864 Hox Genes and Neural Crest Speciﬁcation Figure 4. Hoxb1þ cells generated neuronal cells, glia progenitors, and melanocytes after delamination. Immunoﬂuorescent detection of Hoxb1 (A–M), Tuj1 (A–E), P0 (F–I), and MelEM (J–M) on transverse sections of neural tubes 72h PE of Hoxb1. Boxed areas in (C), (H), (L) are shown in (D, E), (I), and (M), respectively, in higher magniﬁcation. Hoxb1þ cells generated neuronal cells (arrows in [C–E]), glial progenitors (arrowheads in [H, I]), and melanocytes (arrowheads in [L, M]). Tuj1þ and P0þ cells were seen also delaminating inside the lumen of the neural tube (boxed areas in [C, H]), whereas Hoxb1þ/P0þ and Hoxb1þ/MelEmþ cells migrated contralaterally as well. Abbreviations: DAPI, 40,6-diamidino-2- phenylindole; PE, postelectroporation. with Hoxb1 did not affect Hoxb1-induced NC formation and delamination (data not shown). These results suggested that Wnt signaling was not required for Hoxb1-mediated NC induction, whereas Bmp sig- naling was required for strong HNK-1 induction, maintenance of the Snail2 upregulation, and EMT, as well as Hoxb1-medi- ated dorsal cell fate switch. Hoxb1 Interacts with Notch Signaling to Induce NC Active Notch signaling has been associated with maintenance of the neural progenitor state [62, 63] and with induction of NC [64, 65]. Treatment of HH10-11 chick embryos with the c-secretase inhibitor DAPT at 50 lM completely repressed expression of Msx1/2 within 24 hours (Fig. 6G–6H and data not shown). Hoxb1 remained equally effective in repressing the expression of Pax3 and Pax7 in the presence of DAPT (data not shown) and it was able to reactivate expression of Msx1/2 mainly at dorsal levels of the neural tube in a cell au- tonomous manner (Fig. 6G–6I). However, in the presence of DAPT, Hoxb1-mediated induction of HNK-1 was conﬁned to dorsal levels of the neural tube (Fig. 6A–6C), induction of Snail2 was completely abolished (Fig. 6D–6F) and the elec- troporated side of the neural tube retained normal thickness (data not shown). Prompted by the ﬁnding that the NICD on its own induces a small but consistent ventral shift in the expression domain of Msx1/2 (data not shown), we examined whether Hoxb1-induced NC fate commitment entailed poten- tiation of Notch signaling. Coelectroporation of a CSL-de- pendent Notch luciferase reporter [30] with Hoxb1 and assay of the luciferase activity 24h PE showed that Hoxb1 potenti- ated Notch signaling (Fig. 6S) apparently elevating it above a required threshold to induce NC. We then examined whether higher levels of Notch signaling could further increase Hoxb1-driven NC induction. When coelectroporated with NICD, Hoxb1 still repressed expression of both Pax3 and Pax7 and reduced neurogenesis on the electroporated side (data not shown). Surprisingly, coelectroporation of Hoxb1 and NICD strongly repressed Msx1/2 expression (Fig. 6P–6R) and failed to induce Snail2 expression (Fig. 6M–6O). Further- more, induction of HNK-1 was nearly abolished and persisted only occasionally at dorsal levels (Fig. 6J–6L). Thus, Hoxb1- induced switch of spinal cord progenitors to NC fates required intermediate levels of Notch signaling. Notch signaling maintains the neural progenitor state and Hoxb1-mediated commitment to NC cell fate may entail repression of selected effectors. Thus, we assayed expression