Figure 5

Small diameter Gfrα1+ motor neurons are selectively lost in the muscle spindle mutant Egr3^KO and ErbB2^FLOX/-myf5^Cre/+ animals. (A,B) Lamina IX confocal images from a P20 Egr3 wild type (A) and Egr3^KO mutant (B). (A₁, B₁) Gfrα1-TLZ (red) and (A₂,B₂) superimposed with ChAT (blue). Small MNs intensely labeled with Gfrα1-TLZ are frequent in wild type but mostly absent in Egr3^KO mutants. Somatic shrinkage is apparent in the few small Gfrα1-TLZ MNs found in Egr3^KO mutants (arrowheads in B₁). (C) Average size distribution of ChAT+ (gray bars) and Gfrα1-TLZ (white bars) MNs in P20 Egr3^KO animals (n = 3 animals; error bars indicate SEM). Superimposed curves represent the wild-type distributions of small and large MNs. ChAT+ and Gfrα1-TLZ+ populations in Egr3^KO mutants are both unimodal corresponding with large MNs. Small MNs are mostly absent (arrow). (D) Number of ChAT+ neurons sampled per ventral horn in 70-μm thick sections of P20 animals. Egr3^KO (n = 3 animals) and Erb2Δ animals (n = 5) show significant depletions compared to controls (asterisks indicate P < 0.001, one-way ANOVA; P < 0.01 post-hoc Tukey-tests). (E) Percentages of the total ChAT+ population represented by 'small' (<485 μm²) MNs labeled with ChAT (gray bars) or Gfrα1-TLZ (white bars) in Egr3^KO and Erb2Δ mutants. Both animals show a large depletion of small MNs compared to control (asterisks indicate P < 0.001, one-way ANOVA; P < 0.01 when compared to control using post-hoc Tukey-tests). In ErbΔ2 animals the reduction is not as pronounced as in Egr3^KO animals despite a larger depletion in total ChAT+ MNs (D). Inset shows the average size distribution histogram in ErbΔ2 animals (gray bars, ChAT+; white bars, Gfrα1+) suggesting partial depletion of both large and small MNs. (F) Average size distribution of MNs in Egr3^KO animals with different combinations of markers: ChAT+ (gray bars), Hb9::GFP+ (green bars), Gfrα1-TLZ+/Hb9::GFP- (dark blue bars) and Gfrα1-TLZ+/Hb9::GFP+ (light blue bars). Superimposed are the fitted distributions for different types of MNs in the wild-type. (G) Average number of MNs per ventral horn in each category. Asterisks denote significant differences (P < 0.001, t-test) between wild-type (gray bars) and Egr3^KO mutants (white bars). Significant differences were observed in all MNs and in small MNs (<485 μm²) labeled with either ChAT or Gfrα1-TLZ. Hb9::GFP+ MNs are not significantly depleted in Egr3^KO mutants, though Gfrα1-TLZ expression is downregulated in surviving large diameter MNs (F). (H) Time course of size differentiation and depletion of small MNs in Egr3^KO animals. Gray bars indicate size distribution in wild type (WT; n = 3 animals for each age) and black bars in age matched Egr3^KO animals (n = 2 at P0, n = 4 at P5 and P10). At P0, MN sizes are unimodal and there is a small depletion in ChAT+ neurons distributed in all size bins. At P5 there is initial differentiation of small vs. large MNs and in the Egr3^KO mutant there is a larger depletion of ChAT+ MNs concentrated in the small bins. At P10 the size distribution in the wild type resolves into two discrete peaks for the small and large population and in the Egr3^KO mutant the depleted neurons are clearly in the small size bins. Histogram at the right show the percentage depletions calculated in Egr3^KO mutants of different ages. Scale bars: (A₁, B₁) 100 μm.