Single Molecule-based FliFISH Validates Radial and Heterogeneous Gene Expression Patterns in Pancreatic Islet β Cells
figureposted on 2021-03-08, 20:18 authored by Fangjia Li, Dehong Hu, Cailin Dieter, Charles Ansong, Lori Sussel, Galya Orr
Single cell RNA sequencing (scRNA-Seq) technologies have greatly enhanced our understanding of islet cell transcriptomes and have revealed the existence of β cell heterogeneity. However, comparison of scRNA-Seq datasets from different groups have highlighted inconsistencies in gene expression patterns, primarily due to variable detection of lower abundance transcripts. Furthermore, such analyses are unable to uncover the spatial organization of heterogeneous gene expression. Here we used fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) to quantify transcripts in single cells in mouse pancreatic islet sections. We compared the expression patterns of Insulin 2 (Ins2) with Mafa and Ucn3 – two genes expressed in β cells as they mature, as well as Rgs4 – a factor with variably reported expression in the islet. This approach accurately quantified transcripts across a wide range of expression levels - from single copies to over hundred copies per cell in one islet. Importantly, fliFISH allowed evaluation of transcript heterogeneity in the spatial context of an intact islet. These studies confirm the existence of a high degree of heterogeneous gene expression levels within the islet and highlight relative and radial expression patterns that likely reflect distinct β cell maturation states along the radial axis of the islet.