No abstract

Although single cell RNA sequencing studies have begun providing compendia of cell expression profiles 1 – 9 , it has proven more difficult to systematically identify and localize all molecular types in individual organs to create a full molecular cell atlas. Here we describe droplet- and plate-based single cell RNA sequencing (scRNAseq) applied to ~75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, which have allowed us to define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 of 45 previously known cell types or subtypes and 14 new ones. This comprehensive molecular atlas elucidates the biochemical functions of lung cell types and the cell-selective transcription factors and optimal markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signaling interactions including sources and targets of chemokines in immune cell trafficking and expression changes on lung homing; and identifies the cell types directly affected by lung disease genes and respiratory viruses. Comparison to mouse identified 17 molecular types that appear to have been gained or lost during lung evolution and others whose expression profiles have been substantially altered, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions, and interactions are achieved in development and tissue engineering and altered in disease and evolution.

The mammalian lung is a highly branched network, in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types remain obscure, in part due to the limited number of lineage markers1-3 and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations1–5. We used microfluidic single cell RNA sequencing (RNA-seq) on 198 individual cells at 4 different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or prior purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell type diversity in the distal lung and led to the discovery of many novel cell type markers and transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full lifecycle of the alveolar type 2 cell lineage. This single cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

Molecular genetic studies of Drosophila melanogaster have led to profound advances in understanding the regulation of development. Here we report gene expression patterns for nearly one-third of all Drosophila genes during a complete time course of development. Mutations that eliminate eye or germline tissue were used to further analyze tissue-specific gene expression programs. These studies define major characteristics of the transcriptional programs that underlie the life cycle, compare development in males and females, and show that large-scale gene expression data collected from whole animals can be used to identify genes expressed in particular tissues and organs or genes involved in specific biological and biochemical processes.

Alveoli are gas-exchange sacs lined by squamous alveolar type (AT) 1 cells and cuboidal, surfactant-secreting AT2 cells. Classical studies suggested AT1 arise from AT2 cells, but recent studies propose other sources. Here we use molecular markers, lineage tracing, and clonal analysis to map alveolar progenitors throughout the mouse lifespan. We show that during development AT1 and AT2 cells arise directly from a bipotent progenitor, whereas after birth new AT1 derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. This stem cell function is broadly activated by AT1 injury, and AT2 self-renewal is selectively induced by EGF ligands in vitro and oncogenic KrasG12D in vivo, efficiently generating multifocal, clonal adenomas. Thus, there is a switch after birth, when AT2 cells function as stem cells that contribute to alveolar renewal, repair, and cancer. We propose that local signals regulate AT2 stem cell activity: a signal transduced by EGFR-KRAS controls self-renewal and is hijacked during oncogenesis, while another signal controls reprogramming to AT1 fate.

Mammalian lungs are branched networks containing thousands to millions of airways arrayed in intricate patterns that are crucial for respiration. How such trees are generated during development, and how the developmental patterning information is encoded, have long fascinated biologists and mathematicians. However, models have been limited by a lack of information on the normal sequence and pattern of branching events. Here we present the complete three-dimensional branching pattern and lineage of the mouse bronchial tree, reconstructed from an analysis of hundreds of developmental intermediates. The branching process is remarkably stereotyped and elegant: the tree is generated by three geometrically simple local modes of branching used in three different orders throughout the lung. We propose that each mode of branching is controlled by a genetically-encoded subroutine, a series of local patterning and morphogenesis operations, which are themselves controlled by a more global master routine. We show that this hierarchical and modular program is genetically tractable, and it is ideally suited to encoding and evolving the complex networks of the lung and other branched organs.Many organs are composed of highly ramified tubular networks, each with a distinct architecture tailored to its physiological function. The bronchial tree of the human lung has over 10 5 conducting and 10 7 respiratory airways arrayed in an intricate pattern crucial for oxygen flow [1][2][3][4] . Classical studies of lung structure 5-8 raise the question of how the information required to generate a tree of such complexity is biologically encoded 9 . Individually configuring thousands or millions of branches would require a tremendous amount of patterning information, far more than is biologically plausible, to specify when and where each branch forms during development, and the size, shape, and direction of outgrowth of each branch. One possibility is that the process is not precisely controlled, for example if branching occurs randomly to fill available space. Another is that control is precise but coding is simplified by repeated use of a branching mechanism, as in Mandelbrot's fractal model and other elegant algorithms [10][11][12][13][14][15][16][17] . Even with these attractive models and recent progress in identifying lung development genes 18 , understanding of the program that directs branching remains rudimentary. This is largely due to the complexity of the bronchial tree, which makes it difficult to follow branching dynamics beyond the earliest events [19][20][21] . Although branching of the lung and other organs can occur in culture [22][23][24][25] , it is unlikely these recapitulate the full pattern. Here, we have determined the complete in vivo pattern of branching and branch lineage of the mouse bronchial tree, and show that it is generated using three geometrically distinct local modes of branching coupled in three different sequences. The branch lineage of the mouse bronchial treeThe bronchial tree develops by bran...

Lung alveoli are lined by squamous alveolar epithelial type 1 (AT1) epithelial cells that facilitate gas exchange, and neighboring AT2 cells that synthesize and secrete surfactant. Alveoli are maintained by intermittent activation of rare ‘bifunctional’ AT2 cells that retain surfactant biosynthesis function but also serve as stem cells, generating new AT1 cells and self-renewing throughout adult life. While stem cell proliferation is controlled by EGFR/KRAS signaling, how the stem cells are selected, maintained, and the fates of their daughter cells controlled are unknown. Here we show that expression of the Wnt target gene Axin2 in mouse lung identifies a rare, stable subpopulation of AT2 cells with stem cell activity. Many lie near single fibroblasts that express Wnt5a and other Wnt genes, and genetically targeting Wnt secretion by fibroblasts depletes the Axin2+ AT2 stem cell population. Axin2 turns off when daughter cells leave the Wnt niche and transdifferentiate into AT1 cells, and sustaining Wnt signaling blocks transdifferentiation whereas abrogation of Wnt signaling promotes it, both in vivo and in vitro. Upon severe alveolar epithelial injury, Axin2 is induced throughout the AT2 population, recruiting ‘ancillary’ AT2 cells into a progenitor role. Niche expression of Wnt5a and the Wnt secretion mediator Porcupine is unchanged by injury, but Wnt7b and several other Wnt genes are broadly induced along with Porcupine in AT2 cells, and pharmacologic or genetic inhibition of this autocrine Wnt signaling impairs the AT2 proliferative response. The results support a model in which individual AT2 cells reside in single cell fibroblast niches that provide a short-range paracrine (or "juxtacrine") Wnt signal that selects and maintains alveolar stem cell identity and proliferative capacity, while severe injury induces AT2 autocrine Wnt signals that transiently expand the stem cell pool during repair.

Antagonists of several growth factor signaling pathways play important roles in developmental patterning by limiting the range of the cognate inducer. Here, we describe an antagonist of FGF signaling that patterns apical branching of the Drosophila airways. In wild-type embryos, the Branchless FGF induces secondary branching by activating the Breathless FGF receptor near the tips of growing primary branches. In sprouty mutants, the FGF pathway is overactive and ectopic branches are induced on the stalks of primary branches. We show that FGF signaling induces sprouty expression in the nearby tip cells, and sprouty acts nonautonomously and in a competitive fashion to block signaling to the more distant stalk cells. sprouty encodes a novel cysteine-rich protein that defines a new family of putative signaling molecules that may similarly function as FGF antagonists in vertebrate development.