Figure 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–G, Double staining of spinal cord sections with anti-Olig2 and anti-Nkx2.2 antibodies at HH 14 (B–D) and HH 17 (E–G). H–J, Specific expression of mCAT1-myc in the p3 domain. pNkx2.2-mCAT1-myc was introduced by 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 µm.
Figure 2.
Both V3 interneurons and somatic motoneurons are generated from Nkx2.2-positive progenitors.
A and B, sim1 in situ hybridization (purple) followed by GFP immunohistochemistry (brown). Recombined cells were sim1-positive (arrow in A) or sim1-negatve (arrowhead in A). An arrow in B indicates recombined cell axon outside the spinal cord, suggesting it was a motoneuron axon. C, GFP-positive recombined cells in the HH35 spinal cord, showing a GFP-positive axon extending outside the spinal cord. D-F, Double staining with HB9 and GFP immunohistochemistry, demonstrating a HB9-positive recombind cell. Scale bars in A and B = 50 µm; in C = 200 µm; in F = 20 µm.
Figure 3.
LacZ-labeled cells by electroporation with pNkx2.2-Cre;cAct-xStopx-nLacZ strongly express Nkx2.2 in the ventricular zone.
pNkx2.2-Cre and cAct-xStopx-nLacZ were electroporated at HH 14 in the chick neural tube and an embryo was grown to be HH 21 (E3.5; A–F) or HH 32 (E7; G–O). A, LacZ-staining followed by immunohistochemistry using Nkx2.2 antibody. Sections were arranged in rostral (left) to caudal (right) order. B-F, Triple immunostaining with anti-Nkx2.2, anti-Olig2, and anti-LacZ antibodies. Arrowheads indicate recombined cells. G, LacZ-staining followed by immunohistochemistry using Nkx2.2 antibody. Sections were arranged in rostral (left) to caudal (right) order. Insets show higher magnification pictures of LacZ-positive cells in the Nkx2.2-positive ventricular zone. H-M, Sections at HH 32 were double-immunolabeled using anti-LacZ and anti-Nkx2.2 (H-J), or anti-LacZ and anti-Olig2 (K-M), respectively. Arrowheads indicate recombined cells. N, LacZ-positive cells with Islet1/2 immunoreactivity. O, LacZ-positive cells with HB9 immunoreactivity. Scale bars in A, G, M, O = 100 µm; in F = 50 µm.
Figure 4.
Distribution of Nkx2.2 lineage motoneurons in the spinal cord.
pNkx2.2-Cre and cAct-xStopx-nLacZ were electroporated at HH 14 in the chick neural tube. A and B, LacZ-positive cells of one chick spinal cord at HH 32 (E7) were superimposed and marked with dots on a schematic diagram. Three independent experiments were performed in the brachial spinal cord (A) or thoracic spinal cord (B). C and D, A LacZ-positive cell in the ventral horn in which most of the motoneurons were retrogradely labeled with fluorogold (FG). E–G, GFP-positive recombined cells with brainbow system in the ventral horn were retrogradely labeled with FG (G; arrowheads). GFP-positive axons were extending toward the ventral root (arrows). H and L, Sections of HH 42 spinal cord were subjected to LacZ-staining followed by immunohistochemistry using ChAT antibody. LacZ-positive cells in the ventral horn (H; somatomotor neuron) and close to the central canal (L; preganglionic CT neuron) are shown. Arrowheads indicate double positive cells and the asterisk shows the location of the central canal. I–K and M–O, Expression of ChAT in LacZ-positive cells was also analyzed by double fluorescent labeling (I–K, M–O). P, LacZ/ChAT-positive cells of one chick spinal cord were counted and divided by the total number of LacZ-positive cells and were shown as percentages (P). Five chick embryos were examined in this study. Scale bars in A and L = 200 µm; D, F and O = 50 µm.
Figure 5.
Nkx2.2-positive progenitors contribute to all types of motoneurons.
pNkx2.2-Cre and cAct-xStopx-nLacZ were electroporated at HH 14 in the chick neural tube and an embryo was grown to be HH 32 (E7). A-C, Sections were subjected to LacZ-staining followed by immunohistochemistry using Lim3 antibody for MMC neurons (A), in situ hybridization for raldh2 for LMC neurons (B), or foxP1 for CT neurons (C), respectively. Arrow indicates double positive cells and magnified images of double positive cells are shown in the inset of each figure. D, A schematic representation of our hypothetical mechanism for motoneuron generation from Nkx2.2-positive progenitors. Scale bars indicate 100 µm.