Label-retaining β-cells are derived from the dorsal pancreatic bud. Embryos were injected at the one-cell stage with H2B-RFP mRNA (A, A′, and G) or H2B-RFP mRNA and prkci MO (B, B′, C, and D). (A and B) Confocal projections of 52 hpf Tg(ptf1a:GFP)jh1 pancreata and (A2 and B′) confocal sections from each projection. (C and D) Confocal sections through the principal islet at 52 hpf. (E) Confocal projection of an islet coexpressing Tg(-4.0ins:GFP)zf5 and Tg(insulin:dsRed)m1018 at 46 hpf. (F) Quantification of Tg(ins:dsRed)m1018 expressing, and total β-cells per embryo from 24 to 46 hpf, expressed as mean ± SEM (See Table S1). (G) Confocal section through a Tg(-4.0ins:GFP)zf5 positive islet. (H) Quantification of H2B-RFP label-retaining, and total β-cells per embryo from 52 hpf to 12 dpf, expressed as mean ± SEM (see Table S1). (A and A′) β-cells that do not retain the H2B:RFP label appear by 52 hpf. (B and B2) Insulin expression is restricted to H2B-RFP positive cells in prkci morphants. (B) None of the label-retaining β-cells colocalized with Tg(ptf1a:GFP)jh1 expression, indicating that these cells derive from the dorsal bud. Some label-retaining cells do not express Insulin (arrow) and are likely other endocrine cell types. (C and D) All Islet-1 positive endocrine cells retained the H2B-RFP label when bud fusion was inhibited. (E) Recently differentiated β-cells only express GFP (green, arrow). (F) β-cells that differentiated by 28 hpf (green bars) coexpress dsRed at 46 hpf (yellow bars). (G) At 120 hpf, some β-cells (yellow, arrowhead) retain the H2B-RFP label while others (green, arrow) do not. (H) Yellow bars: The number of H2B-RFP label-retaining β-cells does not change from 52 hpf to 12 dpf. Green bars: The total number of β-cells increases over this period.
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