Figure 6

Beta-cell death regulates the islet’s immune component and inflammation.

(A) Representative confocal images (maximum projection) of primary islets from 6 months old WT and Tg(ins:p35) animals in the transgenic background of Tg(mpeg:mCherry);Tg(tnfα:GFP). Immunostaining against insulin is used to label the beta cells (gray). Tg(mpeg:mCherry) reporter marks the macrophages (red), Tg(tnfα:GFP) marks the tnf-α expressing cells (green) while the nuclei are stained with Hoechst (blue). Scale bar 20 µm. (A’) Insets show high-magnification single planes from the confocal projection of the WT animal (corresponding to the area marked using a yellow dotted line in A). Scale bar, 20 µm. A subset of macrophages express tnf-α (yellow arrow) while others do not (white arrow). (B) Quantification showing the number of mCherry-positive cells per unit islet area in WT and Tg(ins:p35). Error bars are mean ± SD from n = 5 independent samples per group. The horizontal bar represents the mean value. Unpaired two-tailed t-test with Welch’s correction, ***P = 0.000494. (C) Quantification showing the percentage of mCherry and tnfα double-positive cells in the islet of WT and Tg(ins:p35) animals. Error bars are mean ± SD from n = 5 independent samples per group. The horizontal bar represents the mean value. Unpaired two-tailed t-test with Welch’s correction, **P = 0.003271. (D) Beta cells from 6 months old Tg(ins:mCherry);Tg(NF-kB:GFP) and Tg(ins:p35);Tg(ins:mCherry);Tg(NF-kB:GFP) animals were analysed using FACS. The data shows the results from the analysis of islets from n = 5 combined fish per group with two biological replicates. (E) The FlowJo graph shows GFP intensity (along the X-axis) and the distribution of beta cells from WT and Tg(ins:p35) animals, respectively. Horizontal lines indicate the division point between GFP^low and GFP^high levels. Percentage values represent the proportion of cells with GFP^low or GFP^high expression. Source data are available online for this figure.