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Fig. 4. Temporal requirement of Edn1/Ednra signaling for neural crest induction. Stage 12 (A–J), 14 (K–R) or 16 (S) embryos were grafted on the right neural fold with an Edn1 peptide-soaked bead or with the specific Ednra-inhibitor BQ123. Embryos were cultured until stage 14 (A–J) or 18 (K–S), when the expression of marker genes was analyzed. Arrowheads indicate the grafted side. Red circles indicate the position of Edn1- or BQ123-soaked beads. (A–D, K–M) Edn1 peptide increases the expression of neural crest markers Snail2 (A, K) and FoxD3 (B, L). The marker Sox2 (C) and XK81a are reduced (D). (S) No changes in the expression of FoxD3 were observed when Edn1-soaked beads were grafted into stage-16 embryos. (E–H, N–P) BQ123 leads to a reduction in the expression of neural crest markers FoxD3 (E, N) and Snail2 (F, O) on the treated side. BQ123 treatment produces an increase in the expression of Sox2 (P). (I–J, Q–R) Control embryos grafted with BSA-soaked bead. No effect on the expression of neural crest (I, Q; FoxD3) or neural plate (J, R; Sox2) markers is observed. |
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Fig. 3. Ednra is required for early neural crest specification. (A–I) Effect of EdnraMO on neural crest specification. Arrows indicate the injected side. (A'–D') Transverse sections of embryos at the level of cephalic neural crest. (A, B) Ednra-depleted embryos fail to express neural crest markers Snail2 (A) and FoxD3 (B). (A, B, insets) The injected side is recognized by the fluorescence of the lineage tracer FLDx. (C, D) Expression of neural plate marker Sox2 and epidermal marker XK81a is expanded on the EdnraMO-injected side. (E) Embryo processed by double in situ hybridization for Sox2 and XK81a genes showing the reduction of prospective neural crest domain in the injected side. (F) Injection of control morpholino (CoMO) showed no effect. (G) Coinjection of EdnraMO and Ednra′ mRNA rescues Snail2 expression. (H) Single injection of Ednra′ mRNA expanded the Snail2 expression domain. (I) Quantification of rescue experiments. Results are expressed as percentage of embryos normally expressing Snail2 for each treatment (yellow bars), and as percentage of embryos showing reduced Snail2 expression (purple bars). Two different concentrations of Ednra′ mRNA were coinjected for rescue experiments (0.5 ng/embryo and 0.9 ng/embryo). (J–N) Ednra participates in the early neural crest specification. Ednra-injected embryos show increased expression of Snail2 (J) and FoxD3 (K). Expression of the neural plate marker Sox2 (L) and epidermal marker XK81a (M) are reduced on the injected side. (N) Double in situ hybridization for Sox2 and XK81a shows the enlargement of prospective neural crest territory in the injected side. Broken line, dorsal midline; brackets indicate the width of the neural plate (C, L), the width of neural crest domain (E, N) or the width of the neural plate plus the neural crest domain (D, M). |
