862 Hox genes rather than a consequence of general elevation of Hox protein levels. To further test this, we electroporated Hoxb1 in the Hox-free midbrain territory at HH stage 10-11 and assayed for upregulation of HNK-1, Snail2, Msx1/2, and b-III-tubulin. Consistent with the effects observed in the trunk, Hoxb1 expression in the midbrain resulted in upregula- tion of Snail2, Msx1/2, upregulation of HNK-1, and reduced neuronal differentiation indicated by reduced b-III-tubulin expression 24h PE (supporting information Fig. 3). These ﬁndings suggested that anterior Hox genes might induce NC cell character with variable efﬁciencies. Among the genes tested, Hoxb1 showed the highest efﬁciency and to explore the mechanisms involved, we concentrated on analyz- ing its effects in NC induction. Hoxb1 Expression Induces Cell Fate Changes in the Trunk Neural Tube The reduction of b-III-tubulin expression and the ectopic gen- eration of migratory NC cells suggested a switch from spinal cord to NC cell fate. To address this, we examined whether Hoxb1 expression altered the establishment of neural progeni- tor domains in the dorsal neural tube. The Pax3 and Pax7 transcription factors are expressed in premigratory NC cells but also in progenitors that give rise to dorsal interneurons [44, 45]. We found that Hoxb1 repressed expression of both Pax3 and Pax7 in a cell autonomous manner with early down- regulation of Pax7 starting at 12h PE which continued at 24h PE (Fig. 3A–3C, 3G–3I); downregulation of Pax3 started at 24h PE (Fig. 3D–3F, 3J–3L). This ﬁnding and the Hoxb1 induced downregulation of b-III-tubulin 24 hours later (Fig. 1M–1O) suggested that a neuronal to NC cell fate switch had taken place. Wnt and Bmp signaling have been implicated in the induction of NC fates [46–52]. Therefore, we examined whether Hoxb1 driven NC cell induction resulted from an up- regulation of dorsal Wnt and/or Bmp signals. Coelectropora- tion of Hoxb1 with either a Tcf luciferase reporter plasmid or a Bmp luciferase reporter plasmid [29] and assay of the lucif- erase activity 12h PE showed that Hoxb1 did not potentiate either Wnt or Bmp signaling (data not shown). On the other hand, we found that expression of the dorsal signaling mole- cules Wnt1, Wnt3A, Bmp4, and Bmp7 were repressed 24h PE, consistent with a cell fate switch (Fig. 3M–3X). This sug- gested that Hoxb1 induced NC cell fate without enhancing expression of dorsal morphogens. We then examined whether Hoxb1 had a similar effect in medial and ventral markers. The Pax2 transcription factor establishes the identity of dorsomedial GABAergic interneur- ons [53], and its expression was also repressed upon Hoxb1 expression 48h PE (Fig. 3AG–3AI). Expression of the ventral neural progenitor marker Nkx6-1 [54] (Fig. 3M–3O) 24h PE and the motor neuron markers MNR2 and Isl1 [54] (Fig. 3AG–3AL) 48h PE was also repressed. These results were consistent with the observed delamination of cells from medial and ventral levels and concomitant reduced neuronal differentiation. To examine whether Hoxb1 induces established mediators of trunk NC cell induction, survival, and delamination, we examined the expression of Sox9, FoxD3, and Sox10 at 12, 24, and 48h PE. Sox9 is expressed in premigratory NC cell where it promotes cell survival and together with FoxD3 and Snail2 induces trunk NC cells [19]. Electroporation of Hoxb1 resulted in downregulation of Sox9 by 12h PE and downregu- lation of FoxD3 by 24h PE. We found no changes in Sox10 expression at 24h and 48h PE (supporting information Fig. 4 and data not shown). Hox Genes and Neural Crest Speciﬁcation These data showed that a spinal cord to NC cell fate switch initiated by the expression of Hoxb1 in the trunk neu- ral tube. On the other hand, Hoxb1 did not induce Sox9 and Foxd3 expression suggesting that it can bypass aspects of the trunk NC genetic cascade. Hoxb11 NC Cells Are Migratory with a Broad De- velopmental Potential To examine the developmental potential of Hoxb1þ induced NC cells we examined b-III-tubulin, P0 and MelEM expres- sion that mark the neuronal, Schwann and melanocyte line- ages respectively [55–58]. Numerous Tuj1þ/Hoxb1þ cells were found in the periphery 72h PE. In most cases they stayed in close proximity to the neural tube but were also found as far as the sympathetic ganglia where they occasion- ally formed an ectopic ganglion (Fig. 4A–4E). Hoxb1þ/P0þ cells migrated into the periphery and were observed both ipsi- laterally and contralaterally (Fig. 4F–4I). Hoxb1 and MelEM immunoﬂuorescence revealed the presence of Hoxb1þ/ MelEMþ cells in both ipsilateral and contralateral positions (Fig. 4J–4M). These results established that Hoxb1þ cells migrated away form the neural tube in ectopic positions and had a broad de- velopmental potential. These observations are consistent with the notion that Hoxb1þ cells are bona ﬁde NC cells. Bmp but Not Wnt Signaling Is Required for Hoxb1- Dependent NC Cell Induction The downregulation of dorsal signaling molecules observed upon Hoxb1 induction was consistent with a cell fate switch but it did not address the possibility that these signals were required for Hoxb1 driven NC induction. To address this we co-electroporated Hoxb1 with constructs that blocked signal transduction of these pathways. To block canonical Wnt sig- naling, we co-electroporated a dominant negative DN-TCF4 construct, lacking the b-catenin interaction domain [26]. Tcfs lacking this interaction domain assemble alternative com- plexes with transcriptional co-repressors, which act as multi- meric dominant transcriptional repressors [59, 60]. Accord- ingly, DN-TCF4 downregulates Msx1/2 at the dorsal most part of the neural tube can downregulate expression of Msx1/ 2 (supporting information Fig. 5). Co-electroporation of DN- TCF4 with Hoxb1 did not block any of the hallmarks of Hoxb1-driven NC cell formation (supporting information Fig. 5): Snail2 and HNK-1 were still upregulated at 24 hours PE, Pax3 and Pax7 were repressed, the electroporated side was thinner and EMT occurred by 48 hours PE (supporting infor- mation Fig. 5 and data not shown). To block Bmp signaling, we used a noggin expression construct [27] that alone was sufﬁcient to completely block Bmp signaling, as assayed by luciferase assays measuring the activity of a Bmp Response Element [29] at 24 hours PE (data not shown). Hoxb1 was still able to induce Snail2 and HNK-1 expression at 24h PE in the presence of noggin (Fig. 5A–5F), but HNK-1 upregulation was noticeably weaker (compare Fig. 5A–5C with Fig. 1D–1F). Furthermore, Snail2 upregulation was not maintained past 24h PE (Fig. 5G–5I) suggesting that the maintenance of Hoxb1-mediated Snail2 upregulation was Bmp dependent. Consequently, EMT was blocked in Hoxb1/noggin coelectroporated embryos and the neural tube retained its normal size in the electroporated side (data not shown). Moreover, the cell fate switch induced by Hoxb1 was essentially blocked in the presence of noggin (Fig. 5J–5O). On the other hand, constitutive activation of Bmp signaling using CA BMPR IA [61] to coelectroporate