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 Delta^Stu (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 Delta^Stu 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.