Figure 3. The GAP Activity of p190 Is Dispensable for Motor Axon Exiting but Required for Distal Pathfinding.

(A) Schematic of p190 and protein sequence alignment highlighting the catalytic arginine R1284. The targeted mutation R1284A generates GAP-inactive p190. Direct cDNA sequencing demonstrates CGG (Arg-1284) > GCA (Ala-1284) substitution. The synonymous C > T substitution in the third position of Tyr-1283 was introduced to facilitate annealing of allele-specific genotyping primers.

(B–G) Whole-mount images of ISL^MN::fGFP⁺ p190^+/− heterozygotes (controls; B, D, and F) or p190^R1284/− compound heterozygous embryos (C, E, and G), carrying one copy of R1284A allele and one copy of the knockout allele. (B and C) Motor axon exiting defects are absent in p190^R1284/− heterozygotes (C, arrowhead to a residual short ectopic bundle). p190^R1284/− embryos display normal fasciculation of intercostal nerves (E) while the peroneal nerve in the hindlimb is severely stunted (arrow in G).

(H) Quantification of ectopic GFP⁺ motor axon bundles in embryo whole mounts. Mean ± SEM; Mann-Whitney U test, ***p < 0.001 p190^−/− versus p190^R1284/−; ns, p > 0.05 p190^+/− versus p190^R1284/−. p190^R1284A/− embryos only display rare short ectopic bundles, similarly to p190^+/− controls.

(I) Incidence of peroneal nerve defect in p190^R1284/− mutants and p190^R1284/+ controls with color-coded phenotypic classes: normal, green; moderate thinning, blue; severely stunted, red.

Scale bars, 200 μm. See also Figure S3.