo control with (lane 4) or without (lane 5) the RT step.
Fig. 3. HI-Xmsx-1 induces headorganizer marker genes in the ventralendomesoderm. Two ventral blastomeres of 4-cell-stage embryos were coinjected marginally with 100 pg of HI- or TI-Xmsx-1 mRNA and 50 pg of β-galactosidase mRNA as a lineage tracer, then processed for RT-PCR (A) and whole-mount in situ hybridization (B) at stage 10.25. (A) The ventro-vegetal quarter of the embryos injected with the indicated mRNA (top, lanes 1, 2, 3) was dissected at stage 10.25 and RNA was extracted immediately, for early stage, or after being cultured until sibling embryos reached stage 18, for late stage. RNA extracted from each explant was analyzed by RT-PCR. Lane 4 shows the expression of each marker in whole embryos and lane 5 shows the control reactions with no RT step. (B) The injected embryos were stained by red Gal for lineage tracing and then analyzed by whole-mount in situ hybridization for Xotx-2, Xhex and cerberus gene expression. Anteriororganizer genes were induced in the ventralendomesoderm. The dorsal blastopore lip is indicated by black arrowheads. Arrows indicate the RNA-injected ventral marginal zone, in which the nuclei of cells are stained red. AS, antisense probe; S, sense probe.
Fig. 8. Immunostaining of activated Smad1 and Xmsx-1 protein. Gastrula embryos were subjected to immunohistochemistry using anti-phosphoSmad1 (A) and anti-MSX-1 (B) antibodies. Embryos were bisected along the dorsoventral axis. The dorsal blastopore lip is indicated with an arrowhead.
