No abstract

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.

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.

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.