Functional tissue regeneration is required for restoration of normal organ homeostasis after severe injury. While some organs, such as the intestine, harbor active stem cells throughout homeostasis and regeneration1, more quiescent organs like the lung often contain facultative progenitor cells which are recruited after injury to participate in regeneration2,3. Here we show that a Wnt-responsive alveolar epithelial progenitor (AEP) lineage within the alveolar type 2 (AT2) cell population acts as a major facultative progenitor cell in the distal lung. AEPs are a stable lineage during alveolar homeostasis but expand rapidly to regenerate a large proportion of the alveolar epithelium after acute lung injury. AEPs exhibit a distinct transcriptome, epigenome, and functional phenotype with specific responsiveness to Wnt and Fgf signaling. In distinction to other proposed lung progenitor cells, human AEPs (hAEPs) can be directly isolated via expression of the conserved cell surface marker TM4SF1, and hAEPs act as functional human alveolar epithelial progenitor cells in 3D organoids. Together, our results identify the AEP lineage as an evolutionarily conserved alveolar progenitor and a new target for human lung regeneration strategies.

The lung is an architecturally complex organ comprised of a heterogeneous mixture of various epithelial and mesenchymal lineages. We have used single-cell RNA-sequencing and signaling lineage reporters to generate a spatial and transcriptional map of the lung mesenchyme. We find that each mesenchymal lineage has a distinct spatial address and transcriptional profile leading to unique niche regulatory functions. The Mesenchymal Alveolar Niche Cell is Wnt responsive, expresses Pdgfrα, and is critical for alveolar epithelial cell growth and self-renewal. In contrast, the Axin2+ Myofibrogenic Progenitor cell preferentially generates pathologically deleterious myofibroblasts after injury. Analysis of the secretome and receptome of the alveolar niche reveals functional pathways that mediate growth and self-renewal of alveolar type 2 progenitor cells including IL-6/Stat3, Bmp, and Fgf signaling. These studies define the cellular and molecular framework of lung mesenchymal niches and reveal the functional importance of developmental pathways promoting self-renewal versus pathological response to tissue injury.

Alveologenesis is the culmination of lung development and involves the correct temporal and spatial signals to generate the delicate gas exchange interface required for respiration. Using a novel Wnt signaling reporter system, we demonstrate the emergence of a Wnt-responsive alveolar epithelial cell sublineage that arises during alveologenesis called the axin2+ alveolar type 2 cell or AT2Axin2. The number of AT2Axin2 cells increases substantially during late lung development, correlating with a wave of Wnt signaling during alveologenesis. Transcriptome analysis, in vivo clonal analysis, and ex vivo lung organoid assays reveal that AT2sAxin2 promote enhanced AT2 cell growth during generation of the alveolus. Activating Wnt signaling results in expansion of AT2s whereas inhibition of Wnt signaling inhibits AT2 cell development and shunts alveolar epithelial development towards the alveolar type 1 cell lineage. These findings reveal a wave of Wnt-dependent AT2 expansion required for lung alveologenesis and maturation.

Summary Postnatal tissue quiescence is thought to be a default state in the absence of a proliferative stimulus such as injury. Previous studies have demonstrated that certain embryonic development programs are reactivated aberrantly in adult organs to drive repair and regeneration1–3, it is not well understood how quiescence is maintained in organs such as the lung which displays a remarkably low level of cellular turnover4,5. We now demonstrate that quiescence in the adult lung is an actively maintained state and is regulated by hedgehog signaling. Epithelial-specific deletion of sonic hedgehog during postnatal homeostasis in the lung results in a proliferative expansion of the adjacent lung mesenchyme. Hedgehog signaling is initially down-regulated during the acute phase of epithelial injury as the mesenchyme proliferates in response, but returns to baseline during injury resolution as quiescence is restored. Activation of hedgehog during acute epithelial injury attenuates the proliferative expansion of the lung mesenchyme, whereas inactivation of hedgehog signaling prevents the restoration of quiescence during injury resolution. Finally, we show that hedgehog also regulates epithelial quiescence and regeneration in response to injury via a mesenchymal feedback mechanism. These results demonstrate that epithelial-mesenchymal interactions coordinated by hedgehog actively maintains postnatal tissue homeostasis, and deregulation of hedgehog during injury leads to aberrant repair and regeneration in the lung.

The lung alveolus is the functional unit of the respiratory system required for gas exchange. During the transition to air breathing at birth, biophysical forces are thought to shape the emerging tissue niche. However, the intercellular signaling that drives these processes remains poorly understood. Applying a multimodal approach, we identified alveolar type 1 (AT1) epithelial cells as a distinct signaling hub. Lineage tracing demonstrates that AT1 progenitors align with receptive, force-exerting myofibroblasts in a spatial and temporal manner. Through single-cell chromatin accessibility and pathway expression (SCAPE) analysis, we demonstrate that AT1-restricted ligands are required for myofibroblasts and alveolar formation. These studies show that the alignment of cell fates, mediated by biophysical and AT1-derived paracrine signals, drives the extensive tissue remodeling required for postnatal respiration.

During the stepwise specification and differentiation of tissue-specific multipotent progenitors, lineage-specific transcriptional networks are activated or repressed to orchestrate cell specification. The gas-exchange niche in the lung contains two major epithelial cell types, alveolar type 1 (AT1) and AT2 cells, and the timing of lineage specification of these cells is critical for the correct formation of this niche and postnatal survival. Integrating cell-specific lineage tracing studies, spatially specific mRNA transcript and protein expression, and single-cell RNA-sequencing analysis, we demonstrate that specification of alveolar epithelial cell fate begins concomitantly with the proximal–distal specification of epithelial progenitors and branching morphogenesis earlier than previously appreciated. By using a newly developed dual-lineage tracing system, we show that bipotent alveolar cells that give rise to AT1 and AT2 cells are a minor contributor to the alveolar epithelial population. Furthermore, single-cell assessment of the transcriptome identifies specified AT1 and AT2 progenitors rather than bipotent cells during sacculation. These data reveal a paradigm of organ formation whereby lineage specification occurs during the nascent stages of development coincident with broad tissue-patterning processes, including axial patterning of the endoderm and branching morphogenesis.

Abstract-We show that 1 of the type II bone morphogenetic protein (BMP) receptor ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated in hypoxia-induced pulmonary hypertension (PH). Furthermore, heterozygous null Bmp4 lacZ/ϩ mice are protected from the development of hypoxia-induced PH, vascular smooth muscle cell proliferation, and vascular remodeling. This is associated with a reduction in hypoxia-induced Smad1/5/8 phosphorylation and Id1 expression in the pulmonary vasculature. In addition, pulmonary microvascular endothelial cells secrete BMP4 in response to hypoxia and promote proliferation and migration of vascular smooth muscle cells in a BMP4-dependent fashion. These findings indicate that BMP4 plays a dominant role in regulating BMP signaling in the hypoxic pulmonary vasculature and suggest that endothelium-derived BMP4 plays a direct, paracrine role in promoting smooth muscle proliferation and remodeling in hypoxic PH. Key Words: bone morphogenetic proteins Ⅲ endothelial cells Ⅲ hypoxic pulmonary hypertension Ⅲ signaling pathways Ⅲ Smad Ⅲ vascular remodeling Ⅲ vascular smooth muscle cell proliferation C hronic hypoxia is the most common underlying cause of secondary pulmonary hypertension (PH) in humans. 1 This is associated with the development of fixed defects in the pulmonary vasculature, including medial wall thickening and muscularization of the peripheral vasculature, both mimicked by a rodent model of hypoxic PH in rats and mice. 2,3 Numerous studies using these experimental models provide evidence of an imbalance in the secretion of vasoactive agents and mitogens in the pulmonary vasculature, leading to structural changes in the pulmonary vasculature. 4 -6 Genetic studies in patients with familial primary pulmonary hypertension (FPPH) have identified mutations in 1 of the 3 known type II bone morphogenetic protein receptors (BMP-RII), BMPR2. 7-9 BMP-RII is a member of the transforming growth factor type ␤ family of receptors that acts downstream of the BMP family of ligands. 10,11 BMP ligands interact with 2 classes of transmembrane receptors, termed type I receptors, (ALK2, 3, and 6), and the type II receptors (BMP-RII and Act-RIIA and -IIB). Ligand binding induces type I-receptor phosphorylation by type II receptors, leading to activation of downstream signaling including the classical Smad1/5/8 and the alternative p38 and extracellular signalregulated kinase, mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C pathways. 10,11 Evidence of a role for this signaling pathway in hypoxic PH was shown in 2 recent in vivo studies. By manipulating BMP-RII signaling through 2 different approaches, these studies demonstrated that BMP-RII can promote opposite effects on the pulmonary vasculature under different conditions. 12,13 To explore the mechanisms mediating these effects, we looked at the regulation BMP ligands and downstream signaling in a mouse model of hypoxia-induced PH. One of these ligands, BMP4, is widely expressed in the adult...

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