866 Hox Genes and Neural Crest Speciﬁcation Figure 6. Optimal levels of Notch signaling were required for Hoxb1-mediated induction of neural crest cell fates and Hoxb1 itself modulates Notch signaling. Immunoﬂuorescent detection of HNK-1 ([A–C], [J–L]), Snail2 ([D–F], [M–O]), and Msx1/2 ([G–I], [P–R]) on transverse sections of neural tubes, luciferase assays for CSL transcriptional activity (S) and Hes5 detection by in situ hybridization (T–V). All assays were done 24h PE of Hoxb1 in the presence of DAPT (A–I), of Hoxb1 with NICD ([J–R], [V]), of Hoxb1 alone (S, T), or NICD alone (U). Hoxb1 in the presence of DAPT-activated HNK-1 only at dorsal levels ([A–C], asterisks in [B]), failed to activate Snail2 (D–F) but reactivated Msx1/2 at dorsal levels ([G–I], asterisks in [H]). Hoxb1 in the presence of NICD failed to activate HNK-1 (J–L), reactivated Snail2 only marginally at dorsal levels ([M–O], asterisk in [N]) and repressed Msx1/2 ([P–R], arrowheads in [Q]). Hoxb1 electroporation increased CSL transcriptional activity (S) (p < .001). Hoxb1 on its own repressed Hes5 (arrowheads in [T]) in contrast to NICD that activated it (asterisks in [U]). Coelectroporation of Hoxb1 and NICD restored nor- mal levels of Hes5 expression (V). Abbreviations: DAPT, N-[N-(3,5-diﬂuorophenacetyl-L-alanyl)]-S-phenylglycine s-butyl ester; GFP, green ﬂuores- cent protein; HNK-1, human natural killer-1; NICD, Notch intracellular domain; PE, postelectroporation; RLUs, relative light units. is consistent with functional overlap between anterior Hox genes and posterior NC speciﬁers. In this regard, it is worth noting that both FoxD3 and Sox9 were necessary to stabilize Snail2 in ectopic expression experiments [73]. In contrast, Hoxb1-mediated induction of Snail2 occurred more rapidly and lasted longer than that observed after electroporation of Sox9 or FoxD3 [19, 37]. Snail2 is a labile protein, its stability is enhanced through the action of FoxD3 and Sox9 [73]. Our ﬁndings suggest that Hoxb1 can substitute for FoxD3 and Sox9 in this function. The Hoxb1-mediated repression of en- dogenous Sox9 and FoxD3 suggested that Hoxb1 plays a dis- tinct role in promoting delamination and differentiation of premigratory NC cells in the neural tube. Notably, Sox9 is downregulated before delamination [37], whereas maintenance of FoxD3 expression in the cranial NC blocks migration and differentiation [74]. Extended functional redundancy in NC speciﬁcation may have masked the suggested role of anterior Hox genes in this process. Single or double Hox mutants display patterning defects but not NC speciﬁcation or migration defects [20]. Eliminating the Hox-binding partners pbx genes in zebraﬁsh lead to a transformation of the r2-r7 territory into r1 [75]. A similar experiment in frogs whereby the entire paralogous group 1 was knocked down resulted in NC migratory but not speciﬁcation defects concomitant with an expansion of the Gbx2 expression in the r2–r7 territory. Gbx2 has been