Figure 3. Genetic mouse models to label the Foxp2-V1 lineage.

(A) P5 mouse intersection of En1^Cre/+ and Foxp2^Flpo/+ with two reporter alleles (Ai9 R26 ^lsl-tdT and R26 ^RCE:dualEGFP). Foxp2-V1s express EGFP (green) and non-Foxp2-V1s tdTomato (red). A few ‘yellow cells’ correspond with Foxp2-V1 neurons that failed to remove the tdTomato Ai9 reporter. (B) Density contours demonstrate high spatial overlap between V1 neurons expressing Foxp2 (EGFP+) or not (tdT+ and EGFP-) (n=6 ventral horns). (C) Expression of lineage labels are stable throughout postnatal development, suggesting no additional Foxp2 gene upregulation in V1s after P0 (n=6 ventral horns per age in one animal, except for P5 in which 6 animals and 36 ventral horns are included; error bars show SEM). (D) Lineage labeling in P15 mice is uniform across all spinal cord segments from thoracic 13 to sacral 1 (n=6 ventral horns in each segment from 2 mice; error bars show SEM). (E) Foxp2-V1 (EGFP) and non-Foxp2 V1 (tdT) lineage labeling with antibody staining for the transcription factors defining the four major V1 clades: Foxp2, Pou6f2, MafB, and Sp8. The Foxp2-V1 lineage contains all P5 Foxp2-expressing V1s and excludes almost all those expressing the markers of other clades. (F) Percentages of Foxp2-V1s (EGFP) expressing the clade markers at P5. Around half of the Foxp2-V1s maintain expression of the Foxp2 protein at P5, and a minimal number of these cells express transcription factors that define other V1 clades. (G) Percentages of non-Foxp2 lineage V1s (tdT) expressing the different V1 clade markers. Cells outside of the Foxp2-V1 lineage do not express Foxp2 at P5, and this subset contain V1s from the three other clades (for both plots n=6.5 ± 2.6 mice, 4 ventral horns each; error bars show SEMs). (H) FLTG reporter mice reveal lineage-labeled non-V1 Foxp2 cells in the spinal cord. EGFP is expressed in Foxp2-V1s and tdTomato in non-V1 Foxp2 cells. The zoomed images of the highlighted region with and without NeuN-IR demonstrate that non-V1 Foxp2 cells include non-neuronal cell types with astrocyte morphologies. Only neurons (NeuN-IR) are included in the cell density contour plots; non-V1 Foxp2 neurons are located in the medial ventral horn and a few in the deep dorsal horn (n=2 animals at P5, 6 ventral horns in each). (I) Most Foxp2-neurons (red, non-V1s or green, V1s) are in the ventral horn and their number in 30-μm-thick L4-5 sections decreases with age as the neuropil matures and expands in size. Dorsal horn Foxp2-neurons maintain their numbers despite the growth of the spinal cord, suggesting de novo postnatal Foxp2 expression in this population. Each point is one animal analyzed through 6 ventral horns (errors bars are SD). (J) Percentage of Foxp2-lineage-labeled cells that are Foxp2-V1s remains constant throughout postnatal development (n=2 mice, 6 ventral horns each; error bars show SD).

Figure 3—figure supplement 1. Postnatal downregulation of Foxp2 expression.

(A) Confocal image of En1^Cre/+, Foxp2^flpo/+, R26^RCE:dualEGFP genetic labeling (green) immunostained for Foxp2 protein expression (magenta) in a P5 mouse. Low-magnification 2D projection at the left. Higher magnification of the indicated area at the right. Only a proportion of lineage label Foxp2-V1s express Foxp2 protein by P5. (B) Density contours demonstrate that lineage-labeled Foxp2-V1s with or without Foxp2 protein expression at P5 occupy similar areas in the spinal cord at P5 (n=3 mice, 3 ventral horns per animal). (C) Left, Foxp2-V1s make up roughly 60% of all V1s when measured with genetic labeling, but only about 30% of V1s maintain detectable Foxp2 expression at P5 (n is same as B; error bars show SD). Right, around half of Foxp2-V1s maintain detectable levels of Foxp2 at P5 (same data).