title: Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq.
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 are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four 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 by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous 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 previously unknown cell-type markers, including 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 life cycle 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.
authors.affiliation: 1] Departments of Bioengineering and Applied Physics, Stanford University School of Medicine and Howard Hughes Medical Institute, Stanford, California 94305, USA [2].
authors.forename: Barbara
authors.initials: B
authors.lastname: Treutlein
authors.affiliation: 1] Department of Biochemistry, Stanford University School of Medicine and Howard Hughes Medical Institute, Stanford, California 94305, USA [2].
authors.forename: Doug G
authors.initials: DG
authors.lastname: Brownfield
authors.affiliation: Departments of Bioengineering and Applied Physics, Stanford University School of Medicine and Howard Hughes Medical Institute, Stanford, California 94305, USA.
authors.forename: Angela R
authors.initials: AR
authors.lastname: Wu
authors.affiliation: Departments of Bioengineering and Applied Physics, Stanford University School of Medicine and Howard Hughes Medical Institute, Stanford, California 94305, USA.
authors.forename: Norma F
authors.initials: NF
authors.lastname: Neff
authors.affiliation: Departments of Bioengineering and Applied Physics, Stanford University School of Medicine and Howard Hughes Medical Institute, Stanford, California 94305, USA.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Mantalas
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine and Howard Hughes Medical Institute, Stanford, California 94305, USA.
authors.forename: F Hernan
authors.initials: FH
authors.lastname: Espinoza
authors.affiliation: Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Tushar J
authors.initials: TJ
authors.lastname: Desai
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine and Howard Hughes Medical Institute, Stanford, California 94305, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Krasnow
authors.affiliation: Departments of Bioengineering and Applied Physics, Stanford University School of Medicine and Howard Hughes Medical Institute, Stanford, California 94305, USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Quake
mesh_terms: D000818:Animals; D001980:Bronchi / Q000166:cytology; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics*; D004847:Epithelial Cells / Q000145:classification / Q000166:cytology* / Q000378:metabolism*; D005260:Female; D005819:Genetic Markers; D016678:Genome / Q000235:genetics; D023281:Genomics; D008168:Lung / Q000166:cytology* / Q000196:embryology; D051379:Mice; D008810:Mice, Inbred C57BL; D011650:Pulmonary Alveoli / Q000166:cytology; D011659:Pulmonary Gas Exchange; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D013234:Stem Cells / Q000166:cytology; D059467:Transcriptome / Q000235:genetics
chemical_list: D005819:Genetic Markers
references.citation: Nat Biotechnol. 2011 Nov 13;29(12):1120-7
references.pmid: 22081019
references.citation: Nat Methods. 2014 Jan;11(1):41-6
references.pmid: 24141493
references.citation: Genome Res. 1999 Dec;9(12):1198-203
references.pmid: 10613842
references.citation: PLoS One. 2010 Nov 22;5(11):e14085
references.pmid: 21124928
references.citation: Nat Methods. 2013 Nov;10(11):1096-8
references.pmid: 24056875
references.citation: Nature. 2014 Mar 13;507(7491):190-4
references.pmid: 24499815
references.citation: Bioinformatics. 2009 May 1;25(9):1105-11
references.pmid: 19289445
references.citation: Nat Methods. 2012 Mar 04;9(4):357-9
references.pmid: 22388286
references.citation: PLoS One. 2012;7(8):e37046
references.pmid: 22916088
references.citation: Mol Cell Biol. 2004 Aug;24(15):6635-44
references.pmid: 15254231
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D144-9
references.pmid: 22080564
references.citation: Genome Biol. 2013 Apr 17;14(4):R31
references.pmid: 23594475
references.citation: Nat Protoc. 2012 Apr 05;7(5):813-28
references.pmid: 22481528
references.citation: Cell Stem Cell. 2010 May 7;6(5):468-78
references.pmid: 20452321
references.citation: Cell Rep. 2012 Sep 27;2(3):666-73
references.pmid: 22939981
references.citation: Genome Res. 2011 Jul;21(7):1160-7
references.pmid: 21543516
references.citation: Nucleic Acids Res. 2011 Oct;39(18):e120
references.pmid: 21737426
references.citation: Am J Physiol Lung Cell Mol Physiol. 2005 Jan;288(1):L179-89
references.pmid: 15447939
references.citation: Nat Methods. 2005 Oct;2(10):731-4
references.pmid: 16179916
references.citation: Genome Biol. 2009;10(3):R25
references.pmid: 19261174
references.citation: Cell. 2005 Jun 17;121(6):823-35
references.pmid: 15960971
references.citation: Dev Cell. 2012 Sep 11;23(3):482-93
references.pmid: 22975322
references.citation: Nat Protoc. 2009;4(1):44-57
references.pmid: 19131956
references.citation: Am J Respir Cell Mol Biol. 2009 Mar;40(3):340-8
references.pmid: 18757308
references.citation: Nat Methods. 2013 Nov;10(11):1093-5
references.pmid: 24056876
references.citation: Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12592-7
references.pmid: 22797898
references.citation: Nat Biotechnol. 2012 Aug;30(8):777-82
references.pmid: 22820318
references.citation: Am J Physiol Lung Cell Mol Physiol. 2006 Aug;291(2):L191-9
references.pmid: 16510470
references.citation: Nucleic Acids Res. 2009 Jan;37(1):1-13
references.pmid: 19033363
references.citation: Nat Protoc. 2010 Mar;5(3):516-35
references.pmid: 20203668
references.citation: Nature. 2013 Jun 13;498(7453):236-40
references.pmid: 23685454
references.citation: Genome Res. 2011 Sep;21(9):1543-51
references.pmid: 21816910
references.citation: Bioinformatics. 2011 Mar 15;27(6):863-4
references.pmid: 21278185
references.citation: Bioinformatics. 2009 Aug 15;25(16):2078-9
references.pmid: 19505943
references.citation: Nat Methods. 2009 May;6(5):377-82
references.pmid: 19349980
references.citation: CSH Protoc. 2008 May 01;2008:pdb.prot5003
references.pmid: 21356835