Sox9 is associated with two distinct patterning events during snake lung morphogenesis.

The evolutionary forces that allowed species adaptation to different terrestrial environments and led to great diversity in body shape and size required acquisition of innovative strategies of pattern formation during organogenesis. An extreme example is the formation of highly elongated viscera in snakes. What developmental patterning strategies allowed to overcome the space constraints of the snake's body to meet physiological demands? Here we show that the corn snake uses a Sox2-Sox9 developmental tool kit common to other species to generate and shape the lung in two phases. Initially Sox9 was found at low levels at the tip of the primary lung bud during outgrowth and elongation of the bronchial bud, without driving branching programs characteristic of mammalian lungs. Later, Sox9 induction is recapitulated in the formation of an extensive network of radial septae emerging along the elongated bronchial bud that generates the respiratory region. We propose that altogether these represent key patterning events for formation of both the respiratory faveolar and non-respiratory posterior compartments of the snake's lung.

Yap and its subcellular localization have distinct compartment-specific roles in the developing lung.

Although the Hippo-yes-associated protein (Yap) pathway has been implicated in lung development, the specific roles for Yap and its nucleocytoplasmic shuttling in the developing airway and alveolar compartments remain elusive. Moreover, conflicting results from expression studies and differences in the lung phenotypes of Yap and Hippo kinase null mutants caused controversy over the dynamics and significance of Yap subcellular localization in the developing lung. Here, we show that the aberrant morphogenesis of Yap-deficient lungs results from the disruption of developmental events specifically in distal epithelial progenitors. We also show that activation of nuclear Yap is enough to fulfill the Yap requirements to rescue abnormalities in these lungs. Remarkably, we found that Yap nucleocytoplasmic shuttling is largely dispensable in epithelial progenitors for both branching morphogenesis and sacculation. However, if maintained transcriptionally active in airways, nuclear Yap profoundly alters proximal-distal identity and halts epithelial differentiation. Taken together, these observations provide novel insights into the crucial importance of Hippo-Yap signaling in the lung prenatally.

Use of hPSC-derived 3D organoids and mouse genetics to define the roles of YAP in the development of the esophagus.

Balanced progenitor activities are crucial for the development and maintenance of high turn-over organs such as the esophagus. However, the molecular mechanisms regulating these progenitor activities in the esophagus remain to be elucidated. Here, we demonstrated that Yap is required for the proliferation of esophageal progenitor cells (EPCs) in the developing murine esophagus. We found that Yap deficiency reduces EPC proliferation and stratification whereas persistent Yap activation increases cell proliferation and causes aberrant stratification of the developing esophagus. We further demonstrated that the role of YAP signaling is conserved in the developing human esophagus by utilizing 3D human pluripotent stem cell (hPSC)-derived esophageal organoid culture. Taken together, our studies combining loss/gain-of-function murine models and hPSC differentiation support a key role for YAP in the self-renewal of EPCs and stratification of the esophageal epithelium.

A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships.

Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of an HS-mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell. We used this library to (1) determine that the strictly defined fine structure of HS, not its overall degree of sulfation, is more important for FGF2-FGFR1 signaling; (2) define the epitope features of commonly used anti-HS phage display antibodies; and (3) delineate the fine inter-regulation networks by which HS genes modify HS and chain length in mammalian cells at a cell-type-specific level. Our mutant-cell library will allow robust and systematic interrogation of the roles and related structures of HS in a cellular context.

Human airway branch variation and chronic obstructive pulmonary disease.

Susceptibility to chronic obstructive pulmonary disease (COPD) beyond cigarette smoking is incompletely understood, although several genetic variants associated with COPD are known to regulate airway branch development. We demonstrate that in vivo central airway branch variants are present in 26.5% of the general population, are unchanged over 10 y, and exhibit strong familial aggregation. The most common airway branch variant is associated with COPD in two cohorts (n = 5,054), with greater central airway bifurcation density, and with emphysema throughout the lung. The second most common airway branch variant is associated with COPD among smokers, with narrower airway lumens in all lobes, and with genetic polymorphisms within the FGF10 gene. We conclude that central airway branch variation, readily detected by computed tomography, is a biomarker of widely altered lung structure with a genetic basis and represents a COPD susceptibility factor.

Epithelial Notch signaling regulates lung alveolar morphogenesis and airway epithelial integrity.

Abnormal enlargement of the alveolar spaces is a hallmark of conditions such as chronic obstructive pulmonary disease and bronchopulmonary dysplasia. Notch signaling is crucial for differentiation and regeneration and repair of the airway epithelium. However, how Notch influences the alveolar compartment and integrates this process with airway development remains little understood. Here we report a prominent role of Notch signaling in the epithelial-mesenchymal interactions that lead to alveolar formation in the developing lung. We found that alveolar type II cells are major sites of Notch2 activation and show by Notch2-specific epithelial deletion (Notch2(cNull)) a unique contribution of this receptor to alveologenesis. Epithelial Notch2 was required for type II cell induction of the PDGF-A ligand and subsequent paracrine activation of PDGF receptor-α signaling in alveolar myofibroblast progenitors. Moreover, Notch2 was crucial in maintaining the integrity of the epithelial and smooth muscle layers of the distal conducting airways. Our data suggest that epithelial Notch signaling regulates multiple aspects of postnatal development in the distal lung and may represent a potential target for intervention in pulmonary diseases.

Notch3-Jagged signaling controls the pool of undifferentiated airway progenitors.

Basal cells are multipotent airway progenitors that generate distinct epithelial cell phenotypes crucial for homeostasis and repair of the conducting airways. Little is known about how these progenitor cells expand and transition to differentiation to form the pseudostratified airway epithelium in the developing and adult lung. Here, we show by genetic and pharmacological approaches that endogenous activation of Notch3 signaling selectively controls the pool of undifferentiated progenitors of upper airways available for differentiation. This mechanism depends on the availability of Jag1 and Jag2, and is key to generating a population of parabasal cells that later activates Notch1 and Notch2 for secretory-multiciliated cell fate selection. Disruption of this mechanism resulted in aberrant expansion of basal cells and altered pseudostratification. Analysis of human lungs showing similar abnormalities and decreased NOTCH3 expression in subjects with chronic obstructive pulmonary disease suggests an involvement of NOTCH3-dependent events in the pathogenesis of this condition.

Neuroepithelial body microenvironment is a niche for a distinct subset of Clara-like precursors in the developing airways.

Clara cells of mammalian airways have multiple functions and are morphologically heterogeneous. Although Notch signaling is essential for the development of these cells, it is unclear how Notch influences Clara cell specification and if diversity is established among Clara cell precursors. Here we identify expression of the secretoglobin Scgb3a2 and Notch activation as early events in a program of secretory cell fate determination in developing murine airways. We show that Scgb3a2 expression in vivo is Notch-dependent at early stages and ectopically induced by constitutive Notch1 activation, and also that in vitro Notch signaling together with the pan-airway transcription factor Ttf1 (Nkx2.1) synergistically regulate secretoglobin gene transcription. Furthermore, we identified a subpopulation of secretory precursors juxtaposed to presumptive neuroepithelial bodies (NEBs), distinguished by their strong Scgb3a2 and uroplakin 3a (Upk3a) signals and reduced Ccsp (Scgb1a1) expression. Genetic ablation of Ascl1 prevented NEB formation and selectively interfered with the formation of this subpopulation of cells. Lineage labeling of Upk3a-expressing cells during development showed that these cells remain largely uncommitted during embryonic development and contribute to Clara and ciliated cells in the adult lung. Together, our findings suggest a role for Notch in the induction of a Clara cell-specific program of gene expression, and reveals that the NEB microenvironment in the developing airways is a niche for a distinct subset of Clara-like precursors.

Notch signaling prevents mucous metaplasia in mouse conducting airways during postnatal development.

Goblet cell metaplasia and mucus overproduction contribute to the pathogenesis of chronic lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). Notch signaling regulates cell fate decisions and is crucial in controlling goblet cell differentiation in the gut epithelium. Little is known, however, about how endogenous Notch signaling influences the goblet cell differentiation program that takes place in the postnatal lung. Using a combination of genetic and in vitro approaches here we provide evidence of a novel role for Notch in restricting goblet cell differentiation in the airway epithelium during the postnatal period. Conditional inactivation of the essential Notch pathway component Pofut1 (protein O-fucosyltransferase1) in Tgfb3-Cre-expressing mice resulted in an aberrant postnatal airway phenotype characterized by marked goblet cell metaplasia, decreased Clara cell number and increase in ciliated cells. The presence of the same phenotype in mice in which the Notch transcriptional effector Rbpjk was deleted indicated the involvement of the canonical Notch pathway. Lineage study in vivo suggested that goblet cells originated from a subpopulation of Clara cells largely present in proximal airways in which Notch was disrupted. The phenotype was confirmed by a panel of goblet cell markers, showed no changes in cell proliferation or altered expression of proinflammatory cytokines and was associated with significant downregulation of the bHLH transcriptional repressor Hes5. Luciferase reporter analysis suggested that Notch directly repressed MUC5AC transcription in lung epithelial cells. The data suggested that during postnatal life Notch is required to prevent Clara cells from differentiating into goblet cells.

Trinucleotide repeat containing 6a (Tnrc6a)-mediated microRNA function is required for development of yolk sac endoderm.

Tnrc6 family members (Tnrc6a/b/c) are key components of the RNA-induced silencing complex in microRNA (miRNA)-mediated gene suppression. Here, we show that Tnrc6a, also known as GW182, is selectively expressed in the yolk sac endoderm and that gene trap disruption of GW182 leads to growth arrest and apoptosis. We found that targets of miRNAs highly expressed in the yolk sac are significantly derepressed in GW182(gt/gt) mutant mice, although levels of miRNAs are not altered. Specifically, growth arrest and apoptosis phenotype are associated with significant derepression of Cdkn1a (p21), Cdkn1c (P27), Lats1, Lats2, Rb1, Rbl, Bim, and Pten, known targets of miRNAs from miR-17/20/93/106 clusters highly expressed in yolk sac endoderm. Together, these data strongly suggest that GW182 is an essential functional component in the RNA-induced silencing complex for miRNA-mediated gene silencing in vivo, and selectively regulation of miRNA activity plays an important role in the proper development of yolk sac.

A local translation program regulates centriole amplification in the airway epithelium.

Biogenesis of organelles requires targeting of a subset of proteins to specific subcellular domains by signal peptides or mechanisms controlling mRNA localization and local translation. How local distribution and translation of specific mRNAs for organelle biogenesis is achieved remains elusive and likely to be dependent on the cellular context. Here we identify Trinucleotide repeat containing-6a (Tnrc6a), a component of the miRNA pathway, distinctively localized to apical granules of differentiating airway multiciliated cells (MCCs) adjacent to centrioles. In spite of being enriched in TNRC6A and the miRNA-binding protein AGO2, they lack enzymes for mRNA degradation. Instead, we found these apical granules enriched in components of the mRNA translation machinery and newly synthesized proteins suggesting that they are specific hubs for target mRNA localization and local translation in MCCs. Consistent with this, Tnrc6a loss of function prevented formation of these granules and led to a broad reduction, rather than stabilization of miRNA targets. These included downregulation of key genes involved in ciliogenesis and was associated with defective multicilia formation both in vivo and in primary airway epithelial cultures. Similar analysis of Tnrc6a disruption in yolk sac showed stabilization of miRNA targets, highlighting the potential diversity of these mechanisms across organs.

Cellular FLICE-like inhibitory protein deviates myofibroblast fas-induced apoptosis toward proliferation during lung fibrosis.

A prominent feature of fibrotic tissue in general and of lungs in particular is fibroblast proliferation and accumulation. In patients overcoming fibrosis, apoptosis limits this excessive cell growth. We have previously shown resistance to Fas-induced apoptosis of primary lung fibroblasts from mice with bleomycin-induced lung fibrosis, their escape from immune surveillance, and continued accumulation in spite of overexpression of the Fas death receptor. Cellular FLICE-like inhibitory protein (c-FLIP) is a regulator of cell death receptor-induced apoptosis in many cell types. We aimed to determine c-FLIP levels in myofibroblasts from fibrotic lungs and to directly assess c-FLIP's role in apoptosis and proliferation of primary lung myofibroblasts. c-FLIP levels were determined by apoptosis gene array, flow cytometry, Western blot, and immunofluorescence before and after down-regulation with a specific small interfering RNA. Apoptosis was assessed by caspase cleavage in Western blot and by Annexin V affinity labeling after FACS and tissue immunofluorescence. Proliferation was assessed by BrdU uptake, also using FACS and immunofluorescence. We show that myofibroblasts from lungs of humans with idiopathic pulmonary fibrosis and from bleomycin-treated versus normal saline-treated mice up-regulate c-FLIP levels. Using the animal model, we show that fibrotic lung myofibroblasts divert Fas signaling from apoptosis to proliferation and that this requires signaling by TNF receptor-associated factor (TRAF) and NF-κB. c-FLIP down-regulation reverses the effect of Fas activation, causing increased apoptosis, decreased proliferation, and diminished recruitment of TRAF to the DISC complex. This indicates that c-FLIP is essential for myofibroblast accumulation and may serve as a potential target to manipulate tissue fibrosis.

Lacrimal gland development and Fgf10-Fgfr2b signaling are controlled by 2-O- and 6-O-sulfated heparan sulfate.

Heparan sulfate, an extensively sulfated glycosaminoglycan abundant on cell surface proteoglycans, regulates intercellular signaling through its binding to various growth factors and receptors. In the lacrimal gland, branching morphogenesis depends on the interaction of heparan sulfate with Fgf10-Fgfr2b. To address if lacrimal gland development and FGF signaling depends on 2-O-sulfation of uronic acids and 6-O-sulfation of glucosamine residues, we genetically ablated heparan sulfate 2-O and 6-O sulfotransferases (Hs2st, Hs6st1, and Hs6st2) in developing lacrimal gland. Using a panel of phage display antibodies, we confirmed that these mutations disrupted 2-O and/or 6-O but not N-sulfation of heparan sulfate. The Hs6st mutants exhibited significant lacrimal gland hypoplasia and a strong genetic interaction with Fgf10, demonstrating the importance of heparan sulfate 6-O sulfation in lacrimal gland FGF signaling. Altering Hs2st caused a much less severe phenotype, but the Hs2st;Hs6st double mutants completely abolished lacrimal gland development, suggesting that both 2-O and 6-O sulfation of heparan sulfate contribute to FGF signaling. Combined Hs2st;Hs6st deficiency synergistically disrupted the formation of Fgf10-Fgfr2b-heparan sulfate complex on the cell surface and prevented lacrimal gland induction by Fgf10 in explant cultures. Importantly, the Hs2st;Hs6st double mutants abrogated FGF downstream ERK signaling. Therefore, Fgf10-Fgfr2b signaling during lacrimal gland development is sensitive to the content or arrangement of O-sulfate groups in heparan sulfate. To our knowledge, this is the first study to show that simultaneous deletion of Hs2st and Hs6st exhibits profound FGF signaling defects in mammalian development.

Maternal smoking and the retinoid pathway in the developing lung.

BACKGROUND: Maternal smoking is a risk factor for pediatric lung disease, including asthma. Animal models suggest that maternal smoking causes defective alveolarization in the offspring. Retinoic acid signaling modulates both lung development and postnatal immune function. Thus, abnormalities in this pathway could mediate maternal smoking effects. We tested whether maternal smoking disrupts retinoic acid pathway expression and functioning in a murine model. METHODS: Female C57Bl/6 mice with/without mainstream cigarette smoke exposure (3 research cigarettes a day, 5 days a week) were mated to nonsmoking males. Cigarette smoke exposure continued throughout the pregnancy and after parturition. Lung tissue from the offspring was examined by mean linear intercept analysis and by quantitative PCR. Cell culture experiments using the type II cell-like cell line, A549, tested whether lipid-soluble cigarette smoke components affected binding and activation of retinoic acid response elements in vitro. RESULTS: Compared to tobacco-naïve mice, juvenile mice with tobacco toxin exposure had significantly (P < 0.05) increased mean linear intercepts, consistent with an alveolarization defect. Tobacco toxin exposure significantly (P < 0.05) decreased mRNA and protein expression of retinoic acid signaling pathway elements, including retinoic acid receptor alpha and retinoic acid receptor beta, with the greatest number of changes observed between postnatal days 3-5. Lipid-soluble cigarette smoke components significantly (P < 0.05) decreased retinoic acid-induced binding and activation of the retinoic acid receptor response element in A549 cells. CONCLUSIONS: A murine model of maternal cigarette smoking causes abnormal alveolarization in association with altered retinoic acid pathway element expression in the offspring. An in vitro cell culture model shows that lipid-soluble components of cigarette smoke decrease retinoic acid response element activation. It is feasible that disruption of retinoic acid signaling contributes to the pediatric lung dysfunction caused by maternal smoking.

Prominin 1 and Notch regulate ciliary length and dynamics in multiciliated cells of the airway epithelium.

Differences in ciliary morphology and dynamics among multiciliated cells of the respiratory tract contribute to efficient mucociliary clearance. Nevertheless, little is known about how these phenotypic differences are established. We show that Prominin 1 (Prom1), a transmembrane protein widely used as stem cell marker, is crucial to this process. During airway differentiation, Prom1 becomes restricted to multiciliated cells, where it is expressed at distinct levels along the proximal-distal axis of the airways. Prom1 is induced by Notch in multiciliated cells, and Notch inactivation abolishes this gradient of expression. Prom1 was not required for multicilia formation, but when inactivated resulted in longer cilia that beat at a lower frequency. Disruption of Notch resulted in opposite effects and suggested that Notch fine-tunes Prom1 levels to regulate the multiciliated cell phenotype and generate diversity among these cells. This mechanism could contribute to the innate defense of the lung and help prevent pulmonary disease.

Airway basal cells show regionally distinct potential to undergo metaplastic differentiation.

Basal cells are multipotent stem cells of a variety of organs, including the respiratory tract, where they are major components of the airway epithelium. However, it remains unclear how diverse basal cells are and how distinct subpopulations respond to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the adult murine trachea. Among these, we found two major subpopulations, collectively comprising the most uncommitted of all the pools, but with distinct gene expression signatures. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to self-renew and initiate a program of differentiation in response to environmental perturbations in primary cultures and in mouse injury models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are still being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of tracheal cartilage-deficient mouse mutants. Finally, we show that features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide novel insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.

Intravitreal Bevacizumab Is Associated With Prolonged Ventilatory Support in Preterm Infants With Bronchopulmonary Dysplasia.

BACKGROUND: Intravitreal bevacizumab (IVB), an anti-vascular endothelial growth factor (VEGF) antibody, is a widely adopted treatment for retinopathy of prematurity (ROP). Although animal studies have demonstrated that IVB inhibits alveologenesis in neonatal rat lung, the clinical influence of IVB on respiratory outcomes has not been studied. RESEARCH QUESTION: Does IVB affect the respiratory outcome in preterm infants with bronchopulmonary dysplasia? STUDY DESIGN AND METHODS: We retrospectively assessed very low birth weight (VLBW) preterm infants admitted to our neonatal ICU between January 2016 and June 2021. Furthermore, we evaluated the short-term respiratory outcomes after IVB therapy in VLBW preterm infants requiring ventilatory support at 36 weeks' postmenstrual age (PMA). RESULTS: One hundred seventy-four VLBW preterm infants with bronchopulmonary dysplasia were recruited. Eighty-eight infants showed ROP onset before being ventilator free, and 78 infants received a diagnosis of the most severe ROP before being ventilator free. Among them, 32 received a diagnosis with type 1 ROP and received IVB treatment. After adjusting for gestational age, birth body weight, and baseline respiratory status, we discovered that IVB is associated significantly with prolonged ventilatory support and a lower likelihood of becoming ventilator free (hazard ratio, 0.53; P = .03). INTERPRETATION: IVB may have a short-term respiratory adverse effect in patients requiring ventilatory support at 36 weeks' PMA. Therefore, long-term follow-up for respiratory outcomes may be considered in VLBW infants who receive IVB treatment.

Contribution of Trp63CreERT2-labeled cells to alveolar regeneration is independent of tuft cells.

Viral infection often causes severe damage to the lungs, leading to the appearance of ectopic basal cells (EBCs) and tuft cells in the lung parenchyma. Thus far, the roles of these ectopic epithelial cells in alveolar regeneration remain controversial. Here, we confirm that the ectopic tuft cells are originated from EBCs in mouse models and COVID-19 lungs. The differentiation of tuft cells from EBCs is promoted by Wnt inhibition while suppressed by Notch inhibition. Although progenitor functions have been suggested in other organs, pulmonary tuft cells don't proliferate or give rise to other cell lineages. Consistent with previous reports, Trp63CreERT2 and KRT5-CreERT2-labeled ectopic EBCs do not exhibit alveolar regeneration potential. Intriguingly, when tamoxifen was administrated post-viral infection, Trp63CreERT2 but not KRT5-CreERT2 labels islands of alveolar epithelial cells that are negative for EBC biomarkers. Furthermore, germline deletion of Trpm5 significantly increases the contribution of Trp63CreERT2-labeled cells to the alveolar epithelium. Although Trpm5 is known to regulate tuft cell development, complete ablation of tuft cell production fails to improve alveolar regeneration in Pou2f3-/- mice, implying that Trpm5 promotes alveolar epithelial regeneration through a mechanism independent of tuft cells.