(A) Genetic lineage tracing system, used in conjunction with the ablation system, to determine the cellular origin of new β cells. The experimental protocol for the ablation-lineage tracing experiment is shown below. Tam, tamoxifen. (B) Experiment design and possible interpretations of lineage tracing results. For simplicity, the β cell recombination rate (labeling cells by HPAP expression) is presented as 100% and the rate of new β cell accumulation as 25%. (C) Representative confocal image of an islet from a 2-month-old Insulin-rtTA;TET-DTA;Insulin-CreER^TM;Z/AP transgenic mouse exposed to doxycycline to ablate β cells, injected with tamoxifen to label surviving β cells, and treated with BrdU for 2 weeks to label new cells as shown in A. Arrows denote HPAP⁺BrdU⁺ β cells, the progeny of surviving and proliferating β cells; arrowheads mark HPAP^–BrdU⁺ β cells, derived from either non-β cells or nonlabeled β cells. Because the Insulin-rtTA and Insulin-CreER^TM driver strains are distinct transgenes, their efficiency of β cell killing and recombination, respectively, was not expected to be identical. (D) Quantification of the fraction of labeled (HPAP⁺) β cells compared with the fraction of labeled cells among newly born (BrdU⁺) β cells in 5 mice (n denotes number of β cells counted per mouse). The similar percentages of labeled cells indicates that new β cells are derived primarily by proliferation of surviving β cells.