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Fig. 1. BMP inhibition in vivo is not sufficient for neural induction. In all panels, Sox2 expression marks prospective neural tissue, K81 expression marks prospective epidermis, XBra expression marks mesoderm, at the late gastrula stage 13, and Slug marks neural crest progenitors at early neurula stage 15. Marker gene expression is revealed in purple, while β-galactosidase activity resulting from injection of lacZ RNA as a lineage tracer is in red. (A) Sixteen-cell embryos injected with 100 pg CABR RNA in one dorsal-most animal blastomere. BMPR activation represses the neural marker Sox2 and activates the epidermal marker K81. (B) Eight-cell embryos injected animally in the two left blastomeres with lacZ (250 pg/blastomere) and tBR (400 pg/blastomere) RNAs showing neural plate expansion and epidermis suppression (u, uninjected side). (C-G) Sixteen-cell embryos injected in one ventral-most animal blastomere with lacZ (C), lacZ and tBR (400 pg, D), or lacZ and Smad6 (1 ng in E,G; 4 ng in F) RNAs. In whole embryos, BMP inhibition by Smad6 represses epidermis, but does not induce neural tissue. Ectopic neural crest could form in embryos that received 1 ng (arrow), but not 4 ng, Smad6 RNA. (G) Animal caps explanted at blastula stage 9 express Sox2 in response to Smad6 injection in ventral ectoderm cells. The inset in the lower panel illustrates that Sox2 expression (purple) is restricted to the injected region (red). (A,B) Dorsal views, anterior towards the top. (C-F) Ventral views, anterior towards the top, except right panels in E,F, which are lateral views, anterior towards the top. Scale bars: 500 μm. |
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Fig. 3. FGFR signalling is required for neural induction. (A) Four-cell embryos were treated until fixation with various concentrations of SU5402, as indicated. Phenotypic classes were defined according to the severity of axial deficiencies. Class V embryos lacked muscle, NCAM-positive neural tissue and neural crests [as revealed by 12.101 (B) and 4d (C) immunostaining at tailbud stage, or by Slug RNA hybridisation at neurula stage (D)]. Class V embryos at gastrula stages lacked prospective posterior and axial mesoderm (XBra, Xcad3, ADMP; E-G), prospective haematopoietic mesoderm (Xnr2, H), but contained prospective endodermal tissue (sox17α, I). (J-M) Sox2 (J,K) and opl (L,M) expression are shown at early (J,L) or late (K,M) gastrula stages in control and class V embryos. No neural precursors are present in class V embryos. (N,O) Thirty-two-cell embryos were injected with 250 pg lacZ RNA in one A1 blastomere (animal-most, dorsal-most). In untreated controls, injected cells populate mostly the eye and the brain and some head epidermis (N). In the presence of 120 μM SU5402, embryos were class V, and the injected cells were now found entirely in the epidermis below the cement gland (O). These cells expressed the marker K81, indicating that prospective neural cells were converted into epidermal progenitors in absence of FGF activity. SU5402 is not toxic to ectoderm cells as they remain alive, and expressβ -galactosidase and K81. (A-C) Lateral view, anterior towards the left. (N) Lateral view, anterior towards the right. (D,G,J-M) Dorsal view, anterior towards the top. In M, the embryo is slightly tilted upwards. (E,F,H) Vegetal view, dorsal towards the top. (I) Hemisectioned embryo, dorsal towards the right. O, frontal view. |
