FIGURE 3

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