Integrated Single-Cell RNA-Sequencing Analysis of Aquaporin 5-Expressing Mouse Lung Epithelial Cells Identifies GPRC5A as a Novel Validated Type I Cell Surface Marker

Abstract

Molecular and functional characterization of alveolar epithelial type I (AT1) cells has been challenging due to difficulty in isolating sufficient numbers of viable cells. Here we performed single-cell RNA-sequencing (scRNA-seq) of tdTomato⁺ cells from lungs of AT1 cell-specific Aqp5-Cre-IRES-DsRed (ACID);R26tdTomato reporter mice. Following enzymatic digestion, CD31^-CD45^-E-cadherin⁺tdTomato⁺ cells were subjected to fluorescence-activated cell sorting (FACS) followed by scRNA-seq. Cell identity was confirmed by immunofluorescence using cell type-specific antibodies. After quality control, 92 cells were analyzed. Most cells expressed 'conventional' AT1 cell markers (<i>Aqp5</i>, <i>Pdpn</i>, <i>Hopx</i>, <i>Ager</i>), with heterogeneous expression within this population. The remaining cells expressed AT2, club, basal or ciliated cell markers. Integration with public datasets identified three robust AT1 cell- and lung-enriched genes, <i>Ager</i>, <i>Rtkn2</i> and <i>Gprc5a</i>, that were conserved across species. GPRC5A co-localized with HOPX and was not expressed in AT2 or airway cells in mouse, rat and human lung. GPRC5A co-localized with AQP5 but not pro-SPC or CC10 in mouse lung epithelial cell cytospins. We enriched mouse AT1 cells to perform molecular phenotyping using scRNA-seq. Further characterization of putative AT1 cell-enriched genes revealed GPRC5A as a conserved AT1 cell surface marker that may be useful for AT1 cell isolation.