(A) Diagram showing dorsal (D) and ventral (V) limb innervation by spinal motor nerves in control (WT or Fz3+/−; top) and Fz3−/− (bottom) embryos. In Fz3−/− limbs, the dorsal nerve is thinned distal to the plexus at the base of the limb (highlighted by arrows). SC, spinal cord. (B–F′) NF immunostaining of whole-mount fore- and hindlimbs from E13.5 Fz3+/− and Fz3−/− embryos. (B′–F′), magnified view of boxed regions in (B–F). Arrows (control) and arrowheads (mutant) indicate dorsal nerves, which are moderately thinner in Fz3−/− forelimbs and substantially thinner in Fz3−/− hindlimbs. Asterisks indicate the points at which axons stall in Fz3−/− limbs. The red to blue color code represents depth within the Z-stack, oriented here along the dorsal to ventral axis. Scale bars: (B–F), 500 µm; (B′–F′), 100 µm. (G–H′) NF immunostaining of 1 mm-thick sections in the dorsoventral plane encompassing fore- or hindlimbs from E12.5 Fz3+/− and Fz3−/− embryos. Arrows (control) and arrowheads (Fz3−/−) indicate dorsal nerves, which are thinner in Fz3−/− limbs. Asterisks indicate the points at which axons stall in Fz3−/− limbs. Red to blue color code represents depth within the Z-stack, oriented here along the rostrocaudal axis. P, proximal; D, distal. Scale bars, 500 µm. (I–L′′) GFP (green) and NF (red) double immunostaining of whole-mount forelimbs and hindlimbs from E12.5 Hb9-EGFP;Fz3+/+ and Hb9-EGFP;Fz3−/− embryos (I–L). Motor axons (labeled with GFP) are selectively eliminated from the dorsal nerve in mutant limbs (arrowheads and arrows). (I′–L′) are enlarged views of the NF immunostaining in the boxed regions in (I–L), with color representing depth along the dorsoventral axis. Similarly, (I′′–L′′) are enlarged views of the GFP immunostaining. P, proximal; D, distal; d, dorsal; i, intermediate; v, ventral. Scale bars: (J) and (L), 500 µm; (J′′) and (L′′), 100 µm. (M) Diagrams showing the locations of nerve diameter measurements quantified in (N), traced from images (B′) and (D′). The locations of two dorsal nerve measurements (DDistal, dorsal nerve diameter measured immediately distal to the stalling point; DProximal, dorsal nerve diameter measured immediately proximal to the stalling point) and one ventral nerve measurement (V) are shown. Scale bar, 100 µm. (N) The diameters of motor nerves at the locations shown in (M) were measured from NF immunostained whole-mount forelimbs (top) and hindlimbs (bottom) from two litters at E12.5 and one litter at E13.5. Bars, mean ± SEM. p-values are shown for the indicated pair-wise comparisons (student’s t-test).