scholarly journals Early lineage specification defines alveolar epithelial ontogeny in the murine lung

2019 ◽  
Vol 116 (10) ◽  
pp. 4362-4371 ◽  
Author(s):  
David B. Frank ◽  
Ian J. Penkala ◽  
Jarod A. Zepp ◽  
Aravind Sivakumar ◽  
Ricardo Linares-Saldana ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Single Cell ◽  
Branching Morphogenesis ◽  
Lineage Tracing ◽  
Transcriptional Networks ◽  
Alveolar Type ◽  
Sequencing Analysis ◽  
Alveolar Cells ◽  
Lineage Specification ◽  
Alveolar Epithelial

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.

Modulation of ion conductance and active transport by TGF-β1 in alveolar epithelial cell monolayers

2003 ◽  
Vol 285 (6) ◽  
pp. L1192-L1200 ◽  
Author(s):  
Brigham C. Willis ◽  
Kwang-Jin Kim ◽  
Xian Li ◽  
Janice Liebler ◽  
Edward D. Crandall ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Active Transport ◽  
Alveolar Epithelial Cell ◽  
Alveolar Type ◽  
Type I ◽  
Ion Conductance ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers ◽  
Tgf Β1

Transforming growth factor-β1 (TGF-β1) may be a critical mediator of lung injury and subsequent remodeling during recovery. We evaluated the effects of TGF-β1 on the permeability and active ion transport properties of alveolar epithelial cell monolayers. Rat alveolar type II cells plated on polycarbonate filters in defined serum-free medium form confluent monolayers and acquire the phenotypic characteristics of alveolar type I cells. Exposure to TGF-β1 (0.1-100 pM) from day 0 resulted in a concentration- and time-dependent decrease in transepithelial resistance ( Rt) and increase in short-circuit current ( Isc). Apical amiloride or basolateral ouabain on day 6 inhibited Isc by 80 and 100%, respectively. Concurrent increases in expression of Na+-K+-ATPase α1- and β1-subunits were observed in TGF-β1-treated monolayers. No change in the α-subunit of the rat epithelial sodium channel (α-rENaC) was seen. Exposure of confluent monolayers to TGF-β1 from day 4 resulted in an initial decrease in Rt within 6 h, followed by an increase in Isc over 72-96 h. These results demonstrate that TGF-β1 modulates ion conductance and active transport characteristics of the alveolar epithelium, associated with increased Na+-K+-ATPase, but without a change in α-rENaC.

56 Mammalian pre-implantation embryos at the single cell level: The bovine as a model for early human embryonic development

2020 ◽  
Vol 32 (2) ◽  
pp. 153
Author(s):  
N. Ortega ◽  
V. Ahola ◽  
A. Plaza-Reyes ◽  
J. Schell ◽  
P. Kumar ◽  
...  
Keyword(s):  
Single Cell ◽  
Human Embryo ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bovine Embryo ◽  
Sequencing Analysis ◽  
Bovine Embryos ◽  
Primitive Endoderm ◽  
Lineage Specification ◽  
Early Human

Single-cell transcriptomics and gene editing on human pre-implantation embryos have proved that mechanisms previously identified and well characterised in mouse pre-implantation development may not hold true in the human embryo. However, ethical and legal concerns limit the availability of surplus human embryos for research, resulting in the need for novel animal models. Bovine embryos share morphological and temporal resemblance with human early development; still, key molecular and cellular developmental mechanisms need to be further explored. In the present study, we performed a comparative single-cell RNA sequencing analysis across multiple pre-implantational developmental stages of invitro-derived bovine, human (Petropoulos et al. 2016 Cell 165, 1012-1026; https://doi.org/10.1016/j.cell.2016.03.023), and mouse embryos (Cheng et al. 2019 Cell Rep. 26, 2593-2607, https://doi.org/10.1016/j.celrep.2019.02.031; Posfai et al. 2017 eLife 6, e22906, https://doi.org/10.7554/eLife.22906; Chen et al. 2016 Genome Res. 26, 1342-1354). In total 497 blastomeres corresponding to embryonic days (E) E4-E8 were prepared using Smart sEqn 2, Illumina HiSEqn 2500 sequencing with 50-bp single-end reads. Embryonic stage was defined by cell numbers and morphology as multicellular stage (Mu, ~E4), morulae (M; E5), early blastocysts (EB, ~6), mid-expanded blastocysts, (MB, ~E7), and late expanded/hatched blastocysts (LB, ~E8). We found top differentially expressed genes elucidating how lineage specification and pluripotency is controlled in the early bovine embryo. Our transcriptomic data showed that the first lineage segregation in the pre-implantation bovine embryo occurs after cavitation at E6 in the early blastocyst, suggesting a similarity with the early human blastocyst. In addition, E7 showed distinctive and more mature inner cell mass (ICM) and trophectoderm (TE) profiles. Different from the mouse, where the first lineage segregation of TE-ICM is suggested to occur at the morula stage due to CDX2-OCT4 antagonism, we found that the expression of OCT4 protein cattle expanded blastocysts is not restricted to the ICM, similar to what has been reported to occur in the human embryo. Interestingly, we also found that the second lineage segregation, which segregates the epiblast from the primitive endoderm within the ICM, starts in bovine at E8 expanded hatched late blastocysts, suggesting a potential difference with humans, where TE-ICM and epiblast-primitive endoderm has previously been reported to occur almost concurrently. We are now complementing our findings with genome editing in bovine embryos by generating knockout embryos of different target genes part of an evolutionarily conserved signaling pathway to study their effects in the early pre-implantation embryo. We aim to elucidate how early lineage specification and pluripotency establishment occurs while offering theoretical support for efficient derivation and culture of bovine embryonic stem cells.

scholarly journals Simultaneous isolation of endothelial and alveolar epithelial type I and type II cells during mouse lung development in the absence of a transgenic reporter

2020 ◽  
Vol 318 (4) ◽  
pp. L619-L630 ◽  
Author(s):  
Yves Donati ◽  
Sanja Blaskovic ◽  
Isabelle Ruchonnet-Métrailler ◽  
Josefina Lascano Maillard ◽  
Constance Barazzone-Argiroffo
Keyword(s):  
Epithelial Cell ◽  
De Novo ◽  
Morphological Changes ◽  
Alveolar Epithelial Cell ◽  
Mouse Lung ◽  
Cell Populations ◽  
Type I ◽  
Type Ii ◽  
Alveolar Cells ◽  
Alveolar Epithelial

Mouse lung developmental maturation and final alveolarization phase begin at birth. During this dynamic process, alveolar cells modify their morphology and anchorage to the extracellular matrix. In particular, alveolar epithelial cell (AEC) type I undergo cytoplasmic flattening and folding to ensure alveoli lining. We developed FACS conditions for simultaneous isolation of alveolar epithelial and endothelial cells in the absence of specific reporters during the early and middle alveolar phase. We evidenced for the first time a pool of extractable epithelial cell populations expressing high levels of podoplanin at postnatal day (pnd)2, and we confirmed by RT-qPCR that these cells are already differentiated but still immature AEC type I. Maturation causes a decrease in isolation yields, reflecting the morphological changes that these cell populations are undergoing. Moreover, we find that major histocompatibility complex II (MHCII), reported as a good marker of AEC type II, is poorly expressed at pnd2 but highly present at pnd8. Combined experiments using LysoTracker and MHCII demonstrate the de novo acquisition of MCHII in AEC type II during lung alveolarization. The lung endothelial populations exhibit FACS signatures from vascular and lymphatic compartments. They can be concomitantly followed throughout alveolar development and were obtained with a noticeable increased yield at the last studied time point (pnd16). Our results provide new insights into early lung alveolar cell isolation feasibility and represent a valuable tool for pure AEC type I preparation as well as further in vitro two- and three-dimensional studies.

scholarly journals Modulation of T1α expression with alveolar epithelial cell phenotype in vitro

1998 ◽  
Vol 275 (1) ◽  
pp. L155-L164 ◽  
Author(s):  
Zea Borok ◽  
Spencer I. Danto ◽  
Richard L. Lubman ◽  
Yuxia Cao ◽  
Mary C. Williams ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Shape ◽  
Alveolar Epithelial Cell ◽  
Cell Phenotype ◽  
Alveolar Type ◽  
Type I ◽  
Soluble Factors ◽  
Alveolar Epithelial

T1α is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1α expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1α expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1α expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1(DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1α expression was quantified by Northern and Western blotting, respectively. Expression of T1α progressively increases in AEC grown in MDSF ± BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1α expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1α on days 4 and 8. T1α expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1α expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.

scholarly journals Heat-Killed Fusobacterium nucleatum Triggers Varying Heme-Related Inflammatory and Stress Responses Depending on Primary Human Respiratory Epithelial Cell Type

Molecules ◽  
2020 ◽  
Vol 25 (17) ◽  
pp. 3839
Author(s):  
Ryo Koike ◽  
Marni E. Cueno ◽  
Keiko Nodomi ◽  
Muneaki Tamura ◽  
Noriaki Kamio ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Inflammatory Response ◽  
Stress Responses ◽  
Inflammatory Responses ◽  
Fusobacterium Nucleatum ◽  
Alveolar Epithelial Cells ◽  
Cell Type ◽  
Alveolar Cells ◽  
Epithelial Cell Type ◽  
Alveolar Epithelial

Fusobacterium nucleatum (Fn) is generally an opportunistic oral pathogen that adheres to mammalian mucosal sites, triggering a host inflammatory response. In general, Fn is normally found within the human oral cavity; however, it was previously reported that Fn is a risk factor for certain respiratory diseases. Surprisingly, this was never fully elucidated. Here, we investigated the virulence potential of heat-killed Fn on primary human tracheal, bronchial, and alveolar epithelial cells. In this study, we measured the secretion of inflammatory- (IL-8 and IL-6), stress- (total heme and hydrogen peroxide), and cell death-related (caspase-1 and caspase-3) signals. We established that the inflammatory response mechanism varies in each epithelial cell type: (1) along tracheal cells, possible Fn adherence would trigger increased heme secretion and regulated inflammatory response; (2) along bronchial cells, potential Fn adherence would simultaneously initiate an increase in secreted H2O2 and inflammatory response (ascribable to decreased secreted heme amounts); and (3) along alveolar cells, putative Fn adherence would instigate the increased secretion of inflammatory responses attributable to a decrease in secreted heme levels. Moreover, regardless of the epithelial cell-specific inflammatory mechanism, we believe these are putative, not harmful. Taken together, we propose that any potential Fn-driven inflammation along the respiratory tract would be initiated by differing epithelial cell-specific inflammatory mechanisms that are collectively dependent on secreted heme.

Src kinase integrates PI3K/Akt and MAPK/ERK1/2 pathways in T3-induced Na-K-ATPase activity in adult rat alveolar cells

2011 ◽  
Vol 301 (5) ◽  
pp. L765-L771 ◽  
Author(s):  
Jianxun Lei ◽  
David H. Ingbar
Keyword(s):  
Atpase Activity ◽  
Src Kinase ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Akt Inhibitor ◽  
Alveolar Cells ◽  
Alveolar Epithelial ◽  
Adult Rat ◽  
Constitutively Active Mutants ◽  
Constitutively Active

We previously reported that the 3,5,3′-triiodo-l-thyronine (T3)-induced increase of Na-K-ATPase activity in rat alveolar epithelial cells (AECs) required activation of Src kinase, PI3K, and MAPK/ERK1/2. In the present study, we assessed the role of Akt in Na-K-ATPase activity and the interaction between the PI3K and MAPK in response to T3 by using MP48 cells, inhibitors, and constitutively active mutants in the MP48 (alveolar type II-like) cell line. The Akt inhibitor VIII blocked T3-induced increases in Na-K-ATPase activity and amount of plasma membrane Na-K-ATPase protein. The Akt inhibitor VIII also abolished the increase in Na-K-ATPase activity induced by constitutively active mutants of either Src kinase or PI3K. Moreover, constitutively active mutants of Akt increased Na-K-ATPase activity in the absence of T3. Thus activation of Akt was required for T3-induced Na-K-ATPase activity in AECs and is sufficient in the absence of T3. Inhibitors of Src kinase (PP1), PI3K (wortmannin), and ERK1/2 (U0126) all blocked the T3-induced Na-K-ATPase activity. PP1 blocked the activation of PI3K and also ERK1/2 by T3, whereas U0126 did not prevent T3 activation of Src kinase or PI3K activity. Wortmannin did not significantly alter T3-increased MAPK/ERK1/2 activity, suggesting that T3-activated PI3K/Akt and MAPK/ERK1/2 pathways acted downstream of the Src kinase. Furthermore, in the absence of T3, a constitutively active mutant of Src kinase increased activities of Na-K-ATPase, PI3K, and MAPK/ERK1/2. A constitutively active mutant of PI3K enhanced Na-K-ATPase activity but did not alter the MAPK/ERK1/2 activity significantly. In summary, in adult rat AECs T3-stimulated Src kinase activity can activate both PI3K/Akt and MAPK/ERK1/2, and activation of Akt is necessary for T3-induced Na-K-ATPase activity.

scholarly journals The Normal and Fibrotic Mouse Lung in Situ Classified by High Dimensional Single Cell Analysis on Routinely Processed Tissue

2021 ◽  
Author(s):  
Roberta Ciccimarra ◽  
Maddalena M. Bolognesi ◽  
Matteo Zoboli ◽  
Giorgio Cattoretti ◽  
Fabio F. Stellari ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Progressive Increase ◽  
Mouse Lung ◽  
Alveolar Type ◽  
Type I ◽  
Alveolar Cells ◽  
Antibody Staining ◽  
Fibrotic Process

Abstract Single cell classification is elucidating homeostasis and pathology in tissues and whole organs. We applied in situ spatial proteomics by multiplex antibody staining to routinely processed mouse lung, healthy and during a fibrosis model. With a limited validated antibody panel (24) we classify the normal constituents (alveolar type I and II, bronchial epithelia, endothelial, muscular, stromal and hematopoietic cells) and by quantitative measurements, we show the progress of lung fibrosis over a 4 weeks course, the changing landscape and the cell-specific quantitative variation of a multidrug transporter. An early decline in AT2 alveolar cells and a progressive increase in stromal cells seems at the core of the fibrotic process.

IL-23 amplifies the epithelial-mesenchymal transition of mechanically conditioned alveolar epithelial cells in RA-ILD through mTOR/S6 signaling

2021 ◽  
Author(s):  
Chujie Zhang ◽  
Shaohua Wang ◽  
Jessica Lau ◽  
Anja C. Roden ◽  
Eric L. Matteson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Lung Fibrosis ◽  
Epithelial Mesenchymal Transition ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type I ◽  
Sequencing Analysis ◽  
Mesenchymal Transition ◽  
Caveolin 1 ◽  
Alveolar Epithelial

Epithelial-mesenchymal transition (EMT) creates an environment facilitating fibrosis following alveolar epithelial cell injury. IL-23 has important roles in chronic autoimmune conditions like rheumatoid arthritis (RA), but its role in the interstitial lung disease that affects RA patients is unclear. This study aimed to determine the pro-fibrogenic role of IL-23 on somatic alveolar type I (ATI) epithelial cells. Primary ATI cells were isolated from rats and cultured on plastic dishes for 1-3 weeks. After prolonged culture (≥14 days) on rigid culture dishes, primary ATI cells gradually acquired a mesenchymal phenotype, identified by decreased expression of caveolin-1, and reorganization of F-actin cytoskeleton, indicating the initiation of EMT by matrix stiffness. To determine how IL-23 promotes EMT in vitro, transitioning ATI cells, cultured on a stiff substrate for ≥14 days were stimulated with IL-23. The EMT phenotype was significantly enhanced by IL-23 which upregulated α-SMA, collagen I/III protein, and decreased caveolin-1. Furthermore, IL-23 significantly promoted cell invasion as well as apoptotic resistance on transitioning ATI cells. Mechanistically, IL-23 induced EMT was mTOR/S6 signaling dependent and reversible by rapamycin. Transcriptional sequencing analysis of human lung fibrosis biopsy tissue revealed key roles for IL-23 in RA-ILD. This result was further validated by significantly upregulated IL-23 expression at the mRNA level in RA-ILD lung sections. Notably, transitioning ATI epithelial cells were abundantly detected in RA-ILD tissue. Taken together, these data support a role for IL-23 in the pathogenesis of RA lung fibrosis by promoting EMT in alveolar epithelial cells through mTOR/S6 signaling.

Polarized distribution of Na(+)-H+ antiport activity in rat alveolar epithelial cells

1994 ◽  
Vol 266 (2) ◽  
pp. L138-L147 ◽  
Author(s):  
R. L. Lubman ◽  
E. D. Crandall
Keyword(s):  
Epithelial Cell ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelium ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Tissue Resistance ◽  
Recovery From Acidification ◽  
Epithelial Cell Monolayers

In this study, we investigated the polarized distribution of Na(+)-H+ antiport activity in alveolar epithelial cell monolayers. Rat alveolar type II pneumocytes were grown on detachable tissue culture-treated Nuclepore filters. The membrane filters, with their adherent intact alveolar epithelial cell monolayers, were mounted in a cuvette designed to contain two fluid compartments separated by the monolayer. Cells were loaded with the pH-sensitive dye 2',7'-biscarboxyethyl-5,6-carboxylfluorescein and intracellular pH (pHi) measured spectrofluorometrically. Monolayers were studied at ambient temperature on days 3–4 in culture, coincident with the development of high tissue resistance (RT > or = 2000 omega.cm2). Cells were incubated in HCO(3-)-free Na+ buffer [(in mM) 140 NaCl, 6 HEPES, pH 7.4] and acidified by NH3 prepulse. Rates of realkalinization (JH+) were calculated as the product of the initial rate of recovery (dpHi/dt) and the intracellular buffer capacity (beta i). Under control conditions, recovery occurred with an initial JH+ of 28.4 mM/min. When 100 microM dimethylamiloride (DMA), an amiloride analogue with enhanced specificity for inhibiting the Na(+)-H+ antiporter, was present in the basolateral fluid, recovery was inhibited by > 90%. Conversely, when the monolayers were acidified in Na+ buffer containing DMA (100 microM) in the apical fluid, acidification and recovery were identical to control. Recovery from acidification was inhibited by basolateral DMA with a one-half maximal inhibitory concentration (IC50) of 100 nm and by basolateral amiloride with an IC50 of 10 microns. Recovery was completely inhibited by omission of Na+ from the basolateral fluid, but omission of Na+ from apical fluid had no effect. We conclude that Na(+)-H+ antiport activity is located exclusively on the basolateral surface of these alveolar epithelial cell monolayers, where it most likely represents the high-amiloride affinity isoform of the Na(+)-H+ antiporter, NHE-1. The Na(+)-H+ antiporter, asymmetrically distributed to the basolateral surface of the polarized alveolar epithelium, contributes to intracellular homeostasis in alveolar pneumocytes and may also play a role in signal transduction in these cells.

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