Alveolar epithelial progenitor cells require Nkx2-1 to maintain progenitor-specific epigenomic state during lung homeostasis and regeneration.

Lung epithelial regeneration after acute injury requires coordination cellular coordination to pattern the morphologically complex alveolar gas exchange surface. During adult lung regeneration, Wnt-responsive alveolar epithelial progenitor (AEP) cells, a subset of alveolar type 2 (AT2) cells, proliferate and transition to alveolar type 1 (AT1) cells. Here, we report a refined primary murine alveolar organoid, which recapitulates critical aspects of in vivo regeneration. Paired scRNAseq and scATACseq followed by transcriptional regulatory network (TRN) analysis identified two AT1 transition states driven by distinct regulatory networks controlled in part by differential activity of Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids was sufficient to cause transition to a proliferative stressed Krt8+ state, and AEP-specific deletion of Nkx2-1 in adult mice led to rapid loss of progenitor state and uncontrolled growth of Krt8+ cells. Together, these data implicate dynamic epigenetic maintenance via Nkx2-1 as central to the control of facultative progenitor activity in AEPs.

Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wntless (Wls), a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulates the expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and ß-catenin-deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/ß-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.NEW & NOTEWORTHY Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. In this study, we focused on the role of ion channels in the differentiation and patterning of the large airways of the developing respiratory tract. We identify a mechanism by which Wnt-beta-catenin signaling controls levels of ion channel-encoding genes to promote tracheal differentiation.

EMC3 regulates mesenchymal cell survival via control of the mitotic spindle assembly.

Eukaryotic cells transit through the cell cycle to produce two daughter cells. Dysregulation of the cell cycle leads to cell death or tumorigenesis. Herein, we found a subunit of the ER membrane complex, EMC3, as a key regulator of cell cycle. Conditional deletion of Emc3 in mouse embryonic mesoderm led to reduced size and patterning defects of multiple organs. Emc3 deficiency impaired cell proliferation, causing spindle assembly defects, chromosome mis-segregation, cell cycle arrest at G2/M, and apoptosis. Upon entry into mitosis, mesenchymal cells upregulate EMC3 protein levels and localize EMC3 to the mitotic centrosomes. Further analysis indicated that EMC3 works together with VCP to tightly regulate the levels and activity of Aurora A, an essential factor for centrosome function and mitotic spindle assembly: while overexpression of EMC3 or VCP degraded Aurora A, their loss led to increased Aurora A stability but reduced Aurora A phosphorylation in mitosis.

Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in non-contractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wls, a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulated expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and ß-catenin deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/ß-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.

PRDM3/16 Regulate Chromatin Accessibility Required for NKX2-1 Mediated Alveolar Epithelial Differentiation and Function.

Differential chromatin accessibility accompanies and mediates transcriptional control of diverse cell fates and their differentiation during embryogenesis. While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Here, we demonstrate that the paired domain zinc finger transcriptional regulators PRDM3 and PRDM16 regulate chromatin accessibility to mediate cell differentiation decisions during lung morphogenesis. Combined deletion of Prdm3 and Prdm16 in early lung endoderm caused perinatal lethality due to respiratory failure from loss of AT2 cell function. Prdm3/16 deletion led to the accumulation of partially differentiated AT1 cells and loss of AT2 cells. Combination of single cell RNA-seq, bulk ATAC-seq, and CUT&RUN demonstrated that PRDM3 and PRDM16 enhanced chromatin accessibility at NKX2-1 transcriptional targets in peripheral epithelial cells, all three factors binding together at a multitude of cell-type specific cis-active DNA elements. Network analysis demonstrated that PRDM3/16 regulated genes critical for perinatal AT2 cell differentiation, surfactant homeostasis, and innate host defense. Lineage specific deletion of PRDM3/16 in AT2 cells led to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16.

PI3K signaling specifies proximal-distal fate by driving a developmental gene regulatory network in SOX9+ mouse lung progenitors.

The tips of the developing respiratory buds are home to important progenitor cells marked by the expression of SOX9 and ID2. Early in embryonic development (prior to E13.5), SOX9+progenitors are multipotent, generating both airway and alveolar epithelium, but are selective progenitors of alveolar epithelial cells later in development. Transcription factors, including Sox9, Etv5, Irx, Mycn, and Foxp1/2 interact in complex gene regulatory networks to control proliferation and differentiation of SOX9+progenitors. Molecular mechanisms by which these transcription factors and other signaling pathways control chromatin state to establish and maintain cell-type identity are not well-defined. Herein, we analyze paired gene expression (RNA-Seq) and chromatin accessibility (ATAC-Seq) data from SOX9+ epithelial progenitor cells (EPCs) during embryonic development in Mus musculus. Widespread changes in chromatin accessibility were observed between E11.5 and E16.5, particularly at distal cis-regulatory elements (e.g. enhancers). Gene regulatory network (GRN) inference identified a common SOX9+ progenitor GRN, implicating phosphoinositide 3-kinase (PI3K) signaling in the developmental regulation of SOX9+ progenitor cells. Consistent with this model, conditional ablation of PI3K signaling in the developing lung epithelium in mouse resulted in an expansion of the SOX9+ EPC population and impaired airway epithelial cell differentiation. These data demonstrate that PI3K signaling is required for epithelial patterning during lung organogenesis, and emphasize the combinatorial power of paired RNA and ATAC seq in defining regulatory networks in development.

Studying how lungs develop has helped us understand and treat often-devastating lung diseases. This includes diseases like cystic fibrosis which result from spelling mistakes known as mutations in a person's genetic code. However, not all lung diseases involve mutations. Many other diseases, in both adults and children, are caused by genes failing to switch on or off at some point during lung development. DNA is surrounded by various proteins which package it into a compressed structure known as chromatin. Cells can control which genes are turned on or off by modifying how tightly packed parts of the genetic code are within chromatin. Changes in chromatin accessibility, also known as 'epigenetic' changes, are a normal part of development, and guide cells towards specific jobs or identities as an organ matures. However, how this happens in the developing lung is poorly understood. Here, Khattar, Fernandes et al. set out to determine how chromatin accessibility shapes development of the tissue lining the lungs, focusing on a group of progenitor cells which produce the protein SOX9. These cells are initially found at the tips of the early lung, where they go on to develop into the cells that line the whole of the mature organ. Initial experiments used large-scale genetic techniques to measure gene activity and chromatin accessibility simultaneously in progenitor cells extracted from the lungs of mice. Khattar, Fernandes et al. were then able to predict the signaling pathways that shape the lung lining based on which genes were surrounded by unpacked chromatin, and determine the proteins responsible for these epigenetic changes. This included the signaling pathway Phosphatidylinositol 3 kinase (PI3K) which is involved in a number of cellular processes. Additional experiments in mice confirmed that the PI3K pathway became active very early in lung development and remained so until adulthood. In contrast, mice lacking a gene that codes for a key part of the PI3K pathway had defective lungs which failed to develop a proper lining. The data generated in this study will provide an important resource for future studies investigating how epigenetic changes drive normal lung development. Khattar, Fernandes et al. hope that this knowledge will help researchers to better understand the cause of human lung diseases, and identify already available 'epigenetic drugs' which could be repurposed to treat them.

Maladaptive functional changes in alveolar fibroblasts due to perinatal hyperoxia impair epithelial differentiation.

Infants born prematurely worldwide have up to a 50% chance of developing bronchopulmonary dysplasia (BPD), a clinical morbidity characterized by dysregulated lung alveolarization and microvascular development. It is known that PDGFR alpha-positive (PDGFRA+) fibroblasts are critical for alveolarization and that PDGFRA+ fibroblasts are reduced in BPD. A better understanding of fibroblast heterogeneity and functional activation status during pathogenesis is required to develop mesenchymal population-targeted therapies for BPD. In this study, we utilized a neonatal hyperoxia mouse model (90% O2 postnatal days 0-7, PN0-PN7) and performed studies on sorted PDGFRA+ cells during injury and room air recovery. After hyperoxia injury, PDGFRA+ matrix and myofibroblasts decreased and PDGFRA+ lipofibroblasts increased by transcriptional signature and population size. PDGFRA+ matrix and myofibroblasts recovered during repair (PN10). After 7 days of in vivo hyperoxia, PDGFRA+ sorted fibroblasts had reduced contractility in vitro, reflecting loss of myofibroblast commitment. Organoids made with PN7 PDGFRA+ fibroblasts from hyperoxia in mice exhibited reduced alveolar type 1 cell differentiation, suggesting reduced alveolar niche-supporting PDGFRA+ matrix fibroblast function. Pathway analysis predicted reduced WNT signaling in hyperoxia fibroblasts. In alveolar organoids from hyperoxia-exposed fibroblasts, WNT activation by CHIR increased the size and number of alveolar organoids and enhanced alveolar type 2 cell differentiation.

BMP4 and Wnt signaling interact to promote mouse tracheal mesenchyme morphogenesis.

Tracheobronchomalacia and complete tracheal rings are congenital malformations of the trachea associated with morbidity and mortality for which the etiology remains poorly understood. Epithelial expression of Wls (a cargo receptor mediating Wnt ligand secretion) by tracheal cells is essential for patterning the embryonic mouse trachea's cartilage and muscle. RNA sequencing indicated that Wls differentially modulated the expression of BMP signaling molecules. We tested whether BMP signaling, induced by epithelial Wnt ligands, mediates cartilage formation. Deletion of Bmp4 from respiratory tract mesenchyme impaired tracheal cartilage formation that was replaced by ectopic smooth muscle, recapitulating the phenotype observed after epithelial deletion of Wls in the embryonic trachea. Ectopic muscle was caused in part by anomalous differentiation and proliferation of smooth muscle progenitors rather than tracheal cartilage progenitors. Mesenchymal deletion of Bmp4 impaired expression of Wnt/ß-catenin target genes, including targets of WNT signaling: Notum and Axin2. In vitro, recombinant (r)BMP4 rescued the expression of Notum in Bmp4-deficient tracheal mesenchymal cells and induced Notum promoter activity via SMAD1/5. RNA sequencing of Bmp4-deficient tracheas identified genes essential for chondrogenesis and muscle development coregulated by BMP and WNT signaling. During tracheal morphogenesis, WNT signaling induces Bmp4 in mesenchymal progenitors to promote cartilage differentiation and restrict trachealis muscle. In turn, Bmp4 differentially regulates the expression of Wnt/ß-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

YAP regulates alveolar epithelial cell differentiation and AGER via NFIB/KLF5/NKX2-1.

Ventilation is dependent upon pulmonary alveoli lined by two major epithelial cell types, alveolar type-1 (AT1) and 2 (AT2) cells. AT1 cells mediate gas exchange while AT2 cells synthesize and secrete pulmonary surfactants and serve as progenitor cells which repair the alveoli. We developed transgenic mice in which YAP was activated or deleted to determine its roles in alveolar epithelial cell differentiation. Postnatal YAP activation increased epithelial cell proliferation, increased AT1 cell numbers, and caused indeterminate differentiation of subsets of alveolar cells expressing atypical genes normally restricted to airway epithelial cells. YAP deletion increased expression of genes associated with mature AT2 cells. YAP activation enhanced DNA accessibility in promoters of transcription factors and motif enrichment analysis predicted target genes associated with alveolar cell differentiation. YAP participated with KLF5, NFIB, and NKX2-1 to regulate AGER. YAP plays a central role in a transcriptional network that regulates alveolar epithelial differentiation.

IGF1R controls mechanosignaling in myofibroblasts required for pulmonary alveologenesis.

Ventilation throughout life is dependent on the formation of pulmonary alveoli, which create an extensive surface area in which the close apposition of respiratory epithelium and endothelial cells of the pulmonary microvascular enables efficient gas exchange. Morphogenesis of the alveoli initiates at late gestation in humans and the early postnatal period in the mouse. Alveolar septation is directed by complex signaling interactions among multiple cell types. Here, we demonstrate that IGF1 receptor gene (Igf1r) expression by a subset of pulmonary fibroblasts is required for normal alveologenesis in mice. Postnatal deletion of Igf1r caused alveolar simplification, disrupting alveolar elastin networks and extracellular matrix without altering myofibroblast differentiation or proliferation. Moreover, loss of Igf1r impaired contractile properties of lung myofibroblasts and inhibited myosin light chain (MLC) phosphorylation and mechanotransductive nuclear YAP activity. Activation of p-AKT, p-MLC, and nuclear YAP in myofibroblasts was dependent on Igf1r. Pharmacologic activation of AKT enhanced MLC phosphorylation, increased YAP activation, and ameliorated alveolar simplification in vivo. IGF1R controls mechanosignaling in myofibroblasts required for lung alveologenesis.

Pretreatment of aged mice with retinoic acid supports alveolar regeneration via upregulation of reciprocal PDGFA signalling.

OBJECTIVES: Idiopathic pulmonary fibrosis (IPF) primarily affects the aged population and is characterised by failure of alveolar regeneration, leading to loss of alveolar type 1 (AT1) cells. Aged mouse models of lung repair have demonstrated that regeneration fails with increased age. Mouse and rat lung repair models have shown retinoic acid (RA) treatment can restore alveolar regeneration. Herein, we seek to determine the signalling mechanisms that become activated on RA treatment prior to injury, which support alveolar differentiation. DESIGN: Partial pneumonectomy lung injury model and next-generation sequencing of sorted cell populations were used to uncover molecular targets regulating alveolar repair. In vitro organoids generated from epithelial cells of mouse or patient with IPF co-cultured with young, aged or RA-pretreated murine fibroblasts were used to test potential targets. MAIN OUTCOME MEASUREMENTS: Known alveolar epithelial cell differentiation markers, including HOPX and AGER for AT1 cells, were used to assess outcome of treatments. RESULTS: Gene expression analysis of sorted fibroblasts and epithelial cells isolated from lungs of young, aged and RA-pretreated aged mice predicted increased platelet-derived growth factor subunit A (PDGFA) signalling that coincided with regeneration and alveolar epithelial differentiation. Addition of PDGFA induced AT1 and AT2 differentiation in both mouse and human IPF lung organoids generated with aged fibroblasts, and PDGFA monoclonal antibody blocked AT1 cell differentiation in organoids generated with young murine fibroblasts. CONCLUSIONS: Our data support the concept that RA indirectly induces reciprocal PDGFA signalling, which activates regenerative fibroblasts that support alveolar epithelial cell differentiation and repair, providing a potential therapeutic strategy to influence the pathogenesis of IPF.

Aurothioglucose enhances proangiogenic pathway activation in lungs from room air and hyperoxia-exposed newborn mice.

Bronchopulmonary dysplasia (BPD), a long-term respiratory morbidity of prematurity, is characterized by attenuated alveolar and vascular development. Supplemental oxygen and immature antioxidant defenses contribute to BPD development. Our group identified thioredoxin reductase-1 (TXNRD1) as a therapeutic target to prevent BPD. The present studies evaluated the impact of the TXNRD1 inhibitor aurothioglucose (ATG) on pulmonary responses and gene expression in newborn C57BL/6 pups treated with saline or ATG (25 mg/kg ip) within 12 h of birth and exposed to room air (21% O2) or hyperoxia (>95% O2) for 72 h. Purified RNA from lung tissues was sequenced, and differential expression was evaluated. Hyperoxic exposure altered ~2,000 genes, including pathways involved in glutathione metabolism, intrinsic apoptosis signaling, and cell cycle regulation. The isolated effect of ATG treatment was limited primarily to genes that regulate angiogenesis and vascularization. In separate studies, pups were treated as described above and returned to room air until 14 days. Vascular density analyses were performed, and ANOVA indicated an independent effect of hyperoxia on vascular density and alveolar architecture at 14 days. Consistent with RNA-seq analyses, ATG significantly increased vascular density in room air, but not in hyperoxia-exposed pups. These findings provide insights into the mechanisms by which TXNRD1 inhibitors may enhance lung development.

Neutrophil extracellular traps activate IL-8 and IL-1 expression in human bronchial epithelia.

Neutrophil extracellular traps (NETs) provide host defense but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute to human airway cell inflammation. Primary normal human bronchial epithelial cells (HBEs) grown at air-liquid interface and wild-type (wt)CFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 h in vitro. Cytokines were measured in the apical supernatant by Luminex, and the effect on the HBE transcriptome was assessed by RNA sequencing. NETs consistently stimulated IL-8, TNF-α, and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, and GM-CSF). Expression of HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, was increased in response to NETs. NET exposure in the presence of anakinra [recombinant human IL-1 receptor antagonist (rhIL-1RA)] dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. rhIL-36RA limited the increase in expression of proinflammatory cytokine RNAs in HBEs exposed to NETs. NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. The present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.

MEG3 is increased in idiopathic pulmonary fibrosis and regulates epithelial cell differentiation.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease causing fibrotic remodeling of the peripheral lung, leading to respiratory failure. Peripheral pulmonary epithelial cells lose normal alveolar epithelial gene expression patterns and variably express genes associated with diverse conducting airway epithelial cells, including basal cells. Single-cell RNA sequencing of pulmonary epithelial cells isolated from IPF lung tissue demonstrated altered expression of LncRNAs, including increased MEG3. MEG3 RNA was highly expressed in subsets of the atypical IPF epithelial cells and correlated with conducting airway epithelial gene expression patterns. Expression of MEG3 in human pulmonary epithelial cell lines increased basal cell-associated RNAs, including TP63, KRT14, STAT3, and YAP1, and enhanced cell migration, consistent with a role for MEG3 in regulating basal cell identity. MEG3 reduced expression of TP73, SOX2, and Notch-associated RNAs HES1 and HEY1, in primary human bronchial epithelial cells, demonstrating a role for MEG3 in the inhibition of genes influencing basal cell differentiation into club, ciliated, or goblet cells. MEG3 induced basal cell genes and suppressed genes associated with terminal differentiation of airway cells, supporting a role for MEG3 in regulation of basal progenitor cell functions, which may contribute to tissue remodeling in IPF.

Notum attenuates Wnt/ß-catenin signaling to promote tracheal cartilage patterning.

Tracheobronchomalacia (TBM) is a common congenital disorder in which the cartilaginous rings of the trachea are weakened or missing. Despite the high prevalence and clinical issues associated with TBM, the etiology is largely unknown. Our previous studies demonstrated that Wntless (Wls) and its associated Wnt pathways are critical for patterning of the upper airways. Deletion of Wls in respiratory endoderm caused TBM and ectopic trachealis muscle. To understand mechanisms by which Wls mediates tracheal patterning, we performed RNA sequencing in prechondrogenic tracheal tissue of Wlsf/f;ShhCre/wt embryos. Chondrogenic Bmp4, and Sox9 were decreased, while expression of myogenic genes was increased. We identified Notum, a deacylase that inactivates Wnt ligands, as a target of Wls induced Wnt signaling. Notum's mesenchymal ventral expression in prechondrogenic trachea overlaps with expression of Axin2, a Wnt/ß-catenin target and inhibitor. Notum is induced by Wnt/ß-catenin in developing trachea. Deletion of Notum activated mesenchymal Wnt/ß-catenin and caused tracheal mispatterning of trachealis muscle and cartilage as well as tracheal stenosis. Notum is required for tracheal morphogenesis, influencing mesenchymal condensations critical for patterning of tracheal cartilage and muscle. We propose that Notum influences mesenchymal cell differentiation by generating a barrier for Wnt ligands produced and secreted by airway epithelial cells to attenuate Wnt signaling.

Alveolar injury and regeneration following deletion of ABCA3.

Adaptation to air breathing after birth is dependent upon the synthesis and secretion of pulmonary surfactant by alveolar type 2 (AT2) cells. Surfactant, a complex mixture of phospholipids and proteins, is secreted into the alveolus, where it reduces collapsing forces at the air-liquid interface to maintain lung volumes during the ventilatory cycle. ABCA3, an ATP-dependent Walker domain containing transport protein, is required for surfactant synthesis and lung function at birth. Mutations in ABCA3 cause severe surfactant deficiency and respiratory failure in newborn infants. We conditionally deleted the Abca3 gene in AT2 cells in the mature mouse lung. Loss of ABCA3 caused alveolar cell injury and respiratory failure. ABCA3-related lung dysfunction was associated with surfactant deficiency, inflammation, and alveolar-capillary leak. Extensive but incomplete deletion of ABCA3 caused alveolar injury and inflammation, and it initiated proliferation of progenitor cells, restoring ABCA3 expression, lung structure, and function. M2-like macrophages were recruited to sites of AT2 cell proliferation during the regenerative process and were present in lung tissue from patients with severe lung disease caused by mutations in ABCA3. The remarkable and selective regeneration of ABCA3-sufficient AT2 progenitor cells provides plausible approaches for future correction of ABCA3 and other genetic disorders associated with surfactant deficiency and acute interstitial lung disease.

EMC3 coordinates surfactant protein and lipid homeostasis required for respiration.

Adaptation to respiration at birth depends upon the synthesis of pulmonary surfactant, a lipid-protein complex that reduces surface tension at the air-liquid interface in the alveoli and prevents lung collapse during the ventilatory cycle. Herein, we demonstrated that the gene encoding a subunit of the endoplasmic reticulum membrane complex, EMC3, also known as TMEM111 (Emc3/Tmem111), was required for murine pulmonary surfactant synthesis and lung function at birth. Conditional deletion of Emc3 in murine embryonic lung epithelial cells disrupted the synthesis and packaging of surfactant lipids and proteins, impaired the formation of lamellar bodies, and induced the unfolded protein response in alveolar type 2 (AT2) cells. EMC3 was essential for the processing and routing of surfactant proteins, SP-B and SP-C, and the biogenesis of the phospholipid transport protein ABCA3. Transcriptomic, lipidomic, and proteomic analyses demonstrated that EMC3 coordinates the assembly of lipids and proteins in AT2 cells that is necessary for surfactant synthesis and function at birth.

Studying Wnt Signaling During Patterning of Conducting Airways.

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

Endodermal Wnt signaling is required for tracheal cartilage formation.

Tracheobronchomalacia is a common congenital defect in which the walls of the trachea and bronchi lack of adequate cartilage required for support of the airways. Deletion of Wls, a cargo receptor mediating Wnt ligand secretion, in the embryonic endoderm using ShhCre mice inhibited formation of tracheal-bronchial cartilaginous rings. The normal dorsal-ventral patterning of tracheal mesenchyme was lost. Smooth muscle cells, identified by Acta2 staining, were aberrantly located in ventral mesenchyme of the trachea, normally the region of Sox9 expression in cartilage progenitors. Wnt/ß-catenin activity, indicated by Axin2 LacZ reporter, was decreased in tracheal mesenchyme of Wls(f/f);Shh(Cre/+) embryos. Proliferation of chondroblasts was decreased and reciprocally, proliferation of smooth muscle cells was increased in Wls(f/f);Shh(Cre/+) tracheal tissue. Expression of Tbx4, Tbx5, Msx1 and Msx2, known to mediate cartilage and muscle patterning, were decreased in tracheal mesenchyme of Wls(f/f);Shh(Cre/+) embryos. Ex vivo studies demonstrated that Wnt7b and Wnt5a, expressed by the epithelium of developing trachea, and active Wnt/ß-catenin signaling are required for tracheal chondrogenesis before formation of mesenchymal condensations. In conclusion, Wnt ligands produced by the tracheal epithelium pattern the tracheal mesenchyme via modulation of gene expression and cell proliferation required for proper tracheal cartilage and smooth muscle differentiation.

Mesenchymal Wnt signaling promotes formation of sternum and thoracic body wall.

Midline defects account for approximately 5% of congenital abnormalities observed at birth. However, the molecular mechanisms underlying the formation of the ventral body wall are not well understood. Recent studies linked mutations in Porcupine-an O-acetyl transferase mediating Wnt ligand acylation-with defects in the thoracic body wall. We hypothesized that anomalous Wnt signaling is involved in the pathogenesis of defective closure of the thoracic body wall. We generated a mouse model wherein Wntless (Wls), which encodes a cargo receptor mediating secretion of Wnt ligands, was conditionally deleted from the developing mesenchyme using Dermo1Cre mice. Wls(f/f);Dermo1(Cre/+) embryos died during mid-gestation. At E13.5, skeletal defects were observed in the forelimbs, jaw, and rib cage. At E14.5, midline defects in the thoracic body wall began to emerge: the sternum failed to fuse and the heart protruded through the body wall at the midline (ectopia cordis). To determine the molecular mechanism underlying the phenotype observed in Wls(f/f);Dermo1(Cre/+) embryos, we tested whether Wnt/ß-catenin signaling was operative in developing the embryonic ventral body wall using Axin2(LacZ) and BatGal reporter mice. While Wnt/ß-catenin signaling activity was observed at the midline of the ventral body wall before sternal fusion, this pattern of activity was altered and scattered throughout the body wall after mesenchymal deletion of Wls. Mesenchymal cell migration was disrupted in Wls(f/f);Dermo1(Cre/+) thoracic body wall partially due to anomalous ß-catenin independent Wnt signaling as determined by in vitro assays. Deletion of Lrp5 and Lrp6 receptors, which mediate Wnt/ß-catenin signaling in the mesenchyme, partially recapitulated the phenotype observed in the chest midline of Wls(f/f);Dermo1(Cre/+) embryos supporting a role for Wnt/ß-catenin signaling activity in the normal formation of the ventral body wall mesenchyme. We conclude that Wls-mediated secretion of Wnt ligands from the developing ventral body wall mesenchyme plays a critical role in fusion of the sternum and closure of the secondary body wall. Thus, impaired Wls activity in the ventral body wall mesenchyme is a mechanism underlying ectopia cordis and unfused sternum.