Cell-type-specific and disease-associated expression quantitative trait loci in the human lung.

Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis. Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA sequencing of lung tissue from 66 individuals with pulmonary fibrosis and 48 unaffected donors. Using a pseudobulk approach, we mapped expression quantitative trait loci (eQTLs) across 38 cell types, observing both shared and cell-type-specific regulatory effects. Furthermore, we identified disease interaction eQTLs and demonstrated that this class of associations is more likely to be cell-type-specific and linked to cellular dysregulation in pulmonary fibrosis. Finally, we connected lung disease risk variants to their regulatory targets in disease-relevant cell types. These results indicate that cellular context determines the impact of genetic variation on gene expression and implicates context-specific eQTLs as key regulators of lung homeostasis and disease.

An integrated cell atlas of the lung in health and disease.

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1+ profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

Cell type-specific and disease-associated eQTL in the human lung.

Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis (PF). Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA-sequencing of lung tissue from 67 PF and 49 unaffected donors. Employing a pseudo-bulk approach, we mapped expression quantitative trait loci (eQTL) across 38 cell types, observing both shared and cell type-specific regulatory effects. Further, we identified disease-interaction eQTL and demonstrated that this class of associations is more likely to be cell-type specific and linked to cellular dysregulation in PF. Finally, we connected PF risk variants to their regulatory targets in disease-relevant cell types. These results indicate that cellular context determines the impact of genetic variation on gene expression, and implicates context-specific eQTL as key regulators of lung homeostasis and disease.

Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyze the transcriptomes of 611,398 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observe a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD AT2 cells express higher levels of genes linked directly to the efficiency of viral replication and the innate immune response. Additionally, we identify basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyzed the transcriptomes of 605,904 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observed a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD epithelial cells expressed higher levels of genes linked directly to the efficiency of viral replication and innate immune response. Additionally, we identified basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis.

Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a KRT5- /KRT17 + pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis.

Ager-CreERT2: A New Genetic Tool for Studying Lung Alveolar Development, Homeostasis, and Repair.

The alveolar region of the lung is composed of two major epithelial cell types: cuboidal alveolar type 2 cells (AT2 cells), which produce surfactant proteins, and large, thin, alveolar type 1 cells (AT1 cells), specialized for efficient gas exchange. AT1 cells cover more than 95% of the alveolar surface and constitute a major barrier to the entry of pathogenic agents. Relatively few genetic tools are available for studying the development of AT1 cells, the function of genes expressed in them, and the effect of specifically killing them in vivo in the adult lung. One distinguishing feature of AT1 cells is the high level of expression of the gene Ager, encoding the advanced glycation endproduct-specific receptor, a member of the immunoglobulin superfamily of cell surface receptors. In this paper, we report the generation of a novel Ager-CreERT2 allele in which Cre recombinase is inserted into the first coding exon of the endogenous gene. After treatment with tamoxifen the allele enables Ager+ progenitor cells to be efficiently lineage labeled during late embryonic development and AT1 cells to be killed in the adult lung using a Rosa26-diphtheria toxin A allele. Significantly, adult mice in which approximately 50% of the AT1 cells are killed survive the loss; repair is associated with increased proliferation of SFTPC+ (surfactant protein C-positive) AT2 cells and the upregulation of Ager expression. The Ager-CreERT2 allele thus expands the repertoire of genetic tools for studying AT1 turnover, physiology, and repair.

Niche-mediated BMP/SMAD signaling regulates lung alveolar stem cell proliferation and differentiation.

The bone morphogenetic protein (BMP) signaling pathway, including antagonists, functions in lung development and regeneration of tracheal epithelium from basal stem cells. Here, we explore its role in the alveolar region, where type 2 epithelial cells (AT2s) and Pdgfrα+ type 2-associated stromal cells (TASCs) are components of the stem cell niche. We use organoids and in vivo alveolar regrowth after pneumonectomy (PNX) - a process that requires proliferation of AT2s and differentiation into type 1 cells (AT1s). BMP signaling is active in AT2s and TASCs, transiently declines post-PNX in association with upregulation of antagonists, and is restored during differentiation of AT2s to AT1s. In organoids, BMP4 inhibits AT2 proliferation, whereas antagonists (follistatin, noggin) promote AT2 self-renewal at the expense of differentiation. Gain- and loss-of-function genetic manipulation reveals that reduced BMP signaling in AT2s after PNX allows self-renewal but reduces differentiation; conversely, increased BMP signaling promotes AT1 formation. Constitutive BMP signaling in Pdgfrα+ cells reduces their AT2 support function, both after PNX and in organoid culture. Our data reveal multiple cell-type-specific roles for BMP signaling during alveolar regeneration.

Lung organoids: current uses and future promise.

Lungs are composed of a system of highly branched tubes that bring air into the alveoli, where gas exchange takes place. The proximal and distal regions of the lung contain epithelial cells specialized for different functions: basal, secretory and ciliated cells in the conducting airways and type II and type I cells lining the alveoli. Basal, secretory and type II cells can be grown in three-dimensional culture, with or without supporting stromal cells, and under these conditions they give rise to self-organizing structures known as organoids. This Review summarizes the different methods for generating organoids from cells isolated from human and mouse lungs, and compares their final structure and cellular composition with that of the airways or alveoli of the adult lung. We also discuss the potential and limitations of organoids for addressing outstanding questions in lung biology and for developing new drugs for disorders such as cystic fibrosis and asthma.

Identifying direct targets of transcription factor Rfx2 that coordinate ciliogenesis and cell movement.

Recently, using the frog Xenopus laevis as a model system, we showed that transcription factor Rfx2 coordinates many genes involved in ciliogenesis and cell movement in multiciliated cells (Chung et al., 2014). To our knowledge, it was the first paper to utilize the genomic resources, including genome sequences and interim gene annotations, from the ongoing Xenopus laevis genome project. For researchers who are interested in the application of genomics and systems biology approaches in Xenopus studies, here we provide additional details about our dataset (NCBI GEO accession number GSE50593) and describe how we analyzed RNA-seq and ChIP-seq data to identify direct targets of Rfx2.

Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT).

The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development.

Coordinated genomic control of ciliogenesis and cell movement by RFX2.

The mechanisms linking systems-level programs of gene expression to discrete cell biological processes in vivo remain poorly understood. In this study, we have defined such a program for multi-ciliated epithelial cells (MCCs), a cell type critical for proper development and homeostasis of the airway, brain and reproductive tracts. Starting from genomic analysis of the cilia-associated transcription factor Rfx2, we used bioinformatics and in vivo cell biological approaches to gain insights into the molecular basis of cilia assembly and function. Moreover, we discovered a previously un-recognized role for an Rfx factor in cell movement, finding that Rfx2 cell-autonomously controls apical surface expansion in nascent MCCs. Thus, Rfx2 coordinates multiple, distinct gene expression programs in MCCs, regulating genes that control cell movement, ciliogenesis, and cilia function. As such, the work serves as a paradigm for understanding genomic control of cell biological processes that span from early cell morphogenetic events to terminally differentiated cellular functions. DOI: http://dx.doi.org/10.7554/eLife.01439.001.

RFX2 is broadly required for ciliogenesis during vertebrate development.

In Caenorhabditis elegans, the RFX (Daf19) transcription factor is a major regulator of ciliogenesis, controlling the expression of the many essential genes required for making cilia. In vertebrates, however, seven RFX genes have been identified. Bioinformatic analysis suggests that Rfx2 is among the closest homologues of Daf19. We therefore hypothesize that Rfx2 broadly controls ciliogenesis during vertebrate development. Indeed, here we show that Rfx2 in Xenopus is expressed preferentially in ciliated tissues, including neural tube, gastrocoel roof plate, epidermal multi-ciliated cells, otic vesicles, and kidneys. Knockdown of Rfx2 results in cilia-defective embryonic phenotypes and fewer or truncated cilia are observed in Rfx2 morphants. These results indicate that Rfx2 is broadly required for ciliogenesis in vertebrates. Furthermore, we show that Rfx2 is essential for expression of several ciliogenic genes, including TTC25, which we show here is required for ciliogenesis, HH signaling, and left-right patterning.

Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut.

Individual cell shape changes are essential for epithelial morphogenesis. A transcriptional network for epithelial cell shape change is emerging in Drosophila, but this area remains largely unexplored in vertebrates. The distinction is important as so far, key downstream effectors of cell shape change in Drosophila appear not to be conserved. Rather, Shroom3 has emerged as a central effector of epithelial morphogenesis in vertebrates, driving both actin- and microtubule-based cell shape changes. To date, the morphogenetic role of Shroom3 has been explored only in the neural epithelium, so the broad expression of this gene raises two important questions: what are the requirements for Shroom3 in non-neural tissues and what factors control Shroom3 transcription? Here, we show in Xenopus that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut and that Shroom3 transcription in the gut requires the Pitx1 transcription factor. Moreover, we show that Pitx proteins directly activate Shroom3 transcription, and we identify Pitx-responsive regulatory elements in the genomic DNA upstream of Shroom3. Finally, we show that ectopic expression of Pitx proteins is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrate new breadth to the requirements for Shroom3 in morphogenesis, and they also provide a cell-biological basis for the role of Pitx transcription factors in morphogenesis. More generally, these results provide a foundation for deciphering the transcriptional network that underlies epithelial cell shape change in developing vertebrates.

Pax6-dependent Shroom3 expression regulates apical constriction during lens placode invagination.

Embryonic development requires a complex series of relative cellular movements and shape changes that are generally referred to as morphogenesis. Although some of the mechanisms underlying morphogenesis have been identified, the process is still poorly understood. Here, we address mechanisms of epithelial morphogenesis using the vertebrate lens as a model system. We show that the apical constriction of lens epithelial cells that accompanies invagination of the lens placode is dependent on Shroom3, a molecule previously associated with apical constriction during morphogenesis of the neural plate. We show that Shroom3 is required for the apical localization of F-actin and myosin II, both crucial components of the contractile complexes required for apical constriction, and for the apical localization of Vasp, a Mena family protein with F-actin anti-capping function that is also required for morphogenesis. Finally, we show that the expression of Shroom3 is dependent on the crucial lens-induction transcription factor Pax6. This provides a previously missing link between lens-induction pathways and the morphogenesis machinery and partly explains the absence of lens morphogenesis in Pax6-deficient mutants.