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Fig. 3. Early Induction of the NC by DLMZ requires Wnt activation and BMP inhibition. (A-F) The DLMZ was dissected from stage 10.5 embryos and conjugated with animal caps taken from embryos injected at the eight-cell stage with 1 ng of dd2 or 0.6 ng of dnTCF3 mRNA together with FLDx. The conjugates were cultured until the equivalent of stage 15 and the expression of Snail2 (B-D) or Sox2 (E) was analysed. (B) Control conjugate of DLMZ with uninjected animal cap. (C) Conjugate of DLMZ with an animal cap injected with dd2 mRNA. (D) Conjugate of DLMZ with animal cap injected with dnTCF3 mRNA. (E) Sox2 expression showing continued presence of neural plate (68%, n=19). FDX injected animal caps also express Sox2 (data not shown, 63%, n=11). (F) Summary of the expression of Snail2 in conjugates. Each experiment was repeated three times with at least 26 explants each. (G-L) The DLMZ was dissected from stage 10.5 embryos injected at the eight-cell stage in the equatorial region with 1 ng of dnWNT8 mRNA or 2 ng of morpholinos against chordin (cho MO), and conjugated with animal caps taken from uninjected embryos. The conjugates were cultured until the equivalent of stage 15 and the expression of Snail2 (H-J) or Sox2 (K) was analysed. (H) Control conjugate. (I) Conjugate containing DLMZ injected with dnWnt8 mRNA. (J) Conjugate containing DLMZ injected with cho MO. (K) Sox2 expression showing inhibition of neural plate by cho MO (0% of expression, n=30), when compared with controls (75% of expression, n=20; not shown). (L) Summary of the expression of Snail2 in conjugates. Each experiment was repeated three times with at least 30 explants each. (M,N) In situ hybridisation of Wnt8 in a stage 10.25 gastrula (M) or in DLMZ (N). (O,P) In situ hybridisation of chordin in a stage 10.25 gastrula (O) or in DLMZ (P); inset in N shows LMZ. Lines in M and O indicate how the DLMZ was dissected. |
