Gpr56 signals through Gα_12/13 and RhoA to promote radial sorting. (A–I) TEM images of the pLLn of WT, gpr56^stl13/+, and gpr56^stl13/stl13 larvae injected with (A–C) phenol red control (n = 6 animals per genotype), (D–F) 0.5 ng 3MOs targeting Gα_12/13 signaling (3MO = gna12-MO, gna13a-MO, and gna13b-MO; n = 6 WT, n = 5 gpr56^stl13/+, and n = 5 gpr56^stl13/stl13 animals examined), or (G–I) 1 ng 3MO (n = 3 animals per genotype). (J–L) In a separate series of experiments, animals were injected with constitutively active rhov14 (rhov14: n = 3 animals per genotype; control: n = 3 each WT and gpr56^stl13/+, n = 4 gpr56^stl13/stl3). Sorted axons are pseudocolored orange. Myelinated axons are pseudocolored blue. (A–L) Bar, 1 µm. (M) Two-way ANOVA analysis revealed a significant interaction between genotype and treatment for 3MO experiments (****, P < 0.0001). Tukey’s multiple comparisons test was also performed and found a significant reduction in the number of sorted axons between control WT embryos and control heterozygous (****, P < 0.0001) as well as control mutant embryos (****, P < 0.0001). A significant decrease in sorted axon number was observed between 0.5-ng 3MO-treated WT and -treated heterozygous (***, P < 0.001) and -treated mutant embryos (****, P < 0.0001). No significant comparisons were found in the 1-ng 3MO condition. (N) Total axon number is unaffected by genotype or treatment (two-way ANOVA). (O) Quantification of the number of axons sorted 1:1 in phenol red controls (P < 0.03, unpaired Student’s t test) compared with (P) rhov14-injected embryos (no significant differences). (Q and R) Total axon number was unchanged by test condition (unpaired Student’s t test). Data reflect two independent experimental replicates. (M–R) Error bars represent SEM.