Figure 1

Genetic strategy to identify and define Renshaw cells. (A) Triple transgenic En1-Cre :: Mafb-GFP :: Ai9 R26 lsl-tdT animals label Mafb-expressing cells with GFP and the V1 lineage with tdTomato (tdT). (B) 2D projection of a triple fluorescence confocal image stack (50 µm thick) showing En1-tdT (V1 interneurons, red), Mafb-GFP (green), and calbindin immunoreactivity (CB-IR, blue) at P15. (C) Venn diagram illustrates co-localization possibilities between En1-tdT, Mafb-GFP, and CB-IR; colors match cell plots obtained from a 6-month old animal (12 superimposed 50 µm thick sections). V1-MafB cells appear as distinct dorsal and ventral groups; the ventral group is CB-IR and corresponds to Renshaw cells. The Renshaw cell area (RCA) was anatomically defined as the bottom 45% of the deep ventral horn. This region includes the vast majority of triple-labeled cells (● orange circles); less than one triple-labeled cell per hemicord was observed outside this region across all time points. Graphs show quantification of triple-labeled cells at P5, P15, and 6 months of age inside or outside the RCA (n = 2–4 animals per group, errors bars = S.D.; see Supplemental Table 3). Within the RCA, 98% of CB-IR cells co-express En1-tdT and Mafb-GFP by P15. Asterisks indicate significance level after One-Way ANOVAs across all conditions. ****p < 0.0001, n.s. non-significant. For pair-wise comparisons and details of statistics, refer to Supplemental Table 3.