TMEM63A-mediated Ca2+ signaling in OLs regulates stable myelin deposition and targeted sheath growth in zebrafish (A) Tmem63a wild-type (left; T63a+/+) and Tmem63a zebrafish mutant alleles (middle; T63a−/−) with resulting mutant mRNA. ∗ Represents premature stop codon in exon 10. (B) T63a+/+ and T63a−/− zebrafish spinal cords at 3 days post-fertilization (3 dpf) expressing a transgenic myelin reporter. Lateral views with anterior to the left. Yellow brackets indicate hypomyelination in T63a−/−. D, dorsal; V, ventral white matter. (C) Ventral and dorsal white-matter volume in ∼640 μm long T63a+/+ (left) and T63a−/− hemi-spinal cords at 3 dpf. (D) Individual myelinating OL with all their sheaths. (E) Number of stabilized sheaths per OL. (F) Internodes in T63a+/+ and T63a−/− spinal cords at 4 dpf. (G and H) Internode length in μm (G) and relative frequency distribution (H). (I and J) Time-series projection of GCaMP6s signals and intensity vs. time traces of four CaMs T63a+/+ (I) and T63a−/− (J) OLs during 20 min time-lapse acquisitions at 2.5 s/frame at 3–4 dpf. Region of interests (ROIs) (red) and corresponding traces, with Ca2+ events highlighted in red (I). No Ca2+ events detected in (J). (K) Proportion of T63a+/+ and T63a−/− fish exhibiting Ca2+ events during 20 min. (L–N) Number (L), duration (M), and amplitude (N) of Ca2+ events per 500 μm2 myelinated area during 20 min. (O) Myelin sheath growth binned by 0–1 Ca2+ events vs. multiple Ca2+ events during 20 min, displayed as shortening (negative growth) and lengthening (positive growth). (P) T63a+/+ and T63a−/− OLs retracting individual myelin sheaths during an 18-h time-lapse acquisition. (Q) Number of retracted sheaths. (R and S) Growth of individual myelin sheaths in μm/h (R) and cumulative Gaussian distribution (S). (T and U) Electron micrographs of T63a+/+ (T) and T63a−/− (U) zebrafish spinal cords at 10 dpf. Overview images (left) and higher magnification images of small-diameter (middle) and large-diameter axons (right). Insets, small-diameter axons (0.2–0.3 μm; white outline). (V and W) g ratios of individual myelinated axons as a function of axon diameter (V) and binned axon diameter (W) in T63a+/+ and T63a−/− spinal cords at 10 dpf. Slopes, non-significant; intercepts, ∗∗p < 0.01 (V). (X) Relative frequency distribution of axon diameters of the 100 smallest myelinated axons. Data represent mean ± SEM (C, E, Q, and W) and median with interquartile range (G, L–N, and R). ns, non-significant; ∗p < 0.05 (including S); ∗∗p < 0.01 (including X), ∗∗∗p < 0.001 (including H), ∗∗∗∗p < 0.0001. See also Figure S6, Videos S4, S5, and S6, and Table S3 (sample sizes and statistical tests).
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