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Morphological development of the lung requires complex signal crosstalk between the mesenchymal and epithelial progenitors. Elucidating the genetic cascades underlying signal crosstalk is essential to understanding lung morphogenesis. Here, we identified Nolz1 as a mesenchymal lineage-specific transcriptional regulator that plays a key role in lung morphogenesis. Nolz1 null mutation resulted in a severe hypoplasia phenotype, including a decreased proliferation of mesenchymal cells, aberrant differentiation of epithelial cells and defective growth of epithelial branches. Nolz1 deletion also downregulated Wnt2, Lef1, Fgf10, Gli3 and Bmp4 mRNAs. Mechanistically, Nolz1 regulates lung morphogenesis primarily through Wnt2 signaling. Loss-of-function and overexpression studies demonstrated that Nolz1 transcriptionally activated Wnt2 and downstream β-catenin signaling to control mesenchymal cell proliferation and epithelial branching. Exogenous Wnt2 could rescue defective proliferation and epithelial branching in Nolz1 knockout lungs. Finally, we identified Fgf9 as an upstream regulator of Nolz1. Collectively, Fgf9-Nolz1-Wnt2 signaling represents a novel axis in the control of lung morphogenesis. These findings are relevant to lung tumorigenesis, in which a pathological function of Nolz1 is implicated. [ABSTRACT FROM AUTHOR]

Introduction –Clinical observations and animal models suggest a critical role for the dynamic regulation of transmural pressure and peristaltic airway smooth muscle contractions for proper lung development. However, it is currently unclear how such mechanical signals are transduced into molecular and transcriptional changes at the cell level. To connect these physical findings to a mechanotransduction mechanism, we identified a known mechanosensor, TRPV4, as a component of this pathway.Methods –Embryonic mouse lung explants were cultured on membranes and in submersion culture to modulate explant transmural pressure. Time-lapse imaging was used to capture active changes in lung biology, and whole-mount images were used to visualize the organization of the epithelial, smooth muscle, and vascular compartments. TRPV4 activity was modulated by pharmacological agonism and inhibition.Results –TRPV4 expression is present in the murine lung with strong localization to the epithelium and major pulmonary blood vessels. TRPV4 agonism and inhibition resulted in hyper- and hypoplastic airway branching, smooth muscle differentiation, and lung growth, respectively. Smooth muscle contractions also doubled in frequency with agonism and were reduced by 60% with inhibition demonstrating a functional role consistent with levels of smooth muscle differentiation. Activation of TRPV4 increased the vascular capillary density around the distal airways, and inhibition resulted in a near complete loss of the vasculature.Conclusions –These studies have identified TRPV4 as a potential mechanosensor involved in transducing mechanical forces on the airways to molecular and transcriptional events that regulate the morphogenesis of the three essential tissue compartments in the lung.

Abstract Background The epithelial-mesenchymal signaling involving SHH-FOXF1, TBX4-FGF10, and TBX2 pathways is an essential transcriptional network operating during early lung organogenesis. However, precise regulatory interactions between different genes and proteins in this pathway are incompletely understood. Methods To identify TBX2 and TBX4 genome-wide binding sites, we performed chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) in human fetal lung fibroblasts IMR-90. Results We identified 14,322 and 1,862 sites strongly-enriched for binding of TBX2 and TBX4, respectively, 43.95% and 18.79% of which are located in the gene promoter regions. Gene Ontology, pathway enrichment, and DNA binding motif analyses revealed a number of overrepresented cues and transcription factor binding motifs relevant for lung branching that can be transcriptionally regulated by TBX2 and/or TBX4. In addition, TBX2 and TBX4 binding sites were found enriched around and within FOXF1 and its antisense long noncoding RNA FENDRR, indicating that the TBX4-FGF10 cascade may directly interact with the SHH-FOXF1 signaling. Conclusions We highlight the complexity of transcriptional network driven by TBX2 and TBX4 and show that disruption of this crosstalk during morphogenesis can play a substantial role in etiology of lung developmental disorders.

Background: The epithelial-mesenchymal signaling involving SHH-FOXF1, TBX4-FGF10, and TBX2 pathways is an essential transcriptional network operating during early lung organogenesis. However, precise regulatory interactions between different genes and proteins in this pathway are incompletely understood.Methods: To identify TBX2 and TBX4 genome-wide binding sites, we performed chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) in human fetal lung fibroblasts IMR-90.Results: We identified 14,322 and 1,862 sites strongly-enriched for binding of TBX2 and TBX4, respectively, 43.95% and 18.79% of which are located in the gene promoter regions. Gene Ontology, pathway enrichment, and DNA binding motif analyses revealed a number of overrepresented cues and transcription factor binding motifs relevant for lung branching that can be transcriptionally regulated by TBX2 and/or TBX4. In addition, TBX2 and TBX4 binding sites were found enriched around and within FOXF1 and its antisense long noncoding RNA FENDRR, indicating that the TBX4-FGF10 cascade may directly interact with the SHH-FOXF1 signaling.Conclusions: We highlight the complexity of transcriptional network driven by TBX2 and TBX4 and show that disruption of this crosstalk during morphogenesis can play a substantial role in etiology of lung developmental disorders. [ABSTRACT FROM AUTHOR]

Small‐cell lung cancer (SCLC) accounts for approximately 15% of lung cancer cases; however, it is characterized by easy relapse and low survival rate, leading to one of the most intractable diseases in clinical practice. Despite decades of basic and clinical research, little progress has been made in the management of SCLC. The current standard first‐line regimens of SCLC still remain to be cisplatin or carboplatin combined with etoposide, and the adverse events of chemotherapy are by no means negligible. Besides, the immunotherapy on SCLC is still in an early stage and novel studies are urgently needed. In this review, we describe SCLC development and current therapy, aiming at providing useful advices on basic research and clinical strategy. [ABSTRACT FROM AUTHOR]

Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wild-type lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis.

Abstract The highly conserved transcription factor Grainyhead-like 2 (Grhl2) exhibits a dynamic expression pattern in lung epithelium throughout embryonic development. Using a conditional gene targeting approach to delete Grhl2 in the developing lung epithelium, our results demonstrate that Grhl2 plays multiple roles in lung morphogenesis that are essential for respiratory function. Loss of Grhl2 leads to impaired ciliated cell differentiation and perturbed formation of terminal saccules. Critically, a substantial increase in Sox9-positive distal tip progenitor cells was observed following loss of Grhl2 , suggesting that Grhl2 plays an important role in branching morphogenesis. Gene transcription profiling of Grhl2 -deficient lung epithelial cells revealed a significant down regulation of Elf5 , a member of the Ets family of transcription factors. Furthermore, ChIP and comparative genomic analyzes confirmed that Elf5 is a direct transcriptional target of Grhl2. Taken together, these results support the hypothesis that Grhl2 controls normal lung morphogenesis by tightly regulating the activity of distal tip progenitor cells. Highlights • Grhl2 is a critical transcriptional regulator of respiratory function. • Loss of Grhl2 results in an increase in Sox9-positive distal tip progenitor cells. • Elf5, a transcription factor enriched in lung epithelial tip progenitor cells is downregulated in Grhl2 -deficient lungs. • Genomic studies confirmed Elf5 as a novel direct target of Grhl2 in lung epithelium. [ABSTRACT FROM AUTHOR]

Embryonic lung development requires reciprocal endodermal-mesodermal interactions; mediated by various signaling proteins. Wnt/β-catenin is a signaling protein that exhibits the pivotal role in lung development, injury and repair while aberrant expression of Wnt/β-catenin signaling leads to asthmatic airway remodeling: characterized by hyperplasia and hypertrophy of airway smooth muscle cells, alveolar and vascular damage goblet cells metaplasia, and deposition of extracellular matrix; resulting in decreased lung compliance and increased airway resistance. The substantial evidence suggests that Wnt/β-catenin signaling links embryonic lung development and asthmatic airway remodeling. Here, we summarized the recent advances related to the mechanistic role of Wnt/β-catenin signaling in lung development, consequences of aberrant expression or deletion of Wnt/β-catenin signaling in expansion and progression of asthmatic airway remodeling, and linking early-impaired pulmonary development and airway remodeling later in life. Finally, we emphasized all possible recent potential therapeutic significance and future prospectives, that are adaptable for therapeutic intervention to treat asthmatic airway remodeling. [ABSTRACT FROM AUTHOR]

Background/Aims: The human embryo develops in a hypoxic environment. In this way, cells have to rely on the glycolytic pathway for energy supply, leading to an intracellular accumulation of monocarboxylates such as lactate and pyruvate. These acids have an important role in cell metabolism and their rapid transport across the plasma membrane is crucial for the maintenance of intracellular pH homeostasis. This transport is mediated by a family of transporters, designated by monocarboxylate transporters (MCTs), namely isoforms 1 and 4. MCT1/4 expression is regulated by the ancillary protein CD147.The general aim of this study was to characterize the expression pattern of MCT1/4, CD147 and the glucose transporter GLUT1 during human fetal lung development and elucidate the role of MCTs in lung development. Methods: The expression pattern of MCT1/4 and GLUT1 was characterized by immunohistochemistry and fetal lung viability and branching were evaluated by exposing rat fetal lung explants to CHC, an inhibitor of MCT activity. Results: Our findings show that all the biomarkers are differently expressed during fetal lung development and that CHC appears to have an inhibitory effect on lung branching and viability, in a dose dependent way. Conclusion: We provide evidence for the role of MCTs in embryo lung development, however to prove the dependence of MCT activity further studies are waranted.

Morphological development of the lung requires complex signal crosstalk between the mesenchymal and epithelial progenitors. Elucidating the genetic cascades underlying signal crosstalk is essential to understanding lung morphogenesis. Here, we identified Nolz1 as a mesenchymal lineage-specific transcriptional regulator that plays a key role in lung morphogenesis. Nolz1 null mutation resulted in a severe hypoplasia phenotype, including a decreased proliferation of mesenchymal cells, aberrant differentiation of epithelial cells and defective growth of epithelial branches. Nolz1 deletion also downregulated Wnt2, Lef1, Fgf10, Gli3 and Bmp4 mRNAs. Mechanistically, Nolz1 regulates lung morphogenesis primarily through Wnt2 signaling. Loss-of-function and overexpression studies demonstrated that Nolz1 transcriptionally activated Wnt2 and downstream β-catenin signaling to control mesenchymal cell proliferation and epithelial branching. Exogenous Wnt2 could rescue defective proliferation and epithelial branching in Nolz1 knockout lungs. Finally, we identified Fgf9 as an upstream regulator of Nolz1. Collectively, Fgf9-Nolz1-Wnt2 signaling represents a novel axis in the control of lung morphogenesis. These findings are relevant to lung tumorigenesis, in which a pathological function of Nolz1 is implicated., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

• Cdyl −/− mice died of respiratory distress immediately at birth because of distinct abnormalities in distal lung morphogenesis. • Cdyl gene deletion led to an arrest in alveolar epithelium cell differentiation and to the excessive proliferation of immature AEC-II cells. • Microarray analysis showed that Cdyl gene deletion influenced the Cks1 gene expression. • We validated that Cdyl repressed the transcriptional activity of Cks1 in vitro. CDYL is a chromodomain protein that has been identified as a transcriptional co-repressor that is primarily involved in the formation of repressor complexes which coordinate histone modifications to repress gene transcription. However, most functions and mechanisms of action of the CDYL protein are unknown. In this study, we show that Cdyl −/− mice died of respiratory distress immediately at birth because of distinct abnormalities in distal lung morphogenesis which was characterized by thickened septal and expiratory alveolus atelectasis. Furthermore, Cdyl deletion in mice led to excessive proliferation of immature epithelial cells and an arrest in alveolar epithelium cell differentiation in late gestation which were associated with decreased secretion of mature surfactant proteins in alveolus. Microarray analysis showed that Cdyl gene deletion influenced the expression of genes regulating neuroactive ligand-receptor interactions, cell adhesion, and cell cycle. We validated that Cdyl repressed the transcriptional activity of Cks1 in vitro. In conclusion, Cdyl gene participates in the perinatal respiratory epithelium differentiation and maturation that is important for normal lung function at birth. [ABSTRACT FROM AUTHOR]

Pten is a multifunctional tumor suppressor. Deletions and mutations in the Pten gene have been associated with multiple forms of human cancers. Pten is a central regulator of several signaling pathways that influences multiple cellular functions. One such function is in cell motility and migration, although the precise mechanism remains unknown. In this study, we deleted Pten in the embryonic lung epithelium using Gata5-cre mice. Absence of Pten blocked branching morphogenesis and ERK and AKT phosphorylation at E12.5. In an explant model, Pten Δ/Δ mesenchyme-free embryonic lung endoderm failed to branch. Inhibition of budding in Pten Δ/Δ explants was associated with major changes in cell migration, while cell proliferation was not affected. We further examined the role of ERK and AKT in branching morphogenesis by conditional, endodermal-specific mutants which blocked ERK or AKT phosphorylation. MEK DM/+ ; Gata5-cre (blocking of ERK phosphorylation) lung showed more severe phenotype in branching morphogenesis. The inhibition of budding was also associated with disruption of cell migration. Thus, the mechanisms by which Pten is required for early endodermal morphogenesis may involve ERK, but not AKT, mediated cell migration. [ABSTRACT FROM AUTHOR]

Abstract The lung is the product of a set of complex developmental interactions between two distinct tissues, the endodermally derived epithelium and the mesoderm. Each tissue contributes to lung development by fine-tuning the spatial and temporal pattern of gene expression for a distinct array of signaling molecules, transcriptional molecules and molecules related to the extracellular matrix. Morphoregulatory transcriptional factors such as NKX2.1 have the crucial role of connecting the cell–cell crosstalk to the activation or repression of gene expression through which processes such as cellular proliferation, migration, differentiation and apoptosis can be controlled. Although none of the factors participating in lung development are exclusively lung-specific, their unique combinations and interactions constitute the basis for emergence of lung structural and functional specificities. An understanding of the individual molecules and their unique interactions in the context of lung development is necessary for the construction of a morphogenetic map for this vital organ as well as for the development of rational and innovative approaches to congenital and induced lung disease.

Background: Parasympathetic signaling has been inferred to regulate epithelial branching as well as organ regeneration and tumor development. However, the relative contribution of local nerve contact versus secreted signals remains unclear. Here, we show a conserved (vertebrates to invertebrates) requirement for intact local nerves in airway branching, persisting even when cholinergic neurotransmission is blocked. Results: In the vertebrate lung, deleting enhanced green fluorescent protein (eGFP)-labeled intrinsic neurons using a two-photon laser leaves adjacent cells intact, but abolishes branching. Branching is unaffected by similar laser power delivered to the immediately adjacent non-neural mesodermal tissue, by blocking cholinergic receptors or by blocking synaptic transmission with botulinum toxin A. Because adjacent vasculature and epithelial proliferation also contribute to branching in the vertebrate lung, the direct dependence on nerves for airway branching was tested by deleting neurons in Drosophila embryos. A specific deletion of neurons in the Drosophila embryo by driving cell-autonomous RicinA under the pan-neuronal elav enhancer perturbed Drosophila airway development. This system confirmed that even in the absence of a vasculature or epithelial proliferation, airway branching is still disrupted by neural lesioning. Conclusions: Together, this shows that airway morphogenesis requires local innervation in vertebrates and invertebrates, yet neurotransmission is dispensable. The need for innervation persists in the fly, wherein adjacent vasculature and epithelial proliferation are absent. Our novel, targeted laser ablation technique permitted the local function of parasympathetic innervation to be distinguished from neurotransmission. [ABSTRACT FROM AUTHOR]

Hoxb5 and Hoxa5 transcription factor proteins uniquely impact lung morphogenesis at the developmental time point when extremely preterm infants are born. The effect of O exposure (0.4 FiO) used in preterm infant care on these Hox proteins is unknown. We used ex vivo fetal mouse lung organ cultures to explore the effects of 0.4 FiO on lung airway and vascular formation in the context of Hoxb5 and Hoxa5 expression and regulation. Compared to room air, 48 h (h) 0.4 FiO adversely attenuated airway and microvasculature formation while reducing lung growth and epithelial cell volume, and increasing mesenchymal volume. 0.4 FiO decreased pro-angiogenic Hoxb5 and VEGFR2 while not altering protein levels of angiostatic Hoxa5. Lungs returned to RA after 24 h 0.4FiO had partial structural recovery but remained smaller and less developed. Mesenchymal cell apoptosis increased and proliferation decreased with time in O while epithelial cell proliferation significantly increased. Hoxb5 overexpression led to prominent peri-airway VEGFR2 expression and promoted lung vascular and airway patterning. Hoxa5 overexpression had the opposite effects. We conclude that 0.4 FiO exposure causes a profound loss of airway and lung microvascular development that occurs partially via reduction in pro-angiogenic Hoxb5 while angiostatic Hoxa5 expression is maintained. [ABSTRACT FROM AUTHOR]

The mammalian genome contains two ERK/MAP kinase genes, Mek1 and Mek2, which encode dual-specificity kinases responsible for ERK/MAP kinase activation. In order to define the function of the ERK/MAPK pathway in the lung development in mice, we performed tissue-specific deletions of Mek1 function on a Mek2 null background. Inactivation of both Mek genes in mesenchyme resulted in several phenotypes, including giant omphalocele, kyphosis, pulmonary hypoplasia, defective tracheal cartilage and death at birth. The absence of tracheal cartilage rings establishes the crucial role of intracellular signaling molecules in tracheal chondrogenesis and provides a putative mouse model for tracheomalacia. In vitro, the loss of Mek function in lung mesenchyme did not interfere with lung growth and branching, suggesting that both the reduced intrathoracic space due to the dysmorphic rib cage and the omphalocele impaired lung development in vivo. Conversely, Mek mutation in the respiratory epithelium caused lung agenesis, a phenotype resulting from the direct impact of the ERK/MAPK pathway on cell proliferation and survival. No tracheal epithelial cell differentiation occurred and no SOX2-positive progenitor cells were detected in mutants, implying a role for the ERK/MAPK pathway in trachea progenitor cell maintenance and differentiation. Moreover, these anomalies were phenocopied when the Erk1 and Erk2 genes were mutated in airway epithelium. Thus, the ERK/MAPK pathway is required for the integration of mesenchymal and epithelial signals essential for the development of the entire respiratory tract. [ABSTRACT FROM AUTHOR]

Background/Aims: The human embryo develops in a hypoxic environment. In this way, cells have to rely on the glycolytic pathway for energy supply, leading to an intracellular accumulation of monocarboxylates such as lactate and pyruvate. These acids have an important role in cell metabolism and their rapid transport across the plasma membrane is crucial for the maintenance of intracellular pH homeostasis. This transport is mediated by a family of transporters, designated by monocarboxylate transporters (MCTs), namely isoforms 1 and 4. MCT1/4 expression is regulated by the ancillary protein CD147.The general aim of this study was to characterize the expression pattern of MCT1/4, CD147 and the glucose transporter GLUT1 during human fetal lung development and elucidate the role of MCTs in lung development. Methods: The expression pattern of MCT1/4 and GLUT1 was characterized by immunohistochemistry and fetal lung viability and branching were evaluated by exposing rat fetal lung explants to CHC, an inhibitor of MCT activity. Results: Our findings show that all the biomarkers are differently expressed during fetal lung development and that CHC appears to have an inhibitory effect on lung branching and viability, in a dose dependent way. Conclusion: We provide evidence for the role of MCTs in embryo lung development, however to prove the dependence of MCT activity further studies are waranted. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Establishment of the functional pulmonary vasculature requires intimate interaction between the epithelium and mesenchyme. Previous genetic studies have led to inconsistent conclusions about the contribution of epithelial Wnts to pulmonary vasculature development. This discrepancy is possibly due to the functional redundancy among different Wnts. Here, we use Shh-Cre to conditionally delete Gpr177 (the mouse ortholog of Drosophila Wntless, Wls), a chaperon protein important for the sorting and secretion of Wnt proteins. Deletion of epithelial Gpr177 reduces Wnt signaling activity in both the epithelium and mesenchyme, resulting in severe hemorrhage and abnormal vasculature, accompanied by branching defects and abnormal epithelial differentiation. We then used multiple mouse models to demonstrate that Wnt/β-catenin signaling is not only required for the proliferation and differentiation of mesenchyme, but also is important for the maintenance of smooth muscle cells through the regulation of the transcription factor Kruppel-like factor 2 (Klf2). Together, our studies define a novel mechanism by which epithelial Wnts regulate the normal development and maintenance of pulmonary vasculature. These findings provide insight into the pathobiology of congenital lung diseases, such as alveolar capillary dysplasia (ACD), that have abnormal alveolar development and dysmorphic pulmonary vasculature. [ABSTRACT FROM AUTHOR]

Purpose Esophageal atresia and tracheo-esophageal fistula (EA-TEF) result from abnormal division of the foregut into esophagus and trachea thus, it may influence airway branching and lung development. The present study examined lung morphogenesis in fetuses with EA-TEF focusing in the expression of FGF10 and its receptor FGFR2 IIIb. Methods Pregnant rats received either 1.75 mg/kg i.p. adriamycin or vehicle on E7, E8 and E9. Embryos were recovered at E15, E18 and E21 and lungs processed for immunohistochemistry and RT-PCR. Three groups were studied: control, adriamycin-exposed with EA-TEF, and adriamycin-exposed without EA-TEF. Comparisons were performed with Mann–Whitney or t tests (significance level, 5 %). Results Lung weight at E15 and E18 were significantly lower in adriaEA fetuses in which the relative mRNA levels of FGF10 were significantly higher. These differences disappeared near term. The receptor FGFR2 IIIb messenger was only significantly increased in adria noEA fetuses at E15. Immunohistochemical study was consistent with these findings. Conclusions Abnormal expression of FGF10 during earlier stages of development, when the lungs are smaller than controls, suggests a compensatory response aimed at ‘‘catching up’’ delayed tracheobronchial branching. Whether similar changes take place in the human condition and influence respiratory physiology remain to be determined. [ABSTRACT FROM AUTHOR]

The human lung consists of multiple cell types derived from early embryonic compartments. The morphogenesis of the lung, as well as the injury repair of the adult lung, is tightly controlled by a network of signaling pathways with key transcriptional factors. Lung cancer is the third most cancer-related death in the world, which may be developed due to the failure of regulating the signaling pathways. Sox (sex-determining region Y (Sry) box-containing) family transcriptional factors have emerged as potent modulators in embryonic development, stem cells maintenance, tissue homeostasis, and cancerogenesis in multiple processes. Recent studies demonstrated that the members of the Sox gene family played important roles in the development and maintenance of lung and development of lung cancer. In this context, we summarize our current understanding of the role of Sox family transcriptional factors in the morphogenesis of lung, their oncogenic potential in lung cancer, and their potential impact in the diagnosis, prognosis, and targeted therapy of lung cancer. [ABSTRACT FROM AUTHOR]

The airways of the mammalian lung are lined with highly specialized epithelial cell types that are the targets of airborne toxicants and injury. Notch signaling plays an important role in the ontogeny of airway epithelial cells, but its contributions to recruitment, expansion or differentiation of resident progenitor/stem cells, and repair and re-establishment of the normal composition of airway epithelium following injury have not been addressed. In this study, the role of a specific Notch receptor, Notch1, was investigated by targeted inactivation in the embryonic lung epithelium using the epithelial-specific Gata5-Cre driver line. Notch1-deficient mice are viable without discernible defects in pulmonary epithelial cell-fate determination and differentiation. However, in an experimental model of airway injury, activity of Notch1 is found to be required for normal repair of the airway epithelium. Absence of Notch1 reduced the ability of a population of cells distinguished by expression of PGP9.5, otherwise a marker of pulmonary neuroendocrine cells, which appears to serve as a reservoir for regeneration of Clara cells. Hairy/enhancer of split-5 ( Hes5) and paired-box-containing gene 6 ( Pax6) were found to be downstream targets of Notch1. Both Hes5 and Pax6 expressions were significantly increased in association with Clara cell regeneration in wild-type lungs. Ablation of Notch1 reduced Hes5 and Pax6 and inhibited airway epithelial repair. Thus, although dispensable in developmental ontogeny of airway epithelial cells, normal activity of Notch1 is required for repair of the airway epithelium. The signaling pathway by which Notch1 regulates the repair process includes stimulation of Hes5 and Pax6 gene expression. S TEM C ELLS 2012;30:946-955 [ABSTRACT FROM AUTHOR]

Pulmonary ErbB4 deletion leads to a delay in fetal lung development, alveolar simplification, and lung function disturbances in adult mice. We generated a model of intrauterine infection in ErbB4 transgenic mice to study the additive effects of antenatal LPS administration and ErbB4 deletion during fetal lung development. Pregnant mice were treated intra-amniotically with an LPS dose of 4 μg at E17 of gestation. Lungs were analyzed 24 h later. A significant influx of inflammatory cells was seen in all LPS-treated lungs. In heterozygote control lungs, LPS treatment resulted in a delay of lung morphogenesis characterized by a significant increase in the fraction of mesenchyme, a decrease in gas exchange area, and disorganization of elastic fibers. Surfactant protein (Sftp)b and Sftpc were upregulated, but mRNA of Sftpb and Sftpc was downregulated compared with non-LPS-treated controls. The mRNA of Sftpa1 and Sftpd was upregulated. In ErbB4-deleted lungs, the LPS effects were more pronounced, resulting in a further delay in morphological development, a more pronounced inflammation in the parenchyma, and a significant higher increase in all Sftp. The effect on Sftpb and Sftpc mRNA was somewhat different, resulting in a significant increase. These results imply a major role of ErbB4 in LPS-induced signaling in structural and functional lung development. [ABSTRACT FROM AUTHOR]

Abstract: Background/Purpose: Iroquois homeobox (Irx) genes have been implicated in the early lung morphogenesis of vertebrates. Irx1-3 and Irx5 gene expression is seen in fetal lung in rodents up to day (D) 18.5 of gestation. Fetal lung in Irx knockdown mice shows loss of mesenchyme and dilated airspaces, whereas nitrofen-induced hypoplastic lung displays thickened mesenchyme and diminished airspaces. We hypothesized that the Irx genes are up-regulated during early lung morphogenesis in the nitrofen-induced hypoplastic lung. Methods: Pregnant rats were exposed either to olive oil or nitrofen on D9. Fetal lungs harvested on D15 were divided into control and nitrofen groups; and the lungs harvested on D18 were divided into control, nitrofen without congenital diaphragmatic hernia (CDH[−]), and nitrofen with CDH (CDH[+]). Irx gene expression levels were analyzed by reverse transcriptase polymerase chain reaction. Immunohistochemistry was performed to evaluate protein expression of Irx family. Results: Pulmonary Irx1-3 and Irx5 messenger RNA expression levels were significantly up-regulated in nitrofen group compared with controls at D15. On D15, Irx immunoreactivity was increased in nitrofen-induced hypoplastic lung compared with controls. Conclusion: Overexpression of Irx genes in the early lung development may cause pulmonary hypoplasia in the nitrofen CDH model by inducing lung dysmorphogenesis with thickened mesenchyme and diminished airspaces. [ABSTRACT FROM AUTHOR]

Abstract: Lung development depends on accurate and precise patterning of a pulmonary anlagen, consisting of both endodermally and mesodermally derived progenitor cells. In this process, the need to establish communication and control among individual cells is paramount. Transforming growth factor-β (TGFβ) and Wingless/int (Wnt) signaling pathways serve this need. The individual functional repertoire of the two pathways is further expanded by cross-talk and integration of signaling at multiple levels taking advantage of their hard-wired multi-component signal transduction platforms. Cross-talk creates the possibility for both specificity and versatility in signaling during development and during repair of injured tissue. Understanding the mechanics and the physiological implications of this cross-talk is necessary for therapeutic or preventive targeting of either TGFβ or Wnt signaling pathways. [Copyright &y& Elsevier]

Clara cells, together with ciliated and pulmonary neuroendocrine cells, make up the epithelium of the bronchioles along the conducting airways. Clara cells are also known as progenitor or stem cells during lung regeneration after injury. The mechanisms of Clara cell differentiation are largely unknown. Transforming growth factor beta (TGFβ)is a multifunctional molecule with roles in normal development and disease pathogenesis. In this study, we deleted the TGFβ type I receptor Alk5 in the embryonic lung epithelium using Gata5-Cre mice. Absence of Alk5 blocked Clara cell differentiation but had no effect on ciliated or pulmonary neuroendocrine cells. Hairy/Enhancer of Split-1, which is expressed in Clara cell putative 'progenitors' was found to be a downstream target of Alk5 in vivo and in vitro. Loss of Alk5-mediated signaling also stimulated Pten gene expression and inhibited ERK phosphorylation in vivo. Using lung epithelial cells, we show that Alk5-regulated Hes1 expression is stimulated through Pten and the MEK/ERK and PI3K/AKT pathways. Thus, the signaling pathway by which TGFβ/ALK5 regulates Clara cell differentiation may entail inhibition of Pten expression, which in turn activates ERK and AKT phosphorylation. [ABSTRACT FROM AUTHOR]

Abstract: Transforming growth factor-beta is a multifunctional growth factor with roles in normal development and disease pathogenesis. One such role is in inhibition of lung branching morphogenesis, although the precise mechanism remains unknown. In an explant model, all three TGFβ isoforms inhibited FGF10-induced morphogenesis of mesenchyme-free embryonic lung endoderm. Inhibition of budding by TGFβ was partially abrogated in endodermal explants from Smad3 −/− or conditional endodermal-specific Smad4 Δ/Δ embryonic lungs. Endodermal explants from conditional TGFβ receptor II knockout lungs were entirely refractive to TGFβ-induced inhibition. Inhibition of morphogenesis was associated with dedifferentiation of endodermal cells as documented by a decrease in key transcriptional factor, NKX2.1 protein, and its downstream target, surfactant protein C (SpC). TGFβ reduced the proliferation of wild-type endodermal cells within the explants as assessed by BrdU labeling. Gene expression analysis showed increased levels of mRNA for Pten, a key regulator of cell proliferation. Conditional, endodermal-specific deletion of Pten overcame TGFβ''s inhibitory effect on cell proliferation, but did not restore morphogenesis. Thus, the mechanisms by which TGFβ inhibits FGF10-induced lung endodermal morphogenesis may entail both inhibition of cell proliferation, through increased Pten, as well as inhibition or interference with morphogenetic mediators such as Nkx2.1. Both of the latter are dependent on signaling through TβRII. [Copyright &y& Elsevier]

Abstract: There is increasing evidence that epithelial–vascular interactions are essential for tissue patterning. Here we identified components of the molecular cross talk between respiratory epithelial cells and pulmonary capillaries necessary for the formation of the gas exchange surface of the lung. Selective inactivation of the Vegf-A gene in respiratory epithelium results in an almost complete absence of pulmonary capillaries, demonstrating the dependence of pulmonary capillary development on epithelium-derived Vegf-A. Deficient capillary formation in Vegf-A deficient lungs is associated with a defect in primary septae formation, a morphogenetic process critical for distal lung morphogenesis, coupled with suppression of epithelial cell proliferation and decreased hepatocyte growth factor (Hgf) expression. Lung endothelial cells express Hgf, and selective deletion of the Hgf receptor gene in respiratory epithelium phenocopies the malformation of septae, confirming the requirement for epithelial Hgf signaling in normal septae formation and suggesting that Hgf serves as an endothelium-derived factor that signals to the epithelium. Our findings support a mechanism for primary septae formation dependent on reciprocal interactions between respiratory epithelium and the underlying vasculature, establishing the dependence of pulmonary capillary development on epithelium-derived Vegf-A, and identify Hgf as a putative endothelium-derived factor that mediates the reciprocal signaling from the vasculature to the respiratory epithelium. [Copyright &y& Elsevier]