Figure 3.
Microglia screen myelin sheaths and phagocytose myelin in the zebrafish spinal cord. (A) Maximum projection of a microglia (green) making contact with myelin sheaths (magenta) in a 4 dpf Tg (mpeg1:EGFP; sox10:mRFP) zebrafish spinal cord. (B−F) Microglia scanning behavior along myelin sheaths in the spinal cord was analyzed by confocal time-lapse imaging of 3–4 dpf Tg (mpeg1:EGFP; mbp:mCherry-CAAX) or Tg (mpeg1:EGFP; sox10:mRFP) larvae over 14.5–15 h. Tile scans were acquired every 20 min. Individual microglia were followed through all time frames in which they were contacting myelin sheaths of one hemi-spinal cord (15 microglia from n = 6 larvae). (B) Example of the movement of a single microglia followed over 10 h. Top: Individual time frames are color-coded to highlight microglia movement over time. Bottom: Binary image of the microglia in time frame 1. For the analysis of the microglia motility along the spinal cord, tracks were manually drawn in the Manual Tracking plugin in Fiji by marking soma position in each time frame (overlaid as a yellow line in this image). A: anterior, P: posterior, D: dorsal, V: ventral. (C) Mean velocity of microglia movement along the spinal cord. (D) Mean duration of breaks in motility taken by individual microglia. (E) Tracking profiles (as shown in B) were plotted as the Euclidean distance of the microglia soma in each frame from the position of the soma in the first frame. The shape of the resulting curves was used to classify microglia movements into patterns. Colors identify individual microglia. Asterisk marks sheath displayed in B. (F) Microglia presence per time frame was added up in quadrants of the spinal cord (1 to 14, anterior to posterior) and displayed as heatmap to visualize preferential screening behavior. D: dorsal, V: ventral. (G) Confocal time-lapse imaging of a microglia taking up a myelin fragment over 90 min. Tilescans were acquired every 3 min. Images show maximum projections. For better visualization, only a part of the hemi-spinal cord was projected. Details show the myelin fragment inside the microglial process at distinct time points (top row: merged images, bottom row: 552 nm channel). (H and I) Myelin fragments accumulate inside microglia. (H) Example image from a 10 dpf Tg (mpeg1:EGFP; sox10:mRFP) spinal cord. To quantify myelin fragments within microglia, microglia were manually segmented in Imaris and the resulting surfaces were used to mask the 552 nm channel. (I) Quantification shows the sum of myelin fragment volumes within microglia in Tg (mpeg1:EGFP; mbp:mCherry-CAAX) spinal cords (n = 5 larvae per time point). One-way ANOVA with post-hoc Tukey’s test: 3 vs. 7 dpf: P = 0.0345, 3 vs. 14 dpf: P = 0.0046, 5 vs. 14 dpf: P = 0.0089, all other comparisons were non-significant. (J) Colocalization of mbp:mCherry-CAAX+ fragments with microglial lysosomes, labeled by transiently expressed mpeg1:KalTA4;UAS:EGFP-Rab7. (K) Microglia engulfing an enlarged, seemingly unwrapping myelin sheath (top row) and surrounding round mbp-mCherry-CAAX–positive structures budding off from the ventral Mauthner axon (bottom row). (L) Examples of myelin abnormalities (marked by arrows) observed in wt and csf1rDM Tg (mbp:EGFP-CAAX) larvae (top row: myelinosomes budding off from the Mauthner axon, bottom row left: sheath degeneration, bottom row middle + left: “myelin flaps,” which may represent locally unwrapped myelin). Quantification of the number of myelin abnormalities in 10 dpf dorsal and ventral spinal cord of wt (black) and csf1rDM (blue) larvae, normalized by the myelinated area (wt: n = 10 larvae, csf1rDM: n = 11 larvae). Two-sided Student’s t test: P < 0.0001. (M) Maximum projections of wt and csf1rDM Tg (mbp:EGFP-CAAX) ventral spinal cords showing myelin outfoldings budding off the Mauthner axon (arrows). Quantification of the number of myelin outfoldings in 10 dpf ventral spinal cord of wt and csf1rDM larvae, normalized by the myelinated area (wt: n = 10 larvae, csf1rDM: n = 11 larvae). Two-sided Student’s t test: P < 0.0001. (N) Examples of myelin ultrastructure in SEM cross-sections of the wt and csf1rDM zebrafish spinal cord at P18. Arrows indicate an outfolding (purple), a myelin fragment (green), and a minor outfolding (blue). (O) Quantification shows the percentage of myelin aberrations, categorized by subtypes, in single SEM cross-sections of the wt and csf1rDM zebrafish spinal cord at P18 (n = 4 larvae). One-way ANOVA with post-hoc Tukey’s test: fragments, wt vs. csf1rDM: P = 0.0068, minor outfoldings, wt vs. csf1rDM: P = 0.0001, all other comparisons were non-significant. Data represent means ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Scale bars: 5 µm (G, J–M), 10 µm (A, H), 50 µm (B). See also Video 4.

Microglia screen myelin sheaths and phagocytose myelin in the zebrafish spinal cord. (A) Maximum projection of a microglia (green) making contact with myelin sheaths (magenta) in a 4 dpf Tg (mpeg1:EGFP; sox10:mRFP) zebrafish spinal cord. (B−F) Microglia scanning behavior along myelin sheaths in the spinal cord was analyzed by confocal time-lapse imaging of 3–4 dpf Tg (mpeg1:EGFP; mbp:mCherry-CAAX) or Tg (mpeg1:EGFP; sox10:mRFP) larvae over 14.5–15 h. Tile scans were acquired every 20 min. Individual microglia were followed through all time frames in which they were contacting myelin sheaths of one hemi-spinal cord (15 microglia from n = 6 larvae). (B) Example of the movement of a single microglia followed over 10 h. Top: Individual time frames are color-coded to highlight microglia movement over time. Bottom: Binary image of the microglia in time frame 1. For the analysis of the microglia motility along the spinal cord, tracks were manually drawn in the Manual Tracking plugin in Fiji by marking soma position in each time frame (overlaid as a yellow line in this image). A: anterior, P: posterior, D: dorsal, V: ventral. (C) Mean velocity of microglia movement along the spinal cord. (D) Mean duration of breaks in motility taken by individual microglia. (E) Tracking profiles (as shown in B) were plotted as the Euclidean distance of the microglia soma in each frame from the position of the soma in the first frame. The shape of the resulting curves was used to classify microglia movements into patterns. Colors identify individual microglia. Asterisk marks sheath displayed in B. (F) Microglia presence per time frame was added up in quadrants of the spinal cord (1 to 14, anterior to posterior) and displayed as heatmap to visualize preferential screening behavior. D: dorsal, V: ventral. (G) Confocal time-lapse imaging of a microglia taking up a myelin fragment over 90 min. Tilescans were acquired every 3 min. Images show maximum projections. For better visualization, only a part of the hemi-spinal cord was projected. Details show the myelin fragment inside the microglial process at distinct time points (top row: merged images, bottom row: 552 nm channel). (H and I) Myelin fragments accumulate inside microglia. (H) Example image from a 10 dpf Tg (mpeg1:EGFP; sox10:mRFP) spinal cord. To quantify myelin fragments within microglia, microglia were manually segmented in Imaris and the resulting surfaces were used to mask the 552 nm channel. (I) Quantification shows the sum of myelin fragment volumes within microglia in Tg (mpeg1:EGFP; mbp:mCherry-CAAX) spinal cords (n = 5 larvae per time point). One-way ANOVA with post-hoc Tukey’s test: 3 vs. 7 dpf: P = 0.0345, 3 vs. 14 dpf: P = 0.0046, 5 vs. 14 dpf: P = 0.0089, all other comparisons were non-significant. (J) Colocalization of mbp:mCherry-CAAX+ fragments with microglial lysosomes, labeled by transiently expressed mpeg1:KalTA4;UAS:EGFP-Rab7. (K) Microglia engulfing an enlarged, seemingly unwrapping myelin sheath (top row) and surrounding round mbp-mCherry-CAAX–positive structures budding off from the ventral Mauthner axon (bottom row). (L) Examples of myelin abnormalities (marked by arrows) observed in wt and csf1rDM Tg (mbp:EGFP-CAAX) larvae (top row: myelinosomes budding off from the Mauthner axon, bottom row left: sheath degeneration, bottom row middle + left: “myelin flaps,” which may represent locally unwrapped myelin). Quantification of the number of myelin abnormalities in 10 dpf dorsal and ventral spinal cord of wt (black) and csf1rDM (blue) larvae, normalized by the myelinated area (wt: n = 10 larvae, csf1rDM: n = 11 larvae). Two-sided Student’s t test: P < 0.0001. (M) Maximum projections of wt and csf1rDM Tg (mbp:EGFP-CAAX) ventral spinal cords showing myelin outfoldings budding off the Mauthner axon (arrows). Quantification of the number of myelin outfoldings in 10 dpf ventral spinal cord of wt and csf1rDM larvae, normalized by the myelinated area (wt: n = 10 larvae, csf1rDM: n = 11 larvae). Two-sided Student’s t test: P < 0.0001. (N) Examples of myelin ultrastructure in SEM cross-sections of the wt and csf1rDM zebrafish spinal cord at P18. Arrows indicate an outfolding (purple), a myelin fragment (green), and a minor outfolding (blue). (O) Quantification shows the percentage of myelin aberrations, categorized by subtypes, in single SEM cross-sections of the wt and csf1rDM zebrafish spinal cord at P18 (n = 4 larvae). One-way ANOVA with post-hoc Tukey’s test: fragments, wt vs. csf1rDM: P = 0.0068, minor outfoldings, wt vs. csf1rDM: P = 0.0001, all other comparisons were non-significant. Data represent means ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Scale bars: 5 µm (G, J–M), 10 µm (A, H), 50 µm (B). See also Video 4.

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