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. Author manuscript; available in PMC: 2014 Oct 22.
Published in final edited form as: Curr Biol. 2013 Jun 20;23(13):1242–1250. doi: 10.1016/j.cub.2013.05.037

Figure 2. Nutrients regulate β-cell differentiation.

Figure 2

(A) Tg(TP1:CreERT2);Tg(ubi3C:loxp-GFP-loxp:mCherry) larvae were treated with 4-OHT at 14dpf for 16 h, fixed at 17dpf, and stained for 2F11 to mark IPD cells. 4-OHT treatment resulted in the mosaic labeling of individual IPD cells (mCherry+;GFP- cells) (arrowheads). (B) Experimental setup for the lineage tracing of β-cells from IPD cells. Tg(insulin:loxP:mCherry-STOP:loxP:H2BGFP)+ β-cells that originated from IPD cells with Tg(TP1:CreERT2) activity exhibit H2BGFP expression instead of mCherry. In a single progenitor scenario (1), each SI would be composed of H2BGFP+ or mCherry+ cells, whereas in a multiple progenitor scenario (2), SIs would be mosaic, containing both H2BGFP+ and mCherry+ cells. (C-F) Tg(TP1:CreERT2);Tg(insulin:loxP:mCherry-STOP:loxP:H2BGFP) larvae treated with limiting concentrations of 4-OHT at 16dpf for 16h and analyzed at 40dpf. (C) A mosaic SI composed of H2BGFP+ (arrowheads) and mCherry+ β-cells. (D) Three individual H2BGFP+ β-cells (arrowheads) located in the periphery of a SI suggesting that they have originated from three different IPD cells. (E) A PI showing two peripheral H2BGFP+ groups of β-cells (arrowheads), indicating several cell cycles after differentiation. (F) A single H2BGFP+ β-cell (arrowhead) in proximity to an SI. 15 of 20 animals treated with 4-OHT contained lineage traced cells, whereas no lineage traced cells were observed in vehicle-treated controls (n=7 animals, 80 SIs and 7 PIs). (G) Tg(TP1:H2BmCherry);Tg(ins:GFP) animals were examined at 30dpf. A projection of several planes shows an SI (arrow). The white arrowhead points to a single β-cell in the periphery of the SI. The yellow arrowhead points to a single β-cell outside the SI, extending a long cellular process towards it. Both of these β-cells exhibit higher levels of Tg(TP1:H2BmCherry) fluorescence compared to the rest of the β-cells in the SI, suggesting that they recently differentiated and did not undergo proliferation. (H-I) Tg(ins:Kaede) animals were maintained on a low-calorie diet until 20dpf (H) or switched to a high-calorie diet (I) from 15 to 20dpf (see Experimental Procedures). Whereas there is only a single Tg(ins:Kaede)+ SI (arrowhead) posterior to the PI (arrow) in H, multiple SIs (arrowheads) have formed in I. (J) Quantification of the number of SIs posterior to the PI (n=17 animals for each group). The high-calorie diet induced the formation of more SIs compared to the low-calorie diet (****p<0.0001). Error bars = s.e.m. Scale bars, 20 μm. See also Figure S2.