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Fig. 5. Spatial distribution of Xp54nrb mRNA during Xenopus early development. Whole mount in situ hybridization is performed on embryos from stage 3 to stage 21. Photomicrographs of whole-mounts and sec- tions through the corresponding whole mounts for stage 3 (A), stage 6.5 (B), stage 11 (C,D), stage 14 (E,F) and stage 21 (G,H) are shown. (A,B) Xp54nrb transcript is detected at the animal pole of cleaving embryos (animal views). (C) Xp54nrb expression in mid-gastrula is found in the ectoderm and mesoderm with a strong expression in dorsal side (lateral view, dorsal on the left). This pattern is confirmed by section analysis (D) which showed labelled of the ectoderm (ec) and in the involuting meso- derm (mes). (E) At early neural stage, the expression is restricted to the developing nervous system (anterior view, dorsal side is up) with a strong expression underlying the neural plate. (F) Transverse section analysis of the stage 14 shows that Xp54nrb is expressed in the sensorial layer of the neuroectoderm (Nes) and absent in the epithelial layer of the neuroecto- derm (Nep), in the notochord (n) and in the somitic mesoderm (ms). (G) At neurula (stage 21, anterior view, dorsal side is up) Xp54nrb expression is high in the anterior neural territories; optic field (of), mesencephalon (Me) and rhombencephalon (Rh) are labelled. Transverse section analysis (H) in a posterior position shows that only the neural tube (nt) is stained at this level. Abbreviations: bl; blastoporal lip, d; dorsal, Ec; ectoderm, end;endoderm, mes; mesoderm, Me; mesencephalon n; notochord, Nes; sensorial layer of the neuroectoderm, Nep; epithelial layer of the neuroectoderm, of; optic field, nt; neural tube, Rh; rhombencephalon, sm; somitic mesoderm, v; ventral. Scale bars 0.5 mm in (A,B), 0.3 mm in (C,D,E,G,H) and 0.1 mm in (F). Black bars in (E,G) represent the position of the corresponding sections in (F,H) respectively.
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Fig. 6. Spatial distribution of Xp54nrb mRNA during Xenopus early organogenesis. Photomicrographs of whole-mounts and sections through the corresponding whole mounts for stage 23 (A, B) and stage 35 (C-F) embryos are shown. (A) Whole mount and section analysis (B) at stage 23 show Xp54nrb expressed in optic cup (oc), otic vesicle (ov), branchial arches (ba) and mesencephalon (Me) (whole mount, lateral view, anterior is left). (C) Anterior view at stage 35 shows similar labelling patterns to the stage 23 embryo with neuroretina, encephalon and spinal chord labelled. (D) In the transverse section at the trunk level, only the ventricular zone of the spinal cord is stained (white arrowheads). (E) Section at the head level through the eye (F), enlargement of E) the dorsal part of D the spinal chord, and the inner layer of the neuroretina are labelled.
No staining is detected in the lens or in the ciliary marginal zone (CMZ, black arrowhead in F). Abbreviations: ba; branchial arches, cg; cement gland, CMZ, ciliary marginal zone, G, ganglion cell layer, I, inner nuclear cell layer, L, lens, Me; mesencephalon, O, outer nuclear cell layer, ov; otic vesicle, oc; optic cup, Rh; rhombencephalon, RPE retinal pigmented epithelium, Te; telencephalon, Scale bars 0.5 mm (in A-D), and 0.1 mm (in E, F).
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Fig. 7. Morpholino MoXp54 blocks Xp54nrb-GFP translation, but not human p54nrb-GFP translation. (A) Western blot. Two-cell-stage embryos are injected into the animal pole of both blastomeres. Lysates from stage 13 whole embryos are analysed by immunoblotting with anti- GFP antibody. Anti a-tubulin antibody attests equivalent loading. ui; non injected embryo extract (Lane1). Embryos were injected with 4 ng of control morpholino (MoC, lanes 2, 3, and 4) or 4 ng of morpholino against Xp54nrb (MoXp54, lanes 5, 6, and 7) and with 50 pg GFP mRNA (lanes 2, 5), 2ng of Xp54nrb-GFP mRNA (lanes 3, 6), or 2 ng hp54nrb-GFP mRNA (lanes 4, 7) respectively. GFP protein is expressed in embryos injected with Moc or with MoXp54.The Xp54nrb-GFP fusion protein is translated in embryos injected with MoC (lane 3) but not translated in embryos injected with MoXp54 (lane 6). While the morpholino against Xp54nrb inhibits Xp54nrb-GFP expression (lane 6), the human tagged p54nrb-GFP is still synthesised (lane 7). (B) Micrographs of stage 13 embryos showing GFP fluorescence. Left top panel: embryo injected with 4 ng MoXp54 and 50 pg GFP mRNA, Left lower panel: embryo injected with 4 ng MoXp54 and 1 ng Xp54nrb-GFP mRNA (Xp54-GFP), Right top panel: embryo injected with 4 ng MoC and 1 ng Xp54nrb-GFP mRNA, Right lower panel, embryo injected with 4 ng MoXp54 and 1 ng human p54nrb-GFP mRNA (p54-GFP). Xp54nrb morpholino (MoXp54) allows human p54nrb-GFP expression but blocks Xp54nrb-GFP signals.
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Fig. 8. Morpholino MoXp54 impairs expression pattern of proneural genes. Embryos are injected at the two-cell stage into one blastomere with either 4 ng of a control morpholino (MoC), 4 ng of Xp54nrb morpholino (MoXp54) or co-injected with 4 ng of MoXp54 and 2 ng of human p54nrb mRNA. Embryos are raised to stage 14 or to stage 17, fixed and analysed by whole mount in situ hybridization for the expression of POU2 (A-D,M), Zic3 (E-H,N) and Sox2 (I-L,O). The side of injection is visualized by the b-galactosidase enzymatic reaction with Red-Gal substrate. Injected side is to the right.The embryos are shown in anterior views, dorsal is up.The patterns observed are representative of the embryos analysed.
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