Extended Data Figure 8: Ngn3 activation is required for insulin expression in dedifferentiated δ-cells.

a, qPCR for Ngn3 mRNA after β-cell ablation reveals a transitory fivefold upregulation of Ngn3 transcripts 6 weeks after β-cell ablation when β-cell ablation is performed before puberty, but not in adult mice. Controls: n_1-month-old = 3; n_{1.5-month-old} = 3; n_2-month-old = 6; n_{2.5-month-old} = 3; n_{2.5-month-old} = 3; n_3-month-old = 3; n_{3.5-month-old} = 3; n_4-month-old = 3; DT (2-week-old): n_0.5 mpa = 3; n_1 mpa = 3; n_1.5 mpa = 6; n_2 mpa = 3; DT (2-month-old): n_0.5 mpa = 3; n_1 mpa = 3; n_1.5 mpa = 3; n_2 mpa = 3. Each individual sample (mouse) was run in triplicate, in each of three independent reactions. Built-in Welch’s test (P = 0.0112, 0.0178). b, Ngn3 transcriptional activity can be monitored in Ngn3-YFP knock-add-on mice because Ngn3 promoter activity results in YFP expression. In non-ablated age-matched control pups, or in ablated adults, no islet YFP⁺ cells were found (data not shown), yet when β-cells are ablated at 2 weeks of age, 86% of insulin⁺ cells also express YFP⁺ at 1.5 mpa. Control: n = 3, 6,358 insulin⁺-cells scored; DT: n = 3, 675 insulin⁺-cells scored; Welch’s test (P = 0.0010). c, At 1.5 mpa, 81% of YFP⁺ cells co-express insulin, but no glucagon, Sst or PP (data not shown). Two weeks later, YFP⁺ cells are almost absent, reflecting the downregulation of Ngn3 expression reported in a, and suggesting that insulin⁺ cells originate from cells transiently activating Ngn3 expression after ablation. Control: n_1-month-old = 3; n_{1.5-month-old} = 3; n_2-month-old = 3; n_{2.5-month-old} = 3; absent YFP⁺ cells in all control conditions; DT: n_0.5 mpa = 3, 31 YFP⁺ cells; n_1 mpa = 3, 123 YFP⁺ cells; n_1.5 mpa = 3, 729 YFP⁺ cells; n_2 mpa = 3; 47 YFP⁺ cells. Welch’s test and ANOVA (P < 0.0001). d, Irreversible lineage tracing of Ngn3-expressing cells at 1 and 1.5 mpa upon tamoxifen (TAM) administration in Ngn3-CreERT; R26-YFP; RIP-DTR mice; immunofluorescence analyses reveal that in the absence of β-cell ablation, there is no YFP induction (controls). In ablated mice, nearly all insulin⁺ cells are YFP⁺ with time (arrows). At early time-points (1 mpa), YFP⁺/hormone-negative cells are found: these are probably differentiating cells before insulin expression. e, f, In β-cell-ablated Ngn3-CreERT; R26-YFP; RIP-DTR pups, 91% of insulin⁺ cells co-express YFP⁺ (control: n = 3, 3,472 insulin⁺-cells scored, DT: n = 3, 489 insulin⁺-cells scored) (e) and inversely, 93% of the YFP⁺ cells are insulin⁺ (f) (control: n = 3; absent YFP⁺-cells in all control conditions; DT: n = 3, 478 YFP⁺-cells scored). g, Experimental design to block Ngn3 upregulation in β-cell-ablated prepubescent mice by administrating DOX to mice bearing five mutant alleles: Ngn3-tTA^+/+; TRE-Ngn3^+/+; RIP-DTR. In these mice the Ngn3 coding region is replaced by a DOX-sensitive transactivator gene (tTA); the endocrine pancreas develops normally because Ngn3 expression is allowed in the absence of DOX by the binding of tTA to the promoter of the TRE-Ngn3 transgene. Pups were given DT at 2 weeks of age and then DOX 2 weeks later, to block Ngn3 upregulation. They were euthanized when Ngn3 peaks after ablation (2-month-old). h, Islets from non-ablated (no DT) and ablated (DT) mice, exposed (Ngn3 inhibition) or not (normal Ngn3 expression) to DOX treatment from 4 weeks of age. β-Cell regeneration is efficient in absence of DOX (as previously shown), but decreases after Ngn3 blockade, resulting in the appearance of glucagon/insulin bihormonal cells. i, Sharply decreased regeneration by blocking Ngn3 expression in DOX-treated mice reveals the requirement of Ngn3 for efficient β-cell regeneration in pups. DT: n = 266 islets scored, 3 mice; DT+DOX: n = 167, 4 mice. Welch’s test (inter-islet P < 0.0001; inter-animal P = 0.0352), Mann–Whitney (P < 0.0001). j, Glucagon⁺/insulin⁺ bihormonal cells appear in DOX-treated β-cell-ablated pups (Ngn3 inhibition), suggesting a switch to an ‘adult-like’, less efficient, mechanism of regeneration. Control+DOX: n = 3, 9,233 insulin⁺-cells scored; DT: n = 3, 1,385 insulin⁺-cells scored; DT+DOX: n = 4, 141 insulin⁺-cells scored. Welch’s test (P = 0.0081), ANOVA (P < 0.0001). k, Combined double lineage tracing of δ-cells (Tomato⁺) and Ngn3-expressing cells (YFP⁺) shows by immunofluorescence that nearly all insulin⁺ cells express both reporters, but no Sst (arrows). Sst⁺ cells (arrowheads) are YFP- and insulin-negative. Scale bars, 20 µm. Error bars show s.d.