Fig. 4

Immune cells infiltrate the islet during development and persist throughout adult life.

(a) Confocal images of pancreata from 15, 21 and 45 dpf animals. Beta-cells were labeled using an insulin antibody (grey), leukocytes were labeled using an L-plastin antibody (magenta) and Tg(ptf1a:GFP) marks the acinar cells (green). Immune cells are present within the islet at 45 dpf (arrows). (b) Confocal images of whole islets from Tg(tnfα:GFP) animals at 1 ypf. Islets were labeled using TSQ (Zn²⁺ labeling dye) (blue), leukocytes were labelled with an L-plastin antibody (magenta) and Tg(tnfα:GFP) marks cells expressing tnfα (green). Scale bars 20 µm. (b’) Insets show high-magnification single planes from the confocal stacks (corresponding to the area marked using a white dotted-line in b). Scale bar 10 µm. (c–c’) Confocal image of a one ypf islet showing a single plane. The TgBAC(tnfα:GFP) line marks the tnfα-positive cells (green), whereas Tg(mpeg1:mCherry) marks the macrophages (red). The L-plastin antibody marks all leukocytes (grey) and TSQ (Zn2 +labeling dye) was used to mark the islet (n = 5). Scale bar, 10 µm. (d) Confocal images showing islets at five dpf. The tnfα cDNA was expressed under the insulin promoter. The plasmid was injected in Tg(NF-kB:GFP) embryos at the one-cell-stage and the islets were analyzed at 5 dpf. Beta-cells were labeled with an insulin antibody (red). Tg(NF-kB:GFP) reporter expression is shown in green. (e) The graph shows the percentage of GFP and insulin double-positive cells in un-injected controls (n = 7) and ins:tnfα injected animals (n = 8) at five dpf. Horizontal bars represent mean values (two-tailed t-test, *p<0.05).