Gouti, Briscoe, Gavalas 867 Figure 7. Expression of Hoxa2 in the trunk neural tube induced a neuronal progenitor to neural crest cell fate switch and limited epithelial-to- mesenchymal transition. Immunoﬂuorescent detection of HNK-1 ([A–C], [G–I]), Hoxa2 (D–F), Tuj1 (J–L), Pax7 (M–O), Pax3 (P–R), Msx1/2 (S–U), and cadherin 6B (V–X) was performed on transverse sections of neural tubes 24h ([A–F], [M–X]) and 48h PE (G–L) with Hoxa2. Hoxa2 cell autonomously upregulated HNK-1 ([A–C] and [G–I], asterisks in [B] and [H]). Hoxa2þ cells delaminated 24h PE ([D–F], arrows in [D, E]). Hoxa2 strongly repressed neuronal differentiation 48h PE ([J–L], arrowheads in [K]). Hoxa2 cell autonomously repressed Pax7 ([M–O], arrow- heads in [N]), Pax3 ([P–R], arrowheads in [Q]) and cadherin 6B ([V–X], arrowheads in [W]), whereas it upregulated Msx1/2 ([S–U], asterisks in [T]). Abbreviations: GFP, green ﬂuorescent protein; HNK-1, human natural killer-1; PE, postelectroporation. recently identiﬁed as a NC inducer accounting for the pres- ence of NC in the transformed territory [76, 77]. However, an r4-speciﬁc triple Hox loss-of-function mutation resulted in loss of the expression of all r4 NC-derived molecular markers and the structures derived from r4 NC populating the second arch. Reciprocal grafts between mutant and wt embryos showed that these defects were cell autonomous to the mutant r4 suggesting a role for anterior Hox genes in NC speciﬁca- tion [21, 22]. Here, we show by gain-of-function experiments that anterior Hox genes had a similar but variable capacity in inducing NC cell fates when expressed in the trunk. Hoxb4, expressed in the postotic region of the hindbrain, next to RA generating axial mesoderm [78] failed to induce NC character. These ﬁndings further support the existence of functional speciﬁcity among Hox genes [79–81]. Anterior Hox Genes May Sensitize Cells to NC-Inducing Signals A gradient of Bmp activity speciﬁes the border of the neural plate as the presumptive NC territory and subsequently a www.StemCells.com combination of Wnt, Fgf, and RA signaling transforms the border of the neural plate into NC [4, 5]. Wnt, Fgf, and RA signals have been implicated in the acquisition of caudal neu- ral identity [82–86]. The localization of the tissues expressing these signals [87], the expression patterns of RA degrading enzymes [88, 89], and the expression patterns of Wnt signal- ing antagonists [90] generate a gradient of posteriorizing, and therefore, NC-inducing activities that leave the hindbrain region at a relative deﬁcit of these signals [91]. We hypothe- sized that Hox homeobox genes expressed in the anterior neu- ral plate compensate by sensitizing cells to NC-inducing sig- nals. Consistent with this, Noggin blocked the Hoxb1 NC- inducing capacity, at least partly by abolishing Hoxb1-medi- ated Snail2 stabilization, suggesting an important input of Bmp signaling in the late steps of the NC genetic cascade. On the other hand, blocking Notch signaling with DAPT or constitutively activating Notch signaling with NICD had a similar effect suggesting that intermediate levels of Notch sig- naling are necessary for Hoxb1-mediated NC speciﬁcation and EMT. We propose that Hoxb1 may promote NC speciﬁ- cation by elevating Notch signaling above a possible threshold