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Fig. 1. Xenopus laevis Ihh and Hh pathway components are expressed in the neural crest. (A, B, D–F and H) Dorsal views of Xenopus laevis embryos, anterior side is on the left. (C and G) Lateral views. (A) Ihh transcripts are first detected since the late gastrula stage (stage 12.5) in the lateral domains of the neural plate (arrowheads). (B) During neurulation Ihh is expressed in the neural plate and in the neural plate border (arrowheads). No expression is observed in the neural plate midline (small arrow). No staining was observed in the sense probe control (inset). The line (I) indicates the site of transverse section shown in I. (C) Stage 23 embryos show Ihh expression in the cranial neural crest migratory streams (arrowheads), mandibular (m), hyoid (h) and branchial (b), in the optic (Op) and otic (Ot) vesicles, and in the somites (s, arrow). (D) Ptc1 in situ hybridization in a stage14 embryo. Ptc1 expression is observed in the neural folds (arrowheads) and in the lateral neural plate, while expression is absent in the midline (arrow). No staining was observed using the Ptc1 sense probe (inset). The line (J) indicates the site of transverse section shown in J. (E) Smo transcripts are observed in the neural plate and neural plate border in stage 12.5 embryos. (F) Stage 14 embryos show Smo expression in the neural plate and neural folds (arrowheads). The line (K) indicates the site of transverse section shown in K. (G) In stage 22 embryos Smo is expressed in the neural crest migratory streams (arrowheads), optic vesicle and somites. (H) Gli3 transcription factor is expressed in stage 13 embryos at the lateral border of the neural plate and neural folds (arrowheads). The line (L) indicates the site of transverse section shown in L. (I–L) Transverse section of embryos displayed in B, D, F, and H showing Ihh (I), Ptc1 (J), Smo (K), and Gli3 (L) expression. Asterisks indicate neural crest tissue; np, neural plate; n, notochord. (M–O) Double in situ hybridizations for Ihh (purple) and FoxD3 (turquoise), Gli2 (purple) and FoxD3 (turquoise), and Gli3 (purple) and FoxD3 (turquoise). (M’–O’) Transverse sections of embryos displayed in M–O showing that Ihh, Gli2, and Gli3 expressions overlap with FoxD3 in the neural crest territory. (P) Schematic diagram summarizing the expression of Ihh, Gli2, Gli3 and neural crest markers FoxD3, Snail1/2 in the left half of a midneurula stage transversally sectioned embryo. Yellow (Ihh), red (Gli2) and green (Gli3) lines encircle the areas of expression for each gene. NP, neural plate; N notochord; NNE, non-neural ectoderm; M, mesoderm; E, ectoderm.
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Fig. 2. Analysis of Ihh and Hh members' expression. (A) RT-PCR analysis of the expression of Ihh, Smo, Ptc1, Gli3 and MyoD in neural crest, intermediate mesoderm and non-neural ectoderm explants. Explants were dissected out from stage16 embryos (see Material and methods). EF1α, loading control. (B) Ptc1 expression levels were analyzed as a readout of Hedgehog signaling activity in different embryonic regions, as indicated in C. Each Ptc1 expression level has been normalized to the level of ODC, and presented as a ratio to the neural crest Ptc1 expression level. (C) Schematic drawing depicting a transverse section of a typical midneurula embryo indicating the dissected explants: 1, NC, neural crest explants; 2, IM, intermediate mesodermal explants; 3, NNE, non-neural ectoderm; 4, NP, neural plate; 5, N, notochord.
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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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Fig. 6. Ihh gain- and loss-of-function produce no changes in cell proliferation and apoptosis. Dorsal views of Xenopus laevis embryos, anterior side is on the left. (A and C) The mitotic nuclei were visualized by whole-mount immunostaining using anti-phospho-Histone H3 antibody. White arrows indicate the injected side. (B and D) Apoptotic nuclei were labeled by TUNEL. Ihh- and IhhMO-injected sides show no changes in cell proliferation of apoptosis compared with the respective control sides. Black arrowhead indicates the injected side.
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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. 8. Ihh signaling is necessary for neural crest maintenance. (A) Neural crest explants (NC) were prepared by dissection of the neural crest only, or by including the underlying mesoderm (NC + M) in the explants. Groups of explants were fixed immediately after excision (left), at stages 12, 14 or 17. Groups of explants (NC only or NC + M) were cultured until stages 16, 18 or 23 in the presence or absence of cyclopamine. (B, F and J) NC explants removed from embryos at different stages express FoxD3 when fixed at the moment of dissection. (C and D) NC explants dissected from stage 12 embryos and cultured until stage 16 express FoxD3 at similar levels regardless of the presence of the mesoderm. (D) The NC explants isolated at stage 12 and cultured until stage 16 in the presence of 20 μM cyclopamine lose FoxD3 expression. (G) NC explants dissected from stage 14 embryos show a mild inhibition of FoxD3 expression when they were cultured in the absence of mesodermal tissue. (H) A strong expression of FoxD3, similar to control explants, was observed in NC + M explants. (I) Culture of NC + M explants dissected from stage 14 embryos in 20 μM cyclopamine abolishes the expression of FoxD3 neural crest marker. (K) NC explants dissected at stage 17 without mesoderm and cultured until stage 23 lose FoxD3 expression compared with NC + M explants (L), revealing that signals from the mesoderm are required for the maintenance of specification. (M) Cyclopamine treatment of NC + M explants isolated from stage 17 embryos produced inhibition of FoxD3 expression.
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Fig. 9. Ihh signaling is required for neural crest migration. The anterior side of the embryos is on the left. (A) Stage 18 embryos were grafted on the right neural crest region with a 20 μM cyclopamine-soaked bead. Embryos were cultured until stages 21–23 and the expression pattern of marker genes was analyzed. FoxD3 expressing neural crest cells show arrested migration and accumulated laterally to the hindbrain. The leading edge of migration is indicated by dashed lines. (C) Embryos grafted with control BSA-soaked beads show normal neural crest migration compared with the control untreated side (D). (E) Schematic drawing indicating the experimental procedure for the analysis of Ihh participation during neural crest migration. One-cell stage embryos were microinjected with CoMO, IhhMO, α3000CRIhh construct and lineage tracer. When embryos reached stage 17, neural crest explants containing the underlying mesodermal tissue (NC + M) or neural crest tissue alone (NC) were grafted into wild type or IhhMO injected host embryos. (F and F’) CoMO-injected NC + M explants show normal migration when transplanted into wild type host embryos. (G and G’) IhhMO-injected NC explants grafted into normal wild type host embryos show normal neural crest migration. (H and H’) IhhMO- and α3000CRIhh-coinjected NC + M explants grafted into wild type embryos show normal neural crest migration. The CRIhh construct rescued neural crest and produced a normal migratory cell population. The mesodermal source of Ihh is able to support the migration. (I and I’) IhhMO-injected NC explants grafted onto IhhMO-injected host embryos show no migration. (J and J’) IhhMO- and α3000CRIhh-coinjected NC + M explants grafted onto wild type embryos show normal neural crest migration. (K and K’) Wild type NC explants grafted into IhhMO-injected host embryos show normal migration. The neural crest production of Ihh is able to sustain normal cell migration.
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Fig. 10. Ihh participates in the formation of neural crest derivatives. (A–F) Craniofacial cartilages, Alcian blue staining. (A) IhhMO-injected side of stage 45 embryos (arrowhead) present a marked reduction of Meckel's, ceratohyal and ceratobranchial cartilages. (B) The coinjection of IhhMO and specific Snail2 promoter-driven (α3000) expression of CRIhh rescues normal cartilages morphology. (C) Schematic representation of IhhMO effects on Xenopus head cartilages. (D) Stage 23 embryos were grafted on the right branchial arch region with 20 μM cyclopamine-soaked beads, cultured until stage 46 and stained with Alcian blue. The ceratobranchial cartilage was the most affected by this treatment. (E) Control BSA-soaked beads grafted in the branchial region of stage 23 embryos produced no effect on craniofacial cartilages. (F) Control morpholino (CoMO)-injected embryos show no changes in cartilage morphology. (G–I) IhhMO produced no effects on pigment cell development (G–H) or Trp2 expression (I) compared with their respective non-injected sides (G’, J and K). (A–F) Anterior side is at the top. M, Meckel's cartilage; Ir, infrarrostral cartilage; CH, ceratohyal cartilage; BH, basihyal cartilage; CB, ceratobranchial cartilage.
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