Figure 2Molecular, Structural, and Axonal Connectivity Analyses of L5 NOS1+ Columns in the Mid-Fetal Human FOp
(A and B) Triple immunofluorescent staining for NOS1 (green), BCL11B (blue), a marker of L5 subcortical projection neurons, and SATB2 (red), a marker of upper layer corticocortical projection neurons, in 20 PCW FOp L5. NOS1+ and BCL11B+ pyramidal neurons formed alternating columns (outlined) separated by clusters of SATB2+ neurons. (C) Analysis of columnarity in FOp L5 neurons. NOS1+ neurons were significantly more columnar in organization compared to NOS1− neurons. *P< 0.05. Error bars represent the 5th and 95th percentiles of thirty measurements. (D) NOS1 immunostaining (brown) and FEZF2 in situ hybridization (blue) of FOp L5 at 18 PCW. NOS1+ neurons co-expressed FEZF2 (open arrowheads). (E, F, and G) Immunofluorescent staining for NOS1 (green) and FOXP2, synaptophysin (SYP), or FOS (red in E, F, and G). NOS1+ neurons co-expressed FOXP2 and FOS (arrowheads in E and G), and were encircled by SYP punta (arrowheads in F). (H, I, and J) Retrograde axonal tracing at 20 PCW. Retrograde travel of Fast DiI inserted into dorsal internal capsule (red arrowhead) and Fast DiA inserted into the corpus callosum (green arrowhead) were examined after seven months in incubation. In the FOp L5 (I), DiI-labeled subcortical projection neurons formed columns similar those composed of NOS1+ neurons. In the ACC (J), DiA-labeled corticocortical projection neurons did not exhibit obvious columnar organization.