Figure 6

Ngn3⁺ cells are required while Ngn3 in pre-existing β cells is dispensable for the β cell proliferation in PDL pancreas. (a) Description of the transgenic mouse model and strategy used for selective ablation of Ngn3⁺ cells by DT. Tamoxifen injection in bigenic Ngn3Cre^ERT−/+;R26^iDTR+/+ mice results in permanent heritable expression of the DTR gene in Ngn3⁺ cells and cells derived thereof. Eight-week-old bigenic mice and monogenic Ngn3Cre^ERT−/−;R26^iDTR+/+ littermates underwent PDL surgery, s.c. TAM administration (*TAM, 4 mg/injection), and i.v. DT (# daily injections, 125 ng/injection). (b) Ngn3 mRNA expression measured by qRT-PCR in duodenum, and PDL head and tail pancreas of mono- (n=3) or bigenic mice (n=4; *P<0.05 versus the monogenic tissue). Data were normalized to cyclophilin A mRNA and expressed relative to the Ngn3 level in monogenic duodenum. (c) Total insulin content (μg) of PDL head and tail pancreas of mono- or bigenic mice (n=3, **P<0.01). (d) Percentage of Ki67⁺ β cells in PDL head and tail pancreas of mono- (n=3) or bigenic (n=4) mice (**P<0.02). (e) Percentage of Ki67⁺ β cells in small (1–20) or large (21–200) cell clusters of PDL tail (n=3–4, *P<0.05, ns: P>0.05 by two-way ANOVA). Data are expressed as the mean±S.E.M. See also Supplementary Figure S8. (f) Following TAM injection and wash out, RIP^CreERT;Ngn3^+/+ and RIP^CreERT;Ngn3^lox/lox mice underwent PDL surgery. Immunofluorescence staining for Ki67 and insulin in PDL tail tissues is shown. β cell proliferation was quantified and expressed as the mean±S.E.M. (n=6, ns: P>0.05; a total of 30250 β cells were evaluated). Scale bar represents 50 μm. (g) RIP^CreERT;R26^YFP;Ngn3^+/lox mice were treated with TAM as in (f). Islets were isolated and dissociated to sort the islet cells on the basis of TSQ (Dapi, not shown) and YFP fluorescence (FITC). The window (left panel) comprises all the TSQ^high (endocrine) cells. The gating indicates YFP⁺ β cells (green dots). YFP⁻ islet cells (red dots) were also collected. YFP⁻ islet cells had a wild-type (Ngn3⁺) and a floxed (Ngn3^lox) allele, whereas YFP⁺ β cells had lost the Ngn3^lox allele. PCR results for two separate isolations are shown (right panel)