Fig. 3.
β-Cell expression of Kir6.2DN induces β-cell differentiation. A: schematic representation of the Tg(−1.2ins:Kir6.2DN-GFPTE-ON; LR) transgene used to express a dominant-negative murine Kir6.2, a KATP channel subunit, in β-cells under the regulation of the dually inducible tetracycline and ecdysone-dependent transcription factor (TE-ON) system. Transgenic fish were marked with an α-crystalline-driven tagRFP. B: RT-PCR of nontransgenic larvae and Tg(−1.2ins:Kir6.2DN-GFPTE-ON; LR) larvae either without or with doxycycline (Dox) and tebufenozide (Tbf) induction. Expression of Kir6.2DN was only observed after induction. C: total free glucose level of 6 dpf Tg(−1.2ins:H2BmCherry) (Kir6.2DN−) and Tg(−1.2ins:Kir6.2DN-GFPTE-ON; LR); Tg(−1.2ins:H2BmCherry) (Kir6.2DN+) larvae treated with or without doxycycline and tebufenozide. Significantly decreased free glucose levels were observed in Dox + Tbf-induced Tg(−1.2ins:Kir6.2DN-GFPTE-ON; LR); Tg(−1.2ins:H2BmCherry) larvae. D: β-cell number in 6 dpf Tg(−1.2ins:Kir6.2DN-GFPTE-ON; LR);Tg(−1.2ins:H2BmCherry) larvae increased significantly in animals induced to express Kir6.2DN in β-cells in the absence of feeding; however, β-cell number in these induced double-transgenic larvae was not significantly different from sibling controls following overnutrition either using 20 mM glucose or 5% egg yolk. All values are means ± SE; n are shown inside of the bars. Groups labeled with different letters are significantly different from each other (P < 0.05). E: transgenic larvae were incubated and labeled with EdU-containing medium for 24 h starting at 5 dpf. EdU-positive cells are revealed with green fluorescence, and β-cells are marked by red fluorescence by nuclear mCherry. Double-transgenic Tg(−1.2ins: Kir6.2DN-GFPTE-ON; LR); Tg(−1.2ins:H2BmCherry) larvae (b) and double-transgenic Tg(−1.2ins: GCKV91L; LR);Tg(−1.2ins:H2BmCherry) (c) larvae at 6 dpf showed little evidence of overlapping colabeling. The images are confocal projections, and scale bars indicate 20 μm.