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Fig. 1. Expression patterns of the neural markers used in this study, as detected by whole-mount in situ hybridization at stage 30/31 (A-H) or stage 22/23 (I-L). (A-K) lateral views; (L) dorsal view.
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Fig. 2. Specification assays on dorsal ectoderm (DE) of stage 10.5 embryos. DE was explanted as outlined in the scheme, grown to stage 12.5. (J), to stage 22/23 (A-I) or to stage 30/31 (K-M), and finally processed for in situ hybridization with probes for nrp1, Xotx2, XBF-1, Xrx1, Xvax1b, Xemx1, Xkrox20, XhoxB9, cardiac actin and chd as indicated.
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Fig. 3. Characterization of different fragments of dorsal mesendoderm used in recombination experiments. Fragments were reproducibly dissected from stage 10.5 (A-F) or stage 11 (G,H), as shown in the scheme, cultured to stage 12.5, and assayed for expression of the organizer marker genes Xhex (A), chd (B), gsc (C,E,G) and Xnot (D,F,H).
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Fig. 4. Tissue recombination induction assays. Conjugates were made by recombining stage 9 animal caps with the involuted anterior dorsal mesendoderm (ADME, green) of stage 10.5 embryo (A-E, upper scheme on the left) or with the presumptive notochordal fragment (brown) of stage 11 embryo (F-J, lower scheme on the left). Conjugates were grown to stage 22/23 and assayed by in situ hybridization for expression of the neural markers nrp1 (A,F), Xotx2 (B,G), Xrx1 (C,H), XBF-1 (D,I), Xemx1 (E,J). (K) RT-PCR analysis of the expression of neural markers in similar conjugates: AC, animal caps; WE, whole embryo; `green' and `brown' correspond to the colored fragments in the schemes.
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Fig. 5. The involuted ADME of stage 10.5 embryo acts on stage 10.5 DE to elicit proper Xemx1 expression at stage 22/23. (A) DE (violet) and involuted ADME (green) were explanted and recombined at stage 10.5 (upper scheme), grown to stage 22/23 and assayed for Xemx1 expression. (B) DE and the underlying involuted ADME (red) were explanted together at stage 10.5 (lower scheme), grown to stage 22/23 and assayed for Xemx1 expression.
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Fig. 6. The ADE promotes Xemx1 expression and downregulates Xnkx2.1 expression in explants of DE. (A,B) DE (violet in upper schematic) was explanted from stage 10.5 embryos, cultured to stage 22/23 (A) or 30/31 (B) and assayed for expression of Xemx1 (A) or Xnkx2.1 (B). (C,D) DE (violet in lower schematic) was explanted from stage 10.5 embryos together with the ADE (yellow in lower schematic), grown to stage 22/23 (C) or 30/31 (D), and assayed for expression of Xemx1 (C) or Xnkx2.1 (D).
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Fig. 7. Injection of chordin or of Smad7 mRNA cannot induce expression of the dorsal telencephalic markers Xemx1 and eomes in animal cap assays. Animal caps from stage 9 embryos injected with 600 pg chordin mRNA (A-D), or with 200 pg Smad7 mRNA (E-H), or from uninjected embryos (I-L) were dissected, grown in pairs to stage 30/31 and assayed for expression of Xrx1, Xemx1, eomes and XK81 as indicated. (I-L) are uninjected control caps.
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Fig. 8. The involuted ADME of stage 10.5 embryo can trigger expression of the dorsal telencephalic markers Xemx1 and eomes in chd injected animal caps. Animal caps (blue in schematic) from stage 9 uninjected (A-F) or injected (G-K) embryos were explanted, conjugated in pairs either without (A-C;G-I) or with (D-F,J,K) the ADME (red) from a stage 10.5 gastrula and grown to stage 30/31. Injected animal caps never express either Xemx1 (G) or eomes (H), but show activation of a control neural marker, Xrx1 (I). chd-injected caps conjugated with the ADME show activation of both Xemx1 (J) and eomes (K), whereas no activation of these genes or of Xrx1 is detected in conjugates between ADME and uninjected animal caps (D-F). Uninjected caps never show expression of Xemx1, eomes or Xrx1 (A-C).
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Fig. 9. cerberus, but not chd, mRNA triggers Xemx1 and eomes expression in injected animal caps. Animal caps were injected with amounts indicated of cerberus (A-C) or chd (D-F), or a combination of chd and cerδC1 (G,H) mRNA, or were uninjected (I,J). At stage 30/31 they were assayed for expression of Xrx1 (A,D), Xemx1 (B,E,G,I) and eomes (C,F,H,J).
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Fig. 11. FGF effect on neuralized animal caps. As shown in the scheme, animal caps were dissected from stage 10.5 embryos injected either with chordin (660 pg)+cer-S (2 ng) mRNA (A-O), or with cerberus mRNA (2 ng) (A′-N′), and recombined in pairs either without addition of FGF-soaked beads (A,F,K,A′,E′,I′,M′) or with FGF-soaked beads (B-E,G-J,L-O,B′-D′,F′-H′,J′-L′,N′). After reaching stage 30/31, they were processed by in situ hybridization for the expression of Xnkx2.1 (A-E,A′-D′), eomes (F-J,E′-H′), Xemx1 (K-O,I′-L′) or Xnkx2.4 (M′,N′). Concentrations used for FGF8 were 100 ng/μl (B,G,L,B′,F′,J′), 200 ng/μl (C,H,M,C′,G′,K′,N′) or 400 ng/μl (D′,H′,L′); concentrations used for bFGF were 100 ng/μl (D,I,N) or 200 ng/μl (E′,J′,O′).
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Fig. 12. FGF signaling is required for telencephalic gene expression. As shown in the schematic, the early dorsal blastopore lip (brown) of a stage 10-10+ gastrula was sandwiched either between two uninjected stage 9 animal caps (A-C) or animal caps injected with δXFGFR-4a (320 pg/blastomere) (blue) in all four animal blastomeres of eight-cell stage embryos (D-F). Conjugates were grown to stage 30/31 and assayed for expression of Xnkx2.1 (A,D), Xemx1 (B,E) or Sox2 (C,F).
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