Chronological analysis with fluorescent timer reveals unique features of newly generated ß-cells

Abstract

Although numerous studies have uncovered the molecular mechanisms regulating pancreas development, it remains to be clarified how ß-cells arise from progenitors and how recently specified ß-cells are different from preexisting ß-cells. To address these questions, we developed a mouse model in which the insulin 1 promoter drives DsRed-E5 Timer fluorescence that shifts its spectrum over time. In transgenic embryos, green fluorescent ß-cells were readily detected by FACS and could be distinguished from mature ß-cells only until postnatal day 0, suggesting that ß-cell neogenesis occurs exclusively during embryogenesis. Transcriptome analysis with green fluorescent cells sorted by FACS demonstrated that newly differentiated ß-cells highly expressed progenitor markers, such as Sox9, Neurog3, and Pax4, showing the progenitor-like features of newborn ß-cells. Flow cytometric analysis of cell cycle dynamics showed that green fluorescent cells were mostly quiescent, and differentiated ß-cells were mitotically active. Thus, the precise temporal resolution of this model enables us to dissect the unique features of newly specified insulin-producing cells, which could enhance our understanding of ß-cell neogenesis for future cell therapy.