Gouti, Briscoe, Gavalas 12h PE and 24h PE (data not shown), and was detected in mi- gratory cells 48h PE (Fig. 1I–1K). It is worth noting that although NC speciﬁers, such as the group E Sox genes, can also induce Snail2 expression on their own, they do so transi- ently and without subsequent cell delamination [19, 37]. Con- sistent with NC cell fate induction, Msx1/2þ cells were detected ectopically located in more ventral positions of the neural tube at 24h PE (Fig. 1L–1N). EMT involves loss of the apical basal polarity, a change in the expression of cell adhesion molecules, such as cadherins, as well as a radical reorganization of the cytoskeleton. This is in- compatible with high proliferation rates, thus proliferation rates in NC cells are low and Snail genes induce EMT by blocking proliferation [38]. Thus, we examined whether electroporation of Hoxb1 and Snail2 induction were accompanied by cell adhesion changes and reduction of proliferation. Cadherin 6B is distrib- uted apically in the neuroepithelium [39] and the induction of migratory NC cells relies upon the Snail2-dependent repression of its expression [40]. N-cadherin is also distributed apically in the neuroepithelium [41] and, similarly to cadherin 6B NC cell migration depends upon its downregulation [42]. Consistent with the initiation of an EMT after Hoxb1 electroporation, we found that both cadherin 6B and N-cadherin were downregulated in Hoxb1þ cells 24h PE (Fig. 1O–1Q, 1R–1T). On the other hand, cadherin-7 is expressed in migratory NC cells [39] and, consist- ent with induction of EMT, expression of Hoxb1 resulted in cad- herin-7 upregulation 24h PE (Fig. 1U–1W). The reduced size of the electroporated side of the neural tube (supporting information Fig. 1) could be due to cells undergoing EMT and leaving the neural tube, but it could also be due to a reduction in proliferation rates or cell death or both. To address this, dividing cells in electroporated embryos were labeled by addition of BrdU 4h PE and collected 6h PE. Hoxb1þ cells in the electroporated side showed a marked reduction in BrdU incorporation suggesting that Hoxb1 induced cell cycle withdrawal (supporting information Fig. 1). To inves- tigate whether excessive cell death was also induced, we per- formed cleaved caspase-3 immunoﬂuorescence assays at 24h PE but found similarly minimal staining on both Hoxb1 electro- porated and nonelectroporated sides (data not shown). These results argue that expression of Hoxb1 in the trunk neural tube induces key features of the NC genetic cascade including the swift and sustained expression of Snail2, induc- tion of Msx1/2 expression, alteration of the cadherin expres- sion proﬁle, and a reduction in proliferation rates. Anterior Hox Transcription Factors Can Induce NC Cell Character with Variable Efﬁciencies To determine whether other anterior Hox patterning genes are also able to induce NC cell speciﬁcation, we introduced sev- eral anterior Hox expression vectors into the developing trunk neural tube of chick embryos via in ovo electroporation at HH stage 10-11. NC cell fate was examined using the premi- gratory and migratory avian NC cell marker human natural killer-1 (HNK-1), a surface epitope that has been used widely to identify NC cells [43]. We found that Hoxb1 and Hoxa2 strongly induced HNK-1 24h PE in a cell autonomous manner within the neural tube and disrupted the morphology of the neuroepithelium (Fig. 2D–2F, 7). Compared with Hoxb1, Hoxa1, and Hoxb2 had a relatively weaker capacity to induce HNK-1 (supporting information Fig. 1), whereas the more posterior Hoxb4 was not able to upregulate HNK-1 expression (supporting information Fig. 2). Ectopic HNK-1þ cells were present as early as 12h PE of Hoxb1 (Fig. 2A–2C) and appeared to be delaminating from the basal surface of the neural tube at 24h PE primarily in dorsal but also medial and www.StemCells.com 861 Figure 2. Expression of Hoxb1 unregulated HNK-1 and reduced neu- ronal differentiation. Immunohistochemical detection of HNK-1 (A–I), Hoxb1 (J–L), and Tuj1 (M–O) was performed on transverse sections of neural tubes 12h PE (A–C), 24h PE (D–F), and 48h PE (G–O) with Hoxb1. Hoxb1 upregulated HNK-1 ([A–I], asterisks in [B, E, H]) in a cell autonomous manner. Conversely, it strongly repressed neuronal dif- ferentiation as seen by Tuj1 immunoﬂuorescence at 48h PE ([M–O], arrowheads in [N]). Electroporated cells delaminating ectopically could be seen as early as 24h PE (arrows in [D, E]) and then migrating away from the neural tube at 48h PE (arrows in [G, H, J, K, M, N]). Abbrevi- ations: DAPI, 40,6-diamidino-2-phenylindole; GFP, green ﬂuorescent protein; HNK-1, human natural killer-1; PE, postelectroporation. ventral positions (Fig. 2D–2I). By 48h PE electroporated cells were observed migrating into the periphery (Fig. 2G–2L). The induction of NC markers correlated with reduced neuronal differentiation, as judged by b-III-tubulin expression (Fig. 2M–2O), suggesting a spinal cord to NC cell fate switch. The differential effects of the tested Hox genes regarding HNK-1 induction suggested that effects are speciﬁc to distinct