Gouti, Briscoe, Gavalas 865 Figure 5. Bmp signaling was not required for Hoxb1-induced HNK- 1 and Snail2 upregulation but was required for maintenance of Snail2 upregulation and repression of dorsal cell fates. Immunoﬂuorescent detection of HNK-1 (A–C), Snail2 (D–I), Pax7 (J–L), and Pax3 (M– O) on transverse sections of neural tubes 24 ([A–F], [J–O]) and 48h PE (G–I) with Hoxb1 and noggin. Hoxb1 induced expression of HNK-1 ([A–C], asterisks in [B]) and Snail2 ([D–F], asterisks in [E]) in the presence of noggin at 24h PE but failed to maintain Snail2 expression at 48h PE (G–I). Hoxb1 no longer repressed Pax7 (J–L) and Pax3 (M–O) in the presence of noggin. Abbreviations: HNK-1, human natural killer-1; PE, postelectroporation. of Hes5, a key effector gene of Notch signaling in the neuroe- pithelium [66] contributing to progenitor maintenance and expressed in the neural tube [67]. Electroporation of Hoxb1 repressed Hes5 expression and that repression was released by NICD coelectroporation (Fig. 6T–6V). This observation also www.StemCells.com provides a likely mechanism for the reduced mitotic rate fol- lowing Hoxb1 electroporation. These ﬁndings suggested that Hoxb1-mediated induction of NC fate but not cell fate switch is dependent upon optimal levels of Notch signaling. Hoxb1 itself potentiates Notch sig- naling raising it above a required threshold and induces cell fate commitment by repressing Hes5. Hoxa2 Has a Similar Potential to Hoxb1 to Induce NC Cell Fates Hoxa2 plays a pivotal role in patterning the NC of the second branchial arch of the head [68], but a role in NC cell induc- tion has not been documented. Hoxa2 was able to upregulate HNK-1 expression in the trunk to a similar extent as Hoxb1 24h PE (Fig. 7A–7C). Furthermore, at 24h PE Hoxa2þ cells were occasionally observed leaving the neural tube from both dorsal and medial positions (Fig. 7D–7F) but HNK-1 upregu- lation was not sustained at high levels 48h PE. Similar to Hoxb1, b-III-tubulin expression was substantially reduced 48h PE (Fig. 7G–7I) and there was a repression of both Pax7 (Fig. 7M–7O) and Pax3 (Fig. 7P–7R) expression by 24h PE establishing that a spinal cord to NC cell fate switch was tak- ing place. In addition, in Hoxa2 electroporated embryos expression of Msx1/2 was extended to medial levels of the neural tube (Fig. 7S–7U) and the neural tube itself was thin- ner (data not shown). Cell adhesion properties also changed, consistent with NC cell fate (Fig. 7V–7X). Hoxa2 on its own did not induce high levels of Snail2 expression at either 24h or 48h PE (data not shown). This might explain the lower lev- els of EMT in the Hoxa2 electroporated embryos. Therefore, Hoxa2 has the capacity to induce NC fates but weaker capacity than Hoxb1 to induce EMT. DISCUSSION Several studies have demonstrated the role of Hox genes in patterning NC cells, particularly, in the cranial region of the body [20]. Despite indications from loss-of-function studies and ES-derived NS cells that anterior Hox genes may partici- pate in NC induction [21, 23] direct evidence was lacking. Here, we provide evidence that anterior Hox genes can mobi- lize the NC genetic cascade leading up to and including EMT. Among the genes examined, Hoxb1 had the strongest potential for inducing NC cell fates by interacting with the Bmp and Notch signaling pathways. We propose that anterior Hox genes sensitize anterior neural cells to NC-inducing sig- nals and participate in key aspects of the cranial NC genetic cascade. Differences Between Trunk and Cranial NC and the Role of Anterior Hox Genes Several lines of evidence suggest differences in the NC genetic cascade in the trunk and cranial region [3]. Snail2 is necessary and sufﬁcient for NC generation and delamination in cranial regions but not in the trunk [14, 36]. Loss of Msx1 and Msx2 resulted in reduced NC survival and NC patterning defects speciﬁcally in cranial and cardiac NC [31]. Sox9 and Sox10 play a central role early in the trunk NC genetic cas- cade [3, 12] but less so in the early steps of cranial NC speci- ﬁcation and delamination [69–72]. Collectively, these studies suggested that the Msx1/2 and Snail genes have a critical role in early speciﬁcation and survival of cranial NC cells, whereas Sox9 and Sox10 play a correspondingly more promi- nent role in the trunk NC. Accordingly, Hoxb1 induced up- regulation of Msx1/2 and Snail2 but not Sox9 or Sox10. This