Transcriptional control of lung alveolar type 1 cell development and maintenance by NK homeobox 2-1

Abstract

The extraordinarily thin alveolar type 1 (AT1) cell constitutes nearly the entire gas exchange surface and allows passive diffusion of oxygen into the blood stream. Despite such an essential role, the transcriptional network controlling AT1 cells remains unclear. Using cell-specific knockout mouse models, genomic profiling, and 3D imaging, we found that NK homeobox 2-1 (Nkx2-1) is expressed in AT1 cells and is required for the development and maintenance of AT1 cells. Without Nkx2-1, developing AT1 cells lose 3 defining features—molecular markers, expansive morphology, and cellular quiescence—leading to alveolar simplification and lethality. NKX2-1 is also cell-autonomously required for the same 3 defining features in mature AT1 cells. Intriguingly, Nkx2-1 mutant AT1 cells activate gastrointestinal (GI) genes and form dense microvilli-like structures apically. Single-cell RNA-seq supports a linear transformation of Nkx2-1 mutant AT1 cells toward a GI fate. Whole lung ChIP-seq shows NKX2-1 binding to 68% of genes that are down-regulated upon Nkx2-1 deletion, including 93% of known AT1 genes, but near-background binding to up-regulated genes. Our results place NKX2-1 at the top of the AT1 cell transcriptional hierarchy and demonstrate remarkable plasticity of an otherwise terminally differentiated cell type.

Quantitative single-cell interactomes in normal and virus-infected mouse lungs

“…We thus hypothesized that, by combining cells of a cell type readily identifiable in scRNA-seq, we could achieve comparable sensitivity to bulk RNA-seq of the same purified cell type. To test this, we used as a standard our published bulk RNA-seq data of FACS-purified alveolar type 1 (AT1) and alveolar type 2 (AT2) cells (Little et al, 2019) and evaluated scRNA-seq gene dropouts as a function of expression level ( Fig. 1A and S1A).…”

“…The sections were washed again in PBS then mounted with Aqua-Poly/Mount (18606, Polysciences) and imaged on a Nikon A1plus confocal microscope or an Olympus FV1000 confocal microscope. For cell lineage counting, 3 images with airway located in the center were taken from 2 Rosa Sun1GFP/+ ; Shh Cre/+ lungs, in which all epithelial cells were labeled with GFP (Little et al, 2019) to allow costaining for endothelial and immune cells. Endothelial cells (ERG + nuclei) and alveolar epithelial cells (GFP + nuclei) excluding the airways were counted using Fiji's 'Find Maxima' function.…”

“…The dropout rate is the percentage of genes not detected by scRNA-seq for a given range of bulk RNA-seq expression values. ScRNA-seq data are from postnatal day (P) 7 (GSE144769); bulk RNA-seq data are from P5 for AT1 cells and P8 for AT2 cells, as published (Little et al, 2019). (D) ScRNA-seq average gene expression for the 4 EC subpopulations, color/symbol-coded as in (C), as a function of bulk RNA-seq gene expression of total ECs.…”

Redundant and additive functions of the four Lef/Tcf transcription factors in lung epithelial progenitors

“…To identify changes in the epithelium, we used our published fluorescence-activated cell sorting protocol to separate the immune, endothelial, epithelial, and mesenchymal cells using CD45, ICAM2, and ECAD ( Fig. 4A) (22). A total of 2,071 and 4,062 epithelial cells from control and quadruple mutant lungs, respectively, were sequenced; both genotypes consisted of 3 epithelial cell types -SOX9 progenitors, SOX2 progeny, and neuroendocrine cells, the last of which were marked by Ascl1 and were among the first to mature in the lung epithelium (23) (Fig.…”

“…Cell dissociation and FACS was performed as previously described (22). Embryonic mouse lobes were dissected free of extra-pulmonary airways in iced Leibovitz media (Gibco, 21083), minced with forceps, and digested in Leibovitz with 2 mg/mL Collagenase Type I (Worthington, CLS-1, LS004197), 2 mg/mL Elastase (Worthington, ESL, LS002294), 0.5 mg/mL DNase I (Worthington, D, LS002007) for 20 min at 37 °C.…”