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. 2019 Jul 17;8:e46912. doi: 10.7554/eLife.46912

Figure 4. Ca2+-ATP-P2ry12 signalling stimulates microglial interactions with AKT1 cells.

The β-actin:GCaMP6f transgenic line was used to monitor and measure in vivo calcium (Ca2+) levels in control and AKT1 expressing cells. The mpeg1:EGFP transgenic line was used to quantify microglial interactions with control and AKT1 cells. (A) Treating larvae with MK801 and MK5 to inhibit NMDA receptor signalling led to a significant reduction of Ca2+ levels in treated AKT1 cells compared to untreated AKT1 cells. Quantification of the mean relative fluorescence intensity (∆F/F0) of Ca2+ levels in control and in AKT1 expressing cells is shown (control (WT): 0.0081 ± 0.006, n = 25; AKT1 (WT): 0.1316 ± 0.016, n = 32; control (MK801 +MK5): 0.0085 ± 0.004, n = 16; AKT1 (MK801 +MK5): 0.0211 ± 0.007, n = 16). (B) The percentage of microglial cells interacting with AKT1 cells was significantly reduced in larvae treated with MK801 and MK5 compared to untreated larvae. (Control (WT): 18.89 ± 1.32, n = 10; AKT1 (WT): 43.75 ± 3.95, n = 10; Control (MK801 +MK5): 17.59 ± 1.89, n = 21; AKT1 (MK801 +MK5): 24.94 ± 1.36, n = 20). (C) The percentage of microglial cells interacting with AKT1 cells was significantly reduced in larvae treated with CBX compared to untreated larvae (Control (DMSO): 17.11 ± 3.02%, n = 10; AKT1 (DMSO): 41.92 ± 2.09%, n = 10; Control (CBX): 12.42 ± 1.42%, n = 13; AKT1 (CBX): 26.99 ± 2.19%, n = 9).(D) The percentage of microglial cells interacting with AKT1 cells was significantly reduced in p2ry12 crispant larvae compared to WT larvae (Control (WT): 14.82 ± 2.19%, n = 10; AKT1 (WT): 40.01 ± 3.66%, n = 11; Control (ctrl-gRNA): 17.38 ± 3.09%, n = 6; AKT1 (ctrl-gRNA): 44.42 ± 1.46%, n = 7; Control (p2ry12-/-): 12.33 ± 2.97%, n = 7; AKT1 (p2ry12-/-): 20.88 ± 2.29%, n = 10).

Figure 4—source data 1. Quantfications of microglial interactions with control and AKT1+ cells upon interference with Ca2+ - ATP -P2ry12 signalling..
elife-46912-fig4-data1.xlsx (17.2KB, xlsx)
DOI: 10.7554/eLife.46912.016

Figure 4.

Figure 4—figure supplement 1. CRISPR/Cas9-mediated mutation of the p2ry12 gene.

Figure 4—figure supplement 1.

Acute mutation of the p2ry12 gene was mediated through the injection of Cas9 and the p2ry12 guide RNA into one-cell stage embryos. (A) Restriction fragment length polymorphism (RFLP) analysis was carried out on single embryos to confirm the efficiency of the guide RNA in mutating the p2ry12 gene. Injection of a control guide did not mutate the p2ry12 locus (lower picture). (B) Injection of Cas9 and the p2ry12 guide RNA into experimental controls (RFP only) and AKT1-induced samples caused efficient mutation of the p2ry12 gene. (C) The Tg(mpeg1:mCherry; p2ry12:p2ry12-GFP) double transgenic fish was utilized to facilitate in vivo observations of P2ry12 knockout. Macrophages and microglia express mCherry under the mpeg1 promoter while microglia express in addition P2ry12-GFP under the control of the p2ry12 promoter. The injection of Cas9 and the control guide RNA did neither impact on mCherry expression nor on P2ry12-GFP expression. Upon injection of Cas9 and the p2ry12 guide RNA, expression of P2ry12-GFP was effectively abolished. Representative images at 5 dpf are shown. Images were captured using an Andor spinning disk confocal microscope with a 20x/0.75 objective. Scale bars represent 20 µm.