Fig. 4

Decrease in the number of beta cell fluorescent nuclei and quantitative analysis of bystander effect in Tg(-1.2ins:TKGFP) larvae treated with Ganciclovir. (A) Tg(-1.2ins:TKGFP) larvae have a decreased number of beta cells when treated with GCV (right, gray column) compared to untreated controls (left, white column); a comparable amount of larvae (+ GCV n = 20; controls, n = 18) was analyzed in repeated experiments. (B, C) Projection of a confocal stack image in a representative untreated (B) and GCV treated (C) larva after 7 days of prodrug administration. Scale bar: 10 μm. (D, E) Beta cell specific apoptosis after ganciclovir treatment. Representative double immunostaining of pancreatic tissue with cleaved-caspase 3 (red) and insulin (green) antisera. The apoptotic beta cell is marked by an arrowhead. (F, G) Comparison of an untreated (D), and a treated group of 7 days larvae (E) for their glucagon expression as revealed by in situ hybridization; glucagon mRNA levels are not affected by the GCV treatment indicating an absence of bystander effect. (H, I) Representative confocal projections of GCV-untreated (E) or GCV-treated larvae (F) displaying a comparable number of fluorescent alpha cells detected with an anti-glucagon antiserum (in red). The nuclei of beta cells expressing TKGFP (in green) are decreased in number in GCV-treated larvae. (J) Table comparing the number of alpha and delta cells in the pancreas of 5dpf Tg(-1.2ins:TKGFP) embryos treated with the prodrug and showing non significant differences with controls. (K, L) Representative pictures of embryos untreated (K) or treated (L) with ganciclovir and stained for nuclei (blue, DAPI) or somatostatin and glucagon (red).