Figure 4

β cells derived from Ngn3⁺ cells preferentially locate and proliferate in small β cell clusters of PDL pancreas. (a) Description of the transgenic mouse model and the labeling strategy for heritable tracing of Ngn3 expressing cells. Ngn3=Ngn3 gene promoter, R26=Rosa26 locus, loxP=loxP sequence, STOP=transcriptional STOP sequence, YFP=yellow fluorescent protein. Tamoxifen injection (TAM, *4 mg/injection) of bigenic Ngn3^CreERT;R26^YFP mice results in Cre-mediated removal of the lox-P flanked STOP sequence, and permanent expression of the YFP reporter gene in cells with an active Ngn3 promoter and their descendants. Eight-week-old male bigenic mice underwent PDL surgery and were TAM-injected (s.c., starting on day of surgery: 4 mg TAM, repeated once every-other-day, five injections in total). (b) YFP-staining in small clusters of Insulin⁺CK19⁺ cells associated with CK19⁺ duct cells of PDL tail. (c) YFP⁺INS⁺ β cells in islets of PDL tail. (d) Quantification of the percentage YFP⁺ β cells in PDL head and tail with the indicated range of Ngn3 mRNA expression level in PDL tail 14 days after ligation (n=3, *P<0.05, **P<0.01 by two-way ANOVA; a total of 21295 β cells evaluated). Ngn3 mRNA was expressed relative to cyclophilin A mRNA and normalized to Ngn3 expression in duodenum. (e) Quantification of the percentage of YFP⁺ β cells in small (1–20 insulin⁺ cells) and large (21–200 insulin⁺ cells) clusters in PDL tail day 14 and 35 (three mice per experimental condition, ***P<0.001 versus large clusters, by two-way ANOVA). (f) Quantification of percentage IdU-labeling (proliferation) in YFP⁺ β cells in small and large clusters (n=3, *P<0.05, versus large clusters). (See also Supplementary Figure S5). Scale bars represent 50 μm