Gautier-Courteille C et al. (2004), EDEN-BP-dependent post-transcriptional regulati...

XB-ART-2726

Development 2004 Dec 01;13124:6107-17. doi: 10.1242/dev.01528.

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EDEN-BP-dependent post-transcriptional regulation of gene expression in Xenopus somitic segmentation.

Le Clainche C , Barreau C , Audic Y , Graindorge A , Maniey D , Osborne HB , Paillard L .

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EDEN-BP is a Xenopus RNA-binding protein that triggers deadenylation [poly(A) tail shortening], and thereby translational repression and degradation, of a subset of maternal mRNAs soon after fertilization. We show here that this factor is expressed in the presomitic mesoderm of older embryos, the site where somitic segmentation takes place. Inhibiting EDEN-BP function using either antisense morpholino oligonucleotides or neutralizing antibodies leads to severe defects in somitic segmentation, but not myotomal differentiation. This is associated with defects in the expression of segmentation markers belonging to the Notch signalling pathway in the presomitic mesoderm. We show by a combination of approaches that the mRNA encoding XSu(H), a protein that plays a central role in Notch signalling, is regulated by the EDEN-BP pathway. Accordingly, XSu(H) is overexpressed in EDEN-BP knock-down embryos, and overexpressing XSu(H) causes segmentation defects. We finally give data indicating that, in addition to XSu(H), other segmentation RNAs are a target for EDEN-BP. These results show that EDEN-BP-dependent post-transcriptional regulation of gene expression is required for the process of somitic segmentation.

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Species referenced: Xenopus laevis
Genes referenced: cdk1 celf1 dlc esr-5 hes5.6 hes5.7 myl4 notch1 rbpj
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	Fig. 1. Expression pattern of EDEN-BP during Xenopus early development. Embryos were collected at different stages: neurula, stage 18 (A); tailbud, stage 25 (B,C,G) and stage 27 (D,F); tadpole, stage 31 (E). (A) Dorsal view, (B-G) lateral views; anterior left. Embryos were processed for whole-mount in situ hybridization using an EDEN-BP antisense (A,B,D,E), or sense (C) probe, or for whole-mount immunohistochemistry using an anti-EDEN-BP antiserum (F), or the corresponding pre-immune serum (G). The asterisks in B, D, E and F highlight the higher amount of EDEN-BP mRNA or protein in the presomitic mesoderm.
	Fig. 2. EDEN-BP morpholinos affect somite segmentation in Xenopus embryos. (A) Xenopus embryos were left uninjected (lane 1), or were injected at the two-cell stage into both blastomeres with a mixture of 25 ng of each EDEN-BP morpholino, in the absence (lane 2) or presence of (2 fmol, lane 3; 0.5 fmol, lane 4) EDEN-BP mRNA. Total protein extracts from embryos collected at stage 25 (tailbud) were analyzed by western blotting using an anti-EDEN-BP antiserum (upper panel) and an anti-cdc2 A17 monoclonal antibody (lower panel). (B-Gâ€²) Embryos were injected into one blastomere at the two-cells stage with 50 ng of control morpholinos (C-Mo), 25 ng of each EDEN-BP-Mo (E-Mo), or 25 ng of each EDEN-BP-Mo and 2 fmol of EDEN-BP mRNA (E-Mo+R). They were allowed to develop until stage 26 and were then processed for immunohistochemistry with the myotome-specific 12/101 monoclonal antibody (B-Dâ€²), or for in situ hybridization with a MHC4 probe (E-Gâ€²). Only photographs of the injected sides are shown. Bâ€²-Gâ€² are higher magnifications of B-G, respectively. Asterisks highlight successive somites for embryos showing segmentation.
	Fig. 4. EDEN-BP morpholinos alter presegmentation in the PSM. Embryos were injected in one blastomere at the two-cell stage with 25 ng of each EDEN-BP morpholino. (A-D) Lateral views of stage 25/26 embryos stained for ESR5 (A,B) or X-Delta2 (C,D). (A,C) Non-injected sides, and (B,D) injected sides of the same embryos. TBD, tailbud domain. S1 to S4, Somitomeres 1 to 4. (E-G) Posterior views of stage 20 embryos stained for ESR9, representative of phase I (E), II (F) and III (G) of the dynamic mode of expression of ESR9. (Eâ€²-Gâ€²) Schematic drawings of E-G.
	Fig. 6. Evidence for other targets of EDEN-BP than XSu(H) in the segmentation process. (A-F) Embryos were injected in one blastomere at the two-cell stage with 2 fmol of XSu(H)1 mRNA. They were allowed to develop to stage 28 (A,B) or stage 26 (C-F), and then stained with the 12/101 monoclonal antibody (A,B), or stained with the ESR5 (C,D) or X-Delta2 (E,F) probes. (A,C,E) Injected sides, and (B,D,F) non-injected sides of the same embryos. Asterisks highlight successive somites in B. TBD, tailbud domain; S1 to S4, somitomeres 1 to 4. (G) Embryos were injected with 25ng of each E-Mo, and 0, 1, 2.5 or 5 ng of each X-Mo as indicated. The graph represents the percentage of abnormally segmented embryos, normalized to 100% for the embryos injected with E-Mo only. The total number of embryos examined for each of these conditions is indicated above each bar.
	celf1 (CUGBP Elav-like family member 1) gene expression in Xenopus laevis embryos, NF stage 18, assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	celf1 (CUGBP Elav-like family member 1) gene expression in Xenopus laevis embryos, NF stage 25, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	celf1 ( CUGBP Elav-like family member 1) gene expression in Xenopus laevis embryos, NF stage 27, as assayed by in situ hybridization, lateral view, anterior left, dorsal up
	celf1 (CUGBP Elav-like family member 1) gene expression in Xenopus laevis embryos, NF stage 31, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	Fig. 3. Analysis of embryos injected with anti-EDEN-BP antibodies. (A-D) Embryos were injected into one blastomere at the two-cell stage with control (non-immune, C-Ab) or anti-EDEN-BP (E-Ab) antibodies. They were allowed to develop until stage 39 and then were stained with the myotome-specific 12/101 monoclonal antibody. C and D are higher magnifications of the lower embryos in A and B, respectively. (A,C) Injected sides and (B,D) non-injected sides of the same embryos. (E-G) Hematoxylin and Eosin-stained horizontal sections of stage 39 embryos injected at the two-cell stage with 200 ng of anti-EDEN-BP antibodies. (F,G) Higher magnifications of the somites of the non-injected (F) and injected (G) sides. n, notochord; s, somite. Arrow indicates the injected side. Asterisks highlight successive somites for embryos showing segmentation. Scale bars: 50 Î¼m.