Gouti, Briscoe, Gavalas emerging hindbrain and trunk NC and may account for these differences but extensive functional redundancy in NC speciﬁ- cation may have masked their implication in this process. Loss-of-function of single or multiple Hox genes generally resulted in patterning defects but not NC speciﬁcation or migration defects [20]. However, a rhombomere 4 (r4)-spe- ciﬁc triple Hox loss-of-function mutation resulted in loss of the expression of all r4 NC-derived molecular markers and all the structures normally 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 implying a loss of NC cell speciﬁcation [21, 22]. In a screen designed to identify potential Hoxb1 target genes and processes in embryonic stem cell-derived neural stem (NS) cells, we found that Msx1/2 and Snail1 as well as dorsal progenitor markers were upregulated in response to Hoxb1 expression [23]. These ﬁndings raised the possibility that anterior Hox genes may be directly involved in NC speci- ﬁcation. Here, we investigated this issue and found that, when expressed in the neural tube of the trunk region of the devel- oping chick embryo, anterior Hox genes can induce NC cell fates to variable extents. Hoxb1 can impose a neural to NC cell fate switch accompanied by a reduction in proliferation and changes in cell adhesion that lead to EMT. Hoxb1þ cells delaminated from both dorsal and medial levels of the neural tube and generated ectopic neuronal cells, glia progenitors, and melanocytes. Hoxb1-induced EMT and NC cell fate switch were dependent upon Bmp signaling and optimal levels of Notch signaling. Hoxb1 itself potentiated Notch signaling and repressed Hes5 to induce NC speciﬁcation and EMT. Hoxa2 had a similar capacity in inducing NC cell character but a weaker one in inducing EMT. Other anterior Hox genes, such as Hoxa1 and Hoxb2, but not the posterior gene Hoxb4, also had the capacity to induce NC cell character. These ﬁndings suggest that patterning genes participate in NC speciﬁcation by interacting with signaling pathways and regulating the expres- sion of key genes involved in NC speciﬁcation and EMT. MATERIALS AND METHODS Differentiation of Mouse Embryonic Stem Cells (ESC) and Immunoﬂuorescence Differentiation of mouse ESC and immunoﬂuorescence were performed as described earlier [23]. SB216763 (S3442, Sigma, Athens, Greece, www.sigmaaldrich.com/greece.html) was added at a ﬁnal concentration of 10 lM at day 4 of the neuroepithelial selection stage until the end of the second day of the expansion phase at which point cells were analyzed. Chick In Ovo Electroporation Fertilized chick eggs were electroporated at Hamburger Ham- ilton (HH) [24] stage 10–11. Plasmid DNA concentration was 1.5–lg/ll. As a control, pCAGGS-IRES-NLS-GFP was included at 0.5 lg/ll. Electroporation was carried out using a BTX ECM830 electroporator delivering ﬁve 20 V pulses of 50 millisecond duration each. Analysis in the spinal cord was carried out at cervical and upper thoracic levels. Expression Vectors The coding regions of mouse Hoxb1, Hoxb2, Hoxa1, Hoxa2, and Hoxb4 cDNAs were inserted into the pCAGGS-IRES- NLS-GFP expression vector [25] upstream of the IRES. The coding regions of Hoxb2, Hoxa1, and Hoxa2 were fused in- frame with the hemagluttinin (HA) tag. The cDNAs were www.StemCells.com 859 veriﬁed by sequencing and expression was veriﬁed after elec- troporation by immunoﬂuorescence with antibodies recognizing the HA tag, Hoxb1, or Hoxb4 proteins. Wnt and Bmp signaling were blocked using the N-terminal deleted transcription factor 4 (DN-TCF4) and noggin chick expression vectors, respectively [26, 27]. Notch signaling was activated using an Notch intracel- lular domain (NICD) chick expression vector [28]. These plas- mids were used at a ﬁnal concentration of 2 lg/ll. N-[N-(3,5-Diﬂuorophenacetyl-L-alanyl)]-S-phenylgly- cine s-Butyl Ester (DAPT) Treatment, Luciferase, and BrdU Incorporation Assays Embryos were treated 6h postelectroporation (PE) with 25 ll of the c-secretase inhibitor DAPT at a concentration of 50 lM and collected 24h PE. To assay Wnt, Bmp, and Notch signal- ing activity the TOPﬂash (Millipore, Athens, Greece, www.millipore.com/ofﬁces/cp3/gr), 2 BRE [29], and 12 RBP-J [30] luciferase reporter plasmids respectively, were elec- troporated in the presence of a renilla luciferase expression plas- mid (PROMEGA, Southampton, UK, www.promega.com/uk) and embryos were harvested 12h PE for chemiluminescence analysis. For BrdU incorporation, chick embryos were labeled by addition of 25 ll of 10 mM BrdU 4h PE and collected 6h PE. Statistical Analysis All quantitative data were expressed as mean 6 SD and signiﬁ- cance levels were calculated using the Student’s t test. For the BrdU analysis, the data shown represent the percentage of GFPþ in the electroporated area that are labeled with BrdU and the per- centage of cells labeled with BrdU on the nonelectroporated side. All results presented in this report were from experiments repeated in at least ﬁve embryos for each stage analyzed. In Situ Hybridization and Immunoﬂuorescence In situ hybridization probes and antibodies used are listed in supporting information Materials and Methods. Whole mount in situ hybridization was followed by cryosectioning and im- munoﬂuorescence using standard methods. Images were acquired using a Leica (Mannheim, Germany, www.leica- microsystems.com) TCS SP5 confocal microscope. RESULTS Hoxb1 Induces Key Features of NC Fates in Trunk Neural Tube To understand the cellular processes and genes that Hox genes and Hoxb1 in particular may control, we generated mouse ESC that allowed inducible expression of Hoxb1. Its timely induction in ESC-derived NS cells resulted in the speciﬁcation of NS cells toward a hindbrain-speciﬁc identity. Molecular analyses sug- gested that Hoxb1þ embryonic stem (ES)–derived NS cells exhibited a preference for dorsal neural tube fates [23]. This could be attributed at least partly to an upregulation of endoge- nously generated dorsal signals such as Bmp4, Wnt1, and Wnt3A, which resulted in the upregulation of the NC genes Snail1 and Msx1 [23]. The homeodomain transcription factor Msx1 is a key inducer of NC cells and, together with Msx2, plays a crucial role in patterning and survival of cranial NC [31, 32]. To probe the possibility that Hoxb1 may synergize with NC- inducing signals to initiate the NC genetic program, we induced dorsal neural tube identity in Hoxb1 and Hoxb1þ ES-derived NS cells by blocking glycogen synthase kinase 3b (GSK3b) ac- tivity using 10 lM of the speciﬁc antagonist SB216763 [33]. We found that although glycogen synthase kinase 3b (GSK3b)