Figure 2
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Figure 2
(A) Whole mount immunodetection in six dpf Tg(sst1.1:GFP); Tg(ins:NTR-P2A-mCherry) larvae showing β-cells (mCherry, red), sst1.1-expressing cells (GFP, green) and double positive bihormonal cells (asterisks) in the main islet in control (CTL) and 3 days after NFP-mediated ablation (3 dpt). Representative confocal images (single optical planes). dpf: days post-fertilization. (B) Quantification of bihormonal cells co-labeled by mCherry and GFP based on confocal images of 6 dpf larvae. Unpaired two-tailed t-test (with Welch correction); ***p < 0.001; Mean ± SD. (C) Whole mount fluorescent in situ hybridization performed on 6 dpf Tg(ins:NTR-P2A-mCherry) larvae with an ins antisense RNA probe (green) combined with either a sst1.1 or a sst2 probe (red). NFP-mediated ablation was performed from 3 to 4 dpf. Representative confocal images of the main islet (single optical planes). (D–G) β-cell tracing with Tg(ins:CRE-ERT2); Tg(ubb:loxP-CFP-loxP-zsYellow); Tg(sst1.1:GFP); Tg(ins:NTR-P2A-mCherry) larvae. (D) Experimental design: CRE recombination was performed by treatment with 4-OHT treatment at six dpf to induce the expression of the lineage tracer zsYellow (gray) in β-cells (INS, red). β-cell ablation (NFP) was then performed at seven dpf and the lineage tracer was analysed in the main islet at 14 dpf (7 dpt). (E-E’) Confocal images showing immunodetection of GFP (green), zsYellow (gray), and INS (red) antibodies. After ablation, traced β-cells are evidenced by double zsYellow+ Ins + staining (gray arrowheads) and bihormonal cells by double Ins + GFP + staining (white asterisks). (E’) Close-up showing two bihormonal cells, one zsYellow+ (derived from a pre-existing β-cell) (yellow arrowhead) and one zsYellow- (asterisk). (F–H) Quantification (CTL, n = 6; NFP, n = 8) based on the confocal images. (F) In CTL non-ablated islets, ZsYellow marked efficiently the Ins+ β-cells (84 ± 19 zsYellow+ Ins + cells out of 89 ± 20 total Ins+ β-cells, representing 94% of the total β-cells). ZsYellow was not detected in sst1.1:GFP+ cells, showing a good specificity. (G) 7 days after ablation (NFP), 47.3 ± 8 Ins + cells were detected and 5.8 ± 4 of them (12%) expressed zsYellow. (H) 42 ± 7.5 Ins + cells are also GFP+ bihormonal and 10% of them (4 ± 3 cells) are labeled with zsYellow. Mean ± SD.
Bihormonal cell formation is age- and ablation model-independent and mostly do not derive from escaping β-cells.