Molecular phenotyping of single pancreatic islet leader beta cells by “Flash-Seq”

Abstract

AbstractAimsSpatially-organised increases in cytosolic Ca2+within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including “leaders” which initiate Ca2+waves. Whether leader cells possess unique molecular features, or localisation, is unknown.Main methodsHigh speed confocal Ca2+imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected “hub” cells. To explore transcriptomic differences between beta cell 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”).Key findingsDistinct Ca2+wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29% of islets, though “hub” cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj<0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Functionally validating these findings, cilia number and length tended to be lower in leadervsfollower cells. Leader cells were also located significantly closer to delta cells in Euclidian space than were follower cells.SignificanceThe existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. Specifically, these data raise the possibility of altered signalling from delta cells towards somatostatin receptors present on leader cell cilia.