Exploring the Perilipin2 cell lineage during murine lung alveolarization

Abstract

Disrupted alveolar morphology characterizes various pulmonary structural diseases, such as bronchopulmonary dysplasia, chronic obstructive pulmonary disease and lung fibrosis. Understanding the process of lung alveolarization might help to develop new therapeutic strategies for pulmonary structural diseases. Cells expressing the protein Perilipin2 (Plin2); synonym: Adipocyte differentiation-related protein (ADRP, synonym), known as lipofibroblasts have been demonstrated to participate in lung alveolarization. Aim of the present study was 1) to analyze function of cells of the Plin2 cell lineage (Plin2lin) during alveolarization using a cell depletion approach in transgenic mice and (2) to characterize molecular signatures of Plin2lin cells during alveolarization using a single cell sequencing approach of murine lineage traced cells of the Plin2lin cell lineage. Using a cre-recombinase (cre) stop loxP Diphtheria toxin A (DTA) system in mice, the current work demonstrated that tamoxifen administration and following cell specific cre recombination in order to ablate Plin2-expressing cells during alveolarization caused to a strong liver failure. Ablation of Plin2lin cells in the lung could not be detected. In contrast, using a cre driven cell specific activation of the reporter gene green fluorescent protein (GFP), Plin2lin cells in the lung could be labelled successfully. To elucidate molecular signatures and contribution of Plin2lin cells to mouse lung cell populations during alveolarization, single-cell RNA sequencing (scRNAseq) of lineage-traced cells was achieved. ScRNAseq was performed on about 8791 and 9095 Plin2lin cells from postnatal day (P) 1 at P7 and P14. Seven major clusters from the Plin2lin were identified. Strikingly, these clusters of the Plin2lin were annotated as epithelial, mesenchymal and vascular cell populations. High-quality imaging on 150 μm precision-cut lung slices from P7 and P14 lungs confirmed cell type markers of annotated cell types. Moreover, two subpopulations of lipofibroblasts were identified: Transcription factor 21 (Tcf21) -low expressing and Tcf21-high expressing mesenchymal lipofibroblasts. Comparison of differentially expressed genes of the two different identified lipofibroblast subpopulations revealed possible target candidate genes, which might contribute to better targeting and better understanding of lipofibroblast subtypes during alveolarization. Generated data serve as an important basis for further studies to gain insights into possible functions of lipofibroblast populations in alveolarization.