Impaired myelin sheath formation and sheath stabilization in the CCM−/− triple knockout.
(A) Confocal images of OLs in wild-type, cntn1b−/−mag−/− dKO, cadm4−/−mag−/− dKO, and CCM−/− tKO zebrafish spinal cord at 4 dpf, labeled with the fluorescent reporter Tg(mbp:EGFP-CAAX). Arrowheads indicate membrane stubs (A′), and an asterisk indicates cell body ensheathment (A″) in CCM−/− tKO. Scalebar is 10 µm in (A) and 5 µm in (A′ and A″). (B–E) Quantification of the total number of processes per OL in the dorsal spinal cord at 4 dpf (B), and of the percentages of processes connected to myelin sheaths (C), myelin stubs (D), and cell body ensheathments (E). Data was collected from a total of 16 (wt) and 20–22 (rest of the genotypes) individual oligodendrocytes from 9 to 11 fish per genotype. (F) Quantification of myelin sheath length in the dorsal spinal cord at 4 dpf. Data was collected from 160 to 180 individual sheaths, and sampled from 3 to 7 fish per genotype. (G) Selected frames from confocal time-lapse recording of OL in wild type, cntn1b−/−mag−/− dKO, cadm4−/−mag−/− dKO, and CCM−/− tKO in the zebrafish spinal cord. OLs were sparsely labeled using myrf:EGFP-CAAX and tracked from the OPC stage to the myelinating OLs stage over 13 hours between 2.5 and 3 dpf. Scale bars are 10 µm. Maximum intensity projections shown. (H) Quantification of the total number of OPC processes identified at the beginning of the time-lapse imaging. (I) Quantification of the total number of early-myelinating OL processes, approximately 2 hours from the OPC stage, when they form nascent myelin sheaths (indicated by arrowheads). (J) Quantification of the maximum number of sheaths formed by newly-differentiated OLs during the 13-h time lapse. (K) Quantification of the percentage of retracted myelin sheaths of the maximum myelin sheaths formed by individual newly-differentiated OLs during the time-lapse imaging. (L) Quantification of the number of stable OL sheaths at the end of the time-lapse imaging. Data was collected from a total of 8–11 individual OLs, and sampled from 4 to 7 fish per genotype for (H–L). Images show standard deviation projections (G). Graphs indicate mean values ± SD (B, H–L), and median with interquartile range (C–F). Data was analyzed by one-way ANOVA with Tukey’s multiple comparisons test (B, H, I), non-parametric Kruskal–Wallis test with Dunn’s multiple comparisons test (C–F), and Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (J–L). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The data underlying this Figure can be found in S1 Data.
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