Airway Smooth Muscle Differentiation Research Articles

CPLANE protein INTU regulates growth and patterning of the mouse lungs through cilia-dependent Hh signaling

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs.

Plasticity in airway smooth muscle differentiation during mouse lung development.

It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.

Patterning the embryonic pulmonary mesenchyme

SummarySmooth muscle guides the morphogenesis of several epithelia during organogenesis, including the mammalian airways. However, it remains unclear how airway smooth muscle differentiation is spatiotemporally patterned and whether it originates from transcriptionally distinct mesenchymal progenitors. Using single-cell RNA-sequencing of embryonic mouse lungs, we show that the pulmonary mesenchyme contains a continuum of cell identities, but no transcriptionally distinct progenitors. Transcriptional variability correlates with spatially distinct sub-epithelial and sub-mesothelial mesenchymal compartments that are regulated by Wnt signaling. Live-imaging and tension-sensors reveal compartment-specific migratory behaviors and cortical forces and show that sub-epithelial mesenchyme contributes to airway smooth muscle. Reconstructing differentiation trajectories reveals early activation of cytoskeletal and Wnt signaling genes. Consistently, Wnt activation induces the earliest stages of smooth muscle differentiation and local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our single-cell approach uncovers the principles of pulmonary mesenchymal patterning and identifies a morphogenetically active mesenchymal layer that sculpts the airway epithelium.

Plasticity in Airway Smooth Muscle Differentiation During Mouse Lung Development

Plasticity in Airway Smooth Muscle Differentiation During Mouse Lung Development

Smooth Muscle Differentiation Is Essential for Airway Size, Tracheal Cartilage Segmentation, but Dispensable for Epithelial Branching.

Airway smooth muscle is best known for its role as an airway constrictor in diseases such as asthma. However, its function in lung development is debated. A prevalent model, supported by invitro data, posits that airway smooth muscle promotes lung branching through peristalsis and pushing intraluminal fluid to branching tips. Here, we test this model invivo by inactivating Myocardin, which prevented airway smooth muscle differentiation. We found that Myocardin mutants show normal branching, despite the absence of peristalsis. In contrast, tracheal cartilage, vasculature, and neural innervation patterns were all disrupted. Furthermore, airway diameter is reduced in the mutant, counter to the expectation that the absence of smooth muscle constriction would lead to a more relaxed and thereby wider airway. These findings together demonstrate that during development, while airway smooth muscle is dispensable for epithelial branching, it is integral for building the tracheal architecture and promoting airway growth.

Airway Smooth Muscle Differentiation is Essential for Promoting Airway Size, Tracheal Cartilage Segmentation, But Dispensable for Epithelial Branching

Airway smooth muscle is best known for its role as an airway constrictor in the pathogenesis of lung diseases such as asthma. However, its function in lung development is poorly understood. A prevalent model, supported by in vitro data, posits that airway smooth muscle promotes lung branching through peristalsis and pushing intraluminal fluid to the tips of the branching epithelium. Here, we test this model in vivo by inactivating Myocardin in the lung mesenchyme, thereby preventing airway smooth muscle cell differentiation. We found that Myocardin mutants show normal branching, despite absence of peristalsis. In contrast, tracheal cartilage, vasculature, and neural innervation patterns were all disrupted in the mutant. Furthermore, airway diameter is reduced in the mutant, counter to the expectation that the absence of smooth muscle constriction would lead to a more relaxed and thereby wider airway. These findings together demonstrate that during development, while airway smooth muscle is dispensable for epithelial branching, it is integral for building the tracheal architecture and promoting airway growth.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Grape expectations (Fibroblast growth factor 9 signaling inhibits airway smooth muscle differentiation in mouse lung by Lan Yi, Eric T. Domyan, Mark Lewandoski, Xin Sun, Dev Dyn 238:123–137) Mammalian lungs have a characteristic “bunch of grapes” morphology: the lungs are stems, its proximal end adjacent to bronchioles, and distal end closest to grape-shaped terminal sacs. Evidence suggests that localization of differentiated airway smooth muscle cells (SMCs) in the proximal, but not distal, lung is critical for organ function. Previous work showed that development of SMCs in the mesenchyme is regulated by FGF9 signaling from inner epithelial and outer mesothelial layers. With the low hanging fruit already picked, Yi and colleagues further tease apart the genetics of SMC development and differentiation. They show that knockout mice lacking Fgf9, or Fgf receptors 1 and 2 (Fgfr1;2) from lung mesenchyme both have ectopic airway SMCs in distal mesenchyme, suggesting FGF9 signals through FGFR1;2 to keep differentiated SMCs from the distal lung. Where do ectopic SMCs come from? Reduced Fgf10 expression in the distal mesenchyme, a marker for SMC precursors, suggests ectopic SMCs arise from precocious differentiation of progenitors. They further present genetic and pharmacological data demonstrating that SHH works in a pathway parallel to FGF9 to promote SMC differentiation. Tell it through the grapevine, their work suggests that FGF9 has dual functions: first to promote SMC precursor proliferation, and second to inhibit SMC differentiation from the distal lung. Wringing out the truth (Dynamic organization and plasticity of sponge bodies by Mark J. Snee, Paul M. Macdonald, Dev Dyn 238:918–930) Sponge bodies, cytoplasmic structures found in Drosophila egg chamber germline cells, are so named for their resemblance to a porous cleaning sponge. These ill-defined structures are thought to be akin to P-bodies, sites of mRNA storage, degradation, and posttranscriptional regulation. This idea is bolstered by Snee and Macdonald's discovery that sponge bodies bear four of six posttranscriptional regulators tested; of these regulators, Bru is enriched in nurse cells, and Orb in the oocyte. Close observation revealed that bodies exchange these components quickly, within 1 min. after entry into the oocyte from interconnected nurse cells. This finding is the first indication that sponge bodies are dynamic. The second indication is the bodies' dramatic morphological transformation under varying environmental conditions. Porous, “dispersed bodies” predominate when females are given food that favor egg-laying, while “reticulated bodies” predominate in virgins, and in females fed more standard fare. The third indication is that photobleaching experiments suggest Stau::GFP, and presumably associated oskar mRNA, moves transiently through sponge bodies as it makes its way to the posterior egg chamber as it matures. While some scrubbing sponges are made from cellulose, these data cleanly show that these sponge bodies are plastic. Magnetic method (Ex vivo magnetofection: A novel strategy for the study of gene function in mouse organogenesis by Terje Svingen, Dagmar Wilhelm, Alexander N. Combes, Brett Hosking, Vincent R. Harley, Andrew H. Sinclair, Peter Koopman, Dev Dyn 238:956–964) Most developmental biologists are familiar with the headaches of mouse transgenics: it is expensive, time-consuming, and technically challenging. Other gene transfer techniques, such as electroporation, can also be impractical or ineffective depending on experimental conditions. Koopman and colleagues have found that magnetofection is a viable alternative for their organ system, explanted genital ridges. With this technique, DNA is bound to magnetic particles, injected into the tissue of interest, and pulled into cells when placed next to a magnetic field. As proof-of-principle, genetic and morphological signs of sex reversal were triggered by magnetofection-induced ectopic expression of Sry in XX genital ridges, and by small interfering RNAs (siRNAs) against Sox9 in XY ridges. Using magnetofection, the authors misexpressed the Sertoli cell–specific Tmem184a in XX genital ridges, which is thought to promote the spermatogonia pathway in germ cells. Germ cells were prevented from entering meiosis—a prerequisite for female germ cell development—providing a mechanism of action for the gene. Although only 20% of genital ridges expressed introduced constructs, efficiency may improve with further experimental refinement. For those seeking a new way to alter gene expression in a developmental system, the pull of magnetofection may be hard to resist.

Fibroblast growth factor 9 signaling inhibits airway smooth muscle differentiation in mouse lung.

In mammalian lungs, airway smooth muscle cells (airway SMCs) are present in the proximal lung adjacent to bronchi and bronchioles, but are absent in the distal lung adjacent to terminal sacs that expand during gas exchange. Evidence suggests that this distribution is essential for the formation of a functional respiratory tree, but the underlying genetic mechanism has not been elucidated. In this study, we test the hypothesis that fibroblast growth factor 9 (Fgf9) signaling is essential to restrict SMC differentiation to the proximal lung. We show that loss of Fgf9 or conditional inactivation of Fgf receptors (Fgfr) 1 and 2 in mouse lung mesenchyme results in ectopic SMCs. Our data support a model where FGF9 maintains a SMC progenitor population by suppressing differentiation and promoting growth. This model also represents our findings on the genetic relationship between FGF9 and sonic hedgehog (SHH) in the establishment of airway SMC pattern.

Integrin-linked kinase regulates smooth muscle differentiation marker gene expression in airway tissue

Phenotypic changes in airway smooth muscle occur with airway inflammation and asthma. These changes may be induced by alterations in the extracellular matrix that initiate signaling pathways mediated by integrin receptors. We hypothesized that integrin-linked kinase (ILK), a multidomain protein kinase that binds to the cytoplasmic tail of beta-integrins, may be an important mediator of signaling pathways that regulate the growth and differentiation state of airway smooth muscle. We disrupted signaling pathways mediated by ILK in intact differentiated tracheal muscle tissues by depleting ILK protein using ILK antisense. The depletion of ILK protein increased the expression of the smooth muscle differentiation marker genes myosin heavy chain (SmMHC), SM22alpha, and calponin and increased the expression of SmMHC protein. Conversely, the overexpression of ILK protein reduced the mRNA levels of SmMHC, SM22alpha, and calponin and SmMHC protein. Analysis by chromatin immunoprecipitation showed that the binding of the transcriptional regulator serum response factor (SRF) to the promoters of SmMHC, SM22alpha, and calponin genes was increased in ILK-depleted tissues and decreased in tissues overexpressing ILK. ILK depletion also increased the amount of SRF that localized within the nucleus. ILK depletion and overexpression, respectively, decreased and increased the activation of its downstream substrate protein kinase B (PKB/Akt). The pharmacological inhibition of Akt activity also increased SRF binding to the promoters of smooth muscle-specific genes and increased expression of smooth muscle proteins, suggesting that ILK may exert its effects by regulating the activity of Akt. We conclude that ILK is a critical regulator of airway smooth muscle differentiation. ILK may mediate signals from integrin receptors that control airway smooth muscle differentiation in response to alterations in the extracellular matrix.

Mast Cells Promote Airway Smooth Muscle Cell Differentiation via Autocrine Up-Regulation of TGF-β1

Asthma is a major cause of morbidity and mortality worldwide. It is characterized by airway dysfunction and inflammation. A key determinant of the asthma phenotype is infiltration of airway smooth muscle bundles by activated mast cells. We hypothesized that interactions between these cells promotes airway smooth muscle differentiation into a more contractile phenotype. In vitro coculture of human airway smooth muscle cells with beta-tryptase, or mast cells with or without IgE/anti-IgE activation, increased airway smooth muscle-derived TGF-beta1 secretion, alpha-smooth muscle actin expression and agonist-provoked contraction. This promotion to a more contractile phenotype was inhibited by both the serine protease inhibitor leupeptin and TGF-beta1 neutralization, suggesting that the observed airway smooth muscle differentiation was driven by the autocrine release of TGF-beta1 in response to activation by mast cell beta-tryptase. Importantly, in vivo we found that in bronchial mucosal biopsies from asthmatics the intensity of alpha-smooth muscle actin expression was strongly related to the number of mast cells within or adjacent to an airway smooth muscle bundle. These findings suggest that mast cell localization in the airway smooth muscle bundle promotes airway smooth muscle cell differentiation into a more contractile phenotype, thus contributing to the disordered airway physiology that characterizes asthma.

EMBRYONIC LUNG GROWTH IS NORMAL IN A cftr-KNOCKOUT MOUSE MODEL

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in embryonic lung growth remains uncertain. The authors used an established embryonic lung culture model to investigate the impact of cftr knockout on lung growth, airway peristalsis, and airway smooth muscle (ASM) distribution. Lung area, perimeter, lung bud count, and frequency of contraction were similar in wild-type (cftr +/+) and cftr knockout mice (cftr −/−). The percentage of mitotic cells was also consistent between genotypes in mesenchyme and epithelium. Smooth muscle distribution surrounding the airway appeared normally distributed in all genotypes. These data suggest that normal embryonic lung growth, ASM differentiation and airway peristalsis are CFTR independent.