| |
Fig. 7. Temporal requirement of Ihh signaling during early neural crest development. Dorsal views of Xenopus laevis embryos, anterior side is on the left. Stage 11.5 (A–C), 12 (D–H) or 14 (J, K) embryos were grafted on the right neural fold with a cyclopamine-soaked bead. Embryos were cultured until stage 13 (A–C), 14 (D–H) or 17 (J and K), when the expression pattern of marker genes was analyzed. Arrowheads indicate the grafted side. (A–C) Early treatment of neural folds with cyclopamine leads to a reduction in the expression of neural crest markers Snail1 (A), Snail2 (B) and Msx1 (C). (D–H) Cyclopmine-soaked beads grafted in stage 12 also produced a decrease in the expression of neural crest markers FoxD3, Snail2 (D and E) and an expansion of the neural plate (F) and prospective epidermis (G) on the treated side. Cyclopamine-soaked beads grafted on the right side of embryos produced no change in the expression of the midline marker Nkx6.2 (double in situ hybridization). (J and K) Cyclopamine-loaded beads grafted at stage 14 produced a less intense decrease in the expression of FoxD3 and Snail2 markers on the treated side. (I and L) No changes in the expression of FoxD3 were observed when BSA-soaked beads were grafted on stage12 or stage14 embryos. (M) Neural plate border explants were dissected out at stage 11 and incubated until stage 13 in 3/8 NAM solution or 3/8 NAM solution containing 20 μM cyclopamine, and the expression of Pax3 and Sox10 was analyzed by RT-PCR. (N) Quantification of the gel is shown in M, where the results are expressed as Relative Intensity (sample/EF1α × 10). |
