A single-cell atlas of mouse lung development

Abstract

Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages – wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.

A CXCL12 morphogen gradient uncovers lung endothelial heterogeneity and promotes distal vascular growth

“…Subpopulations aCap and gCap have been recently described as capillary 1 cells (CAP1), proliferating CAP1 cells, and capillary 2 cells (CAP2) (Sun et al, 2022). In concordance with previous data, in our study both gCap and aCap populations arose after E15.5 and are apparent by E18.5 (Gillich et al, 2020; Negretti et al, 2021; Vila Ellis et al, 2020; Zepp et al, 2021). We validated the transcriptional profile and localized these populations at the tissue-level with RNA ISH for Cxcl12, Apln , and Gpihbp1 transcripts in combination with DsRed expression by IHC (Fig.…”

“…4C and 4F). Since Cxcl12 -DsRed + endothelium was void of venous identity in the lung, we reanalyzed previously published single-cell sequencing data on endothelium from the developing mouse lung for the CXCL12 signaling axis distribution (Negretti et al, 2021). While Cxcr4 remained localized in arterial and capillary endothelium, Ackr3 is expressed not only in the capillary endothelium but also robustly in venous endothelium (Fig.…”

“…CXCL12 is also expressed at lower levels in the vascular smooth muscle and adventitial fibroblasts. While this study did not assess endothelial or mesenchymal-specific deletion of CXCl12, the majority of CXCL12 is expressed in ECs (Negretti et al, 2021; Zepp et al, 2021). In addition, expression of the receptors for CXCL12 indicates an endothelial-dependent signaling mechanism for vascular assembly and growth.…”

“…For the Cxcl12 knockout dataset, but not the dataset of Cxcl12+ cells only, ambient background RNA were cleaned from the scRNA-seq data with "SoupX" as described previously (Schuler et al, 2021;Young and Behjati, 2020). The following genes were used to estimate the non-expressing cells, calculate the contamination fraction, and adjust the gene expression counts: Dcn, Bgn, Aspn,Ecm2,Fos,Lyz1,Lyz2,Mgp,Postn,Scgb1a1.…”

“…Growing evidence indicates that the endothelium of the pulmonary circulation is heterogeneous. Studies using single-cell RNA sequencing analysis on the developing and adult lung have uncovered distinct endothelial subpopulations comprising the arteries, veins, capillaries, and lymphatics (Asosingh et al, 2021; Gillich et al, 2020; Guo et al, 2019; He et al, 2018; Negretti et al, 2021; Niethamer et al, 2020; Rodor et al, 2021; Saygin et al, 2020; Schupp et al, 2021; Travaglini et al, 2020; Vila Ellis et al, 2020; Zepp et al, 2021). In addition, insights from these studies suggest functional specialization of endothelium.…”