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First published online 17 November 2004
doi: 10.1242/dev.01528

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

Carole Gautier-Courteille, Christophe Le Clainche^*, Carine Barreau, Yann Audic, Antoine Graindorge, Dominique Maniey, H. Beverley Osborne and Luc Paillard

CNRS UMR 6061, IFR 97, Faculté de Médecine, Université Rennes 1, 2 avenue Léon Bernard, CS 34317, 35043 Rennes Cedex, France

View larger version (65K): [in a new window]   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.

View larger version (96K): [in a new window]   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-G') 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-D'), or for in situ hybridization with a MHC4 probe (E-G'). Only photographs of the injected sides are shown. B'-G' are higher magnifications of B-G, respectively. Asterisks highlight successive somites for embryos showing segmentation.

View larger version (89K): [in a new window]   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 µm.

View larger version (50K): [in a new window]   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. (E'-G') Schematic drawings of E-G.

View larger version (41K): [in a new window]   Fig. 5. XSu(H) mRNA is regulated by EDEN-BP. (A) Radiolabelled GbORF-mosEDEN (lanes 1-4), ESR5 3'UTR (lanes 5-8), X-Delta2 3'UTR (lanes 9-12) and XSu(H) 3'UTR (lanes 13-16) transcripts were incubated in Xenopus egg extracts in the absence or the presence of the indicated molar excess of unlabelled RNA with an EDEN (+E, GbORF-mosEDEN) or with the EDEN deleted (–E, GbORF). After UV-cross-linking and RNase treatment, radiolabelled proteins were resolved by denaturing electrophoresis and revealed by autoradiography. The position of the 51 kDa molecular weight marker is indicated on the left. The asterisks indicate the position of the EDEN-BP signal. (B) Embryo extracts were treated for immunoprecipitation with anti-EDEN-BP antibodies (lanes 7-8), or pseudo-immunoprecipitations with beads alone (lanes 3-4) or non-immune serum (lanes 5-6). RNA present in the supernatant (S) or pellet (P) was identified by RT-PCR using primers specific for XSu(H) (upper panel) or EF1 (lower panel). Lanes 1 and 2 show products from PCR performed on the input fraction, either with (lane 2) or without (lane 1) previous reverse transcription. Positions of molecular weight markers are indicated on the left. (C) A capped, radiolabelled, polyadenylated transcript corresponding to GbXSu(H) was injected into two-cell embryos in the presence of 150 ng of the control (C-Ab, lanes 6-10) or anti-EDEN-BP (E-Ab, lanes 1-5) immunoglobulins, and incubated for the indicated times. RNA was extracted, and analyzed by electrophoresis and autoradiography. Positions of A+ (A65) and A– (A0) RNAs are indicated. (D) Quantification of C. The amount of deadenylated GbXSu(H) transcript, expressed as a percentage of the total signal, is plotted against time. (E) Relative quantities of XSu(H) and EF1 mRNA were quantified by real-time RT-PCR in stage 25 embryos that had been previously injected in both blastomeres at the 2-cell stage with EDEN-BP-Mo (E-Mo), EDEN-BP-Mo and 2 fmol of EDEN-BP mRNA (E-Mo+R), or that had been left uninjected (NI). Results, expressed as the ratio between XSu(H) and EF1 mRNAs, are shown for five independent embryos. Statistical analysis showed that the ratios are significantly different between EDEN-BP-Mo-injected embryos, and non-injected or morpholino and RNA-injected embryos (Student's t-test, P<0.05).

View larger version (68K): [in a new window]   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.