Blastocyst complementation reveals that NKX2-1 establishes the proximal-peripheral boundary of the airway epithelium.

BACKGROUND: Distinct boundaries between the proximal conducting airways and more peripheral-bronchial regions of the lung are established early in foregut embryogenesis, demarcated in part by the distribution of SOX family and NKX2-1 transcription factors along the cephalo-caudal axis of the lung. We used blastocyst complementation to identify the role of NKX2-1 in the formation of the proximal-peripheral boundary of the airways in mouse chimeric embryos. RESULTS: While Nkx2-1-/- mouse embryos form primordial tracheal cysts, peripheral pulmonary structures are entirely lacking in Nkx2-1-/- mice. Complementation of Nkx2-1-/- embryos with NKX2-1-sufficient embryonic stem cells (ESCs) enabled the formation of all tissue components of the peripheral lung but did not enhance ESC colonization of the most proximal regions of the airways. In chimeric mice, a precise boundary was formed between NKX2-1-deficient basal cells co-expressing SOX2 and SOX9 in large airways and ESC-derived NKX2-1+ SOX9+ epithelial cells of smaller airways. NKX2-1-sufficient ESCs were able to selectively complement peripheral, rather than most proximal regions of the airways. ESC complementation did not prevent ectopic expression of SOX9 but restored ß-catenin signaling in Nkx2-1-/- basal cells of large airways. CONCLUSIONS: NKX2-1 and ß-catenin function in an epithelial cell-autonomous manner to establish the proximal-peripheral boundary along developing airways.

Lineage-specific regulation of inducible and constitutive mast cells in allergic airway inflammation.

Murine mast cells (MCs) contain two lineages: inducible bone marrow-derived mucosal MCs (MMCs) and constitutive embryonic-derived connective tissue MCs (CTMCs). Here, we use RNA sequencing, flow cytometry, and genetic deletion in two allergic lung inflammation models to define these two lineages. We found that inducible MCs, marked by ß7 integrin expression, are highly distinct from airway CTMCs at rest and during inflammation and unaffected by targeted CTMC deletion. ß7High MCs expand and mature during lung inflammation as part of a TGF-ß-inducible transcriptional program that includes the MMC-associated proteases Mcpt1 and Mcpt2, the basophil-associated protease Mcpt8, granule components, and the epithelial-binding αE integrin. In vitro studies using bone marrow-derived MCs (BMMCs) identified a requirement for SCF in this this TGF-ß-mediated development and found that epithelial cells directly elicit TGF-ß-dependent BMMC up-regulation of mMCP-1 and αE integrin. Thus, our findings characterize the expansion of a distinct inducible MC subset in C57BL/6 mice and highlight the potential for epithelium to direct MMC development.

Fusion of airways during avian lung development constitutes a novel mechanism for the formation of continuous lumena in multicellular epithelia.

BACKGROUND: During development of the avian lung, the initially terminally branched epithelial tree later forms a continuous network of airways. This occurs via a large-scale epithelial fusion event, wherein airways that originate proximally collide with those that originate distally to form one continuous lumen. RESULTS: Here, we found that prior to fusion, the epithelium of the embryonic chicken lung undergoes a shape change to permit the initiation and extension of new branches which contain the cells that initiate contact. These changes in epithelial shape coincide with the differentiation of smooth muscle cells that wrap the airways. From these nascent epithelial branches, individual cells form cytoskeletal protrusions that extend toward and form a bridge with their target airway. Additional cells then join the fusion site, forming a bilayered epithelium. During this process, the basement membrane around the prefusion epithelium degrades and then reforms after fusion. The epithelial bilayer then undergoes apoptosis, clearing the path between the two lumens. CONCLUSIONS: The process of airway epithelial fusion in the developing chicken lung constitutes a novel mechanism for the generation of complex multicellular tubes and suggests a conserved role for smooth muscle in the shaping of airway epithelia.

Chloride channels regulate differentiation and barrier functions of the mammalian airway.

The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function.

May patients receiving 5-alpha-reductase inhibitors be in higher risk of COVID-19 complications?

COVID-19 pandemic is a major challenge for global and national healthcare providers. Number of new cases is continuously increasing with an emerging trend showing worse prognosis in males in comparison to females. Based on this observation, our proposed hypothesis is that 5-alpha-reductase inhibitors, that are commonly used for BPH treatment, may be one of the factors contributing to poorer prognosis in males.

Mesenchymal proteases and tissue fluidity remodel the extracellular matrix during airway epithelial branching in the embryonic avian lung.

Reciprocal epithelial-mesenchymal signaling is essential for morphogenesis, including branching of the lung. In the mouse, mesenchymal cells differentiate into airway smooth muscle that wraps around epithelial branches, but this contractile tissue is absent from the early avian lung. Here, we have found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme. Before branching, the basement membrane wraps the airway epithelium as a spatially uniform sheath. After branch initiation, however, the basement membrane thins at branch tips; this remodeling requires mesenchymal expression of matrix metalloproteinase 2, which is necessary for branch extension but for not branch initiation. As branches extend, tenascin C (TNC) accumulates in the mesenchyme several cell diameters away from the epithelium. Despite its pattern of accumulation, TNC is expressed exclusively by epithelial cells. Branch extension coincides with deformation of adjacent mesenchymal cells, which correlates with an increase in mesenchymal fluidity at branch tips that may transport TNC away from the epithelium. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis.

Preterm birth: Born too soon for the developing airway epithelium?

Birth prior to term interrupts the normal development of the respiratory system and consequently results in poor respiratory outcomes that persist throughout childhood. The mechanisms underpinning these poor respiratory outcomes are not well understood, but intrinsic abnormalities within the airway epithelium may be a contributing factor. Current evidence suggests that the airway epithelium is both structurally and functionally abnormal after preterm birth, with reports of epithelial thickening and goblet cell hyperplasia in addition to increased inflammation and apoptosis in the neonatal intensive care unit. However, studies focusing on the airway epithelium are limited and many questions remain unanswered; including whether abnormalities are a direct result of interrupted development, a consequence of exposure to inflammatory stimuli in the perinatal period or a combination of the two. In addition, the difficulty of accessing airway tissue has resulted in the majority of evidence being collected in the pre-surfactant era which may not reflect contemporary preterm birth. This review examines the consequences of preterm birth on the airway epithelium and explores the clinical relevance of currently available models whilst highlighting the need to develop a clinically relevant in vitro model to help further our understanding of the airway epithelium in preterm birth.

FGF10 is an essential regulator of tracheal submucosal gland morphogenesis.

Mucus secretion and mucociliary clearance are crucial processes required to maintain pulmonary homeostasis. In the trachea and nasal passages, mucus is secreted by submucosal glands (SMGs) that line the airway, with an additional contribution from goblet cells of the surface airway epithelium. The SMG mucus is rich in mucins and antimicrobial enzymes. Defective tracheal SMGs contribute to hyper-secretory respiratory diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease, however little is known about the signals that regulate their morphogenesis and patterning. Here, we show that Fgf10 is essential for the normal development of murine tracheal SMGs, with gland development arresting at the early bud stage in the absence of FGF10 signalling. As Fgf10 knockout mice are lethal at birth, inducible knockdown of Fgf10 at late embryonic stages was used to follow postnatal gland formation, confirming the essential role of FGF10 in SMG development. In heterozygous Fgf10 mice the tracheal glands formed but with altered morphology and restricted distribution. The reduction in SMG branching in Fgf10 heterozygous mice was not rescued with time and resulted in a reduction in overall tracheal mucus secretion. Fgf10 is therefore a key signal in SMG development, influencing both the number of glands and extent of branching morphogenesis, and is likely, therefore, to play a role in aspects of SMG-dependent respiratory health.

Alteration of cystic airway mesenchyme in congenital pulmonary airway malformation.

Congenital pulmonary airway malformation (CPAM) is the most common congenital lesion detected in the neonatal lung, which may lead to respiratory distress, infection, and pneumothorax. CPAM is thought to result from abnormal branching morphogenesis during fetal lung development, arising from different locations within the developing respiratory tract. However, the pathogenic mechanisms are unknown, and previous studies have focused on abnormalities in airway epithelial cells. We have analyzed 13 excised lung specimens from infants (age < 1 year) with a confirmed diagnosis of type 2 CPAM, which is supposed to be derived from abnormal growth of intrapulmonary distal airways. By examining the mesenchymal components including smooth muscle cells, laminin, and elastin in airway and cystic walls using immunofluorescence staining, we found that the thickness and area of the smooth muscle layer underlining the airway cysts in these CPAM tissue sections were significantly decreased compared with those in bronchiolar walls of normal controls. Extracellular elastin fibers were also visually reduced or absent in airway cystic walls. In particular, a layer of elastin fibers seen in normal lung between airway epithelia and underlying smooth muscle cells was missing in type 2 CPAM samples. Thus, our data demonstrate for the first time that airway cystic lesions in type 2 CPAM occur not only in airway epithelial cells, but also in adjacent mesenchymal tissues, including airway smooth muscle cells and their extracellular protein products. This provides a new direction to study the molecular and cellular mechanisms of CPAM pathogenesis in human.

Wnt/Fgf crosstalk is required for the specification of basal cells in the mouse trachea.

Basal progenitor cells are crucial for the establishment and maintenance of the tracheal epithelium. However, it remains unclear how these progenitor cells are specified during foregut development. Here, we found that ablation of the Wnt chaperone protein Gpr177 (also known as Wntless) in mouse tracheal epithelium causes a significant reduction in the number of basal progenitor cells accompanied by cartilage loss in Shh-Cre;Gpr177loxp/loxp mutants. Consistent with the association between cartilage and basal cell development, Nkx2.1+p63+ basal cells are co-present with cartilage nodules in Shh-Cre;Ctnnb1DM/loxp mutants, which maintain partial cell-cell adhesion but not the transcription regulation function of ß-catenin. More importantly, deletion of Ctnnb1 in the mesenchyme leads to the loss of basal cells and cartilage, concomitant with reduced transcript levels of Fgf10 in Dermo1-Cre;Ctnnb1loxp/loxp mutants. Furthermore, deletion of Fgf receptor 2 (Fgfr2) in the epithelium also leads to significantly reduced numbers of basal cells, supporting the importance of Wnt/Fgf crosstalk in early tracheal development.

Single-cell branching morphogenesis in the Drosophila trachea.

The terminal cells of the tracheal epithelium in Drosophila melanogaster are one of the few known cell types that undergo subcellular morphogenesis to achieve a stable, branched shape. During the animal's larval stages, the cells repeatedly sprout new cytoplasmic processes. These grow very long, wrapping around target tissues to which the terminal cells adhere, and are hollowed by a gas-filled subcellular tube for oxygen delivery. Our understanding of this ramification process remains rudimentary. This review aims to provide a comprehensive summary of studies on terminal cells to date, and attempts to extrapolate how terminal branches might be formed based on the known genetic and molecular components. Next to this cell-intrinsic branching mechanism, we examine the extrinsic regulation of terminal branching by the target tissue and the animal's environment. Finally, we assess the degree of similarity between the patterns established by the branching programs of terminal cells and other branched cells and tissues from a mathematical and conceptual point of view.

Platelets play an essential role in murine lung development through Clec-2/podoplanin interaction.

Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-ß depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-ß signaling, thus regulating normal lung development.

ß-Catenin maintains lung epithelial progenitors after lung specification.

The entire lung epithelium arises from SRY box 9 (SOX9)-expressing progenitors that form the respiratory tree and differentiate into airway and alveolar cells. Despite progress in understanding their initial specification within the embryonic foregut, how these progenitors are subsequently maintained is less clear. Using inducible, progenitor-specific genetic mosaic mouse models, we showed that ß-catenin (CTNNB1) maintains lung progenitors by promoting a hierarchical lung progenitor gene signature, suppressing gastrointestinal (GI) genes, and regulating NK2 homeobox 1 (NKX2.1) and SRY box 2 (SOX2) in a developmental stage-dependent manner. At the early, but not later, stage post-lung specification, CTNNB1 cell-autonomously maintained normal NKX2.1 expression levels and suppressed ectopic SOX2 expression. Genetic epistasis analyses revealed that CTNNB1 is required for fibroblast growth factor (Fgf)/Kirsten rat sarcoma viral oncogene homolog (Kras)-mediated promotion of the progenitors. In silico screening of Eurexpress and translating ribosome affinity purification (TRAP)-RNAseq identified a progenitor gene signature, a subset of which depends on CTNNB1. Wnt signaling also maintained NKX2.1 expression and suppressed GI genes in cultured human lung progenitors derived from embryonic stem cells.

Transcription Factors Regulating Embryonic Development of Pulmonary Vasculature.

Lung morphogenesis is a highly orchestrated process beginning with the appearance of lung buds on approximately embryonic day 9.5 in the mouse. Endodermally derived epithelial cells of the primitive lung buds undergo branching morphogenesis to generate the tree-like network of epithelial-lined tubules. The pulmonary vasculature develops in close proximity to epithelial progenitor cells in a process that is regulated by interactions between the developing epithelium and underlying mesenchyme. Studies in transgenic and knockout mouse models demonstrate that normal lung morphogenesis requires coordinated interactions between cells lining the tubules, which end in peripheral saccules, juxtaposed to an extensive network of capillaries. Multiple growth factors, microRNAs, transcription factors, and their associated signaling cascades regulate cellular proliferation, migration, survival, and differentiation during formation of the peripheral lung. Dysregulation of signaling events caused by gene mutations, teratogens, or premature birth causes severe congenital and acquired lung diseases in which normal alveolar architecture and the pulmonary capillary network are disrupted. Herein, we review scientific progress regarding signaling and transcriptional mechanisms regulating the development of pulmonary vasculature during lung morphogenesis.

Notch Signaling: Linking Embryonic Lung Development and Asthmatic Airway Remodeling.

Lung development is mediated by assorted signaling proteins and orchestrated by complex mesenchymal-epithelial interactions. Notch signaling is an evolutionarily conserved cell-cell communication mechanism that exhibits a pivotal role in lung development. Notably, both aberrant expression and loss of regulation of Notch signaling are critically linked to the pathogenesis of various lung diseases, in particular, pulmonary fibrosis, lung cancer, pulmonary arterial hypertension, and asthmatic airway remodeling; implying that precise regulation of intensity and duration of Notch signaling is imperative for appropriate lung development. Moreover, evidence suggests that Notch signaling links embryonic lung development and asthmatic airway remodeling. Herein, we summarized all-recent advances associated with the mechanistic role of Notch signaling in lung development, consequences of aberrant expression or deletion of Notch signaling in linking early-impaired lung development and asthmatic airway remodeling, and all recently investigated potential therapeutic strategies to treat asthmatic airway remodeling.

Differential effects of late gestation maternal overnutrition on the regulation of surfactant maturation in fetal and postnatal life.

KEY POINTS: Offspring of overweight and obese women are at greater risk for respiratory complications at birth. We determined the effect of late gestation maternal overnutrition (LGON) in sheep on surfactant maturation, glucose transport and fatty acid metabolism in the lung in fetal and postnatal life. There were significant decreases in surfactant components and numerical density of surfactant producing cells in the alveolar epithelium due to LGON in the fetal lung. However, there were no differences in the levels of these surfactant components between control and LGON lambs at 30 days of age. The reduced capacity for surfactant production in fetuses as a result of LGON may affect the transition to air breathing at birth. There was altered glucose transport and fatty acid metabolism in the lung as a result of LGON in postnatal life. However, there is a normalisation of surfactant components that suggests accelerated maturation in the lungs after birth. ABSTRACT: With the increasing incidence of obesity worldwide, the proportion of women entering pregnancy overweight or obese has increased dramatically. The fetus of an overnourished mother experiences numerous metabolic changes that may modulate lung development and hence successful transition to air breathing at birth. We used a sheep model of maternal late gestation overnutrition (LGON; from 115 days' gestation, term 147 ± 3 days) to determine the effect of exposure to an increased plane of nutrition in late gestation on lung development in the fetus (at 141 days' gestation) and the lamb (30 days after birth). We found a decrease in the numerical density of surfactant protein positive cells, as well as a reduction in mRNA expression of surfactant proteins (SFTP-A, -B and -C), a rate limiting enzyme in surfactant phospholipid synthesis (phosphate cytidylyltransferase 1, choline, α; PCYT1A), and glucose transporters (SLC2A1 and SLC2A4) in the fetal lung. In lambs at 30 days after birth, there were no differences between Control and LGON groups in the surfactant components that were downregulated in the LGON fetuses. However, mRNA expression of SFTP-A, PCYT1A, peroxisome proliferator activated receptor-γ, fatty acid synthase and fatty acid transport protein were increased in LGON lambs compared to controls. These results indicate a reduced capacity for surfactant production in late gestation. While these deficits are normalised by 30 days after birth, the lungs of LGON lambs exhibited altered glucose transport and fatty acid metabolism, which is consistent with an enhanced capacity for surfactant synthesis and restoration of surfactant maturity in these animals.

Identification of FOXJ1 effectors during ciliogenesis in the foetal respiratory epithelium and embryonic left-right organiser of the mouse.

Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary. We compared genome-wide expression profiles of undifferentiated E14.5 vs. abundantly ciliated E18.5 micro-dissected airway epithelia as well as Foxj1+ vs. Foxj1-deficient foetal (E16.5) lungs of the mouse using microarray hybridisation. 326 genes deregulated in both screens are candidates for FOXJ1-dependent, ciliogenesis-associated factors at the endogenous onset of motile ciliogenesis in the lung, including 123 genes that have not been linked to ciliogenesis before; 46% of these novel factors lack known homologues outside mammals. Microarray screening of Noto+ vs. Noto null early headfold embryos (E7.75) identified 59 of the lung candidates as NOTO/FOXJ1-dependent factors in the embryonic left-right organiser that carries a different subtype of motile cilia. For several uncharacterised factors from this small overlap - including 1700012B09Rik, 1700026L06Rik and Fam183b - we provide extended experimental evidence for a ciliary function.

Decreased expression of monocarboxylate transporter 1 and 4 in the branching airway epithelium of nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Monocarboxylate transporters (MCTs) are crucial for the maintenance of intracellular pH homeostasis in developing fetal lungs. MCT1/4 is strongly expressed by epithelial airway cells throughout lung branching morphogenesis. Functional inhibition of MCT1/4 in fetal rat lung explants has been shown to result in airway defects similar to pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH). We hypothesized that pulmonary expression of MCT1/4 is decreased during lung branching morphogenesis in the nitrofen model of CDH-associated PH. METHODS: Timed-pregnant rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on D15, D18, and D21, and divided into control and nitrofen-exposed group. Pulmonary gene expression levels of MCT1/4 were analyzed by qRT-PCR. Immunofluorescence staining for MCT1/4 was combined with E-cadherin in order to evaluate protein expression in branching airway tissue. RESULTS: Relative mRNA levels of MCT1/4 were significantly reduced in lungs of nitrofen-exposed fetuses on D15, D18, and D21 compared to controls. Confocal laser scanning microscopy confirmed markedly decreased immunofluorescence of MCT1/4 in distal bronchial and primitive alveolar epithelium of nitrofen-exposed fetuses on D15, D18, and D21 compared to controls. CONCLUSION: Decreased expression of MCT1/4 in distal airway epithelium may disrupt lung branching morphogenesis and thus contribute to the development of PH in the nitrofen-induced CDH model.

Structural Development, Cellular Differentiation and Proliferation of the Respiratory Epithelium in the Bovine Fetal Lung.

Fetal bovine lung samples of 11 different gestational ages were assigned to a classical developmental stage based on histological morphology. Immunohistochemistry was used to characterize the morphology of forming airways, proliferation rate of airway epithelium and the presence of epithelial cell types (i.e. ciliated cells, club cells, neuroepithelial cells (NECs) and type II pneumocytes). Typical structural organization of pseudoglandular (84-98 days gestational age [DGA]), canalicular (154-168 DGA) and alveolar (224-266 DGA) stages was recognized. In addition, transitional pseudoglandular-canalicular (112-126 DGA) and canalicular-saccular (182 DGA) morphologies were present. The embryonic stage was not observed. A significantly (P <0.05) higher proliferation rate of pulmonary epithelium, on average 5.5% and 4.4% in bronchi and bronchioles, respectively, was present in the transitional pseudoglandular-canalicular phase (112-126 DGA) compared with all other phases, while from 8 weeks before term (224-266 DGA) proliferation had almost ceased. The first epithelial cells identified by specific marker proteins in the earliest samples available for study (84 DGA) were ciliated cells and NECs. Club cells were present initially at 112 DGA and type II pneumocytes at 224 DGA. At the latest time points (224-226 DGA) these latter cell types were still present at a much lower percentage compared with adult cattle. This study characterized bovine fetal lung development by histological morphology and cellular composition of the respiratory epithelium and suggests that the apparent structural anatomical maturity of the bovine lung at term is not matched by functional maturity of the respiratory epithelium.

Update on Molecular Biology of Lung Development--Transcriptomics.

This article highlights some of the significant advances in our understanding of lung developmental biology made over the last few years, which challenge existing paradigms and are relevant to a fundamental understanding of this process. Additional comments address how these new insights may be informative for chronic lung diseases that occur, or initiate, in the neonatal period. This is not meant to be an exhaustive review of the molecular biology of lung development. For a more comprehensive, contemporary review of the cellular and molecular aspects of lung development, readers can refer to recent reviews by others.