Supplementary Figure 1: cbMg display cellular phenotype associated with active cell clearance.
From: Epigenetic regulation of brain region-specific microglia clearance activity
(a) cbMg show a less ramified / less surveillant morphology. Microglia morphology was evaluated in striatum / cerebellum of brain sections from 4/6mo wild-type mice. Left: Representative projection of 3D z-stacked images showing single IBA1+ microglia (IBA1: green, DAPI: blue) with manual 2D tracing of microglial processes. Scale: 10 μm. Right: Representative microglia reconstruction (Neurolucida) with concentric circles for Sholl analysis. Right: Quantification of regional microglia morphology. Numbers of primary processes: stMg: mean=5.325, SEM=0.2393; cbMg: mean=3.100, SEM=0.1871; p=0.0014, F=1.636, t4=7.849. Sholl intersections at 10 μm interval: stMg: mean 19.83, SEM=2.181; cbMg: mean= 11.08, SEM=2.316; p=0.0195, F=1.127, t4=3.774. Number of branch endings: stMg: mean=23.44, SEM=2.718; cbMg: mean= 11.92, SEM=1.728; p=0.0135, F=2.476, t4=4.219. Combined length of processes (μm): stMg: mean 262.4, SEM=31.47; cbMg: mean= 149.2, SEM=40.91; p=0.033, F=1.69, t4=3.198. Branch order: stMg: mean=85.79, SEM=14.08; cbMg: mean=40.67, SEM=8.296; p=0.0262, F=2.882, t4=3.446. n=35 cells from n=5 mice. Longest branch (μm): stMg: mean=37.29, SEM=1.560; cbMg: mean=36.80, SEM=1.136; p=0.7206, F=1.885, t2=0.4115. Soma size (μm2): stMg: mean=46.47, SEM=2.369; cbMg: mean=47.10, SEM=2.164; p=0.9007, F=1.198, t2=0.1411. n=25 cells from n=3 mice. All t-tests were two-tailed paired. (b,c) Microglia in different brain regions display distinct morphological features. (b) CD68+ lysosome content (green) in P2RY12+ microglia (red) was determined using immunofluorescence analysis from brain sections of 4/6mo wild-type mice (DAPI: blue). Scale: 10 μm. Representative image is shown. (c) Quantification of regional microglial morphological properties. Microglial density (mm-1): olfactory bulb microglia (olfMg): mean=130.6, SEM=14.14; striatum microglia (stMg): mean=166.0, SEM=19.05; cortex microglia (cxMg): mean=122.4, SEM=4.713; dentate gyrus microglia (dgMg): mean=138.1, SEM=12.6; cerebellum microglia (cbMg): mean=73.47, SEM=4.713; p=0.0048, F=7.41. Percent P2RY12+ area covered per microglia: olfMg: mean=1.2, SEM=0.168; stMg: mean=0.909, SEM=0.0375; cxMg: mean=1.16, SEM=0.0878; dgMg: mean=0.924, SEM=0.0323; cbMg: mean=0.3646, SEM=0.07586; p=0.0006, F=12.59. Percent CD68+ lysosomal area / microglia: olfMg: mean=0.02393, SEM=0.001433; stMg: mean=0.01349, SEM=0.0025; cxMg: mean=0.01315, SEM=0.00119; dgMg: mean=0.03087, SEM=0.00295; cbMg: mean=0.03889, SEM=0.00147; p< 0.0001, F=30.22. Ratio of CD68+ lysosomal area / microglia area: olfMg: mean=0.01576, SEM=0.0049; stMg: mean=0.015, SEM= 0.003; cxMg: mean=0.014, SEM=0.002; dgMg: mean=0.0190, SEM=0.0025; cbMg: mean=0.126, SEM=0.0225; p<0.0001, F=22.02. One-way ANOVA with Tukey’s Multiple Comparison post-hoc analysis to compare all pairs of columns. 3-5 sections of whole brain regions from n=3 wild-type mice. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Bar graphs with individual data points show mean ± SEM. (d) cbMg lysosomes contain DAPI+ material. Representative immunofluorescence images of microglia (left) and their 3D reconstruction (right, Imaris, related to Fig.1) from striatum and the granule cell layer and molecular layer of the cerebellum. IBA1+ microglia: green; CD68+ lysosomes:red; and DAPI:blue. Scale: 5 μm. Side scatters of microglia containing DAPI+/CD68+ lysosome are shown underneath each forward scatter for cbMg with the DAPI channel masked for the microglia surface. 3D axes are shown. Arrows point to the box enclosing DAPI+/CD68+ lysosome inside IBA1+ microglia with a zoomed in view. Experiment was repeated 3 times independently with similar results.
