Extended Data Fig. 3: In vivo characterization of AAV-Isl1 and AAV-Lhx3 expression in spinal motor neurons.

a, Distribution of ISL⁺, LHX3⁺, ISL + LHX3⁺, and ISL1-LHX3- cells among CHAT⁺ cells in the L4-L5 ventral horn at P45 from animals treated with low titer (6-9E⁺10 vg/animal) or high titer AAVs (3-4⁺11 vg/animal). Mean percentage of motor neurons in each condition was quantified from 6 hemisections per animal and 15 confocal images per hemisection from 2 (low titer) or 6 (high titer) animals. Error bars represent SEM. b, Quantification of transgene-positive motor neurons in L4-L5 ventral horn at P45 in animals treated with AAV-Isl1 or AAV-Lhx3 under high titer conditions (3.6E⁺11 - 3.8E⁺11 vg/animal) at P1. n = 1–4 animals per treatment per time point. Points represent mean transgene-expressing motor neurons per hemisection per animal, quantified across 6 hemisections per animal from 15 confocal images per hemisection. Error bars represent SEM. c, Distribution of AAV⁺ motor neurons among the high titer animals plotted in c, separated by sex (n = 3 animals per sex). No significant effect of sex was observed by one-way ANOVA (p = 0.3739). d, Quantification of total CHAT⁺ motor neurons per hemisection in the L4-L5 region of the spinal cord in untreated animals or in AAV-mCherry-treated or AAV-Isl1+AAV-Lhx3-treated injected at P1 and analyzed at P45 in 15 confocal sections from 6 70 µm hemisections per animal. Each point represents the mean motor neuron count per hemisection from one animal; triangles represent males, and circles represent females. Horizontal bars represent animal medians per treatment condition. No significant effect of treatment on motor neuron survival was observed by one-way ANOVA (p = 0.1600).