Int J Dev Biol
January 1, 2011;
Antagonistic role of XESR1 and XESR5 in mesoderm formation in Xenopus laevis.
signaling pathway is widely conserved from vertebrates to invertebrates and mediates the specification of numerous cell fates during developmental processes. In the Xenopus gastrula embryo
, 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
. RT-PCR and whole-mount in situ hybridization analyses showed that XESR5
was highly expressed in the marginal zone of the early gastrula embryo
, 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 gastrula embryo
, 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
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
Int J Dev Biol
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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 -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 -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 -gal (A,C), XESR1 and -gal (B) or DN-XESR5 and -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 -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 -gal alone (A). All embryos are shown in vegetal view with the ventral side up. Arrows indicate the injection side.