Fig. 3

Beta-cells in the primary islet become functional during early development. a Glucose responsiveness of larval beta-cells at 4?dpf. Top?islets from Tg(ins:GCaMP6s); Tg(ins:mKO2-nls) animals were mounted ex vivo. Beta-cells (red nuclei) were stimulated with a glucose ramp consisting of sequential incubation with 5 (basal), 10, and 20?mM d-glucose, and depolarized via addition of 30?mM KCl. A representative beta-cell is marked with an arrowhead. Bottom?trace of normalized GCaMP6s-fluorescence intensity over time for the beta-cell indicated in the top panels. In response to glucose stimulation, the cell showed strong fluorescence; indicating glucose-stimulated calcium influx. The fluorescence reached its peak after addition of KCl. 60?±?3% of the recorded beta-cells exhibited glucose-induced calcium influx upon sequential incubation with 10 and 20?mM glucose (n?=?36 cells in five islets) but not at basal concentrations. b Whole-mount, double in situ hybridization for insulin (brown) and ucn3 (purple). At 24?hpf, ucn3 transcripts were not detectable in the embryonic islet (arrow) (enlarged inset). At 72?hpf (3?dpf), beta-cells exhibit double positivity for insulin and ucn3 (arrow) (enlarged inset). Arrowheads point to ucn3-expressing neurons at 72?hpf, as shown previously³⁸. See also Supplementary Fig. 18 for single ucn3 in situ hybridization at 3 and 5?dpf. c The ins promoter drives Cre-ER^T2 expression in beta-cells. CRE-mediated recombination excises the floxed mCherry in Tg(ins:Red-Stop-Green). Beta-cells that undergo successful recombination activate insulin:H2B-GFP-expression. To label DBCs specifically, 4-OHT was applied from 24 to 30?hpf. At 72?hpf, a majority of the traced beta-cells (H2B-GFP-positive) showed Ucn3-immunofluorescence (confocal projection) (n?=?5 islets). Scale bars in a, c, 10?µm; 1?mm in b