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Neural differentiation is induced by inhibition of BMP signaling. Secreted inhibitors of BMP such as Chordin from the Spemann organizer contribute to the initial step of neural induction. Xenopus Smad-interacting protein-1 gene (XSIP1) is expressed in neuroectoderm from the early gastrula stage through to the neurula stage. XSIP1 is able to inhibit BMP signaling and overexpression of XSIP1 induces neural differentiation. To clarify the function of XSIP1 in neural differentiation, we performed a loss-of-function study of XSIP1. Knockdown of XSIP1 inhibited SoxD expression and neural differentiation. These results indicate that XSIP1 is essential for neural induction. Furthermore, loss-of-function experiments showed that SoxD is essential for XSIP1 transcription and for neural differentiation. However, inhibition of XSIP1 translation prevented neural differentiation induced by SoxD; thus, SoxD was not sufficient to mediate neural differentiation. Expression of XSIP1 was also required for inhibition of BMP signaling. Together, these results suggest that XSIP1 and SoxD interdependently function to maintain neural differentiation.
Fig. 1. XSIP1 MO inhibited neural development. (A) Western blot analysis of myc-tagged proteins. SDS-PAGE was performed with protein prepared from embryos injected into the blastomere of the two cell stage with 1 ng of XSIP1-MT mRNA or rXSIP1-MT mRNA either with or without 40 ng of XSIP1 MO. α-Actin served as an internal control. (B) XSIP1 MO specifically inhibited neural differentiation induced by XSIP1 mRNA. All embryos were injected with either 1 ng of XSIP1 or rXSIP1 mRNA alone or with 40 ng of XSIP1 MO. Animal caps were dissected at stage 9 and cultured. When sibling embryos reached stage 28, total RNA from 30 explants was extracted and some marker genes were examined by RT-PCR. (C–E) Embryos injected with XSIP1 MO showed defects in neural and eye development. (C) Uninjected embryo. (D) Forty nanogram of XSIP1 MO was injected into the dorsal-animal pole of the eight-cell-stage embryo. (E) Another embryo similarly injected with 40 ng of XSIP1 MO and 50 pg of linearized pCS2-rXSIP1. (F–I) Transverse sections of control MO (F,G) and XSIP1 MO (H,I) injected embryos. Scale bars indicate 100 μm.
Fig. 2. Injection of XSIP1 MO resulted in the down-regulation of terminal neural marker genes. Whole-mount in situ hybridization analysis of N-tubulin (A,B) and N-CAM (C,D) performed on stage 28 embryos. Embryos were injected with 40 ng of either control MO or XSIP1 MO into one dorsal-animal blastomere of the eight-cell stage, and co-injected with 250 pg of nuclear-localized lacZ as a lineage tracer (red stained). (A,C) Control MO-injected embryo. Injected area (red) and marker expression (blue) overlapped. (B, D) XSIP1 MO-injected embryo. Neural markers, N-tubulin and N-CAM were not expressed in the injected area. Inset; Dorsal view of the same embryos.
Fig. 3. Early neural genes were down-regulated by inhibition of XSIP1 mRNA translation, while mesodermal genes were not influenced. Whole-mount in situ hybridization analysis of Xbra (A,E) and chordin (B,F) expression in stage 11 embryos, which had been injected with either control MO or XSIP1 MO into the dorsal marginal zone of the four-cell-stage embryo. An equivalent analysis of SoxD (C,G) and Zic3 (D,H) expression in stage 11 embryos, although one dorsal-animal blastomere of the eight-cell-stage embryo was injected in these embryos. SoxD and Zic3 expression were down-regulated in the XSIP1 MO-injected area (G,H; arrowhead), while they overlapped in the control MO-injected area (C,D). The dorsal view of all embryos is shown.
Fig. 6. Comparison of expression of XSIP1 and SoxD during embryogenesis. Whole-mount in situ hybridization of XSIP1 (A–C) and SoxD (D–F). (A,D) Dorsal view. (B,E) Anterior view.
Fig. 7. Injection of SoxD MO into Xenopus embryos resulted in a neural defect phenotype. (A) Western blot analysis of HA-tagged proteins. SDS-PAGE was performed with protein prepared from embryos injected with 1 ng SoxD-HA(UTR+) or SoxD-HA(UTR−) mRNA either with or without 40 ng SoxD MO and cultured until stage 9. α-Actin served as an internal control. (B–D) Phenotype of embryos injected with 20 ng SoxD MO. (B) Uninjected embryo. (C) SoxD MO was injected into dorsal-animal blastomeres of eight-cell-stage embryos. (D) Linerized pCS2-SoxD-ORF (100 pg) was co-injected with SoxD MO into dorsal-animal blastomeres of eight-cell-stage embryos. (E–J) Whole-mount in situ hybridization analysis of terminal differentiation markers N-tubulin (E–H) and N-CAM (I,J). Embryos were injected with 40 ng SoxD MO into dorsal-animal blastomeres of eight-cell-stage embryos (F,H,J) and cultured until stage 15 (E,F) or 25 (G–J). (E,F) Dorsal view.
Fig. 8. SoxD is required for XSIP1 expression. Whole-mount in situ hybridization analysis of XSIP1 expression in embryos cultured to stage 11 (A,D), 15 (B,E), and 25 (C,F). (A–C) Uninjected embryos. (D–F) Embryos injected with 40 ng of SoxD MO into dorsal-animal blastomeres of eight-cell-stage embryos.
Fig. 9. XSIP1 is required for SoxD-mediated neural induction. Embryos were injected with 500 pg SoxD mRNA alone (B,E) or co-injected with 40 ng XSIP1 MO (C,F) into the ventral marginal zone of four-cell-stage embryos and cultured until stage 32. (B,E) Ectopic neural tissue was observed on the ventral side (arrow head). (C,F) Ectopic neural tissue was not observed on the ventral side (arrow head).
sox15 ( SRY (sex determining region Y)-box 15) gene expression in Xenopus laevis embryos, NF stage 11, assayed by in situ hybridization, lateral view, animal up.
sox15 ( SRY (sex determining region Y)-box 15) gene expression in a Xenopus laevis embryo, NF stage 20, as assayed by in situ hybridization, dorsal view, anterior down.
sox15 ( SRY (sex determining region Y)-box 15) gene expression in Xenopus laevis embryo, NF stage 30, assayed by in situ hybridization. lateral view, anteriorleft, dorsal up.
