Supplementary Figure 1. Nature Neuroscience: doi: /nn.4547

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1 Supplementary Figure 1

2 Characterization of the Microfetti mouse model. (a) Gating strategy for 8-color flow analysis of peripheral Ly-6C + monocytes from Microfetti mice 5-7 days after TAM treatment. Living cells in the forward and side scatter were gated on CD45 + CD11b +, followed by CD115 + to separate monocytes from granulocytes. Monocytes were distinguished by Ly-6C expression levels. Representative histograms of Confetti (GFP, YFP, RFP and CFP) expression in Microfetti Ly-6C lo monocytes (left). Confetti signals were gated on Confetti-negative WT counterparts. Expression of Confetti reporter in Ly-6C hi (open circle) and Ly-6C lo (colored) monocytes (right). Each symbol represents one mouse. Animals with Confetti-labeled Ly-6C lo monocytes were used for tracking long-term microglial self-renewal during homeostasis (n = 21 mice; data shown as total Confetti expression in Fig. 2b, 1 dead animal excluded). (b) Detection of microglial division in vivo. Three Microfetti mice were examined by two-photon laser scanning microscopy via a cortical window six weeks after TAM treatment (see Supplemental Experimental Procedures). A representative image shows RFP + microglial cells (arrowheads) emerged two days after targeted laser lesion (asterisk). One RFP + microglial cell (arrow) was no longer detected in the same 200 µm depth volume on Day 2. Scale bar, 50 µm (same scale in both panels). (c) Quantification of change in area of coverage by GFP + and RFP + microglia per minute of in vivo imaging over 30 minutes from Cx3cr1 GFP/+ and Microfetti mice, respectively. Each symbol represents one microglial cell. Mann Whitney U-test, P = ns, not significant. Data are represented as mean ± SEM. n = 3 mice per genotype. (d) Representative maximum intensity projection images for c showing two consecutive time points, t1 (red) and t2 (green), with 1 minute interval. Static structures are revealed in the overlay (yellow). Scale bar, 30 µm (same scale in both panels). See also Supplementary Videos 1 and 2.

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4 Supplementary Figure 2 Differential regional stability of the microglial network during steady state. (a) Representative 2D renderings of Confetti + (green, yellow, red, and cyan colored balls) and Iba-1 immunolabeled (magenta balls) microglia from 636 µm x 636 µm x 50 µm confocal volumes of the cortex, hippocampus and cerebellum over 1-36 weeks after TAM treatment of 8 weeks old female Microfetti mice. (b) Confetti + microglial densities in different brain regions over 36 weeks. Microglial density (y-axis) was obtained from dividing same color cell counts by ring volume of each central ring size (x-axis). Cell densities from recorded data (red line) were plotted against densities derived from 10,000 Monte Carlo (MC) simulation of random distribution of Confetti + microglia within each dataset (blue box plots represent simulated density distributions: central mark is the median, box represents the 25 th to 75 th percentiles, whiskers extend up to the 98 th percentile and data points above were regarded as outliers). Measured values (red line) that appear above the 98 th percentile of MC simulations (blue whiskers), for example at 8 weeks after TAM treatment in the hippocampus and cerebellum where r = 40 µm, indicate the non-random appearance of same color Confetti + microglial cells and imply that proliferation occurred. This peak represents a single cell replication event in an arbitrary volume of mm 3. Mean data are shown. n = 5 mice per time point. (c) Quantitative analysis of randomness of microglial cell proliferation events detected in b. A pair of same color Confetti + microglial cells with 50 µm spatial distance was considered a single cell replication event. Distribution of standard deviation (SD) of a MC simulation (10,000 iterations) of locations of proliferation events derived from a uniform distribution are represented as box plots (blue box plots: central mark is the median, box represents the 25 th to 75 th percentiles, whiskers represent the 2 nd and 98 th percentiles, and outliers are plotted in red + signs). Average SD of the distribution of observed replication events to a perfect uniform distribution (black cross) was compared to MC simulation. Where the observed SD falls within the MC range between the whiskers, the locations of proliferation events can be considered randomly distributed.

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6 Supplementary Figure 3 Spatiotemporal analysis of microglial expansion and redistribution in the injured facial nucleus. (a) Coronal brain section of a Cx3cr1 GFP/+ mouse 7d post facial nerve lesion depicts areas analyzed for kinetics of microglial proliferation in the facial nucleus degeneration model. Microglial cells were marked by GFP (green). Neurons were immunolabeled with NeuN (blue) to locate the facial nucleus in the pons. The facial nucleus, a 200 µm broad perinuclear zone, and the parvicellular reticular nucleus, alpha part (PCRtA) were isolated in equal dimensions on lesion and contralateral sides for quantification of GFP + microglia using the Imaris software. (b) Quantification of GFP + microglial cells in contralateral (black) and injured (red) facial nuclei from onset (1 to 2d) to peak (7 and 14d) to resolution of injury (60d) in 8-14 weeks old female Cx3cr1 GFP/+ mice. (c) Representative confocal images of microglia (GFP, green) with DAPI nuclear counterstain (blue) in matched facial nuclei during the time course of injury. (d) Kinetics of microglial proliferation indicated by Ki67 immunohistochemistry. (e) Representative confocal images for d. Microglia took on less ramified and more amoeboid morphology at 2 d after the lesion. GFP + microglia (green), Ki67 (magenta) and DAPI (blue). (f) Kinetics of microglial activation indicated by MHC class II immunohistochemistry. (g) Representative confocal images for f. Microglia were highly activated and assumed a rod-like or amoeboid morphology 7d following nerve transection. GFP + microglia (green), MHC class II (magenta) and DAPI (blue). Data are represented as mean ± SEM in b, d and f. n = 4 (1 d), 5 (2 d), 6 (7 d), 5 (14 d) and 7 (60 d) mice pooled from five experiments, 3-6 sections each. Two-way ANOVA, * P = in b, ** P = in b, in f, *** P < in b, d and f. Scale bars, 50 µm in c, 20 µm in e and g.

7 Supplementary Figure 4 Distribution of EdU + GFP + microglial cells outside of facial nuclei at 7 and 60 d after lesion. (a) Quantification of EdU + GFP + microglial cells external of the facial nuclei (in female Cx3cr1 GFP/+ mice injured at 8-14 weeks old) at peak of microgliosis (7 d) and recovery (60 d). Each black and red symbol represents one paired count. Data are pooled from two experiments and shown as mean ± SEM. n = 8 (7 d) and 9 (60 d) mice. Two-way ANOVA, time point *** P = 0.001, lesion *** P < (b) Representative overview images for a showing lesion and contralateral pons. Dotted lines demarcate the facial nuclei. High magnification insets show EdU + GFP + microglial cells in the intermediate reticular nucleus. Scale bars, 500 µm, 50 µm in inset.

8 Supplementary Figure 5 Graphical abstract of context-dependent microglial renewal and clonal expansion. Microglial renewal occurs randomly and steadily throughout the healthy CNS with regional differences in rate. The onset of neurodegeneration triggers rapid clonal expansion and activation of microglial cells. During recovery the altered microglial network gradually returns to homeostatic cell density and a non-activated state. Excess microglia associated with disease are eliminated via cell egress and local apoptotic cell death to regain steady state microglial interfaces in the CNS.

9 Supplementary Table 1. Quantification of total Iba1 + microglia analyzed in cortex, hippocampus and cerebellum to determine the mean cell-cell distance (NN, nearest neighbor) and microglial distribution over 36 weeks. n = 5 mice per group. Brain region Total Iba1 + microglia analyzed Weeks after TAM Mean NN ± SEM / µm cortex 2,293 2,174 2,064 2,019 2, ± 0.86 hippocampus 2,168 2,076 2,027 2,130 2, ± 1.02 cerebellum 1,181 1,041 1,102 1,041 1, ± 2.14

10 Supplementary Table 2. Quantification of total CX3CR1 + GFP + microglia analyzed and microglial proliferation rates in each brain compartment. Proliferation rates were calculated from BrdU label per day ± SEM. n = 3 mice. Brain region Total GFP + microglia analyzed Daily proliferation rate / % olfactory bulb 12, ± cortex 15, ± hippocampus 8, ± hypothalamus 9, ± midbrain 12, ± cerebellum 7, ± 0.080

11 Supplementary Table 3. Quantification of Iba-1 + microglial density by measurement of nearest neighbor (NN) cell-cell distance. Mean distance ± SEM are shown. Minimum NN distance recorded is indicated in parentheses. Quantification of total microglial cells analyzed in each group is indicated in bold. n = 6 (2 d), 5 (7 d), 6 (14 d), 6 (30 d) and 7 (60 d) mice pooled from two experiments. Brain region contralateral lesion Days post lesion NN /µm ± ± ± ± ± 5.41 (min.) (18.33) (24.00) (19.69) (15.51) (25.26) cells NN /µm ± ± ± ± ± 2.33 (min.) (12.07) (7.82) (9.16) (8.97) (12.09) cells