Figure 1

Figure 1 Genetic overexpression of p35 leads to beta-cell expansion in juvenile zebrafish.

(A) Schematic showing the baculovirus P35 protein and its mechanism of caspase inhibition. RSL indicates the reactive site loop of P35 (B) Diagrammatic representation showing the genetic construct used to generate the transgenic model of caspase inhibition in beta cells. p35 was cloned under the control zebrafish insulin promoter. mCherry expression under the control of the crystalline (cryaa) promoter serves as a marker of transgenic animals. (C) Representative confocal images (maximum projection) of primary islets from Tg(ins:mCherry) and Tg(ins:p35); Tg(ins:mCherry) at 5, 15, and 30 dpf. Immunostaining against insulin is used to mark the beta cells (in red) while the nuclei are stained with Hoechst (in blue) of 5 dpf WT and Tg(ins:p35) larvae. The beta cells of 15 and 30 dpf WT and Tg(ins:p35) animals are marked using Tg(ins:mCherry) reporter in red. Scale bar, 20 µm. (D) Representative confocal images (single plane) of primary islets from WT and clutch mate Tg(ins:p35) animals at 15, 25, and 30 dpf. Immunostaining against insulin (red) and PCNA (green) marks the beta cells and the proliferating cells, respectively. The nuclei are stained using Hoechst. Scale bar, 20 µm. (E) Quantifications of the number of beta cells in Tg(ins:mCherry) and Tg(ins:p35); Tg(ins:mCherry) animals at 5, 15, 20, 23, 27, and 30 dpf. Each dot represents the number of beta cells per islet. Error bars are mean ± SD. Unpaired two-tailed t-test with Welch’s correction, 5 dpf (ns not significant, p = 0.141307), 15 dpf (ns not significant, p = 0.903301), 20 dpf (****p = 0.000025), 23 dpf (****p = 0.000006), 27 dpf (****p = 0.000001), and 30 dpf (****p = 0.0000000194). n = at least 5 independent samples per group. (F) Quantification showing the percentage of insulin and PCNA double-positive cells per islet in WT and Tg(ins:p35) animals at 15, 25, and 30 dpf. n = at least 3 independent samples per group at each developmental stage. Error bars are mean ± SD. Unpaired two-tailed t-test with Welch’s correction, ns not significant. Source data are available online for this figure.