860 Hox Genes and Neural Crest Speciﬁcation Figure 1. Expression of Hoxb1 induced Msx1/2 expression in embryonic stem-derived neural stem cells and induced sustained expression of Snail2 and Msx1/2 and altered cadherin expression in the chick trunk neural tube. Immunoﬂuorescent detection of Msx1/2 (A–D) showed that its expression was slightly enhanced in Hoxb1 cells (compare [A] with [B]) but dramatically upregulated in Hoxb1þ cells after induction of dorsal cell fates (compare [C] with [D]). Immunoﬂuorescent detection of Snail2 (F–K), Msx1/2 (L–N) cadherin 6B (O–Q), N-cadherin (R–T), and cad- herin 7 (U–W) on transverse sections of neural tubes 6h PE (F–H), 24h PE (L–W), and 48h PE (I–K) with Hoxb1. Hoxb1 upregulated Snail2 expression in a cell autonomous manner as early as 6h PE (asterisks in [G]) and maintained it at least until 48h PE (asterisks in [J]). Delaminat- ing Snail2þ cells were seen at medial levels (arrow at [J]). Hoxb1 expression induced cell autonomously Msx1/2 upregulation 24h PE (arrows in [M]). It also repressed apical expression of cadherin 6B (arrowheads in [P]) and N-cadherin (arrowheads in [S]) at dorsal levels of the neural tube, whereas it induced strong upregulation of cadherin 7 (asterisks in [V]). Scale bar ¼ 50 lm (B). Abbreviations: DAPI, 40,6-diamidino-2- phenylindole; GFP, green ﬂuorescent protein; PE, postelectroporation. inhibition resulted in a small increase of Msx1/2 expression in Hoxb1 NS cells, Msx1/2 expression in Hoxb1þ NS cells increased dramatically (Fig. 1A–1E). These data suggested that Hoxb1 participates in NC cell induction in vivo. To further examine this in vivo, we introduced a Hoxb1 expression vector into the developing trunk neural tube of chick embryos via in ovo electroporation at HH stage 10–11, a stage before NC cell delamination at that axial level. The Snail family genes encode zinc ﬁnger transcriptional repress- ors that are key regulators of EMT [34]. In the chick, Snail2 (a.k.a. as slug) is the functional homolog of the mouse Snail1 [14] and is essential for the induction of EMT [14, 35, 36]. We found that, after Hoxb1 electroporation, Snail2 was rap- idly upregulated in a cell autonomous manner as early as 6h PE (Fig. 1F–1H) suggesting that this was a direct effect of Hoxb1 expression. Upregulation of Snail2 was maintained at