Figure 3.
Delayed clearance of ingested myelin debris by myd88−/− phagocytes in zebrafish larvae. (A and B) Confocal z-sections of a spinal cord lesion show NF-κB activation (cyan) in recruited phagocytes (magenta) over time. NF-κB activation was determined by the colocalized signal of NF-κb:eGFP with mpeg1:mCherry. n = 9 or 10, 7, and 10 or 11 animals at 6 hpi, 2 dpi, and 4 dpi, respectively. (C) Confocal maximum intensity z-projections from a 2 h time-lapse video (Video 1) of the spinal cord lesion at 2 dpi showing the myelinated spinal cord and myelin debris (magenta) and phagocyte (cyan) movement in WT larvae. (D and E) Confocal maximum intensity z-projections of the spinal cord lesion at 2 dpi and 4 dpi show the myelinated spinal cord and myelin debris (magenta) in phagocytes (cyan). The amount of internalized myelin was determined by the colocalized signal of mbp:mCherry-CAAX within mpeg1:eGFP. n = 11–15 and 15–17 animals at 2 and 4 dpi, respectively. (F and G) Confocal maximum intensity z-projections of the spinal cord lesion at 4 dpi, showing lysosomes (magenta) in phagocytes (cyan). The amount of lysosomes was determined by the colocalized signal of LysoTracker with mpeg1:eGFP. n = 8–10 and 14–25 animals at 2 and 4 dpi, respectively. (H and I) Confocal maximum intensity z-projections from 2 h time-lapse videos (Videos 2 and 3) of the spinal cord lesion at 3 dpi show the myelinated spinal cord and myelin debris (magenta) and phagocyte (cyan) movement. Arrowheads show actively moving phagocytes, and short arrows show amoeboid stationary phagocytes. n = 6 or 7 animals. Lateral views of the lesion site are shown; anterior is left and dorsal is down. All data are mean ± SEM (error bars). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; n.s. indicates no significance, by two-way ANOVA test with Tukey’s multiple comparisons test in B, D, and E or Sidak’s multiple comparisons test in G and unpaired t test with Welch's correction in J. Scale bars: 20 µm.

Delayed clearance of ingested myelin debris by myd88−/− phagocytes in zebrafish larvae. (A and B) Confocal z-sections of a spinal cord lesion show NF-κB activation (cyan) in recruited phagocytes (magenta) over time. NF-κB activation was determined by the colocalized signal of NF-κb:eGFP with mpeg1:mCherry. n = 9 or 10, 7, and 10 or 11 animals at 6 hpi, 2 dpi, and 4 dpi, respectively. (C) Confocal maximum intensity z-projections from a 2 h time-lapse video (Video 1) of the spinal cord lesion at 2 dpi showing the myelinated spinal cord and myelin debris (magenta) and phagocyte (cyan) movement in WT larvae. (D and E) Confocal maximum intensity z-projections of the spinal cord lesion at 2 dpi and 4 dpi show the myelinated spinal cord and myelin debris (magenta) in phagocytes (cyan). The amount of internalized myelin was determined by the colocalized signal of mbp:mCherry-CAAX within mpeg1:eGFP. n = 11–15 and 15–17 animals at 2 and 4 dpi, respectively. (F and G) Confocal maximum intensity z-projections of the spinal cord lesion at 4 dpi, showing lysosomes (magenta) in phagocytes (cyan). The amount of lysosomes was determined by the colocalized signal of LysoTracker with mpeg1:eGFP. n = 8–10 and 14–25 animals at 2 and 4 dpi, respectively. (H and I) Confocal maximum intensity z-projections from 2 h time-lapse videos (Videos 2 and 3) of the spinal cord lesion at 3 dpi show the myelinated spinal cord and myelin debris (magenta) and phagocyte (cyan) movement. Arrowheads show actively moving phagocytes, and short arrows show amoeboid stationary phagocytes. n = 6 or 7 animals. Lateral views of the lesion site are shown; anterior is left and dorsal is down. All data are mean ± SEM (error bars). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; n.s. indicates no significance, by two-way ANOVA test with Tukey’s multiple comparisons test in B, D, and E or Sidak’s multiple comparisons test in G and unpaired t test with Welch's correction in J. Scale bars: 20 µm.

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