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Fig. 4. The Notch target gene Hairy2A produces an expansion of the neural crest population. Eight-cell embryos were injected in two blastomeres with 1 ng Hairy2A mRNA, the arrowhead indicates the injected side, which was visualized by alkaline phosphatase-mediated FITC inmunodetection. (A) N-tubulin expression is clearly reduced. (B) Immunodetection of the somite antigen 12/101 analyzed at stage 25. Note that there is no difference in the staining between the injected and uninjected side. (C) The domain of Xslug expression is expanded on the injected side, whereas Bmp4 is dramatically repressed (D,E). (E) Corresponds to a higher magnification of D. (F) Xmsx1 expression is increased on the injected side. Each experiment was performed at least twice with a minimum of 35 embryos. The effect seen in each experiment was observed in at least 70% of embryos. |
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Fig. 3. Notch signaling is required for neural crest specification. Two-cell embryos were injected in one blastomere with 0.7 ng of NICDGR (A,E,I,M,Q), 0.7 ng of Su(H)ankGR (B,F,J,N,R), 1 ng of DeltaStu (C,G,K,O,S) or 0.25 ng of Su(H)DBMGR (D,H,L,P,T) mRNA, and the inducible constructs were activated at stage 12. NICDGR and Su(H)ankGR activate Notch signalling, and DeltaStu and Su(H)DBMGR inhibit Notch signalling. The expression of Xslug, Foxd3, Bmp4, Xmsx1 and Hairy2A was analyzed at stage 17 or 18 by in situ hybridization, and the injected sides were visualized by alkaline phosphatase-mediated FITC immunodetection. The injected side is labeled with an arrow and all embryos are presented dorsally with the anterior to the top. (A,B,E,F) Note the expansion of Xslug (A,B) and Foxd3 (E,F) expression on the injected side after activation of Notch signaling. (C,D,G,H) Note the inhibition in Xslug (C,D) and Foxd3 (G,H) expression on the injected side, after inhibition of Notch signaling. (I-L) The domain of expression of Bmp4 is highlighted in the neural folds by the brackets. Scale bar: 80 μm. Note the reduced expression domain after Notch activation (I,J), and the expansion and increase in the intensity of Bmp4 expression on the injected side after Notch inhibition (K,L). (M-P) Expression of Xmsx1. Note the expansion in the Xmsx1 expression domain after Notch activation (M,N) and the reduction of Xmsx1 expression on the injected side after Notch inhibition (O,P). (Q-T) Hairy2A expression. Note the expansion in Hairy2A expression in the injected side after Notch activation (Q,R) and the decrease in Hairy2A expression on the injected side after Notch inhibition (S,T). Each experiment was performed at least twice with a minimum of 45 embryos. The effect seen in each experiment was observed in at least 70% of embryos. |
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Fig. 6. Xiro1 is upstream of Notch signaling in the specification of the neural crest. Embryos were injected with 1 ng HDGR (A) mRNA, and co-injected with 1 ng Hairy2A (B) or 1 ng Su(H)ankGR (C) mRNA. A second set of experiments was performed by injecting two-cell embryos in one blastomere with 0.25 ng Su(H)DBMGR (D) mRNA, and co-injecting 1 ng Hairy2A (E) or 0.7 ng Xmsx1GR (F) mRNA. Finally, a third set of experiments was performed by injecting one blastomere of a two-cell embryo with 1 ng of NICDGR (G) mRNA, and co-injecting 0.7 ng of dnXmsxGR (H). The embryos were treated with dexomethasone at stage 12, and the expression of Xslug was analyzed by in situ hybridization between stage 17 and 19. The injected side was visualized by alkaline phosphatase-mediated FITC inmunodetection and is indicated by an arrowhead. (A) Xslug expression was inhibited by HDGR. (B) The inhibition of Xiro1 activity was rescued by co-injection of Hairy2A, reaching 89% recovery of Xslug expression (n=56). (C) A similar reversion of Xiro1 inhibition was obtained by activating Notch signaling, 93% rescue of Xslug expression was observed (n=47). (D) Xslug expression was inhibited by Su(H)DBMGR. (E) The inhibition of the Notch signaling could be rescued by co-expression of Hairy2A (92% rescue; n=43). (F) The effect of inhibiting Notch signaling could be rescued by co-expression of Xmsx1 (97% rescue; n=39). (G) Expansion of Xslug expression by injecting 1 ng NICDGR. (H) The effect of NICDGR was rescued by blocking msx1 activity with dnXmsxGR, (92% rescue; n=45), whereas the effect of NICDGR was not rescued by the co-injection of GFP mRNA (I; 0% rescue; n=25). |
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Fig. 5. Xiro1 participates in the induction of neural crest cells. Two-cell embryos were injected in one blastomere with 1 ng of the inducible forms of a repressor of Xiro1 (HDGREnR) (A,D,G,J), an activator form of Xiro1 (HDGRE1A) (B,E,H,K), or with a dominant-negative form of Xiro1 (HDGR) (C,F,I,L). The embryos were treated with dexomethasone at stage 12, and the expression of Xslug, Bmp4, Xmsx1 and Hairy2A was analyzed by in situ hybridization. The injected side was visualized by Myc inmunostaining, or alkaline phosphatase-mediated FITC inmunostaining, and is indicated with an arrowhead. (A-C) Xslug expression. (A) An expansion of the Xslug expressing neural crest domain is observed. (B,C) Xslug expression is reduced on the injected side. (D-F) Bmp4 expression. (D) A repression of Bmp4 in the neural fold domain is indicated by the bracket. (E,F) The levels of Bmp4 transcripts are augmented on the injected side and an expansion in the expression domain is also observed. Note that in F, the expression indicated by two small brackets on the uninjected side is transformed into a single big bracket on the injected side. Scale bar: 85 μm. (G-I) Xmsx1 expression. (G) Note the expanded Xmsx1 expression domain. (H,I) A reduction in the expression of Xmsx1 can be seen in the neural fold region. (J-L) Hairy2A expression. (J) An expanded domain of Hairy2A expression is observed in the neural fold, whereas Hairy2A expression is inhibited by the injection of HDGRE1A and HDGR (K,L). Each experiment was performed at least twice with a minimum of 42 embryos. The effect seen in each experiment was observed in at least 65% of embryos. |
