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. Author manuscript; available in PMC: 2021 Jan 6.
Published in final edited form as: Nat Neurosci. 2020 Jul 6;23(9):1055–1066. doi: 10.1038/s41593-020-0654-2

Figure 3. Microglia phagocytose myelin sheaths with minimal phagocytosis of oligodendrocyte somas.

Figure 3.

(a) Strategy for delaying the breakdown of phagocytosed myelin with bafilomycin A1. (b) Microglia in Tg(mpeg1.1:mVenus-CAAX; mbpa:mCherry-CAAX) larvae treated with DMSO or bafilomycin (1 μm) for 1 hour prior to imaging. Arrowheads mark round myelin inclusions inside microglia. (c) Example sum-projected image of a microglia containing mRFP inclusions (top) with morphological segmentation performed using MorphoLibJ to identify inclusions (bottom). (d) Histogram depicting the number of myelin inclusions per microglia in DMSO and bafilomycin-treated larva. (e-g) Morphological segmentation on sum-projected images of microglia in 4 dpf Tg(mpeg1.1:mVenus-CAAX; mbpa:mCherry-CAAX) larvae that were treated with either DMSO or bafilomycin prior to imaging. Each point represents one inclusion (e) or microglia (f, g); points shaded so overlapping points are visible (DMSO, n=22 cells from 22 larvae containing 48 inclusions; bafilomycin, n=22 cells/22 larvae/175 inclusions). (e) Area (μm2) of myelin inclusions inside microglia, Wilcox rank-sum test. (f) Total myelin inclusion area (μm2) per microglia, Wilcox rank-sum test. (g) Number of myelin inclusions per microglia in bafilomycin-treated larvae over the course of developmental myelination (4–8 dpf). Kruskal-Wallis test, n=fish/cells/inclusions, n=10/10/74 4 dpf, 9/9/79 5 dpf, 12/12/101 8 dpf. (h) Max-projection images of spinal cord oligodendrocytes (magenta) in larvae treated with either DMSO or bafilomycin A1 and live-stained with acridine orange (green). (i-k) Quantification of mbpa+ cells (i), acridine orange cells (j), and mbpa+ cells stained by acridine orange (k) in 3.5 hemisegments and analyzed by Wilcox rank-sum test for n=fish/cells, n=14/602 DMSO, 16/742 bafilomycin. (l) Schematic of transgenic oligodendrocyte reporters that label either oligodendrocyte membrane (mCherry-CAAX) or cytosol (TagRFP). (m) Examples of microglia in either Tg(mpeg1.1:mVenus-CAAX; mbpa:mCherry-CAAX) or Tg(mpeg1.1:mVenus-CAAX; mbpa:TagRFP-T) 4 dpf larvae, treated with either DMSO or bafilomycin A1 prior to imaging. Arrowheads mark myelin inclusions. (n) Number of mCherry-CAAX and TagRFP inclusions per microglia in each of the four groups presented in (m), Wilcox rank-sum test (n=fish/cells/inclusions, n=12/12/18 mCherry-CAAX DMSO, 10/10/74 mCherry-CAAX bafilomycin, 6/6/0 TagRFP DMSO, 8/8/1 TagRFP bafilomycin). Scale bars, 10 μm.