GPR56 regulates mature myelin ultrastructure and domain organization. (A and B) TEM images of WT (n = 3) and Gpr56 mutant mouse (n = 3) SNs on P21. Bar, 2 µm. White arrow denotes myelin outfolding. Black arrowheads denote morphologically WT Remak bundles, which are incompletely formed in Gpr56 mutants (B, white arrowhead/inset). The asterisk marks an axon with no appositions. (C–E) Quantification of (C) total number of myelinated axons (P < 0.45, one-way ANOVA), (D) overall axon number (P < 0.81, one-way ANOVA), and (E) percentage of bundles containing axons that are not fully sheathed in Gpr56 mutants and WT sibling nerves (*, P < 0.03, one-way ANOVA). Error bars represent SEM. (F) Plots of G-ratios in mutants and WT controls (P < 0.002, one-way ANOVA). (G) TEM images of Gpr56^−/− myelinated axons with 0, 1, 2, or 3 appositions. (H) Distribution of the percentage of myelinated axons (mean ± SD) per apposition number in WT (white bars) and Gpr56^−/− SNs (black bars, **, P < 0.002, one-way ANOVA). (I–K) IHC showing GPR56 (green), DAPI (nuclear stain, blue), and MBP (marker of mature myelin) localization on P21 SNs. In WT SNs, GPR56 is detected in pockets (arrowheads) outside of MBP-positive rings but absent from mature axons. (J) GPR56 stain alone. (K) MBP stain alone. Bar, 5 µm.