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The notch signaling pathway is widely conserved from vertebrates to invertebrates and mediates the specification of numerous cell fates during developmental processes. In the Xenopus gastrulaembryo, Xdelta1, one of the Notch ligands, is expressed in the prospective mesoderm prior to Xbra expression. Here, we examined the role of Notch signaling in mesoderm formation. Embryos injected with Xdelta1 morpholino oligo DNA showed a severe gastrulation defect and suppression of Xbra expression, which were completely rescued by co-injection with the active form of Notch. In order to fully understand the role of Notch signaling, we examined the expression of the Notch target genes XESR1 and XESR5. RT-PCR and whole-mount in situ hybridization analyses showed that XESR5 was highly expressed in the marginal zone of the early gastrulaembryo, whereas expression of XESR1 was not detected. Animal cap assays indicated that expression of XESR5 was not induced by Notch signaling but by nodal signaling. To clarify the role of XESR5 in the gastrulaembryo, a dominant negative form of XESR5 was injected into the prospective mesoderm. The truncated form of XESR5 induced the ectopic expression of XESR1, which caused a decrease in Xbra expression and defective gastrulation. In contrast, the truncated form of XESR1 caused an upregulation of XESR5 resulting in an increase in Xbra expression. The antagonistic effect of XESR1 and XESR5 suggests a dual regulation in which XESR5 produces a competent area for mesoderm formation by suppressing the gene expression of XESR1, while XESR1 sharpens the boundary of Xbra expression.
Fig. 1. Comparison of gene expression profiles of Xbra and Xdelta1. (A-D) Gene expression of Xbra (A,B) and Xdelta1 (C,D) at st.9 (A,C) and st.11.5 (B,D). All embryos are shown in lateral view with the dorsal side right. Xdelta1 shows the zygotic expression prior to Xbra expression. (E,F) Double in situ hybridization of Xbra (blue) and Xdelta1 (red) in the st.11.5 embryo. (E) Vegetal view with the dorsal side up. (F) Dorsal view with the animal side up. Gene expression of Xbra and Xdelta1 overlapped with each other in the same marginal zone. Xdelta1 expression was recognized in the Xbra-free dorsal area (arrowheads).
Fig. 2. Effect of Xdelta1-MO on gastrulation and Xbra expression. Xdelta1-MO and tracer mRNAs were in- jected into the lateral side of one blastomere at the 2-cell stage. Gastrulation (A-C) and Xbra expression (D-F) were examined in the injected embryo at st.12 and st.10.5, respectively. All embryos are shown in vegetal view with the ventral side up, and arrows indicate the injection side. GFP (A-C) and ï¢-gal (D-F) were used as tracers of the injection side. In contrast to the normal gastrulation of the GFP-injected control embryo (A), Xdelta1-MO caused the gastrulation defect in the injection side (B), which was completely rescued by co-injection of NICD (C). Xdetla1- MO inhibited Xbra expression (E), which was rescued by co-injection of NICD (F). Injection of ï¢-gal into control embryos showed no effect on Xbra expression (D).
Fig. 3. Spatiotemporal expression profile of XESR1 and XESR5. (A) Quantitative RT-PCR analysis of XESR1 and XESR5 during early development. Initial gene expression of XESR5 was detected at st.10 and gradually increased up to st.20, whereas initial gene expression of XESR1 was recognized from st.11. E: unfertilized egg. Histone H4 was used as the internal marker. -RT: without reverse transcriptase reaction. Num- bers on the photograph indicate the Nieuwkoop and Faber develop- mental stage. (B) Whole-mount in situ hybridization of XESR1 (left column) and XESR5 (right column) in the st.10.5 embryo. Upper row shows the lateral view with the dorsal side right, and lower row shows the vegetal view with the ventral side up.
Fig. 6 (Left). Inhibitory transcriptional regulation of XESR1 and XESR5. Synthesized RNAs of ï¢-gal (A,C), XESR1 and ï¢-gal (B) or DN-XESR5 and ï¢-gal (D) were injected into the lateral side of one blastomere at the 2-cell stage, and gene expression of XESR5 (A,B) or XESR1 (C,D) was examined at st.10.5 by whole-mount in situ hybridization. All embryos are shown in vegetal view with the ventral side up. Arrows indicate the injection side. XESR1 inhibited the gene expression of XESR5 (B, arrow), while DN-XESR5 induced the ectopic expression of XESR1 (D, arrow).
Fig. 7. (Right) Effect of XESR1 and XESR5 on the gene expression of Xbra. Synthesized RNAs of XESR1 (B), DN-XESR1 (C), XESR5 (D) or DN- XESR5 (E) were injected into the lateral side of one blastomere at the 2-cell stage together with ï¢-gal as a lineage tracer, and gene expression of Xbra was examined at st.10.5 by whole-mount in situ hybridization. Control embryo was injected with ï¢-gal alone (A). All embryos are shown in vegetal view with the ventral side up. Arrows indicate the injection side.
