Fig 2: Microglia promote proliferation of AKT1-expressing cells.(A) Treatment with the immunosuppressant Dexamethasone (DEX) and the CSF-1R inhibitor Ki20227 led to reduced microglia numbers and proliferation rates of AKT1-expressing cells in the zebrafish larvae. (i) Quantification of the number of microglia in control and upon AKT1 overexpression in DMSO controls and fish treated with DEX (DMSO Control: 83.8 ± 3.29, n = 34 larvae; DEX Control: 45.5 ± 2.53, n = 36 larvae, p<0.001, N = 3; DMSO AKT1: 154.7 ± 10.38, n = 21 larvae; DEX AKT1: 49.2 ± 5.16, n = 17 larvae, p<0.001, N = 3) and fish treated and Ki20227 (DMSO Control: 83.8 ± 3.29, n = 34 larvae; Ki20227 Control: 26.1 ± 2.55, n = 32 larvae, p<0.001, N = 3; DMSO AKT1: 154.7 ± 10.38, n = 21 larvae; Ki20227 AKT1: 45.1 ± 3.75, n = 22 larvae, p<0.001, N = 3) (ii) Quantification of the level of proliferation of RFP-expressing cells in control and AKT1 overexpression in DMSO controls and fish treated with DEX (DMSO Control: 13.6 ± 1.01%, n = 20; DEX Control: 13.4 ± 1.85%, n = 15 larvae, p=1.00 (n.s.), N = 3; DMSO AKT1: 48.8 ± 2.23%, n = 20 larvae; DEX AKT1: 20.4 ± 3.02%, n = 11 larvae, p<0.001, N = 3) and fish treated with Ki20227 (DMSO Control: 13.6 ± 1.01%, n = 20 larvae; Ki20227 Control: 9.14 ± 1.30%, n = 17 larvae, p=1.00 (n.s.), N = 3; DMSO AKT1: 48.8 ± 2.23%, n = 20 larvae; Ki20227 AKT1: 25.1 ± 2.54%, n = 20 larvae, p<0.001, N = 3) Error bars represent mean ±SEM. (B) irf8-/- zebrafish larvae showed reduced microglia numbers and proliferation rates of AKT1-expressing cells. (i) Quantification of the number of microglia in control and AKT1-positive brains in wt and irf8-/- mutant zebrafish larvae (Control WT: 85.8 ± 2.45, n = 35 larvae; Control irf8-/-: 6.25 ± 2.76, n = 20 larvae, N = 3, p<0.001) (AKT1 WT: 151.4 ± 5.70, n = 48 larvae; AKT1 irf8-/-: 31.0 ± 10.32, n = 8 larvae, p<0.001, N = 3). (ii) Quantification of the level of proliferation of RFP-expressing cells in control and AKT1-positive brains in wt and irf8-/- mutant zebrafish larvae (Control WT: 9.25 ± 0.75%, n = 13; Control irf8-/-: 8.69 ± 0.76%, n = 12, p<1.00 (n.s.), N = 3) (AKT1 WT: 57.1 ± 2.03%, n = 17; AKT1 irf8-/-: 24.3 ± 4.11%, n = 8, p<0.001, N = 3).

Fig 3: Cxcr4b signaling in macrophages is required for the increase in microglia numbers upon AKT1 transformation in the brain.(A) To rescue Cxcr4b expression in macrophages or neural cells in the cxcr4b-/- mutant fish, a cell-specific rescue construct was injected in addition to the NBT driver and lexOP:tagRFP-pA/lexOP:AKT1-lexOP:RFP constructs. Cxcr4b expression was recovered in macrophages through the mpeg1 promoter (‘macrophage’ rescue) via a mpeg1:cxcr4b-pA construct. The expression of cxcr4b in neural cells (‘Neural’ rescue) was rescued through the lexOP:cxcr4b-pA construct. (B) Immunohistochemistry using a Cxcr4b antibody showed that Cxcr4b expression is completely absent in microglia and neural cells in the cxcr4b-/- fish (upper panels). Cxcr4b expression is partially recovered following the respective rescue conditions (lower panels). (C) Quantification of the number of microglia in cxcr4b-/- mutants overexpressing AKT1 before, and after Cxcr4b rescue in macrophages (‘Macrophage’ rescue: 119.5 ± 4.59, n = 28 vs cxcr4b-/- Akt1: 91.4 ± 5.22, n = 17; p<0.001, N = 39) and in neural cells (‘Neural’ rescue: 92.2 ± 4.40, n = 27, p=1 (n.s.), N = 3). (D) Quantification of the level of proliferation of AKT1-expressing cells in cxcr4b-/- mutants overexpressing AKT1 before, and after Cxcr4b rescue in macrophages (‘Macrophage’ rescue: 37.8 ± 1.45%, n = 45 larvae, vs cxcr4b-/- AKT1: 22.1 ± 1.73%, n = 27 larvae p<0.001, N = 3) and in neural cells (‘Neural’ rescue: 23.8 ± 2.08%, n = 22 larvae, vs cxcr4b-/- AKT1: 22.1 ± 1.73%, n = 27 larvae, p=1.00 (n.s.), N = 3). Error bars represent mean ±SEM. Images were captured using a Zeiss LSM710 confocal microscope with a 20X/NA 0.8 objective. Scale bars represent 100 µm.

Fig 4: Detection of p-Akt, t-Akt, p-IKK, t-IKK, p-p65, and t-p65 protein levels in each group.

Fig 5: Infiltration of peripheral leukocytes into the brain parenchyma accounts for increased microglia numbers following AKT1 overexpression.(A) Immunohistochemistry carried out using the a-4C4 antibody for microglia and a-L-plastin antibody for leukocytes to distinguish microglial cells (L-plastin+/4C4+; indicated by yellow arrows) from newly infiltrating leukocytes (L-plastin+/4C4-; indicated by red arrows). This analysis revealed increased infiltration of leukocytes into the brains of AKT1-positive fish. Representative confocal images of the larval zebrafish brain are shown. Upper panels: upon control RFP expression, lower panels: upon AKT1 overexpression. The dotted white line demarcates the brain parenchyma (Bi) Quantification of the number of infiltrated leukocytes (L-plastin+/4C4-) into the brain parenchyma in control and AKT1-positive larvae at 8 dpf (AKT1: 24.1 ± 1.48, n = 17 larvae, and 11.0 ± 0.64, n = 30 larvae in age-matched controls, p<0.001, N = 3). (Bii) Quantification of the total 4C4+ microglia population in control larvae and larvae following AKT1 overexpression at 8 dpf (Control: 85.0 ± 2.64, n = 30 larvae; AKT1: 146.5 ± 6.27, n = 17 larvae, N = 3). (Biii) Quantification of the total L-plastin+ leukocyte population in control larvae and larvae following AKT1 overexpression at 8 dpf (Control: 96.0 ± 2.77, n = 30 larvae; AKT1: 170.5 ± 6.95, n = 17 larvae, p<0.001, N = 3). (Ci) Quantification of the number of infiltrated leukocytes (L-plastin+/4C4-) into the brain parenchyma in control and AKT1-positive larvae at 3 dpf (Control: 6.0 ± 0.56, n = 17 larvae; AKT1: 27.4 ± 3.38, n = 16 larvae, p<0.001, N = 2). (Cii) Quantification of the total 4C4+ microglia population in control larvae and larvae following AKT1 overexpression at 3 dpf (Control: 43.7 ± 2.43, n = 17 larvae; AKT1: 45.6 ± 3.43, n = 16 larvae, p=1, N = 2). (Ciii) Quantification of the total L-plastin+ leukocyte population in control larvae and larvae following AKT1 overexpression at 3 dpf, (Control: 49.7 ± 2.47, n = 17 larvae; AKT1: 73.0 ± 5.47, n = 16 larvae, p<0.001, N = 2). Error bars represent mean ±SEM. Images were captured using a Zeiss LSM710 confocal microscope with a 20X/NA 0.8 objective. Scale bars represent 100 µm.