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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). |
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Fig. 3. Ihh is required for neural crest specification. (A–F) In vivo efficiency of Ihh antisense morpholino oligonucleotide (IhhMO). Dorsal views of Xenopus laevis embryos under a fluorescence stereoscopic microscope, anterior side is on the left. White arrows indicate the injected side. (A’–F’) Fluorescence and clear field images of each embryo are superposed and shown in merged images. (A and A’) Embryo injected with mRNA encoding IhhGFP (1 ng/embryo) showing GFP fluorescence. (B and B’) Embryo injected with IhhGFP mRNA (1 ng/embryo) and CoMO (30 ng/embryo). (C, C’, D and D’) Embryos injected with IhhGFP mRNA (1 ng/embryo) and IhhMO (C and C’, low dose (1), 10 ng/embryo; D and D’, high dose (2), 20 ng/embryo). No embryo shows GFP fluorescence at a high dose of IhhMO. (E and E’) Embryo injected with CRIhhGFP mRNA showing GFP fluorescence. (F and F’) Embryo injected with CRIhhGFP mRNA and a high dose (2) of IhhMO (20 ng/embryo). The expression of CRIhhGFP was not affected by the presence of the morpholino oligonucleotide. (G–V) Analysis of IhhMO effects on neural crest specification. Dorsal views of Xenopus laevis embryos, anterior side is on the left. Arrows indicate the injected side. (G and H) IhhMO-injected embryos show inhibition of FoxD3 and Snail2 neural crest markers. (I and J) Expression of the neural plate marker Sox2 and the epidermal marker XK81a is expanded on the IhhMO-injected side. The brackets indicate the width of neural plate (I), and the width of neural plate plus neural crest domain (J). (K) Double in situ hybridization for Sox2 and XK81a genes led to the reduction in prospective neural crest domain in the injected side. The brackets indicate the width of the neural crest domain. (L and M) Expression of the mesodermal marker Paraxis. No effect was observed in a dorsal blastomere of 8–16 cell embryos (L). The targeting of IhhMO to the mesoderm by microinjection of the vegetative region of blastomere D1.1 produced a reduced Paraxis expression in the injected side (M). (N and O) Co-injection of IhhMO and CRIhh mRNA rescues FoxD3 and Snail2 expression, respectively. (P and Q) The co-injection of CRIhh specifically driven by the Snail2 promoter (α3000CRIhh) rescues the expression of FoxD3 and Snail2 neural crest markers. (R and S) The microinjection of IhhMO into a dorsal blastomere of stage 16 embryos decreases the expression of FoxD3 in the neural crest but produces no effects on the midline markers (double in situ hybridization, white arrowheads, Nkx6.2 and Pintallavis-FoxA4a). (T and U) The effects of IhhMO on the expression of the neural crest marker FoxD3 is rescued by the directed co-injection of CRIhh (Q) or Shh to the neural fold region (U). (V) CoMO-injected embryos show normal expression of FoxD3. |
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Fig. 4. Ihh is required for neural crest specification (continuation). (A–F) Analysis of the overexpression of dominant negative Ihh∆N-C on neural crest specification. (A and B) Ihh∆N-C-injected embryo shows a diminished expression of FoxD3 and Snail2 markers. (C and D) Expression of the neural plate marker Sox2 and the epidermal marker XK81a is expanded on the Ihh∆N-C-injected side. (E and F) FoxD3 expression was rescued in embryos co-injected with wtIhh (E) or NIhh construct (F). Arrowheads indicate the injected side. Brackets indicate the width of the neural plate (C), and the width of the neural plate plus the neural crest domain (D). |
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Fig. 5. Ihh participates in the early neural crest specification. Dorsal views of Xenopus laevis embryos, anterior side is on the left. Injected side is indicated by an arrowhead. wtIhh-injected embryos show increased expression of FoxD3 (A) and Snail2 (B). The expression of the neural plate marker Sox2 (C) and the epidermal marker XK81a (D) appear reduced on the injected side. (E–H) The injection of NIhh mRNA increases the expression of FoxD3 (E) and Snail2 (F) while the markers Sox2 (G) and XK81a are reduced (H). (I) Double in situ hybridization for Sox2 and XK81a genes evidenced the expansion of the prospective neural crest domain in the injected side. The brackets indicate the width of the neural crest domain. (J–L) The Snail2 promoter-driven (α3000CRIhh) overexpression of the CRIhh construct increases the expression of FoxD3 in the neural crest. The same embryo shown in J was hybridized for FoxD3 and is depicted in K. (L) α3000CRIhh-injected embryos show increased FoxD3 expression and normal expression of NKx6.2 in the embryo midline (double in situ hybridization). Brackets indicate the width of the neural plate (C and G), and the width of the neural plate plus the neural crest domain (D and H).
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