The identification of cell type defining genes across human tissues and the functional study of the endothelial adhesion G protein-coupled receptor L4

Abstract

Cell type specific gene expression profiles underlie differences in cell morphology, behaviour, and specialized function. Single cell RNA sequencing can be used to measure gene expression in individual cells, but challenges remain including limited read-depth, artefactual changes due to dissociation from tissue, difficulties in the analysis of fragile or morphologically complex cell types, and bias introduced from the analysis of a limited number of biological replicates. In paper I, we used an integrative correlation analysis to define cell type enriched transcripts from bulk RNAseq, generated from visceral and subcutaneous adipose tissue. We identified depot and sex-specific differences. In Paper II, we expanded our analysis to include cell types in 15 human tissue types, to create a cell type enrichment prediction atlas for all protein coding genes. A cross-tissue comparison identified shared enrichment signatures between cell types in different tissues. We also defined core identity profiles of cell types present in all or most tissue types, including endothelial cells (EC), which can vary in gene enrichment profiles across different vascular beds. The focus of paper III was the functional characterisation of one such highly EC enriched gene, adhesion G protein-coupled rector L4 (ADGRL4). EC have a major role in various biological processes, including the regulation of inflammatory responses and haemostasis. The endothelial restricted expression of ADGRL4 is indicative of an important cell type specific role in EC. We depleted ADGRL4 in EC and measured associated changes in proteome and function, under normal and cytokine stimulated conditions. Under inflammatory conditions, ADGRL4 depletion potentiated EC pro-coagulant protein expression and associated thrombin and fibrin formation. Concurrently, ADGRL4 depletion inhibited the expression of inflammation-induced interferon response genes. This indicates that ADGRL4 has a currently unappreciated role in the EC function, with a potential role in the regulation of coagulation during inflammation.