Ryos (n = 4). Some LacZpositive cells in the Title Loaded From File ventral horn were Isl1/2-positive and HB9positive (Fig. 3N, O), indicating that they differentiated into somatic motoneurons. To define the distribution of Nkx2.2-lineage cells, we stained brachial or thoracic Title Loaded From File spinal cord sections by LacZ histochemistry. Fig. 4A and 4B show schematic diagrams of the distribution of LacZ-positive cells in the brachial or thoracic spinal cord of three independent chick embryos. At HH 32 (E7), LacZ-positive cells were mostly present in the gray matter and the ventricular zone. In two of three embryos (Fig. 4A, left and right), there were LacZpositive cells in the ventral horn in the brachial spinal cord. In the thoracic spinal cord, LacZ-positive cells were observed more dorsally near the ventricular zone, suggesting that they were preganglionic motoneurons (Fig. 4B). In order to confirm that recombined cells included motoneurons that send axons outside the spinal cord, retrograde tracer (fluorogold; FG) was used to label motoneurons. Injected FG was spread not only to the wing bud but also widely to the body cavity including peritoneal and thoracic ones, and therefore, somites and other mesenchymal tissues showed FG fluorescence bilaterally in the coronal sections. Within the spinal cord, somatic motoneurons in the ventral horn and preganglionic cells in the intermediate region showedyellowish white FG fluorescence bilaterally. In cases with FG injection, a LacZ-positive cell was located in the ventral horn where most of cells were retrogradely labeled with FG so that this LacZ-positive cells was highly likely labeled with FG also (Fig. 4C, D). To further analyze this, we used the brainbow plasmid which expresses RFP, and GFP after Cre mediated recombination. GFPpositive cells observed in the ventral part of the ventral horn showed GFP immunoreactivity, one of which was retrogradely labeled with FG (Fig.4E, F, and G arrowheads). Therefore, some of the recombined cells were apparently motoneurons that sent axons outside the spinal cord (Fig. 4F arrows). At later stages (HH 42 or E16), some LacZ-positive cells expressed choline acetyltransferase (ChAT), an enzyme for acetylcholine generation and, thus, often used as a marker for mature motoneurons, in the ventral horn and around the central canal (Fig. 4H and 4L). Expression of ChAT in recombined cells were also confirmed by double-immunohistochemistry using anti-LacZ and anti-ChAT antibodies (Fig. 4I , 4M ; arrowheads). We collected 20 mm sections in each 300 mm section from electroporated spinal cord. The ratio of LacZ/ChAT-double positive cells per total LacZpositve cells was calculated from five embryos. A large number of motoneurons were derived from Nkx2.2-positive cells (Fig. 4P; average = 31.368.4 ). ChAT-negative cells might represent interneuron other than motoneurons. These data suggest that a large population of Nkx2.2-progenitors differentiated into functional motoneurons. In order to define the motoneuron subtype from Nkx2.2positive progenitors, we stained LacZ together with subtypespecific markers [20], [21] at HH 32 in brachial or thoracic spinal cords using Lim3 for MMC, raldh2 for LMC, and foxP1 for CT. Nkx2.2-derived cells differentiated into Lim3 positive (Fig. 5A) and raldh2-positive motoneurons (Fig. 5B) in the brachial spinal cord. In the thoracic spinal cord, we observed LacZ/Lim3 double positive MMC (data not shown) as well as LacZ/foxP1 double positive preganglionic neurons (Fig. 5C). Th.Ryos (n = 4). Some LacZpositive cells in the ventral horn were Isl1/2-positive and HB9positive (Fig. 3N, O), indicating that they differentiated into somatic motoneurons. To define the distribution of Nkx2.2-lineage cells, we stained brachial or thoracic spinal cord sections by LacZ histochemistry. Fig. 4A and 4B show schematic diagrams of the distribution of LacZ-positive cells in the brachial or thoracic spinal cord of three independent chick embryos. At HH 32 (E7), LacZ-positive cells were mostly present in the gray matter and the ventricular zone. In two of three embryos (Fig. 4A, left and right), there were LacZpositive cells in the ventral horn in the brachial spinal cord. In the thoracic spinal cord, LacZ-positive cells were observed more dorsally near the ventricular zone, suggesting that they were preganglionic motoneurons (Fig. 4B). In order to confirm that recombined cells included motoneurons that send axons outside the spinal cord, retrograde tracer (fluorogold; FG) was used to label motoneurons. Injected FG was spread not only to the wing bud but also widely to the body cavity including peritoneal and thoracic ones, and therefore, somites and other mesenchymal tissues showed FG fluorescence bilaterally in the coronal sections. Within the spinal cord, somatic motoneurons in the ventral horn and preganglionic cells in the intermediate region showedyellowish white FG fluorescence bilaterally. In cases with FG injection, a LacZ-positive cell was located in the ventral horn where most of cells were retrogradely labeled with FG so that this LacZ-positive cells was highly likely labeled with FG also (Fig. 4C, D). To further analyze this, we used the brainbow plasmid which expresses RFP, and GFP after Cre mediated recombination. GFPpositive cells observed in the ventral part of the ventral horn showed GFP immunoreactivity, one of which was retrogradely labeled with FG (Fig.4E, F, and G arrowheads). Therefore, some of the recombined cells were apparently motoneurons that sent axons outside the spinal cord (Fig. 4F arrows). At later stages (HH 42 or E16), some LacZ-positive cells expressed choline acetyltransferase (ChAT), an enzyme for acetylcholine generation and, thus, often used as a marker for mature motoneurons, in the ventral horn and around the central canal (Fig. 4H and 4L). Expression of ChAT in recombined cells were also confirmed by double-immunohistochemistry using anti-LacZ and anti-ChAT antibodies (Fig. 4I , 4M ; arrowheads). We collected 20 mm sections in each 300 mm section from electroporated spinal cord. The ratio of LacZ/ChAT-double positive cells per total LacZpositve cells was calculated from five embryos. A large number of motoneurons were derived from Nkx2.2-positive cells (Fig. 4P; average = 31.368.4 ). ChAT-negative cells might represent interneuron other than motoneurons. These data suggest that a large population of Nkx2.2-progenitors differentiated into functional motoneurons. In order to define the motoneuron subtype from Nkx2.2positive progenitors, we stained LacZ together with subtypespecific markers [20], [21] at HH 32 in brachial or thoracic spinal cords using Lim3 for MMC, raldh2 for LMC, and foxP1 for CT. Nkx2.2-derived cells differentiated into Lim3 positive (Fig. 5A) and raldh2-positive motoneurons (Fig. 5B) in the brachial spinal cord. In the thoracic spinal cord, we observed LacZ/Lim3 double positive MMC (data not shown) as well as LacZ/foxP1 double positive preganglionic neurons (Fig. 5C). Th.
