Figure 4.

Sim1 expression is essential for the laminar clustering of early-born V3 INs. Ai, Representative image of E9.5-pulsed EdU detection (confirmed by Hoechst staining) and Nkx2.2 immunoreactivity in an E12.5 lumbar spinal section of a Sim1^Cre/taulacz mouse. Scale bars: Ai, 50 μm, insets, 50 μm). Merged image (Mask) shows a colocalization mask of EdU⁺/Hoechst⁺/Nkx2.2⁺ cells using Imaris software. Aii, Representative image of EdU detection (confirmed with Hoechst staining) in V3 INs (TdTom) in a P0 high lumbar spinal section of a Sim1^Cre/taulacz;Rosa.lsl.tdTom mouse. Scale bars: Aii, 100 μm; insets, 50 μm). Bi, Bii, Hemicord cell position plots of EdU⁺/Hoechst⁺/Nkx2.2⁺ cells in E12.5 lumbar segments of Sim1^Cre (Bi) and Sim1^Cre/taulacz (Bii; n = 3 animals for each pulse age and genotype). Biii, The total number of Nkx2.2⁺/EdU⁺ cells from respective pulse times (E9.5, E10.5, E11.5, E12.5) from E12.5 lumbar spinal segments compared between Sim1 control (Sim1^Cre) and knock-out (Sim1^Cre/taulacz) mice (n = 3 animals for each pulse age and genotype, p value > 0.05, unpaired t test with Welch's correction). Error bars, SD. Ci–Civ, Hemicord cell position heat map plots of EdU⁺/Hoechst⁺/tdTom⁺ cells in P0 higher lumbar (L1–L3) segments of Sim1^Cre;Rosa.lsl.tdTom and Sim1^Cre/taulacz;Rosa.lsl.tdTom mice pulsed at E9.5 (Ci), E10.5 (Cii), E11.5 (Ciii), and E12.5 (Civ; n = 4 animals for each EdU pulse age and genotype). D, EdU⁺ V3 hemicord cell position plots of P0 higher lumbar (L1–L3) spinal cord sections were pooled together as either early-born (EdU⁺: E9.5, E10.5, n = 8 animals total for each genotype) or late-born (EdU⁺: E11.5, E12.5, n = 8 animals total for each genotype) groups. Five-by-five grids were superimposed over hemicord cell position plots, producing 25 discrete bins with specific medial-lateral and dorsal-ventral positions. EdU⁺ V3 IN counts within corresponding bins were then compared between Sim1^Cre;Rosa.lsl.tdTom (Sim1 control) and Sim1^Cre/taulacz;Rosa.lsl.tdTom (Sim1 knock-out) cords for early-born and late-born groups: early-born, Sim1 control (n = 8 animals) vs knock-out (n = 8 animals) mice; late-born, Sim1 control (n = 8 animals) vs knock-out (n = 8 animals) mice. Di–Diii, The average count and SD for Sim1 control (top of bin, average ± SD) and Sim1 knock-out (bottom of bin, average ± SD) cords. Statistical significance between Sim1 control and knock-out counts of the same bin were measured with a two-tailed Mann–Whitney U test. Bins with average counts <1 in both control and knock-out mice were considered nonsignificant. Dii, Diii, The p values are presented as a heat map with white bins indicating no change, blue bins indicating gradations of reduced numbers in Sim1 knock-out mice, and red bins indicating gradations of increased numbers in Sim1 knock-out mice.