Transcriptional control of lung alveolar type 1 cell development and maintenance by NK homeobox 2-1 Danielle R. Littlea,b, Kamryn N. Gerner-Mauroa, Per Flodbyc, Edward D. Crandallc, Zea Borokc, Haruhiko Akiyamad, Shioko Kimurae, Edwin J. Ostrina,f, and Jichao Chena,1 aDepartment of Pulmonary Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; bUniversity of Texas Health Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; cDivision of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine and Hastings Center for Pulmonary Research, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033; dDepartment of Orthopedics, Kyoto University, Sakyo, 606-8507 Kyoto, Japan; eLaboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and fDepartment of General Internal Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030 Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved August 30, 2019 (received for review April 18, 2019) The extraordinarily thin alveolar type 1 (AT1) cell constitutes nearly Hippo signaling promotes progenitor differentiation toward the the entire gas exchange surface and allows passive diffusion of AT1 cell fate (12–14). This growing list of AT1 cell regulators oxygen into the blood stream. Despite such an essential role, the highlights both the underlying complexity and the necessity to transcriptional network controlling AT1 cells remains unclear. Using distinguish direct effects on AT1 cells versus those on progenitors, cell-specific knockout mouse models, genomic profiling, and 3D imag- AT2 cells, or tissue morphology, especially in light of the classical Nkx2-1 ing, we found that NK homeobox 2-1 ( )isexpressedinAT1 observation of rapid AT1 cell-like differentiation of cultured cells and is required for the development and maintenance of AT1 AT2 cells (15). A better understanding of AT1 cell development cells. Without Nkx2-1, developing AT1 cells lose 3 defining features— also requires identification of sequence-specific transcription fac- molecular markers, expansive morphology, and cellular quiescence— leading to alveolar simplification and lethality. NKX2-1 is also cell- tors that activate AT1 cell-specific genes and cellular machinery autonomously required for the same 3 defining features in mature that determines its unique morphology. AT1 cells. Intriguingly, Nkx2-1 mutant AT1 cells activate gastrointes- In a previous model to disrupt AT1 cell development, we ec- BIOLOGY tinal (GI) genes and form dense microvilli-like structures apically. topically expressed the airway transcription factor SRY-box 2 DEVELOPMENTAL Single-cell RNA-seq supports a linear transformation of Nkx2-1 mu- (SOX2) in developing AT1 cells. These mutant cells became tant AT1 cells toward a GI fate. Whole lung ChIP-seq shows NKX2-1 airway-like in association with drastic molecular and morpholog- binding to 68% of genes that are down-regulated upon Nkx2-1 ical changes, prompting the question of whether they retained the deletion, including 93% of known AT1 genes, but near-background lung fate, which we addressed by immunostaining for the lung binding to up-regulated genes. Our results place NKX2-1 at the top lineage transcription factor NKX2-1 (5). To our surprise, AT1 of the AT1 cell transcriptional hierarchy and demonstrate remarkable cells in the control lung had nuclear NKX2-1, different from prior plasticity of an otherwise terminally differentiated cell type. reports that NKX2-1 is a lineage factor during lung specification and branching morphogenesis, but is lost in AT1 cells upon al- lung development | alveolar type 1 cell | NK homeobox 2-1 | transcriptional veolar differentiation (16, 17). Intriguingly, NKX2-1 staining in control | alveologenesis SOX2-expressing mutant AT1 cells was diffuse instead of nuclear, ells often undergo morphological and molecular specializa- Significance Ction to fulfill their physiological function, such as neurons, cardiomyocytes, and pancreatic beta cells. The cell type chiefly Gas exchange in the lung relies on passive diffusion of oxygen responsible for the physiological function of the lung—gas and carbon dioxide across an extraordinarily thin epithelium exchange—is the alveolar type 1 (AT1) cell. AT1 cells cover 95% that is nearly entirely made of the poorly understood alveolar of the gas exchange surface and are 0.1 μm thick to allow passive type 1 (AT1) cell. Our study shows that all AT1 cells express and diffusion of oxygen into the blood stream. They have expansive require NK homeobox 2-1 (Nkx2-1) for their development and morphology, covering multiple alveoli, and fold extensively, maintenance. Nkx2-1 mutant AT1 cells lose their characteristic forming close contact with the vasculature (1–5). Unlike the molecular and cellular features and transform toward a gas- AT1 cell, the alveolar type 2 (AT2) cell, the other major cell type trointestinal fate, highlighting remarkable plasticity of an of the alveolar epithelium, is cuboidal and secretes surfactants to otherwise terminally differentiated cell type. This work estab- reduce surface tension (1, 2). lishes NKX2-1 as the first overarching factor in the AT1 cell The unique morphology of AT1 cells develops in 2 steps (5). transcriptional hierarchy and paves the way for unraveling not First, it undergoes flattening from a columnar progenitor. Then, it > only the unique biology of AT1 cells but also cell type-specific expands 10-fold in size and at the same time folds to interdigitate roles of a lineage transcription factor. with the capillaries. Growing evidence indicates that both tissue environment, such as mechanical forces and extracellular matrix, Author contributions: D.R.L. and J.C. designed research; D.R.L. and K.N.G.-M. performed and intracellular factors could impact AT1 cell development. For research; P.F., E.D.C., Z.B., H.A., and S.K. contributed new reagents/analytic tools; D.R.L., example, reducing mechanical tension from fetal breathing by K.N.G.-M., and E.J.O. analyzed data; and D.R.L. and J.C. wrote the paper. depleting the amniotic fluid or lowering the stiffness of cell culture The authors declare no conflict of interest. surface favors AT2 over AT1 cell differentiation (6). Loss of an This article is a PNAS Direct Submission. extracellular matrix protein, COL4A1, also leads to an increased Published under the PNAS license. ratio of AT2 to AT1 cells (7). Inside the epithelial cells, beta- Data deposition: The data reported in this paper have been deposited in the Gene Ex- catenin–mediated canonical WNT signaling as well as fibroblast pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE129628). growth factor signaling inhibit AT1 cell differentiation (6, 8–10) 1To whom correspondence may be addressed. Email: [email protected]. and HDAC3-dependent TGF-beta signaling is required for proper This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. epithelium expansion and AT1 cell spacing (8, 11). More recently, 1073/pnas.1906663116/-/DCSupplemental. genetic analyses of Mst1/2, Lats1/2,andYap/Taz indicate that First published September 23, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1906663116 PNAS | October 8, 2019 | vol. 116 | no. 41 | 20545–20555 Downloaded by guest on September 29, 2021 as expected for a transcription factor, raising the possibility that junction protein E-cadherin (ECAD) (Fig. 1A). We found that NKX2-1 regulates AT1 cell development. throughout postnatal development as well as in the mature lung, In this study, we use precise genetic deletion models and geno- NKX2-1 was present in every single green fluorescent protein mic analyses to show that NKX2-1 is necessary for the initiation, (GFP)-labeled cell, corresponding to both AT1 and AT2 cells, development, and maintenance of AT1 cells. Nkx2-1 mutant AT1 which were further distinguished by an AT2 marker, lysosome- cells lose 3 defining features—AT1 cell-specific markers, mor- associated membrane protein 3 (LAMP3) (Fig. 1B). Thus, in- phology, and quiescence—and aberrantly express gastrointestinal stead of being lost upon AT1 cell differentiation as suggested (GI) genes. This study establishesNKX2-1asakeytranscription previously (16, 17), NKX2-1 is expressed in both developing and factor controlling AT1 cells. mature AT1 cells. Given the importance of NKX2-1 in early development (20– Results 22) and its aberrant localization in the SOX2 ectopic expression NKX2-1 in AT1 Cells Is Required for Alveologenesis. To confirm and model, as described in the introduction (5), we hypothesized that extend knowledge of NKX2-1 expression in AT1 cells, we genet- NKX2-1 had a role in AT1 cell development. To test this, we ically marked all lung epithelial cell nuclei, including those of generated an AT1 cell-specific knockout of Nkx2-1 using an AT1 and AT2 cells, with a ShhCre driver (18) and a nuclear Cre Aqp5Cre driver (23, 24). A portion of these mice had widespread reporter, RosaSun1GFP (19). To visualize the expansive AT1 cells in expression of a GFP reporter across organs, possibly due to early their entirety, we used whole mount immunostaining and confocal recombination during embryogenesis or within the parental stack imaging of strips cut from lobe edges (ref. 5 and Fig. 1A). In germline, and were thus excluded from analysis. In the remaining such preparations, AT1