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).