HRasV12 expression in the proliferating regions of the developing CNS alters microglia morphology. (a) Schematic representation of the zebrafish germline system used to induce HRasV12 expression based on the outcross of the indicated fish lines. Schematic anterior‐posterior dorsal view of the brain representing the main sub‐divisions: telencephalon (T), optic tectum (OT) cerebellum (CB) and tectal proliferation zone (TPZ) in gray. Confocal images showing mCherry and eGFP‐HRasV12 fluorescent signal in the proliferating regions of the developing brain of HRasV12⁺ larvae from 1 to 5 dpf. White dotted lines mark the main brain subdivisions. Scale bar represents 100 μm. (b) Brain volume was assessed using Imaris surface tool to build the segmented images (right panels) of the mCherry signal (left panels) of proliferating regions of the developing brain from 5 dpf HRasV12⁻ (top panels) and HRasV12⁺ (bottom panels). Scale bar represents 100 μm. Brain volumes of 5 dpf HRasV12⁻ and HRasV12⁺ larvae were quantified. HRasV12⁻: n = 10; HRasV12⁺: n = 10; N = 3. (c) Confocal images of microglia (magenta) distribution throughout the developing brain of HRasV12⁻ (top panels) and HRasV12⁺ brains (bottom panels) from 3 to 5 dpf, using 4C4 antibody to specifically label microglia. Scale bar represents 100 μm. Close‐ups of microglia at 3 dpf and 5 dpf under physiological condition (HRasV12⁻) allow to determine their morphology: amoeboid and ramified. Scale bar represents 10 μm. (d) Close‐ups of microglia from 5 dpf HRasV12⁻ (top panels) and HRasV12⁺ (bottom panels) larvae (left panels) and their segmented images in 3D (right panels) using Imaris surface tool, to assess microglia morphology. Scale bar represents 10 μm. The number of amoeboid microglia was quantified within the microglial population of 5 dpf control and HRasV12⁺ larvae. Results are shown as a percentage of total microglia. HRasV12⁻: n = 15; HRasV12⁺: n = 15; N = 3. Error bars represent mean ± SD. Images were captured using a Zeiss LSM710 confocal microscope with a 20X/NA 0.8 objective. All images represent the maximum intensity projections of Z stacks

Pre‐neoplastic cells promote microglia proliferation. (a) Confocal images of the microglial population (magenta) of 5 dpf HRasV12⁻ (top panel) and HRasV12⁺ (lower panel) larvae. Scale bar represents 100 μm. Quantifications revealed a higher number of microglia in HRasV12⁺ brains compared to HRasV12⁻ brains at 5 dpf. HRasV12⁻: n = 15; HRasV12⁺: n = 15; N = 3. Error bars represent mean ± SD. (b) To measure microglia proliferation, the number of 4C4⁺/EdU⁺ cells was measured within the microglial population of 5 dpf control and HRasV12⁺ larvae. Results are expressed as a percentage of total microglia. HRasV12⁻: n = 17; HRasV12⁺: n = 17; N = 3. Error bars represent mean ± SD. Error bars represent mean ± SD. Images were captured using a Zeiss LSM710 confocal microscope with a 20X/NA 0.8 objective. All images represent the maximum intensity projections of Z stacks

HRasV12⁺ cells affect actin cytoskeleton dependent microglial functions. (a) Schematic representation of the phagocytosis assay used to measure microglia phagocytic activity of 3 and 5 dpf HRasV12⁻ and HRasV12⁺ larvae. Zymosan coupled with a fluorochrome was injected into either the telencephalon or the tectum of HRasV12⁻ and HRasV12⁺ larvae at 3 and 5 dpf. Larvae were incubated for 6 h post‐injection at 28.5°C, fixed, then labeled with the 4C4 antibody to visualize microglia. Confocal image of a 5 dpf control larval brain injected with zymosan (white) into tectum (yellow square). Scale bar represents 100 μm. Close‐up of the injection site reveals zymosan phagocytosed by microglia (magenta). Scale bar represents 20 μm. The Imaris surface tool was used to segment and read out the sum of fluorescence from zymosan internalized by microglia (magenta surface) as well as the total amount of injected zymosan within the telencephalon or tectum (gray surface). The percentage of phagocytosis was calculated following the indicated formula. (b) Efficiency of phagocytosis was calculated for 3 and 5 dpf HRasV12⁻ and HRasV12⁺ larvae injected with zymosan into either the telencephalon or the tectum. Results are expressed as a percentage of the total amount of injected zymosan. 3 dpf: HRasV12⁻: n = 10; HRasV12⁺: n = 10; N = 3; 5 dpf (tectum): HRasV12⁻: n = 28; HRasV12⁺: n = 28; N = 3; 5 dpf (telencephalon): HRasV12⁻: n = 13; HRasV12⁺: n = 13; N = 3; Error bars represent mean ± SD. (c,d) Microglia movement in 3D (motility) was tracked using Imaris tracking tool for the full duration of time series (13 h, Δt = 14 min). Examples of microglia tracks displayed as time color‐coded lines from the different conditions. Scale bar represents 20 μm. Track speed mean and track length were calculated in HRasV12⁻ and HRasV12⁺ conditions at 3 (c) and 5 dpf (d). 3 dpf: HRasV12⁻: n = 30; HRasV12⁺: n = 30; N = 3; 5 dpf: HRasV12⁻: n = 30; HRasV12⁺n = 3; N = 3; Error bars represent mean ± SD. Images were captured using a Zeiss LSM880 confocal microscope with a 20X/NA 1.0 objective. All images represent the maximum intensity projections of Z stacks

The actin nucleation promoting factor wasla is less expressed in microglia from HRasV12⁺ larvae. (a) Heatmap of differentially expressed (DE) genes (FDR < 0.05, Fold Change > |2|) from microglia transcriptome comparisons between 5 dpf HRasV12⁻ and HRasV12⁺ larval brains (11 genes), belonging to zebrafish actin cytoskeleton KEGG pathways [Regulation of actin cytoskeleton, Focal adhesion, Adherens junction, Tight junction and mTOR signaling pathway]. The actin nucleation promoting factor wasla is the top ranked gene of the list. See also Table S3. (b) Dot plots of normalized transformed read counts of the representative WASP family genes. Black plots represent non‐DE genes whereas, red plots correspond to DE genes at 5 dpf between control and HRasV12⁺ conditions. (b) The means ± SD of three independent experiments are plotted. (c) Dot plots of normalized transformed read counts of WASL gene expression of monocyte‐derived macrophages (MDMs) and microglia in human non tumor and glioma IDH WT brain samples (Klemm et al., 2020)

Walsa overexpression in microglia restores their morphology and functions. (a) Close‐up confocal images of microglia (top panels), microglial population (middle panels) and examples of microglia tracks displayed as time color‐coded lines (lower panels) from 5 dpf HRasV12⁻ (left panel), HRasV12⁺ (middle panel) and HRasV12⁺; wasla (right panel) brains. Scale bar represents 10, 100 and 20 μm, respectively. (b) The percentage of amoeboid microglia and (c) the total number of microglia from 5 dpf HRasV12⁻, HRasV12⁺ and HRasV12⁺; wasla brains were quantified. HRasV12⁻: n = 15; HRasV12⁺: n = 15; HRasV12⁺; wasla: n = 15; N = 3. Error bars represent mean ± SD. (d) The percentage of phagocytosis within the tectum of 5 dpf HRasV12⁻, HRasV12⁺ and HRasV12⁺; wasla as well as microglia track speed mean and track length (motility) were quantified. Phagocytosis: HRasV12⁻: n = 16; HRasV12⁺: n = 16; HRasV12⁺; wasla: n = 16; N = 3. Motility: HRasV12⁻: n = 30; HRasV12⁺: n = 30; HRasV12⁺; wasla: n = 30; N = 3. Error bars represent mean ± SD. Red dotted lines indicate the number of microglia, track speed mean or track length in control condition. Images were captured using a Zeiss LSM880 confocal microscope with a 20X/NA 0.8 objective. All images represent the maximum intensity projections of Z stacks

Microglial wasla expression is crucial to slow down tumor progression. (a) Kaplan–Meier survival plot of HRasV12⁻, HRasV12⁺ and HRasV12⁺; wasla larvae control over 31 days, n = 50/60, 4/60 and 3/60, respectively. p = .0017 (Gehan–Breslow–Wilcoxon test between HRasV12⁺ and HRasV12⁺; wasla conditions). Error bars represent mean ± SD. (b) Brain and pre‐neoplastic mass volume were measured using the mCherry signal (brain, top panels) and eGFP signal of HRas⁺ cells (pre‐neoplastic mass; bottom panels) of proliferating regions of the developing brain from 5 dpf HRasV12⁻ (left panel), HRasV12⁺ (middle panel) and HRasV12⁺; wasla (right panel) larvae. Scale bar represents 100 μm. Brain and pre‐neoplastic mass volume from 5 dpf HRasV12⁻, HRasV12⁺ and HRasV12⁺; wasla larvae are quantified using Imaris surface tool. HRasV12⁺: n = 9; HRasV12⁺; wasla: n = 6; N = 3. Error bars represent mean ± SD. Red dotted line indicates the brain volume mean in control condition. (c) Close‐up confocal images of microglia (mpeg1:eGFP⁺ cells) from 5 dpf HRasV12⁺ (left panel) and HRasV12⁺; wasla (right panel) brains. Phagosomes are indicated by red asterisks. Scale bar represents 10 μm. The number of phagosomes per microglia from 5 dpf HRasV12⁺ and HRasV12⁺; wasla brains were quantified. HRasV12⁺: n = 80; HRasV12⁺; wasla: n = 80; N = 3. Error bars represent mean ± SD. (d) Mean of GFP fluorescent intensity (MFI) from phagocytosed pre‐neoplastic cells detected by flow cytometry within isolated microglia from 5 dpf HRasV12⁺ and HRasV12⁺; wasla larvae. The means ± SD of two independent experiments are plotted. Images were captured using a Zeiss LSM880 confocal microscope with a 20X/NA 0.8 objective. All images represent the maximum intensity projections of Z stacks