S after JSH-23 site electroporation (HH 17-19; Fig. 1H-J; n = 4) as previously demonstrated in later stages [11]. This was also confirmed by in situ hybridization that showed mCAT1 mRNA was present in Nkx2.2-expressing cells, but not in Olig2-expressing cells, just dorsally to the p3 domain within the ventricular zone (Fig. 1K and L). These observations suggest that electroporated mCAT1 was expressed only in the Nkx2.2-expressing cells in our experimental condition, thus the reliability of this system is also confined to neurogenic stages. To trace the lineage of p3 domain cells, an EGFP-expressing retroviral solution was injected into the neural tube 24 h after electroporation of pNkx2.2-mCAT1-myc (HH 19). We analyzed Aldoxorubicin embryos 24 h after retroviral transduction and found that EGFP positive cells were present in the ventral neural tube (Fig. 2A;In Situ HybridizationChick embryos were harvested and fixed in 4 paraformaldehyde/PBS at 4uC for 16 h, followed by incubation with DEPC-treated 20 sucrose/PBS for 24 h. For lacZ staining, chick embryos were fixed in 2 paraformaldehyde/ PBS at 4uC for 1 h, followed by incubation with DEPC-treated 20 sucrose/PBS for 12 h. Embryos were embedded in OCT compound (Sakura Finetek Japan, Japan) and sections were prepared using a cryostat. Procedures of in situ hybridization were as previously described [11]. The following cDNAs were used as probes: foxP1 (NM_001024827; nt_259-1173), retinaldehye dehydrogenas1e 2 (raldh2: AF181680; nt_225-1089), sim1 (XM_419817; nt_901-1850), and mCAT1 (slc7a1) (Gotoh et al., 2011). Sections were observed under a microscope (BX51; Olympus, Japan).ImmunohistochemistryProcedures of in situ hybridization and immunohistochemistry were as previously described (Gotoh et al., 2011). For immunohistochemical staining after in situ hybridization, sections were treated with heat by microwaving for 5 min in 10 mM citrate buffer (pH 6.0) and were cooled to room temperature before incubation with primary antibodies. The primary antibodies used in this study were as follows; mouse anti-HB9, mouse anti-Nkx2.2, mouse anti-Lim3 (DSHB, University of Iowa, USA), rabbit antiGFP (Invitrogen, USA), rabbit anti-Olig2, goat anti-ChAT (Millipore, USA), chiken anti-LacZ (Abcam, USA), and rabbit anti-Myc (MBL, Japan). Sections were observed under a fluorescent microscope (BX51; Olympus, Japan) or confocal microscope (FV-1000; Olympus, Japan).Nkx2.2+ Progenitors Generate Somatic MotoneuronsNkx2.2+ Progenitors Generate Somatic MotoneuronsFigure 1. Expression of the murine retroviral receptor is specific to Nkx2.2-positive progenitors. A, A schematic diagram of the lineage tracing method of Nkx2.2-positive progenitors. It consists of the electroporation of the retroviral receptor followed by infection by the murine retrovirus. B , Double staining of spinal cord sections with anti-Olig2 and anti-Nkx2.2 antibodies at HH 14 (B ) and HH 17 (E ). H , Specific expression of mCAT1-myc in the p3 domain. pNkx2.2-mCAT1-myc was introduced by 1379592 in ovo electroporation at HH 14, and 24 h after the electroporation, the spinal cord sections were immunostained using Myc (H, arrow) and Nkx2.2 antibodies (I). A merged image of H and I was shown in J. K and L, Expression of mCAT1 mRNA was shown by in situ hybridization (K and L; purple, arrows) followed by immunohistochemistry using Nkx2.2 (K; brown) or Olig2 (L; brown). Scale bars indicate 50 mm. doi:10.1371/journal.pone.0051581.gbrown). EGFP-positive cells that were observed in th.S after electroporation (HH 17-19; Fig. 1H-J; n = 4) as previously demonstrated in later stages [11]. This was also confirmed by in situ hybridization that showed mCAT1 mRNA was present in Nkx2.2-expressing cells, but not in Olig2-expressing cells, just dorsally to the p3 domain within the ventricular zone (Fig. 1K and L). These observations suggest that electroporated mCAT1 was expressed only in the Nkx2.2-expressing cells in our experimental condition, thus the reliability of this system is also confined to neurogenic stages. To trace the lineage of p3 domain cells, an EGFP-expressing retroviral solution was injected into the neural tube 24 h after electroporation of pNkx2.2-mCAT1-myc (HH 19). We analyzed embryos 24 h after retroviral transduction and found that EGFP positive cells were present in the ventral neural tube (Fig. 2A;In Situ HybridizationChick embryos were harvested and fixed in 4 paraformaldehyde/PBS at 4uC for 16 h, followed by incubation with DEPC-treated 20 sucrose/PBS for 24 h. For lacZ staining, chick embryos were fixed in 2 paraformaldehyde/ PBS at 4uC for 1 h, followed by incubation with DEPC-treated 20 sucrose/PBS for 12 h. Embryos were embedded in OCT compound (Sakura Finetek Japan, Japan) and sections were prepared using a cryostat. Procedures of in situ hybridization were as previously described [11]. The following cDNAs were used as probes: foxP1 (NM_001024827; nt_259-1173), retinaldehye dehydrogenas1e 2 (raldh2: AF181680; nt_225-1089), sim1 (XM_419817; nt_901-1850), and mCAT1 (slc7a1) (Gotoh et al., 2011). Sections were observed under a microscope (BX51; Olympus, Japan).ImmunohistochemistryProcedures of in situ hybridization and immunohistochemistry were as previously described (Gotoh et al., 2011). For immunohistochemical staining after in situ hybridization, sections were treated with heat by microwaving for 5 min in 10 mM citrate buffer (pH 6.0) and were cooled to room temperature before incubation with primary antibodies. The primary antibodies used in this study were as follows; mouse anti-HB9, mouse anti-Nkx2.2, mouse anti-Lim3 (DSHB, University of Iowa, USA), rabbit antiGFP (Invitrogen, USA), rabbit anti-Olig2, goat anti-ChAT (Millipore, USA), chiken anti-LacZ (Abcam, USA), and rabbit anti-Myc (MBL, Japan). Sections were observed under a fluorescent microscope (BX51; Olympus, Japan) or confocal microscope (FV-1000; Olympus, Japan).Nkx2.2+ Progenitors Generate Somatic MotoneuronsNkx2.2+ Progenitors Generate Somatic MotoneuronsFigure 1. Expression of the murine retroviral receptor is specific to Nkx2.2-positive progenitors. A, A schematic diagram of the lineage tracing method of Nkx2.2-positive progenitors. It consists of the electroporation of the retroviral receptor followed by infection by the murine retrovirus. B , Double staining of spinal cord sections with anti-Olig2 and anti-Nkx2.2 antibodies at HH 14 (B ) and HH 17 (E ). H , Specific expression of mCAT1-myc in the p3 domain. pNkx2.2-mCAT1-myc was introduced by 1379592 in ovo electroporation at HH 14, and 24 h after the electroporation, the spinal cord sections were immunostained using Myc (H, arrow) and Nkx2.2 antibodies (I). A merged image of H and I was shown in J. K and L, Expression of mCAT1 mRNA was shown by in situ hybridization (K and L; purple, arrows) followed by immunohistochemistry using Nkx2.2 (K; brown) or Olig2 (L; brown). Scale bars indicate 50 mm. doi:10.1371/journal.pone.0051581.gbrown). EGFP-positive cells that were observed in th.
