Nitta KR et al. (2007), The N-terminus zinc finger domain of Xenopus SI...

XB-ART-35924

Int J Dev Biol 2007 Jan 01;514:321-5. doi: 10.1387/ijdb.062252kn.

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The N-terminus zinc finger domain of Xenopus SIP1 is important for neural induction, but not for suppression of Xbra expression.

Nitta KR , Takahashi S , Haramoto Y , Fukuda M , Tanegashima K , Onuma Y , Asashima M .

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Smad-interacting protein-1 (SIP1), also known as deltaEF2, ZEB2 and zfhx1b, is essential for the formation of the neural tube and the somites. Overexpression of Xenopus SIP1 causes ectopic neural induction via inhibition of bone morphogenetic protein (BMP) signaling and inhibition of Xbra expression. Here, we report the functional analyses of 4 domain-deletion mutants of XSIP1. Deletion of the N-terminus zinc finger domain suppressed neural induction and BMP inhibition, but these were not affected by deletion of the other domains (the Smad binding domain, the DNA-binding homeodomain together with the CtBP binding site and the C-terminus zinc finger). Therefore SIP1 does not inhibit BMP signaling by binding to Smad proteins. In contrast, all of the deletion constructs inhibited Xbra expression. These results suggest that the N-terminus zinc finger domain of XSIP1 has an important role in neural induction and that Xbra suppression occurs via a mechanism separate from the neural inducing activity.

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Species referenced: Xenopus
Genes referenced: ctbp2 gemin2 nherf2 nrp1 sox2 tbxt ventx2.2 zeb2

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	Fig. 1. XSIP1 deletion constructs. Numbers indicate amino acid positions for deleted sequences. The stop codon of the pCS2-MT vector was used for MT-XSIP1-ΔCZf.
	Fig. 2. Real-time RT-PCR and wholemount in situ hybridization analyses of animal caps injected with XSIP1 deletion mutants. (A) N-CAM and NRP1 expression levels in animal caps injected with XSIP1 construct mRNA. Animal caps were dissected from embryos injected with 500 pg of XSIP1 construct mRNA and were cultured until the sibling embryos reached stage 32. Expression of N-CAM and NRP1 mRNA was quantified by real-time RT-PCR. The results are represented as percentages relative to the expression levels in animal caps injected with MT-XSIP1 mRNA. (B) Vent2 expression levels in animal caps injected with XSIP1 construct mRNA. Animal caps were dissected from embryos injected with 500 pg of XSIP1 deletion mutant mRNA and were cultured until the sibling embryos reached stage 11. Expression of Vent2 mRNA was quantified by real-time RTPCR. The results are represented as percentages relative to the expression levels in uninjected animal caps. (C) Whole-mount in situ hybridization analysis of Sox2 expression in animal caps injected with XSIP1 deletion mutant mRNA. Animal caps were dissected from embryos injected with 500 pg XSIP1 construct mRNA and were cultured until the sibling embryos reached stage 14. The expression of Sox2 was markedly reduced in animal caps injected with MT-XSIP1-δNZf mRNA.
	Fig. 3. Xbra expression was downregulated by overexpression of XSIP1 deletion mutants. Each mRNA (500 pg) was co-injected with lacZ mRNA into the marginal region of one blastomere of 4-cell-stage embryos. The injected embryos were cultured until the sibling embryos reached stage 11. Xbra expression was downregulated in the region where mRNA was injected (marked by Red-Gal staining). The embryos shown were injected with lacZ alone (A), MT-SIP1 (B), MT-δNZf (C), MT- δSBD (D), MT-δHD-CBS (E), or MT-δCZf (F).