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Fig. 6
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.