No abstract

Pulsatile increases in cytosolic Ca2+ within individual pancreatic beta cells underlie the stimulation of insulin secretion at elevated glucose concentrations. Recent data, obtained by imaging these transients in intact islets, have revealed the existence of subpopulations of beta cells including leaders which initiate Ca2+ waves across the syncytium and are critical for these dynamics. Whether leader cells possess unique molecular features compared to followers is currently unknown. By combining high speed confocal Ca2+ imaging with fluorescence-activated cell labelling and isolation, we addressed this question in the present study. Ca2+ imaging revealed different wave types, with leader cells identified in 73 % (28 of 38 islets imaged). In contrast, scale free, power-law adherent behaviour, and the existence of highly connected hub cells, was observed in 29 % of islets. To identify possible transcriptomic differences between sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing (Flash-Seq). Differentially expressed transcripts (295; padj<0.05) encoded genes involved in transcriptional regulation, cilium biogenesis and others. These findings demonstrate the existence of discrete transcriptomes between beta cells with definable roles islet physiology and coordination, and suggest that these differences may contribute to the functional specializations of these cells.

Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. The molecular mechanisms through which beta cell hierarchy is established remain poorly understood. The neuronatin (Nnat) gene is imprinted in mice and humans and is required for normal insulin synthesis and secretion. Here, we demonstrate that Nnat is differentially expressed in a discrete beta cell population in a developmental-stage and DNA methylation (DNMT3A)-dependent manner. Explored in mice expressing eGFP from a bacterial artificial chromosome-expressed transgene, NNAT+cells displayed a discrete transcriptome, and appear to represent a beta cell population specialised for insulin production. Correspondingly, highly connected hub cells were less abundant in the NNAT+population. These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established.