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First published online October 27, 2004
doi: 10.1242/10.1242/dev.01445

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Neural induction requires BMP inhibition only as a late step, and involves signals other than FGF and Wnt antagonists

Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (57K): [in a new window]   Fig. 1. Smad6 does not induce neural markers in the chick. (A-G) Experiments to test the activity of Smad6. Smad6-IRES-GFP was electroporated into one half of the chick neural tube (A,B). Staining against phospho-Smad1 (A) reveals that activation of Smad1 has been inhibited in the electroporated cells (green in B), while in control GFP electroporated embryos (C,D) phospho-Smad1 is not altered (C). cSmad6 injection into the marginal zone in Xenopus (E) induces a secondary axis (F), while GFP-injected controls appear normal (G). (H-N) Smad6 does not induce neural markers. Electroporation of Smad6 into competent area opaca epiblast at stage 3+ (H) does not induce Brachyury (I,L; light blue), Sox2 (J) or Sox3 (M) (purple). In this and subsequent figures, electroporated cells were visualized by staining with anti-GFP antibody (K, N; brown). (O-Q) Positive controls. cSmad6 or GFP was injected at the two-cell stage and animal caps isolated at early gastrula (O). Smad6 (P) can neuralize animal caps, while GFP cannot (Q), as assessed by Sox3 expression.

View larger version (124K): [in a new window]   Fig. 2. BMP inhibition is not sufficient for neural induction. Inhibition of BMP by misexpression of dnBMPR (A,B), Chordin (C,D), Noggin (G) or dnBMPR+Chordin+Noggin+Smad6 (E,F) is not sufficient to induce Sox2. (H,I) Sections through the embryos in F,G at the levels indicated. All cell pellets produce background staining after Sox2 in situ hybridization (E-I); sections are therefore necessary to show absence of expression in the epiblast.

View larger version (137K): [in a new window]   Fig. 3. BMP inhibits late markers of neural induction. BMP4 was electroporated into the prospective neural plate at stage 3+ and the consequences analysed in time course. After 12 hours and 15 hours of incubation, the early marker Sox3 is not affected (A,B,G,H), while the later marker Sox2 is strongly downregulated in the neural plate (C,D,I,J). By 20 hours after electroporation, both Sox3 (M,N) and Sox2 (O,P) are downregulated. Neither Sox3 nor Sox2 is altered when control GFP is electroporated (E,F,K,L,Q,R).

View larger version (126K): [in a new window]   Fig. 4. BMP inhibition does not induce neural tissue even in combination with FGF8 and/or Wnt antagonists. Neither a source of FGF8 protein (A) nor inhibition of Wnt signalling by NFz8+Dkk+ Crescent (Wnt; pellet of transfected cells) (B,D) induces Sox2 expression in competent area opaca epiblast. The same is seen after misexpression of a combination of FGF8+NFz8+Dkk+Crescent (C,E), FGF8+Smad6 (F,H,I), or of all of these together (FGF8+NFz8+Dkk+Crescent+Cerberus+Smad6) (G,J,K). The histological sections (D-G) show that the epiblast in direct apposition to the source of factors does not express Sox2.

View larger version (114K): [in a new window]   Fig. 5. BMP inhibition in combination with FGF (FGF2, FGF3 or FGF4) and Wnt antagonists does not induce neural tissue directly. (A,B,I,J,Q,R) Beads with FGF2, FGF3 or FGF4 protein cannot induce either brachyury (light blue) or Sox2 (purple). (C-H,K-P) Likewise, FGF2 or FGF3 together with Smad6 and/or Wnt antagonists does not induce brachyury or Sox2. (S-U) Misexpression of Smad6 and FGF4 induces both brachyury and Sox2; (V-X) addition of Wnt antagonists to the combination inhibits induction of both markers.

View larger version (71K): [in a new window]   Fig. 6. BMP inhibition by Smad6 is not sufficient for neural induction in Xenopus ventral epidermis. (A-I) cSmad6 was targeted to the ventral epidermis by injection into the A4 blastomere of 32-cell stage embryos (A). Smad6 does not activate Sox3 expression (B-E), compare with GFP-injected control embryos (F-I). B,C,F,G are dorsal views; D,E,H,I are ventral views of the embryos in B,C,F,G. C,E,G,I show the embryos in B,D,F,H after staining with anti-fluorescein to reveal the lineage tracer FDX which was co-injected with the mRNA. (J-L) To test that the progeny of the A4 blastomere is competent to respond to neural inducing signals from the organizer, a lineage tracer (FDX) was injected into the A4 blastomere. At late blastula/early gastrula stage, the labelled cells are transplanted into the dorsal side of an unlabelled host embryo, which is grown to early neurula stage (J). (K,L) The transplanted cells (FDX in brown in L) contribute to the neural plate and express Sox3 (purple).

View larger version (74K): [in a new window]   Fig. 7. Effects of BMP inhibition in combination with FGF in Xenopus. Embryos were injected into the A4 blastomere at the 32-cell stage. FGF4 in combination with cSmad6 does not induce brachyury in prospective ventral ectoderm cells at stage 10+ (A-C). Neither FGF8 nor FGF4 is able to induce Sox3 in the ventral epidermis cells of early neurula stage embryos (D-I), while misexpression of FGF4 together with Smad6 induces a patch of Sox3 (J-L). (A,D,G,J) Dorsal views; (B,E,H,K) ventral views of same embryos, which are shown again in C,F,I,L after anti-fluorescein staining.