Tushar Desai

All Publications

• Evidence for lung barrier regeneration by differentiation prior to binucleated and stem cell division. The Journal of cell biology Guild, J., Juul, N. H., Andalon, A., Taenaka, H., Coffey, R. J., Matthay, M. A., Desai, T. J. 2023; 222 (12)

  Abstract

  With each breath, oxygen diffuses across remarkably thin alveolar type I (AT1) cells into underlying capillaries. Interspersed cuboidal AT2 cells produce surfactant and act as stem cells. Even transient disruption of this delicate barrier can promote capillary leak. Here, we selectively ablated AT1 cells, which uncovered rapid AT2 cell flattening with near-continuous barrier preservation, culminating in AT1 differentiation. Proliferation subsequently restored depleted AT2 cells in two phases, mitosis of binucleated AT2 cells followed by replication of mononucleated AT2 cells. M phase entry of binucleated and S phase entry of mononucleated cells were both triggered by AT1-produced hbEGF signaling via EGFR to Wnt-active AT2 cells. Repeated AT1 cell killing elicited exuberant AT2 proliferation, generating aberrant daughter cells that ceased surfactant function yet failed to achieve AT1 differentiation. This hyperplasia eventually resolved, yielding normal-appearing alveoli. Overall, this specialized regenerative program confers a delicate simple epithelium with functional resiliency on par with the physical durability of thicker, pseudostratified, or stratified epithelia.

  View details for DOI 10.1083/jcb.202212088

  View details for PubMedID 37843535

• KRAS(G12D) drives lepidic adenocarcinoma through stem-cell reprogramming. Nature Juul, N. H., Yoon, J. K., Martinez, M. C., Rishi, N., Kazadaeva, Y. I., Morri, M., Neff, N. F., Trope, W. L., Shrager, J. B., Sinha, R., Desai, T. J. 2023

  Abstract

  Many cancers originate from stem or progenitor cells hijacked by somatic mutations that drive replication, exemplified by adenomatous transformation of pulmonary alveolar epithelial type II (AT2) cells1. Here we demonstrate a different scenario: expression of KRAS(G12D) in differentiated AT1 cells reprograms them slowly and asynchronously back into AT2 stem cells that go on to generate indolent tumours. Like human lepidic adenocarcinoma, the tumour cells slowly spread along alveolar walls in a non-destructive manner and have low ERK activity. We find that AT1 and AT2 cells act as distinct cells of origin and manifest divergent responses to concomitant WNT activation and KRAS(G12D) induction, which accelerates AT2-derived but inhibits AT1-derived adenoma proliferation. Augmentation of ERK activity in KRAS(G12D)-induced AT1 cells increases transformation efficiency, proliferation and progression from lepidic to mixed tumour histology. Overall, we have identified a new cell of origin for lung adenocarcinoma, the AT1 cell, which recapitulates features of human lepidic cancer. In so doing, we also uncover a capacity for oncogenic KRAS to reprogram a differentiated and quiescent cell back into its parent stem cell en route to adenomatous transformation. Our work further reveals that irrespective of a given cancer's current molecular profile and driver oncogene, the cell of origin exerts a pervasive and perduring influence on its subsequent behaviour.

  View details for DOI 10.1038/s41586-023-06324-w

  View details for PubMedID 37468622

  View details for PubMedCentralID 4013278

• An integrated cell atlas of the lung in health and disease. Nature medicine Sikkema, L., Ramírez-Suástegui, C., Strobl, D. C., Gillett, T. E., Zappia, L., Madissoon, E., Markov, N. S., Zaragosi, L. E., Ji, Y., Ansari, M., Arguel, M. J., Apperloo, L., Banchero, M., Bécavin, C., Berg, M., Chichelnitskiy, E., Chung, M. I., Collin, A., Gay, A. C., Gote-Schniering, J., Hooshiar Kashani, B., Inecik, K., Jain, M., Kapellos, T. S., Kole, T. M., Leroy, S., Mayr, C. H., Oliver, A. J., von Papen, M., Peter, L., Taylor, C. J., Walzthoeni, T., Xu, C., Bui, L. T., De Donno, C., Dony, L., Faiz, A., Guo, M., Gutierrez, A. J., Heumos, L., Huang, N., Ibarra, I. L., Jackson, N. D., Kadur Lakshminarasimha Murthy, P., Lotfollahi, M., Tabib, T., Talavera-López, C., Travaglini, K. J., Wilbrey-Clark, A., Worlock, K. B., Yoshida, M., van den Berge, M., Bossé, Y., Desai, T. J., Eickelberg, O., Kaminski, N., Krasnow, M. A., Lafyatis, R., Nikolic, M. Z., Powell, J. E., Rajagopal, J., Rojas, M., Rozenblatt-Rosen, O., Seibold, M. A., Sheppard, D., Shepherd, D. P., Sin, D. D., Timens, W., Tsankov, A. M., Whitsett, J., Xu, Y., Banovich, N. E., Barbry, P., Duong, T. E., Falk, C. S., Meyer, K. B., Kropski, J. A., Pe'er, D., Schiller, H. B., Tata, P. R., Schultze, J. L., Teichmann, S. A., Misharin, A. V., Nawijn, M. C., Luecken, M. D., Theis, F. J. 2023

  Abstract

  Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1+ profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

  View details for DOI 10.1038/s41591-023-02327-2

  View details for PubMedID 37291214

  View details for PubMedCentralID 5762154

• Alveolar cell fate selection and lifelong maintenance of AT2 cells by FGF signaling. Nature communications Brownfield, D. G., de Arce, A. D., Ghelfi, E., Gillich, A., Desai, T. J., Krasnow, M. A. 2022; 13 (1): 7137

  Abstract

  The lung's gas exchange surface is comprised of alveolar AT1 and AT2 cells that are corrupted in several common and deadly diseases. They arise from a bipotent progenitor whose differentiation is thought to be dictated by differential mechanical forces. Here we show the critical determinant is FGF signaling. Fgfr2 is expressed in the developing progenitors in mouse then restricts to nascent AT2 cells and remains on throughout life. Its ligands are expressed in surrounding mesenchyme and can, in the absence of exogenous mechanical cues, induce progenitors to form alveolospheres with intermingled AT2 and AT1cells. FGF signaling directly and cell autonomously specifies AT2 fate; progenitors lacking Fgfr2 in vitro and in vivo exclusively acquire AT1 fate. Fgfr2 loss in AT2 cells perinatally results in reprogramming to AT1 identity, whereas loss or inhibition later in life triggers AT2 apoptosis and compensatory regeneration. We propose that Fgfr2 signaling selects AT2 fate during development, induces a cell non-autonomous AT1 differentiation signal, then continuously maintains AT2 identity and survival throughout life.

  View details for DOI 10.1038/s41467-022-34059-1

  View details for PubMedID 36414616

• Developmental Insights into Lung Cancer ANNUAL REVIEW OF CANCER BIOLOGY, VOL 5, 2021 Desai, T. J., Jacks, T., Sawyers, C. L. 2021; 5: 351–69

  View details for DOI 10.1146/annurev-cancerbio-070820-032858

  View details for Web of Science ID 000627716400019

• Lung stem cells and therapy for cystic fibrosis Lung Stem Cells in Development, Health and Disease (ERS Monograph) Vaidyanathan, S., McCarra, M., Desai, T. J. edited by Nikolic, M. Z., Hogan, B. L. 2021: 306-321
• Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature Salahudeen, A. A., Choi, S. S., Rustagi, A., Zhu, J., van Unen, V., de la O, S. M., Flynn, R. A., Margalef-Catala, M., Santos, A. J., Ju, J., Batish, A., Usui, T., Zheng, G. X., Edwards, C. E., Wagar, L. E., Luca, V., Anchang, B., Nagendran, M., Nguyen, K., Hart, D. J., Terry, J. M., Belgrader, P., Ziraldo, S. B., Mikkelsen, T. S., Harbury, P. B., Glenn, J. S., Garcia, K. C., Davis, M. M., Baric, R. S., Sabatti, C., Amieva, M. R., Blish, C. A., Desai, T. J., Kuo, C. J. 2020

  Abstract

  The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. We generated long-term feeder-free, chemically defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia.

  View details for DOI 10.1038/s41586-020-3014-1

  View details for PubMedID 33238290

• Advances in Proximity Ligation in situ Hybridization (PLISH). Bio-protocol Nagendran, M., Andruska, A. M., Harbury, P. B., Desai, T. J. 2020; 10 (21): e3808

  Abstract

  Understanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression in intact tissues, which we have now updated. By recording spatial information for every profiled cell, PLISH enables retrospective mapping of distinct cell classes and inference of their in vivo interactions. PLISH has high sensitivity, specificity and signal to noise ratio. It is also rapid, scalable, and does not require expertise in molecular biology so it can be easily adopted by basic and clinical researchers.

  View details for DOI 10.21769/BioProtoc.3808

  View details for PubMedID 33659462

  View details for PubMedCentralID PMC7842654

• SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes NATURE MEDICINE Sungnak, W., Huang, N., Becavin, C., Berg, M., Queen, R., Litvinukova, M., Talavera-Lopez, C., Maatz, H., Reichart, D., Sampaziotis, F., Worlock, K. B., Yoshida, M., Barnes, J. L., HCA Lung Biological 2020

  Abstract

  We investigated SARS-CoV-2 potential tropism by surveying expression of viral entry-associated genes in single-cell RNA-sequencing data from multiple tissues from healthy human donors. We co-detected these transcripts in specific respiratory, corneal and intestinal epithelial cells, potentially explaining the high efficiency of SARS-CoV-2 transmission. These genes are co-expressed in nasal epithelial cells with genes involved in innate immunity, highlighting the cells' potential role in initial viral infection, spread and clearance. The study offers a useful resource for further lines of inquiry with valuable clinical samples from COVID-19 patients and we provide our data in a comprehensive, open and user-friendly fashion at www.covid19cellatlas.org.

  View details for DOI 10.1038/s41591-020-0868-6

  View details for Web of Science ID 000528517900001

  View details for PubMedID 32327758

• Niche Cells and Signals that Regulate Lung Alveolar Stem Cells In Vivo. Cold Spring Harbor perspectives in biology Juul, N. H., Stockman, C. A., Desai, T. J. 2020

  Abstract

  The distal lung is a honeycomb-like collection of delicate gas exchange sacs called alveoli lined by two interspersed epithelial cell types: the cuboidal, surfactant-producing alveolar type II (AT2) and the flat, gas-exchanging alveolar type I (AT1) cell. During aging, a subset of AT2 cells expressing the canonical Wnt target gene, Axin2, function as stem cells, renewing themselves while generating new AT1 and AT2 cells. Wnt activity endows AT2 cells with proliferative competency, enabling them to respond to activating cues, and simultaneously blocks AT2 to AT1 cell transdifferentiation. Acute alveolar injury rapidly expands the AT2 stem cell pool by transiently inducing Wnt signaling activity in "bulk" AT2 cells, facilitating rapid epithelial repair. AT2 cell "stemness" is thus tightly regulated by access to Wnts, supplied by a specialized single-cell fibroblast niche during maintenance and by AT2 cells themselves during injury repair. Two non-AT2 "reserve" cell populations residing in the distal airways also contribute to alveolar repair, but only after widespread epithelial injury, when they rapidly proliferate, migrate, and differentiate into airway and alveolar lineages. Here, we review alveolar renewal and repair with a focus on the niches, rather than the stem cells, highlighting what is known about the cellular and molecular mechanisms by which they control stem cell activity in vivo.

  View details for DOI 10.1101/cshperspect.a035717

  View details for PubMedID 32179507

• An atlas of the aging lung mapped by single cell transcriptomics and deep tissue proteomics. Nature communications Angelidis, I., Simon, L. M., Fernandez, I. E., Strunz, M., Mayr, C. H., Greiffo, F. R., Tsitsiridis, G., Ansari, M., Graf, E., Strom, T., Nagendran, M., Desai, T., Eickelberg, O., Mann, M., Theis, F. J., Schiller, H. B. 2019; 10 (1): 963

  Abstract

  Aging promotes lung function decline and susceptibility to chronic lung diseases, which are the third leading cause of death worldwide. Here, we use single cell transcriptomics and mass spectrometry-based proteomics to quantify changes in cellular activity statesacross30 cell types and chart the lung proteome of young and old mice. We show that aging leads to increased transcriptional noise, indicating deregulated epigenetic control. We observe cell type-specific effects of aging, uncovering increased cholesterol biosynthesis in type-2 pneumocytes and lipofibroblasts and altered relative frequency of airway epithelial cells as hallmarks of lung aging. Proteomic profiling reveals extracellular matrix remodeling in old mice, including increased collagen IV and XVI and decreased Fraser syndrome complex proteins and collagen XIV. Computational integration of the aging proteome with the single cell transcriptomes predicts the cellular source of regulated proteins and creates an unbiased reference map of the aging lung.

  View details for PubMedID 30814501

• High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia. Cell stem cell Vaidyanathan, S. n., Salahudeen, A. A., Sellers, Z. M., Bravo, D. T., Choi, S. S., Batish, A. n., Le, W. n., Baik, R. n., de la O, S. n., Kaushik, M. P., Galper, N. n., Lee, C. M., Teran, C. A., Yoo, J. H., Bao, G. n., Chang, E. H., Patel, Z. M., Hwang, P. H., Wine, J. J., Milla, C. E., Desai, T. J., Nayak, J. V., Kuo, C. J., Porteus, M. H. 2019

  Abstract

  Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

  View details for DOI 10.1016/j.stem.2019.11.002

  View details for PubMedID 31839569

• Automated cell-type classification in intact tissues by single-cell molecular profiling ELIFE Nagendran, M., Riordan, D. P., Harbury, P. B., Desai, T. J. 2018; 7

  View details for DOI 10.7554/eLife.30510

  View details for Web of Science ID 000424629300001

• Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells. Science (New York, N.Y.) Nabhan, A. n., Brownfield, D. G., Harbury, P. B., Krasnow, M. A., Desai, T. J. 2018

  Abstract

  Alveoli, the lung's respiratory units, are tiny sacs where oxygen enters the bloodstream. They are lined by flat AT1 cells, which mediate gas exchange, and AT2 cells, which secret surfactant. Rare AT2s also function as alveolar stem cells. We show that AT2 lung stem cells display active Wnt signaling and many of them are near single, Wnt-expressing fibroblasts. Blocking Wnt secretion depletes these stem cells. Daughter cells leaving the Wnt niche transdifferentiate into AT1s: maintaining Wnt signaling prevents transdifferentiation whereas abrogating Wnt signaling promotes it. Injury induces AT2 autocrine Wnts, recruiting 'bulk' AT2s as progenitors. Thus, individual AT2 stem cells reside in single cell fibroblast niches providing juxtacrine Wnts that maintain them, whereas injury induces autocrine Wnts that transiently expand the progenitor pool. This simple niche maintains the gas exchange surface, and is coopted in cancer.

  View details for PubMedID 29420258

• Automated cell type classification in intact tissues by single-cell molecular profiling. eLife Nagendran, M. n., Riordan, D. P., Harbury, P. B., Desai, T. J. 2018; 7

  Abstract

  A major challenge in biology is identifying distinct cell classes and mapping their interactions in vivo. Tissue-dissociative technologies enable deep single cell molecular profiling but do not provide spatial information. We developed a proximity ligation- in situ hybridization technology (PLISH) with exceptional signal strength, specificity, and sensitivity in tissue. Multiplexed data sets can be acquired using barcoded probes and rapid label-image-erase cycles, with automated calculation of single cell profiles, enabling clustering and anatomical re-mapping of cells. We apply PLISH to expression profile ~2,900 cells in intact mouse lung, which identifies and localizes known cell types, including rare ones. Unsupervised classification of the cells indicates differential expression of 'housekeeping' genes between cell types, and re-mapping of two sub-classes of Club cells highlights their segregated spatial domains in terminal airways. By enabling single cell profiling of various RNA species in situ, PLISH can impact many areas of basic and medical research.

  View details for PubMedID 29319504

• Trinucleotide repeat containing 6c (TNRC6c) is essential for microvascular maturation during distal airspace sacculation in the developing lung. Developmental biology Guo, H., Kazadaeva, Y., Ortega, F. E., Manjunath, N., Desai, T. J. 2017; 430 (1): 214-223

  Abstract

  GW182 (also known asTNRC6) family members are critically involved in the final effector phase of miRNA-mediated mRNA repression. The three mammalian paralogs, TNRC6a, b and c, are thought to be redundant based on Argonaute (Ago) binding, tethering assays, and RNAi silencing of individual members in cell lines. To test this idea, we generated TNRC6a, b and c knockout mice. TNRC6a mutants die at mid-gestation, while b- and c- deleted mice are born at a Mendelian ratio. However, the majority of TNRC6b and all TNRC6c mutants die within 24h after birth, the latter with respiratory failure. Necropsy of TNRC6c mutants revealed normal-appearing airways that give rise to abnormally thick-walled distal gas exchange sacs. Immunohistological analysis of mutant lungs demonstrated a normal distribution of bronchiolar and alveolar cells, indicating that loss of TNRC6c did not abrogate epithelial cell differentiation. The cellular kinetics and relative proportions of endothelial, epithelial, and mesenchymal cells were also not altered. However, the underlying capillary network was simplified and endothelial cells had failed to become tightly apposed to the surface epithelium in TNRC6c mutants, presumably causing the observed respiratory failure. TGFβ family mutant mice exhibit a similar lung phenotype of thick-walled air sacs and neonatal lethality, and qRT-PCR confirmed dynamic downregulation of TGFβ1 and TGFβR2 in TNRC6c mutant lungs during sacculation. VEGFR, but not VEGF-A ligand, was also lower, likely reflecting the overall reduced capillary density in TNRC6c mutants. Together, these results demonstrate that GW182 paralogs are not functionally redundant in vivo. Surprisingly, despite regulating a general cellular process, TNRC6c is selectively required only in the distal lung and not until late in gestation for proper expression of the TGFβ family genes that drive sacculation. These results imply a complex and indirect mode of regulation of sacculation by TNRC6c, mediated in part by dynamic transcriptional repression of an inhibitor of TGFβ family gene expression.

  View details for DOI 10.1016/j.ydbio.2017.07.018

  View details for PubMedID 28811219

• Keeping it together: Pulmonary alveoli are maintained by a hierarchy of cellular programs. BioEssays Logan, C. Y., Desai, T. J. 2015; 37 (9): 1028-1037

  Abstract

  The application of in vivo genetic lineage tracing has advanced our understanding of cellular mechanisms for tissue renewal in organs with slow turnover, like the lung. These studies have identified an adult stem cell with very different properties than classically understood ones that maintain continuously cycling tissues such as the intestine. A portrait has emerged of an ensemble of cellular programs that replenish the cells that line the gas exchange (alveolar) surface, enabling a response tailored to the extent of cell loss. A capacity for differentiated cells to undergo direct lineage transitions allows for local restoration of proper cell balance at sites of injury. We present these recent findings as a paradigm for how a relatively quiescent tissue compartment can maintain homeostasis throughout a lifetime punctuated by injuries ranging from mild to life-threatening, and discuss how dysfunction or insufficiency of alveolar repair programs produce serious health consequences like cancer and fibrosis.

  View details for DOI 10.1002/bies.201500031

  View details for PubMedID 26201286

• Cellular mechanisms of alveolar pathology in childhood interstitial lung diseases: current insights from mouse genetics CURRENT OPINION IN PEDIATRICS Kuo, C. S., Desai, T. J. 2015; 27 (3): 341-347

  Abstract

  Childhood interstitial lung diseases (ILDs) are a diverse class of disorders affecting the alveolar gas exchange region that lack specific treatments and are usually fatal. Here, we integrate recent insights into alveolar cell biology with histopathology from well characterized mutations of surfactant-associated genes. We take a reductionist approach by parsing discrete histological features and correlating each to perturbation of a particular function of the alveolar epithelial type II (AT2) cell, the central driver of disease, to generate a working model for the cellular mechanisms of disease pathogenesis.The application of genetically modified mice and single cell genomics has yielded new insights into lung biology, including the identification of a bipotent alveolar progenitor in development, mapping of adult AT2 stem cells in vivo, and demonstration that latent cooperative interactions with fibroblasts can be pathologically activated by targeted injury of the AT2 cell.As we learn more about individual and cooperative roles for alveolar cells in health, we can dissect how perturbations of specific cellular functions contribute to disease in childhood ILDs. We hope our updated model centered around the AT2 cell as the initiator of disease provides a cellular framework that researchers can build upon and revise as they identify the specific molecular signals within and between alveolar cells that mediate the diverse pathologic features, so that targeted pharmacologic and cell-based treatments for patients can ultimately be engineered.

  View details for DOI 10.1097/MOP.0000000000000227

  View details for Web of Science ID 000354214800013

  View details for PubMedID 25888154

  View details for PubMedCentralID PMC4466102

• Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature Treutlein, B., Brownfield, D. G., Wu, A. R., Neff, N. F., Mantalas, G. L., Espinoza, F. H., Desai, T. J., Krasnow, M. A., Quake, S. R. 2014; 509 (7500): 371-375

  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.

  View details for DOI 10.1038/nature13173

  View details for PubMedID 24739965

• Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature Desai, T. J., Brownfield, D. G., Krasnow, M. A. 2014; 507 (7491): 190-194

  View details for DOI 10.1038/nature12930

  View details for PubMedID 24499815

• Stem cells: Differentiated cells in a back-up role. Nature Desai, T. J., Krasnow, M. A. 2013; 503 (7475): 204-205

  View details for DOI 10.1038/nature12706

  View details for PubMedID 24196710

• Distinct roles for retinoic acid receptors alpha and beta in early lung morphogenesis DEVELOPMENTAL BIOLOGY Desai, T. J., Chen, F., Lu, J. M., Qian, J., Niederreither, K., Dolle, P., Chambon, P., Cardoso, W. V. 2006; 291 (1): 12-24

  Abstract

  Retinoic acid (RA) signaling is required for normal development of multiple organs. However, little is known about how RA influences the initial stages of lung development. Here, we used a combination of genetic, pharmacological and explant culture approaches to address this issue, and to investigate how signaling by different RA receptors (RAR) mediates the RA effects. We analyzed initiation of lung development in retinaldehyde dehydrogenase-2 (Raldh2) null mice, a model in which RA signaling is absent from the foregut from its earliest developmental stages. We provide evidence that RA is dispensable for specification of lung cell fate in the endoderm. By using synthetic retinoids to selectively activate RAR alpha or beta signaling in this model, we demonstrate novel and unique functions of these receptors in the early lung. We show that activation of RAR beta, but not alpha, induces expression of the fibroblast growth factor Fgf10 and bud morphogenesis in the lung field. Similar analysis of wild type foregut shows that endogenous RAR alpha activity is required to maintain overall RA signaling, and to refine the RAR beta effects in the lung field. Our data support the idea that balanced activation of RAR alpha and beta is critical for proper lung bud initiation and endodermal differentiation.

  View details for DOI 10.1016/j.ydbio.2005.10.045

  View details for Web of Science ID 000236128300002

  View details for PubMedID 16427040

• Retinoic acid selectively regulates Fgf10 expression and maintains cell identity in the prospective lung field of the developing foregut DEVELOPMENTAL BIOLOGY Desai, T. J., Malpel, S., Flentke, G. R., Smith, S. M., Cardoso, W. V. 2004; 273 (2): 402-415

  Abstract

  Although respiratory tract defects that result from disruption of retinoic acid (RA) signaling have been widely reported, the mechanism by which endogenous RA regulates early lung morphogenesis is unknown. Here, we provide novel evidence that a major role for RA is to selectively maintain mesodermal proliferation and induce fibroblast growth factor 10 (Fgf10) expression in the foregut region where the lung forms. By using a pan-RAR antagonist (BMS493) in foregut explant cultures, we show that bud initiation is selectively blocked in the prospective respiratory region by failure to induce Fgf10 in the corresponding mesoderm. The RA regulation of Fgf10 expression occurs only in this region, within a defined developmental window, and is not seen in other foregut derivatives such as thyroid and pancreas where Fgf10 is also required for normal development. Furthermore, we show that RA activity is essential in the lung field to maintain lung cell identity in the endoderm; RAR antagonism disrupts expression of thyroid transcription factor 1 (Ttf1), an early marker of the respiratory region in the endoderm, and surfactant protein C (Sp-C) mRNAs. Our observations in mouse foregut cultures are corroborated by data from an in vivo model of vitamin A deficiency in rats. Our study supports RA as an essential regulator of gene expression and cellular activities during primary bud formation.

  View details for DOI 10.1016/j.ydbio.2004.04.039

  View details for Web of Science ID 000223681000018

  View details for PubMedID 15328022

• COPD: Clinical Manifestations, Diagnosis, and Treatment Baum's Textbook of Pulmonary Diseases (Eds: James D. Crapo, Jeffrey Glassroth, Joel Karlinsky, and Talmadge E. King Jr.) Desai TJ, Karlinsky, JB 2004; 7th ed
• Growth factors in lung development and disease: friends or foe? Respiratory research Desai, T. J., Cardoso, W. V. 2002; 3: 2-?

  Abstract

  Growth factors mediate tissue interactions and regulate a variety of cellular functions that are critical for normal lung development and homeostasis. Besides their involvement in lung pattern formation, growth and cell differentiation during organogenesis, these factors have been also implicated in modulating injury-repair responses of the adult lung. Altered expression of growth factors, such as transforming growth factor beta1, vascular endothelial growth factor and epidermal growth factor, and/or their receptors, has been found in a number of pathological lung conditions. In this paper, we discuss the dual role of these molecules in mediating beneficial feedback responses or responses that can further damage lung integrity; we shall also discuss the basis for their prospective use as therapeutic agents.

  View details for PubMedID 11806837

  View details for PubMedCentralID PMC64813