Science. 2018 单细胞Wnt信号通路维持肺泡II型细胞的干性.pdf

A single cell Wnt signaling niche maintains stemness of alveolar type 2 cellsAhmad Nabhan1,Douglas G.Brownfield1,Mark A.Krasnow*,1,and Tushar J.Desai*,2,31Department of Biochemistry and Howard Hughes Medical Institute,Stanford University School of Medicine,Stanford CA,94305-53072Department of Internal Medicine,Division of Pulmonary and Critical Care,Stanford University School of Medicine,Stanford CA,94305-53073Institute for Stem Cell Biology&Regenerative Medicine,Stanford University School of Medicine,Stanford CA,94305-5307AbstractLung 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.*Corresponding authors:Tushar J.Desai(tdesaistanford.edu)and Mark A.Krasnow(krasnowstanford.edu).Author ContributionsANN,TD and MAK designed the experiments and wrote the manuscript.All experiments except fibroblast and AT2 single cell RNA sequencing were performed and analyzed by ANN.DB performed and analyzed single cell RNA sequencing of alveolar fibroblasts and AT2 cells.HHS Public AccessAuthor manuscriptScience.Author manuscript;available in PMC 2018 September 09.Published in final edited form as:Science.2018 March 09;359(6380):11181123.doi:10.1126/science.aam6603.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptIntroductionAlthough there has been great progress identifying adult tissue stem cells,much less is known about their niches and how niche signals control stem cell function and influence the fate of the daughter cells(1,2).The best understood examples come from genetically tractable systems(35)such as Drosophila testis,where the niche is composed of 1015 cells(“the hub”)that provide three short-range signals to the 510 germline stem cells they directly contact(6).These signals promote stem cell adhesion to the niche and inhibit their differentiation,but following polarized stem cell division the daughter cell that leaves the niche escapes these inhibitory signals and initiates the sperm differentiation program.In mammalian systems,stem cells and their niches are typically more complex,with more cells and/or more complex cellular interactions and dynamics.Even in the best-studied tissues such as blood(7),skin(8,9),and intestine(10),there is still an incomplete understanding of niche cells,signals,and the specific aspects of stem cell behavior each signal controls.Here we describe what may be the simplest stem cell niche and control program,which maintains the gas exchange surface of the adult lung.Genetic studies in mice support a hierarchy of adult stem cells that can replenish the alveolar gas exchange surface(11),some of which are active only following massive injury(1214).Under normal homeostatic conditions,the alveolar epithelium is maintained by rare bifunctional alveolar epithelial type 2(AT2)cells,cuboidal cells that retain the surfactant biosynthesis and secretory function of standard(bulk)AT2 cells(15)but also serve as stem cells that renew the alveolar epithelium throughout the lifespan(16,17).Their intermittent activation gives rise to new AT1 cells,the exquisitely thin epithelial cells that mediate gas exchange,and generates clonal alveolar renewal foci that progressively expand over time and together create 7%new alveoli per year(16).Dying alveolar epithelial cells are proposed to provide a mitogenic signal transduced by the EGFR-KRAS pathway that triggers stem cell division(16).However,it is unclear how the stem cells are selected from bulk AT2 cells,how they are maintained throughout life,and how the fate of their daughter cells-stem cell renewal vs.reprogramming to AT1 identity-is controlled.Here we molecularly identify alveolar stem cells as a rare subpopulation of AT2 cells with constitutive Wnt pathway activity,and show that specialized stromal fibroblasts located adjacent to the stem cells express Wnt5a and other Wnts and comprise a single cell Wnt signaling niche.We provide evidence that this short-range paracrine Wnt signal maintains the stem cell and controls the fate of its daughter cells.We also show that following severe epithelial injury,bulk AT2 cells are recruited as ancillary stem cells,not by expanding the stromal Wnt signaling niche but by transiently inducing autocrine Wnt signaling by AT2 cells.ResultsThe Wnt pathway gene Axin2 is expressed in a rare subset of AT2 cellsMolecular readouts of canonical Wnt signaling activity have identified stem cell populations in a variety of tissues(10),and the Wnt pathway has been shown to be active in developing alveolar progenitors(1821).To determine if any AT2 cells in adult mice show active Wnt Nabhan et al.Page 2Science.Author manuscript;available in PMC 2018 September 09.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptsignaling,we examined AT2 cell expression of the Wnt target gene Axin2(22)using an Axin2-Cre-ERT2 knock-in allele(23)crossed to the Cre-dependent membrane GFP reporter Rosa26mTmG(24).After three daily injections of tamoxifen to induce Cre-ERT2 recombinase activity in Axin2-expressing cells at 2 months of age,FACS analysis showed that 1%of purified AT2 cells expressed the GFP reporter(Fig.1d).Immunostaining for GFP and the canonical AT2 marker Surfactant protein C(SftpC)showed that the Axin2-Cre-ERT2 labeled AT2 cells were distributed sporadically throughout the lung(Fig.1ac).Multiplex single molecule fluorescence in situ hybridization for Axin2 and SftpC confirmed a minor population of Axin2-expressing(Axin2+)AT2 cells distributed throughout the lung(Fig.S1).The Axin2+AT2 cells appear to represent a stable subpopulation because the percentage of AT2 cells labeled using Axin2-Cre-ERT2 did not increase when the daily tamoxifen injections were repeated one and two weeks after the initial induction,and the percentage was similar among adult animals induced at different ages(two and four months)(Fig.1i).This AT2 cell subset expressed all extant markers of mature AT2 cells,including proteins and lipids associated with surfactant phospholipid production(Fig.S2),suggesting that the cells are physiologically functional.No marked AT1 or airway epithelial cells were detected under these pulse-labeling conditions(1000 AT1 cells scored in each of 3 mice),although other(non-epithelial)alveolar cells also expressed Axin2(see below).Thus,Axin2+AT2 cells represent a rare and stable subpopulation of mature AT2 cells distributed throughout the alveolar region of the adult lung.Axin2+AT2 cells have alveolar stem cell activityTo determine if the Axin2+AT2 cells have stem cell activity,we followed the fate of the Axin2-Cre-ERT2 labeled AT2 cells during adult life by adding a half or full year chase(Fig.1el).The labeled cells exhibited three principal features of stem cells(25).First,unlike most AT2 cells which are thought to be quiescent(26),79%of Axin2+AT2 cells labeled with a Cre-dependent Rainbow reporter(Fig.1j)generated small clones of labeled cells(2.5 1.2 cells per clone for all labeled cells including singletons,3.0 1.0 cells per clone for the 79%of labeled cells that generated clones,range 2 5 cells,n=24 Rainbow clones scored)in a six month period(Fig.1k,l).Daughter cells remained local(Fig.1k)with some found as doublets(Fig.1g)that presumably represent instances of recent stem cell division(self-duplication);indeed,on occasion an Axin2+AT2 cell was captured in the process of dividing(Fig.1f),an intermediate we never observed for bulk AT2 cells in normal lungs.Second,there was a six-fold expansion of lineage-labeled AT2 cells relative to unlabeled cells during a one-year chase(Fig.1i),indicating that the Axin2+subset contributes substantially more to AT2 cell maintenance than Axin2-negative AT2 cells.Third,the labeled cells gave rise to another alveolar cell type,as shown by appearance over time of AT1 cells(flat RAGE+SftpC-cells)expressing the lineage label(Fig.1h).Like the AT2 daughter cells,daughter AT1 cells were typically found in close physical association with the presumed founder Axin2+AT2 cell.We conclude that the Axin2+cells constitute a rare subpopulation of AT2 cells with stem cell activity,which slowly(about once every 4 months)self-renew and produce new AT2 and AT1 cells during adult life.Nabhan et al.Page 3Science.Author manuscript;available in PMC 2018 September 09.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptIsolated fibroblasts expressing Wnt5a and other Wnts provide a short-range signal to neighboring AT2 stem cellsBecause Wnt ligands are local signals with a typical range of just one or two cells(27),we presumed there must be a nearby Wnt source that continuously activates the Wnt pathway in the Axin2+AT2 cells.Neighboring fibroblasts were an excellent candidate for the source because some share intimate physical connections with AT2 cells(28),such as Pdgfr-expressing lipofibroblasts that support their production of surfactant and formation of alveolospheres in culture(17,29,30).Indeed,we found that the transmembrane protein Porcupine(Porcn),which catalyzes fatty acylation of Wnts and promotes their secretion(31)and is a putative marker of Wnt signaling niches(32),was expressed in rare alveolar stromal cells(Fig.2a),most of which were Pdgfr-expressing fibroblasts(Fig.2c)and at least some of which are found in close association with AT2 cells(Fig.2b).Serial dosing of the Porcupine inhibitor C59(4-(2-methyl-4-pyridinyl)-N-4-(3-pyridinyl)phenylbenzeneacetamide)reduced the pool of Axin2-expressing AT2 cells by 68%(Fig.2d).Targeted deletion of Wntless,another transmembrane protein required for Wnt trafficking and secretion(33),in lung fibroblasts with TBXLME-Cre(34)or Pdgfr-Cre-ER also reduced the pool of Axin2-expressing AT2 cells(Fig.2e,f),confirming the role of PDGFR-expressing fibroblasts as a source of secreted Wnts that maintains the Axin2+AT2 stem cell pool.The remaining Axin2+AT2 cells could be due to incomplete deletion or perdurance of Wntless in PDGFR+fibroblasts and/or to an alternate Wnt source,like the one induced by injury(see below).Single cell RNA sequencing of isolated alveolar fibroblasts revealed that a subset expressed Wnt5a,most of which(74%)also expressed PDGFR(Fig.2g).Many of the Wnt5a-expressing fibroblasts also expressed low levels of one or two other Wnt genes,including Wnt2(see also ref(30),Wnt2b,Wnt4,and Wnt9a,as did other smaller subpopulations of fibroblasts(Fig.2g),whereas AT2 cells themselves did not express Porcupine(Fig.2a)or any Wnt genes(Fig.S3),at least under normal conditions.Multiplexed single molecule in situ hybridization(35)demonstrated that Wnt5a-expressing fibroblasts(Fig.S4)are scattered throughout the alveolar region of the lung,most in close physical association or directly contacting an Axin2+AT2 cell(Fig.2hk).Although Wnt5a protein is sufficient to induce Axin2 expression in AT2 cells(Fig.2l),it is not the only Wnt ligand operative in vivo because other Wnts can also induce Axin2(Fig.2l),and conditional deletion of Wnt5a with Tbx4LME-Cre only reduced the number of Axin2+AT2 cells in vivo by 15%and the effect did not reach statistical significance(p=0.12).We conclude that Wnt5a,along with the other Wnt ligands expressed by the fibroblasts,activate the canonical Wnt pathway in neighboring AT2 cells.This is a very short range signal,because AT1 daughter cells that derive from Axin2+AT2 cells do not express Axin2(n1000 cells scored in 3 lungs at age 4 months),implying that they do not receive enough signal to maintain Axin2 expression once they move away from the Wnt source.Interestingly,some Wnt-expressing fibroblasts themselves(but not other nearby fibroblasts)expressed Axin2(Figs.2g,S4),suggesting that they may provide an autocrine signal as well.Nabhan et al.Page 4Science.Author manuscript;available in PMC 2018 September 09.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptWnt signaling prevents reprogramming of alveolar stem cells into AT1 cellsTo investigate the function of Wnt signaling in Axin2+AT2 stem cells,we deleted-catenin(36),a transducer of canonical Wnt pathway activity,in mature AT2 cells in vivo using a Lyz2-Cre knock-in allele(37)or an inducible SftpC-CreERT2 knock-in allele(12)while simultaneously marking recombined cells with the Rosa26mTmG Cre-reporter allele.We reasoned that only AT2 cells actively undergoing Wnt signaling(i.e.,Axin2+AT2 cells)would be affected by-catenin deletion.The results showed a tripling in the number of AT1 cells(flat RAGE+SftpC-cells)expressing the AT2 cell lineage mark(234%of all AT1 cells scored with Lyz2-Cre driver,vs 81.3%in wild-type-catenin controls,n=100 random fields scored in 2 or 3 biological replicates),while preserving the percentage of lineage-labeled AT2 cells(853%of all AT2 cells scored vs 823%in wild-type-catenin controls,n=500 AT2 cells scored in 3 biological replicates)and alveolar structure(Fig.3a,b,d;Fig.S5a,b,d).Interestingly,27%of the AT2 lineage-marked AT1 cells in this experiment(48 of 172 scored cells in 3 animals)were spatially isolated and not in physical association with a marked founder AT2 cell,implying that the stem cell had directly converted into an AT1 cell,a rare occurrence in control lungs(4%,n=145 scored cells in