Fig. 3

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