Functionally distinct subgroups of oligodendrocyte precursor cells integrate neural activity and execute myelin formation

Abstract

Recent reports have revealed that oligodendrocyte precursor cells (OPCs) are heterogeneous. It remains unclear whether such heterogeneity reflects different subtypes of cells with distinct functions or instead reflects transiently acquired states of cells with the same function. By integrating lineage formation of individual OPC clones, single-cell transcriptomics, calcium imaging and neural activity manipulation, we show that OPCs in the zebrafish spinal cord can be divided into two functionally distinct groups. One subgroup forms elaborate networks of processes and exhibits a high degree of calcium signaling, but infrequently differentiates despite contact with permissive axons. Instead, these OPCs divide in an activity- and calcium-dependent manner to produce another subgroup, with higher process motility and less calcium signaling and that readily differentiates. Our data show that OPC subgroups are functionally diverse in their response to neurons and that activity regulates the proliferation of a subset of OPCs that is distinct from the cells that generate differentiated oligodendrocytes.

Extended Data Fig. 1. Characterisation of OPCs in the zebrafish spinal cord.

a) Confocal image of a Tg(olig1:memEYFP), Tg(olig1:nls-mApple) zebrafish at the level of the spinal cord at 21 dpf (example of 3 animals from 1 experiment). Scale bar: 50 μm. b) Cross-sectional view of the spinal cord showing the distribution of myelin in Tg(mbp:EGFP-CAAX) at 7 dpf (example of 12 animals from 4 experiments). Scale bar: 10 μm. c) Cross-sectional view of the spinal cord showing the distribution of pre- and postsynapses (Tg(elavl3:synaptophysin-RFP), anti-mCherry, anti-gephyrin) at 7 dpf (example of 12 animals from 4 experiments). Scale bar: 10 μm. d) Confocal images of Tg(mbp:nls-EGFP), Tg(olig1:nls-mApple) transgenic animals between 4 and 28 days post fertilization (n numbers as in panel e). Scale bar: 20 μm. e) Cell numbers of OPCs (olig1: nls-mApple-pos., mbp:nls-EGFP-neg.) and myelinating oligodendrocytes (mbp:nls-EGFP-pos.) in the spinal cord. Data are expressed as mean cells/field ± SD at 3/5/7/10/13/16/20/24/28 dpf (OPCs: 29.6±6.4 vs. 28.1±5.2 vs. 22.3±4.3 vs.19.3±4.5 vs. 21.7±5.4 vs. 22.6±4.2 vs. 25.3±4.6 vs. 27.3±5.1 vs. 32.0±5.9; mOLs: 7.3±5.7 vs. 31.1±5.5 vs. 44.7±6.0 vs. 56.6±6.9 vs. 62.6±7.2 vs. 61.5±11.6 vs. 74.1±9.4 vs. 75.7±13.3 vs. 95.2±13.4; n animals / experiments=17/2, 15/3, 15/3, 16/3, 17/2, 17/2, 20/3, 12/3, 13/3). f) Example images of individual OPCs labelled showing a range of morphologies. The soma can be localised within axo-dendritic (top) or neuron-rich areas (middle, bottom). The process network of an individual cell can be restricted to one side of the spinal cord (top and middle cells), but it can also reach to both sides of the spinal cord (bottom cell) (n numbers as in panel g). Scale bar: 10 μm. g) OPC morphometry using three-dimensional process tracing and creation of a volume hull around the reconstructed filaments (n=228 cells from 56 animals between 3 and 16 dpf in 24 experiments). Scale bar: 10 μm.

Extended Data Fig. 2. Analysis of scRNA sequencing clusters.

a) Schematic overview of cell isolation, sorting and sequencing. b) Flow cytometry plots of olig1:memEYFP-sorted cells and wildtype control animals. Dotted lines indicate the gating used (example from two independent experiments). c) TSNE plot showing expression of sox10 (total sample size n=310 cells). Immunohistochemistry for Sox10 on transversal spinal cord sections of 7 dpf Tg(olig1:nls-mApple), Tg(mbp:nls-EGFP) animals and quantification of Sox10-expressing OPCs (olig1:nls-mApple-positive, mbp:nls-EGFP-negative) in neuron-rich and axo-dendritic areas (100% (68/68) vs 100% (49/49) pos cells, n=16 animals / 4 experiments). Dotted lines in the image indicate the outlines of the spinal cord. Scale bar:10 μm. d) TSNE plot showing expression of olig2 and nkx2.2a (sample size as in panel c). e) TSNE plot showing expression of ppp1r14bb, mbpa, and plp1a (sample size as in panel c). f) Confocal images with in situ hybridisations for cspg4, gpr17, myrf, and labelling of EDU incorporated cells on transversal spinal cord sections of 7 dpf Tg(olig1:nls-mApple), Tg(mbp:nls-EGFP) animals (see Fig. 2e, 2i, 2k, 2l for respective n numbers). Scale bar: 10 μm.

Extended Data Fig. 3. Quantification of OPC morphology and position prior to differentiation.

Quantification OPC complexity and soma position from imaging timelines between 3 and 15 dpf. Measured is the last timepoint as OPC prior to differentiation, as assessed by myelin sheath formation (imaging intervals of 1d between 3 and 7 dpf, and 2d between 7 and 15 dpf). N=10/6/3/3/3/2/1/2/2/2/1/5/1 cells at 3/4/5/6/7/8/9/10/11/12/13/14/15 dpf. Data from 23 animals in 6 experiments.

Extended Data Fig. 4. Time-lapse imaging of OPC population dynamics.

a) Overview images of transgenic zebrafish labelling nuclei of OPCs (olig1:nls-mApple) and myelinating oligodendrocytes (mbp:nls-EGFP) at the beginning and end of a timelapse between 3-5 dpf. Dashed boxes indicate the areas shown in panel c (n=3 animals in 2 experiments). Scale bar: 10 μm. b) Quantification of the fates of OPCs found in neuron-rich areas – detailed breakdown of data shown in Fig 4e. c) Zoom-ins and false colouring of the timelapse shown in panel a showing potential behaviours of OPCs in neuron-rich areas by either remaining quiescent (red cell), generation of new OPCs in neuron-rich areas (magenta cells), or the generation of new OPCs in axo-dendritic areas (green cells). The insets at first and last timepoint show the absence of myelin markers (mbp:nls-EGFP) in the cells studied (n=3 animals in 2 experiments). Scale bar: 10 μm.

Extended Data Fig. 5. Cell Fate Analysis of OPCs with their soma in neuron-rich areas.

a) Time series of an individual OPC with its soma in neuron-rich areas that gives rise to myelinating oligodendrocytes by proliferation-mediated generation of daughter OPCs in axo-dendritic areas. Left panel: Confocal images. Middle panel: Reconstructions of the starting cell and individual daughter cells (cells that will differentiate are shown in blue). Right panels: Y-axis rotations showing olig1:nls-mApple cell body positions within the hemi-spinal cord. Dashed lines depict the outline of the spinal cord. One of 8 examples from 7 animals in 6 experiments. Scale bar: 10 μm. b) Graphical summary of cell fates from the data analysed in Figures 5a-d. See also Supplementary Figure 2.

Extended Data Fig. 6. Characterisation of OPC GCaMP reporter lines.

a) Example images of individual olig1:GCaMP-CAAX labelled OPCs in axo-dendritic areas of the zebrafish spinal cord at 4 dpf. The absence of nascent ensheathments indicates that these cells are not early differentiating oligodendrocytes (n=9 independent experiments). Scale bar: 10 μm. b) Dorsal views of Tg(olig1:GCaMP6m), Tg(mbp:KillerRed) transgenic zebrafish at 4 dpf to label OPCs and differentiated oligodendrocytes. Dotted box indicates position of zoom-ins in bottom row (n=3 animals in 1 experiment). Scale bars: 50 μm (top) and 20 μm (bottom). c) Quantification of single and double-positive cells from images as shown in b. d) ΔF/F₀ GCaMP transients of individual cells in two Tg(olig1:GCaMP6m) zebrafish. Green traces depict cells in axo-dendritic areas, grey traces depict cells in neuron-rich areas (total of 8 animals in 8 experiments).

Extended Data Fig. 7. Effects of chronic 4-AP incubation on zebrafish.

a) Minimum intensity projections of a two-minute time-lapse of fish freely swimming in a 3 cm petri dish in different treatment conditions (n=6/7/3/3 animals in control/4-AP/TTX/4-AP+TTX, 3 independent experiments). b) Traces of GCaMP transients Tg(elavl3:h2b-GCaMP6s) zebrafish at 4 dpf and after overnight incubation in 0.1 mM 4-AP, and before / after 10 μM TTX (7 animals per conditions in 2 experiments). c) Confocal images of Tg(mfap4:memCerulean), Tg(olig1:nls-mApple) zebrafish at 4 dpf after treatment with 0.1 mM 4-AP, 0.5 mM 4-AP, or Danieau’s solution as control. Transmitted light images to show spinal cord morphology and tissue integrity following drug treatment. Scale bars: 100 μm. The graph shows that number of macrophages which accumulate in 400 μM length of spinal cord of Tg(mfap4:memCerulean) zebrafish after 1 day of control (2±0.25/2 cells), 0.1 mM (2±1/2 cells), and 0.5 mM (3±0.25/2 cells) 4-AP treatment (median (25%/75% percentiles); p=0.43 (control vs. 0.1mM 4-AP), p=0.03 (control vs. 0.5 mM 4-AP) (Kruskal-Wallis test, test statistic=3.003), n=16/19/8 animals in 3 experiments. d) Representative images of Tg(mbp:nls-EGFP), Tg(olig1:nls-mApple) zebrafish in control and after 2 days of 0.1 mM 4-AP treatment (see Fig 7g for n numbers). Scale bar: 20 μm.

Figure 1. Characteristics of oligodendrocyte precursor cells (OPCs) in zebrafish

a) Top: Image of whole Tg(olig1:memEYFP) transgenic animal at 5 days post-fertilization (dpf). Scale bar: 1mm. Bottom: confocal image of a Tg(olig1:memEYFP), Tg(olig1:nls-mApple) zebrafish at the level of the spinal cord at 5 dpf. Scale bar: 50 μm. Representative images from 4 animals in 2 independent experiments. b) Cross-sectional view of the spinal cord showing the distribution of OPC processes in Tg(olig1:memEYFP) at 7 dpf (n=33 animals / 11 experiments). Scale bar: 10 μm. c) Cross-sectional view of the spinal cord showing the distribution of axons and dendrites at 7 dpf (anti-acetylated tubulin and anti-MAP2; n=7 animals / 2 experiments). Scale bar: 10 μm. d) Top: sparse labelling of olig1:memEYFP expressing OPCs at 4 dpf. Bottom: Tracing of two neighbouring examplary OPCs and the spinal cord outlines (n numbers in panel g). Dotted lines indicate the position of the y-axis rotations shown in e. e) 90° y-axis rotations at the level of the soma of each of the two cells shown in panel d with a BODIPY counterstain to reveal the position of OPC somata in axo-dendritic (cell #1) and neuron-rich (cell#2) regions (n=12 BODIPY stained animals / 4 experiments). Dotted lines indicate axo-dendritic areas. Scale bar: 10 μm. f) High-magnification view showing the proximity of the processes made by the two OPCs shown in panel d within axo-dendritic areas. Quantification shows the percentage of cell processes resident in axo-dendritic areas formed by OPCs with their soma in neuron-rich and axo-dendritic regions at 4-5 dpf (mean 91.3% ± 2.3 SD neuron-rich vs. 98.9% ± 0.7 SD axo-dendritic; n=5 cells/condition from 6 animals). g) Morphology reconstructions of the two OPCs shown in panels d-f. Quantification shows relative cell complexities of individual OPCs at 4 dpf with their soma in different areas. Triangles indicate example cell#1 and #2. Data are expressed as median ± 25/75% interquartile percentiles, whiskers indicate min/max values (2.1±1.5/3.2 for OPCs in neuron-rich areas vs. 0.6±0.4/1.5 in axo-dendritic areas, p<0.001 (Mann-Whitney U test, U=211; n=36/38 cells from 23 animals in 11 experiments). h) Projections of 60 minutes time-lapse imaging to show remodelled and stable processes of OPCs with their soma in different areas. Quantification shows stable processes over time. Dashed lines connect data points of individual cells. Data points connected by continuous lines represent mean ± SD within the groups. At t=60 minutes: 56.3±12.2 % stable processes for OPCs in neuron-rich areas vs. 25.0±5.5 % in axo-dendritic areas; p=0.001 between groups (two-way repeated-measures ANOVA of time-points 0-60 min, F(1,6)=32); n=4 cells per group from 8 animals in 8 experiments). Scale bar: 20 μm. i) Schematic overview depicting the positioning of OPCs in the zebrafish spinal cord.

Figure 2. Single cell RNA sequencing of zebrafish OPCs

a) TSNE plots of olig1:memEYFP sorted cells with oligodendrocyte lineage identity (cells per cluster: #1=110, #2=28, #3=33, #4=18, #5=19; cells came from 2300 animals at 5 dpf (1 experiment). b) Log(TPM) expression levels of key oligodendrocyte lineage markers in the clusters shown in panel a. Data are expressed as median with interquartile ranges and a violin shape to represent data distribution. c) In situ hybridisation for nkx2.2a on transversal spinal cord sections of 7 dpf Tg(olig1:nls-mApple), Tg(mbp:nls-EGFP) animals and quantification of nkx2.2a-expressing OPCs (olig1:nls-mApple-positive, mbp:nls-EGFP-negative) in neuron-rich and axo-dendritic areas (97% (89/92) vs. 98% (46/47) pos. cells, n=12 animals / 3 experiments). Dotted lines in the image indicate the outlines of the spinal cord. Scale bar:10 μm. d) Violin plots as in panel b with relative expression levels of cspg4 and ptprz1b. e) TSNE plots (see panel a for n numbers) of cspg4 expression and quantification of cspg4-positive cells as described in panel c (82% (69/84) vs. 34% (17/50) pos. cells, p<0.001 (two-tailed Fisher’s exact test), n=15 animals / 4 experiments). f) TSNE plots as in panel e of ptprz1b expression and quantification of ptprz1b-positive cells as described in panel c (63% (60/95) vs. 38% (18/47) pos. cells, p=0.007 (two-tailed Fisher’s exact test), n=6 animals / 2 experiments). g) Violin plots as in panel b with relative expression levels of pcna and mki67. h) TSNE plots as in panel e of pcna and mki67 expression. i) Quantification of proliferative OPCs using EDU incorporation (olig1:nls-mApple-positive, mbp:nls-EGFP-negative) in neuron-rich and axo-dendritic areas (30% (25/82) vs. 26% (17/66) pos. cells, p=0.585 (two-tailed Fisher’s exact test), n=3 animals / 2 experiments). j) Violin plots as in panel b with relative expression levels of gpr17 and myrf. k) TSNE plots as in panel e of gpr17 expression and quantification of gpr17-positive cells as described in panel c (35% (44/125) vs. 69% (33/48) pos. cells, p<0.001 (two-tailed Fisher’s exact test), n=13 animals / 3 experiments). l) TSNE plots as in panel e of myrf expression and quantification of myrf-positive cells as described in panel c (26% (7/27) 58% (14/24) pos. cells, p=0.025 (two-tailed Fisher’s exact test), n=6 animals / 2 experiments). m) Gene ontology (GO) terms of top 30 significantly expressed genes in cluster OPC #4. n) GO terms of top 30 significantly expressed genes in cluster OPC #1.

Figure 3. Differentiation properties of individual OPCs

a) Example time-lapses of individually labelled olig1:memEYFP cells with their soma in neuron-rich (left) and axo-dendritic (right) spinal cord between 3 and 5 dpf. Quantification shows proliferation / differentiation fate within 24 h. 38% (5/13) vs. 19% (4/21) cells proliferated in neuron-rich vs. axo-dendritic areas, p=0.254 (two-tailed Fischer’s exact test); 0% (0/13) vs. 81% (17/21) cells differentiated, p<0.001 (two-tailed Fischer’s exact test), n=11 animals / 8 experiments. Scale bar: 20 μm. b) Frequency distribution of OPC soma positioning before differentiation at different developmental stages (% cells in axo-dendritic areas: 90.7% (39/43) at 4 dpf, 86.0% (37/43) at 7 dpf, 81.8%(9/11) at 14 dpf, n=18/16/4 animals in 3/6/3 experiments). c) Myelinating oligodendrocyte numbers with their soma in neuron-rich areas at different developmental stages. Data are expressed as mean percentage ± SD (3.1±6.2 vs. 6.7±4.7 vs. 10.3±3.9 vs 11.8±5.0 vs. 7.8±3.3 vs. 12.7±5.2 vs. 10.6±4.2 vs. 11.5±3.8 vs. 11.3±3.1 at 3/5/7/10/13/16/20/24/28 dpf, n animals/experiments=17/2, 15/3, 15/3, 16/3, 17/2, 17/2, 20/3, 12/3, 13/3). d) Example images of an individual OPC with its soma in neuron-rich areas between 6 and 16 dpf, while a cntn1b:mCherry labelled axon in close proximity becomes ensheathed with mbp:memCerulean labelled myelin (observation from one animal). Dotted boxes indicate the position of magnified views (bottom 3 panels). Scale bar: 20 μm.

Figure 4. Interrelationships between OPC populations

a) Schematic overview delineating possible scenarios of OPC interrelations. b) Example image of two neighbouring OPCs showing that OPCs with their soma in neuron-rich (cell #1) and axo-dendritic areas (cell #2) can be distinguished by the shape of their cell body (n>10 animals / >3 experiments). Scale bar: 10 μm. c) Selection of time-lapse images over 21 hours acquired at 30 minutes intervals in Tg(mbp:nls-EGFP), Tg(olig1:nls-mApple) zebrafish at 3 dpf showing an OPC division in neuron-rich areas that gives rise to a daughter OPC in axo-dendritic areas, followed by another cell division (n=3 animals / 2 experiments). Scale bar: 10 μm. d) Times between two cell divisions by OPCs with their soma in neuron-rich and axo-dendritic areas as shown in panel c. Data expressed as mean ± 95% confidence interval (15.0±0.9 hours vs. 18.0±1.7 hours, n=15/14 cells from 3 animals in 2 experiments). e) Quantification of the fates of OPCs found in neuron-rich areas as shown in panel c (n=86 cells from 3 animals in 2 experiments). f) Quantification of the origin of OPCs found in axo-dendritic areas as shown in panel c (n=77 cells from 3 animals in 2 experiments).

Figure 5. Long-term contribution to myelinating oligodendrocytes through proliferation-mediated generation of new OPCs.

a) Example trees of OPC fates with their cell body in neuron-rich areas (n=114 starting cells from 45 fish of 9 experiments between 3 and 26 dpf, which expanded to 270 cells of which 119 differentiated, see also Extended Data Fig. 5b and Supplementary Fig. 2). b) Frequency distribution of proliferation and differentiation events observed by individual OPCs in neuron-rich areas over at least 4 days, unless all cells of this clone differentiated earlier (n=114 cells, see panel a). c) Quantification of the time between last cell division prior to OPC differentiation as percentage for all cells analysed (left) and as absolute numbers for cells with their cell body in neuron-rich and axo-dendritic areas (right) (n=119 cells, see panel a). d) Quantification of the timing of cell body translocation from neuron-rich to axon-rich areas relative to a cell division (n=98 cells, see panel a). e) Representative images and quantification of increased oligodendrocyte differentiation at 4 dpf upon splitomicin treatment. Data shown as mean ± 95% confidence interval (13.6±0.9 cells/field vs. 20.4±1.0 cells/field, p<0.001 (two-tailed unpaired t-test, t=4.184, df=41), n=14/29 animals / 4 experiments). Scale bar: 20 μm. f) Fates of OPCs in neuron-rich areas between 3-5 dpf in presence and absence of splitomicin (control vs. splitomicin: 64% (21/33) vs. 45% (15/33) quiescent, p=0.22 (two-tailed Fisher’s exact test); 0% (0/33) vs. 18% (6/33) differentiation following division, p= 0.02 (two-tailed Fisher’s exact test); n=23 animals per group / 6 experiments). g) Schematic representing the fates of OPCs following a cell division event.

Figure 6. In vivo calcium imaging of individual OPCs

a) Overview of imaging conditions. b) Example traces of GCaMP transients in cell processes and soma of Tg(olig1:GCaMP6m) animals at 4 dpf (average duration=2.95±0.5 SD; n=6 ROI from 2 animals). c) Projection of three timepoints of two olig1:GCaMP-CAAX expressing cells at 4 dpf showing transients restricted to process subdomains (example from 27 animals in 23 experiments). Scale bar: 20 μm. Measured ROI are indicated by the dotted lines around the reconstructed cells. Active ROI are highlighted in green and magenta. d) ΔF/F₀ GCaMP transients of the different ROI shown in c. e) Different time points of two individual olig1:GCaMP-CAAX expressing cells showing transients spreading throughout the cells (example from cells as in panel c). Scale bar: 10 μm. f) Quantification of different types of GCaMP transients observed during an observation period of 10 minutes in individual olig1:GCamP-CAAX expressing cells (n numbers as in panel c). g) Top: Dorsal widefield view of a zebrafish at 4 dpf (example of 8 animals in 8 experiments). Below: z-projection and z-rotation of Tg(olig1:GCaMP6m) in the spinal cord as indicated by the boxed area above. Bottom: projection of 2 timepoints showing a GCaMP transient restricted to a single cell in the volume. Scale bar: 50 μm. Top right: the inset depicts a single plane image taken as indicated by boxed area showing individual OPCs with their soma in neuron-rich and axo-dendritic areas. Scale bar: 10 μm. Bottom right: ΔF/F₀ GCaMP transients of all cells (green trace depicts active soma). h) Probability of somatic GCaMP transients of OPC in neuron-rich and axo-dendritic areas at 4 dpf (26% (73/285 cells) vs. 19% (9/48 cells), p=0.007 (two-tailed Wilcoxon matched-pairs signed rank test, W=-36); n=8 animals in 8 experiments. i) Quantification of GCaMP amplitudes measured in somata of OPCs in neuron-rich and axo-dendritic areas at 4 dpf. Data are expressed as mean ± SD (3.9±1.5 ΔF/F₀ vs. 1.8±0.4 ΔF/F₀, p<0.001 (two-tailed Welch’s t test, t= 8.894, df= 39.67), n=81/9 cells from 8 animals in 8 experiments).

Figure 7. Manipulation of neural activity and OPC calcium signalling changes proliferation of OPCs in neuron-rich areas.

a) Overview of experimental paradigms. b) Example GCaMP traces in Tg(olig1:GCaMP6m) at 4 dpf before drug application, after treatment with 4-AP, and after addition of TTX to the same animal. Quantification of GCaMP events (ROI active / ROI total) per fish in the different treatment conditions. Data are expressed as median ± interquartile range, whiskers represent min/max values (12.5%±9.5/18.3 active ROI before 4-AP vs. 42.8%±28.6/78.0 after 4-AP, p=0.004 vs. 30%±14.1/44.1 after 4-AP and subsequent TTX, p=0.23 (Friedman with Dunn’s multiple comparison test, test statistics=11.09), n=9 animals in 5 independent experiments). c) Representative confocal images of Tg(olig1:nls-mApple), EDU-labelled OPCs at 4 dpf. Arrows indicate double positive cells. Quantification shows olig1:nls-mApple/EDU double-positive cells in different treatment conditions. Data expressed as mean ± 95% confidence interval (19.1±1.1% in control vs. 30.6±1.5% in 4-AP vs. 14.1±2.2% in 4-AP+TTX; p<0.001 (control vs. 4-AP), p<0.001 (4-AP vs. 4-AP+TTX) (one-way ANOVA with Tukey’s multiple comparisons test, F(3,89)=22.44), n=37/35/9 animals from 3 experiments). Scale bar: 50 μm. d) Percentage increase in EDU-positive OPCs located in neuron-rich and axo-dendritic areas after 4-AP treatment. Data are expressed as mean ± 95% confidence interval (9±3.1% vs. 0.08±3.5% increase, p=0.02 in neuron-rich areas (two-tailed unpaired t-test, t=2.431, df=53), n=28 animals in 3 experiments). e) Myelinating oligodendrocyte numbers with and without 4-AP treatment. Data are expressed as mean ± 95% confidence interval (48.9±2.4 cells/field in control vs. 54.6±1.5 in 4-AP, p=0.047 (two-tailed unpaired t-test, t=2.058, df=35), n=16/21 animals / 4 experiments). f) Confocal images of individual OPCs at 4 dpf and 24h post 4-AP treatment. Scale bar: 20 μm. Pie charts show the frequency of cell divisions observed in the different conditions (15% (9/61 cells) in control vs. 45% (25/55 cells) in 4-AP-treated group), p<0.001 (two-tailed Fisher’s exact test), n=18/24 animals). g) Confocal images of individual CalEx expressing OPCs at 4 dpf and 24h post 4-AP treatment. Scale bar: 20 μm. Pie charts show the frequency of cell divisions observed in the different conditions (20% (7/35 cells) control vs. 24% (8/33 cells) 4-AP, p=0.77 (two-tailed Fisher’s exact test), n=11/24 animals).