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Postnatal organ-specific stem and progenitor cells are an attractive potential donor cell for tissue-engineering because they can be harvested autologous from the recipient and have sufficient potential to regenerate the tissue of interest with less risk for ectopic growth or tumor formation compared to donor cells from embryonic or fetal sources. We describe the generation of tissue-engineered larynx and trachea (TELT) from human and mouse postnatal organoid units (OU) as well as from human fetal OU. Mouse TELT contained differentiated respiratory epithelium lining large lumens, cartilage and smooth muscle. In contrast, human postnatal TE trachea, formed small epithelial lumens with rare differentiation, in addition to smooth muscle and cartilage. Human fetal TELT contained the largest epithelial lumens with all differentiated cell types as well as smooth muscle and cartilage. Increased epithelial cytokeratin 14 was identified in both human fetal and postnatal TELT compared to native trachea, consistent with regenerative basal cells. Cilia in TELT epithelium also demonstrated function with beating movements. While both human postnatal and fetal progenitors have the potential to generate TELT, there is more epithelial growth and differentiation from fetal progenitors, highlighting fundamental differences in these cell populations., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The tuberous sclerosis complex (TSC) proteins are critical negative regulators of the mammalian/mechanistic target of rapamycin complex 1 pathway. Germline mutations of TSC1 or TSC2 cause TSC, affecting multiple organs, including the kidney and lung, and causing substantial morbidity and mortality. The mechanisms of organ-specific disease in TSC remain incompletely understood, and the impact of TSC inactivation on mesenchymal lineage cells has not been specifically studied. We deleted Tsc2 specifically in mesoderm-derived mesenchymal cells of multiple organs in mice using the Dermo1-Cre driver. The Dermo1-Cre-driven Tsc2 conditional knockout mice had body growth retardation and died approximately 3 weeks after birth. Significant phenotypes were observed in the postnatal kidney and lung. Inactivation of Tsc2 in kidney mesenchyme caused polycystic lesions starting from the second week of age, with increased cell proliferation, tubular epithelial hyperplasia, and epithelial-mesenchymal transition. In contrast, Tsc2 deletion in lung mesenchyme led to decreased cell proliferation, reduced postnatal alveolarization, and decreased differentiation with reduced numbers of alveolar myofibroblast and type II alveolar epithelial cells. Two major findings thus result from this model: inactivation of Tsc2 in mesoderm-derived cells causes increased cell proliferation in the kidneys but reduced proliferation in the lungs, and inactivation of Tsc2 in mesoderm-derived cells causes epithelial-lined renal cysts. Therefore, Tsc2-mTOR signaling in mesenchyme is essential for the maintenance of renal structure and for lung alveolarization., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Branching in the embryonic lung is controlled by a variety of morphogens. Mechanics is also believed to play a significant role in lung branching. The relative roles and interactions of these two broad factors are challenging to determine. We considered three hypotheses for explaining why tracheal occlusion triples branching with no overall increase in size. Both hypotheses are based on tracheal occlusion blocking the exit of secretions. (H1) Increased lumen pressure stretches tissues; stretch receptors at shoulders of growing tips increase local rate of branching. (H2) Blocking exit of secretions blocks advective transport of morphogens, leading to (H2a) increased overall concentration of morphogens or (H2b) increased flux of morphogens at specific locations. We constructed and analyzed computational models of tissue stretch and solute transport in a 3D lung geometry. Observed tissue stresses and stretches were predominantly in locations unrelated to subsequent branch locations, suggesting that tissue stretch (H1) is not the mechanism of enhancement of branching. Morphogen concentration in the mesenchyme (H2a) increased with tracheal occlusion, consistent with previously reported results. Morphogen flux at the epithelial surface (H2b) completely changed its distribution pattern when the trachea was occluded, tripling the number of locations at which it was elevated. Our results are consistent with the hypothesis that tracheal occlusion blocks outflow of secretions, leading to a higher number of high-flux locations at branching tips, in turn leading to a large increase in number of branching locations., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Peristalsis begins in the lung as soon as the smooth muscle forms, and persists until birth. Since the prenatal lung is liquid-filled, smooth muscle action can deform tissues and transport fluid far from the immediately adjacent tissues. Stretching of embryonic tissues and sensation of internal fluid flows have been shown to have potent morphogenetic effects. We hypothesize that these effects are at work in lung morphogenesis. To place that hypothesis in a quantitative framework, we analyze a model of the fluid-structure interactions between embryonic tissues and lumen fluid resulting from peristaltic waves that partially occlude the airway. We find that if the airway is closed, deformations are synchronized; by contrast, if the trachea is open, maximal occlusion precedes maximal pressure. We perform a parametric analysis of how occlusion, stretch, and flow depend on tissue stiffnesses, smooth muscle force, tissue shape and size, and fluid viscosity. We find that most of these relationships are governed by simple ratios., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Neonatal mouse hearts fully regenerate after ventricular resection similar to adult zebrafish. We established cryoinjury models to determine if different types and varying degrees of severity in cardiac injuries trigger different responses in neonatal mouse hearts. In contrast to ventricular resection, neonatal mouse hearts fail to regenerate and show severe impairment of cardiac function post transmural cryoinjury. However, neonatal hearts fully recover after non-transmural cryoinjury. Interestingly, cardiomyocyte proliferation does not significantly increase in neonatal mouse hearts after cryoinjuries. Epicardial activation and new coronary vessel formation occur after cryoinjury. The profibrotic marker PAI-1 is highly expressed after transmural but not non-transmural cryoinjuries, which may contribute to the differential scarring. Our results suggest that regenerative medicine strategies for heart injuries should vary depending on the nature of the injury., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Eya1 gene encodes a transcriptional co-activator that acts with Six1 to control the development of different organs. However, Six1-Eya1 interactions and functional roles in mesenchymal cell proliferation and differentiation as well as alveolarization during the saccular stage of lung development are still unknown. Herein, we provide the first evidence that Six1 and Eya1 act together to regulate mesenchymal development as well as alveolarization during the saccular phase of lung morphogenesis. Deletion of either or both Six1 and Eya1 genes results in a severe saccular phenotype, including defects of mesenchymal cell development and remodeling of the distal lung septae and arteries. Mutant lung histology at the saccular phase shows mesenchymal and saccular wall thickening, and abnormal proliferation of α-smooth muscle actin-positive cells, as well as increased mesenchymal/fibroblast cell differentiation, which become more sever when deleting both genes. Our study indicates that SHH but not TGF-β signaling pathway is a central mediator for the histologic alterations described in the saccular phenotype of Eya1(-/-) or Six1(-/-) lungs. Indeed, genetic reduction of SHH activity in vivo or inhibition of its activity in vitro substantially rescues lung mesenchymal and alveolar phenotype of mutant mice at the saccular phase. These findings uncover novel functions for Six1-Eya1-SHH pathway during the saccular phase of lung morphogenesis, providing a conceptual framework for future mechanistic and translational studies in this area., (© 2013 Elsevier Inc. All rights reserved.)

Fibroblast growth factor (FGF) signaling to the epithelium and mesenchyme mediated by FGF10 and FGF9, respectively, controls cecal formation during embryonic development. In particular, mesenchymal FGF10 signals to the epithelium via FGFR2b to induce epithelial cecal progenitor cell proliferation. Yet the precise upstream mechanisms controlling mesenchymal FGF10 signaling are unknown. Complete deletion of Fgf9 as well as of Pitx2, a gene encoding a homeobox transcription factor, both lead to cecal agenesis. Herein, we used mouse genetic approaches to determine the precise contribution of the epithelium and/or mesenchyme tissue compartments in this process. Using tissue compartment specific Fgf9 versus Pitx2 loss of function approaches in the gut epithelium and/or mesenchyme, we determined that FGF9 signals to the mesenchyme via Pitx2 to induce mesenchymal Fgf10 expression, which in turn leads to epithelial cecal bud formation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Six1 is a member of the six-homeodomain family of transcription factors. Six1 is expressed in multiple embryonic cell types and plays important roles in proliferation, differentiation and survival of precursor cells of different organs, yet its function during lung development was hitherto unknown. Herein we show that Six1(-/-) lungs are severely hypoplastic with greatly reduced epithelial branching and increased mesenchymal cellularity. Six1 is expressed at the distal epithelial tips of branching tubules as well as in the surrounding distal mesenchyme. Six1(-/-) lung epithelial cells show increased expression of differentiation markers, but loss of progenitor cell markers. Six1 overexpression in MLE15 lung epithelial cells in vitro inhibited cell differentiation, but increases the expression of progenitor cell markers. In addition, Six1(-/-) embryos and newborn mice exhibit mesenchymal overproliferation, decreased Fgf10 expression and severe defects in the smooth muscle component of the bronchi and major pulmonary vessels. These defects lead to rupture of major vessels in mutant lungs after birth. Treatment of Six1(-/-) epithelial explants in culture with recombinant Fgf10 protein restores epithelial branching. As Shh expression is abnormally increased in Six1(-/-) lungs, we also treated mutant mesenchymal explants with recombinant Shh protein and found that these explants were competent to respond to Shh and continued to grow in culture. Furthermore, inhibition of Shh signaling with cyclopamine stimulated Six1(-/-) lungs to grow and branch in culture. This study provides the first evidence for the requirement of Six1 in coordinating Shh-Fgf10 signaling in embryonic lung to ensure proper levels of proliferation and differentiation along the proximodistal axis of epithelial, mesenchymal and endothelial cells. These findings uncover novel and essential functions for Six1 as a critical coordinator of Shh-Fgf10 signaling during embryonic lung development. We propose that Six1 is hence critical for coordination of proper lung epithelial, mesenchymal and vascular development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The proper level of proliferation and differentiation along the proximodistal axis is crucial for lung organogenesis. Elucidation of the factors that control these processes will therefore provide important insights into embryonic lung development and regeneration. Eya1 is a transcription factor/protein phosphatase that regulates cell lineage specification and proliferation. Yet its functions during lung development are unknown. In this paper we show that Eya1(-/-) lungs are severely hypoplastic with reduced epithelial branching and increased mesenchymal cellularity. Eya1 is expressed at the distal epithelial tips of branching tubules as well as in the surrounding distal mesenchyme. Eya1(-/-) lung epithelial cells show loss of progenitor cell markers with increased expression of differentiation markers and cell cycle exit. In addition, Eya1(-/-) embryos and newborn mice exhibit severe defects in the smooth muscle component of the bronchi and major pulmonary vessels with decreased Fgf10 expression. These defects lead to rupture of the major vessels and hemorrhage into the lungs after birth. Treatment of Eya1(-/-) epithelial explants in culture with recombinant Fgf10 stimulates epithelial branching. Since Shh expression and activity are abnormally increased in Eya1(-/-) lungs, we tested whether genetically lowering Shh activity could rescue the Eya1(-/-) lung phenotype. Indeed, genetic reduction of Shh partially rescues Eya1(-/-) lung defects while restoring Fgf10 expression. This study provides the first evidence that Eya1 regulates Shh signaling in embryonic lung, thus ensuring the proper level of proliferation and differentiation along the proximodistal axis of epithelial, mesenchymal and endothelial cells. These findings uncover novel functions for Eya1 as a critical upstream coordinator of Shh-Fgf10 signaling during embryonic lung development. We conclude, therefore, that Eya1 function is critical for proper coordination of lung epithelial, mesenchymal and vascular development., (Copyright © 2010 Elsevier Inc. All rights reserved.)

A fundamental step in embryonic development is cell differentiation whereby highly specialised cell types are developed from a single undifferentiated, fertilised egg. One of the earliest lineages to form in the mammalian conceptus is the trophoblast, which contributes exclusively to the extraembryonic structures that form the placenta. Trophoblast giant cells (TGCs) in the rodent placenta form the outermost layer of the extraembryonic compartment, establish direct contact with maternal cells, and produce a number of pregnancy-specific cytokine hormones. Giant cells differentiate from proliferative trophoblasts as they exit the cell cycle and enter a genome-amplifying endocycle. Normal differentiation of secondary TGCs is a critical step toward the formation of the placenta and normal embryonic development. Trophoblast development is also of particular interest to the developmental biologist and immunobiologist, as these cells constitute the immediate cellular boundary between the embryonic and maternal tissues. Abnormalities in the development of secondary TGCs results in severe malfunction of the placenta. Herein we review new information that has been accumulated recently regarding the molecular and cellular regulation of trophoblast and placenta development. In particular, we discuss the molecular aspects of murine TGC differentiation. We also focus on the role of growth and transcription factors in TGC development., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

The miR-17 family of microRNAs has recently been recognized for its importance during lung development. The transgenic overexpression of the entire miR-17-92 cluster in the lung epithelium led to elevated cellular proliferation and inhibition of differentiation, while targeted deletion of miR-17-92 and miR-106b-25 clusters showed embryonic or early post-natal lethality. Herein we demonstrate that miR-17 and its paralogs, miR-20a, and miR-106b, are highly expressed during the pseudoglandular stage and identify their critical functional role during embryonic lung development. Simultaneous downregulation of these three miRNAs in explants of isolated lung epithelium altered FGF10 induced budding morphogenesis, an effect that was rescued by synthetic miR-17. E-Cadherin levels were reduced, and its distribution was altered by miR-17, miR-20a and miR-106b downregulation, while conversely, beta-catenin activity was augmented, and expression of its downstream targets, including Bmp4 as well as Fgfr2b, increased. Finally, we identified Stat3 and Mapk14 as key direct targets of miR-17, miR-20a, and miR-106b and showed that simultaneous overexpression of Stat3 and Mapk14 mimics the alteration of E-Cadherin distribution observed after miR-17, miR-20a, and miR-106b downregulation. We conclude that the mir-17 family of miRNA modulates FGF10-FGFR2b downstream signaling by specifically targeting Stat3 and Mapk14, hence regulating E-Cadherin expression, which in turn modulates epithelial bud morphogenesis in response to FGF10 signaling.

Mammalian Sprouty2 (Spry2) is a key regulator of the receptor tyrosine kinase/ERK signaling pathway and involved in many biological processes, including cell growth, migration, and tumor suppression. Here, we demonstrated that the intracellular protein level of Spry2 was significantly down-regulated by tumor necrosis factor-alpha (TNF-alpha) in both murine Swiss 3T3 fibroblasts and MLE15 lung epithelial cells. Although TNF-alpha activates multiple signaling cascades, only the inhibitor of p38 MAPK pathway blocked TNF-alpha-induced Spry2 down-regulation. Moreover, since both the mRNA level and protein half-life of Spry2 were unaltered by TNF-alpha treatment, this indicated the possible involvement of a translational mechanism in mediating the inhibitory effect of TNF-alpha. Importantly, rescue of the TNF-alpha-induced down-regulation of Spry2 by gene overexpression led to reverse of the apoptotic effect of TNF-alpha in Swiss 3T3 cells. To our knowledge, this study is the first that reported the association of Spry2 with TNF-alpha signaling pathway.

Bone morphogenetic proteins (BMPs) play important roles in regulating lung development and function although the endogenous regulatory effects of BMP signaling are still controversial. We found that BMP type I receptor Alk3 is expressed predominantly in airway epithelial cells during development. The function of Alk3 in lung development was determined using an inducible knockout mouse model by crossing epithelial cell-specific Cre transgenic mice SPC-rtTA/TetO-Cre and floxed-Alk3 mice. Abrogation of Alk3 in mouse lung epithelia from either early lung organogenesis or late gestation resulted in similar neonatal respiratory distress phenotypes accompanied by collapsed lungs. Early-induction of Alk3 knockout in lung epithelial cells caused retardation of early lung branching morphogenesis, reduced cell proliferation, and differentiation. However, late gestation induction of the knockout caused changes in cell proliferation and survival, as shown by altered cell biology, reduced expression of peripheral epithelial markers (Clara cell-specific protein, surfactant protein C, and aquaporin-5), and lack of surfactant secretion. Furthermore, canonical Wnt signaling was perturbed, possibly through reduced Wnt inhibitory factor-1 expression in Alk3-knockout lungs. Therefore, our data suggest that deficiency of appropriate BMP signaling in lung epithelial cells results in prenatal lung malformation, neonatal atelectasis, and respiratory failure.

Tracheal occlusion during lung development accelerates growth in response to increased intraluminal pressure. In order to investigate the role of internal pressure on murine early lung development, we cauterized the tip of the trachea, to occlude it, and thus to increase internal pressure. This method allowed us to evaluate the effect of tracheal occlusion on the first few branch generations and on gene expression. We observed that the elevation of internal pressure induced more than a doubling in branching, associated with increased proliferation, while branch elongation speed increased 3-fold. Analysis by RT-PCR showed that Fgf10, Vegf, Sprouty2 and Shh mRNA expressions were affected by the change of intraluminal pressure after 48h of culture, suggesting mechanotransduction via internal pressure of these key developmental genes. Tracheal occlusion did not increase the number of branches of Fgfr2b-/- mice lungs nor of wild type lungs cultured with Fgfr2b antisense RNA. Tracheal occlusion of Fgf10(LacZ/-) hypomorphic lungs led to the formation of fewer branches than in wild type. We conclude that internal pressure regulates the FGF10-FGFR2b-Sprouty2 pathway and thus the speed of the branching process. Therefore pressure levels, fixed both by epithelial secretion and boundary conditions, can control or modulate the branching process via FGF10-FGFR2b-Sprouty2.

Different animal models of pulmonary fibrosis have been developed to investigate potential therapies for idiopathic pulmonary fibrosis (IPF). The most common is the bleomycin model in rodents (mouse, rat and hamster). Over the years, numerous agents have been shown to inhibit fibrosis in this model. However, to date none of these compounds are used in the clinical management of IPF and none has shown a comparable antifibrotic effect in humans. We performed a systematic review of publications on drug efficacy studies in the bleomycin model to evaluate the value of this model regarding transferability to clinical use. Between 1980 and 2006 we identified 240 experimental studies describing beneficial antifibrotic compounds in the bleomycin model. 222 of those used a preventive regimen (drug given < or =7 days after last bleomycin application), only 13 were therapeutic trials (>7 days after last bleomycin application). In 5 studies we did not find enough details about the timing of drug application to allow inter-study comparison. It is critical to distinguish between drugs interfering with the inflammatory and early fibrogenic response from those preventing progression of fibrosis, the latter likely much more meaningful for clinical application. All potential antifibrotic compounds should be evaluated in the phase of established fibrosis rather than in the early period of bleomycin-induced inflammation for assessment of its antifibrotic properties. Further care should be taken in extrapolation of drugs successfully tested in the bleomycin model due to partial reversibility of bleomycin-induced fibrosis over time. The use of alternative and more robust animal models, which better reflect human IPF, is warranted.

Tissue repair is a well-orchestrated biological process involving numerous soluble mediators, and an imbalance between these factors may result in impaired repair and fibrosis. Transforming growth factor (TGF)-beta is a key profibrotic element in this process and it is thought that its three isoforms act in a similar way. Here, we report that TGF-beta3 administered to rat lungs using transient overexpression initiates profibrotic effects similar to those elicited by TGF-beta1, but causes less severe and progressive changes. The data suggest that TGF-beta3 does not lead to inhibition of matrix degradation in the same way as TGF-beta1, resulting in non-fibrotic tissue repair. Further, TGF-beta3 is able to downregulate TGF-beta1-induced gene expression, suggesting a regulatory role of TGF-beta3. TGF-beta3 overexpression results in an upregulation of Smad proteins similar to TGF-beta1, but is less efficient in inducing the ALK 5 and TGF-beta type II receptor (TbetaRII). We provide evidence that this difference may contribute to the progressive nature of TGF-beta1-induced fibrotic response, in contrast to the limited fibrosis observed following TGF-beta3 overexpression. TGF-beta3 is important in "normal wound healing", but is outbalanced by TGF-beta1 in "fibrotic wound healing" in the lung.

Adult respiratory diseases are caused by many factors, including genetic-environmental interaction. Genetic abnormalities can impact early fetal lung development, postnatal lung maturation, as well as adult lung injury and repair. Studies suggest that abnormally developed lung structure and function may contribute as a susceptibility factor for several adult lung diseases. This review focuses on the relationship between lung development and pathogenesis of several lung diseases including COPD, cystic fibrosis (CF), and asthma. COPD with emphysema has been considered to be an accelerated involutional disease of aging smokers. However, since only a proportion (approximately 15%) of smokers get COPD with emphysema, clearly genetic susceptibility must play a significant part in determining both the age of onset and the rapidity of decline in lung function. In mice, interference with key genes either by null mutation, hypomorphism, or gain or loss of function results in phenotypes comprising either neonatal lethal respiratory distress if the structural effect is severe, or reduced alveolarization and/or early onset emphysema if the effect is milder. Reported susceptibility candidate genes are therefore discussed in some detail, including elastin, lysyl oxidase, fibrillin, the transforming growth factor-beta-Smad3 pathway, as well as extracellular matrix proteases. In the case of CF, the Cftr gene has been shown to regulate fetal lung epithelial cell differentiation and maturation. Subtle abnormalities of lung structure and function are found in clinically asymptomatic CF infants. Finally, airway remodeling due to chronic inflammation is important in infants who later acquire asthma.

The key role played by Fgf10 during early lung development is clearly illustrated in Fgf10 knockout mice, which exhibit lung agenesis. However, Fgf10 is continuously expressed throughout lung development suggesting extended as well as additional roles for FGF10 at later stages of lung organogenesis. We previously reported that the enhancer trap Mlcv1v-nLacZ-24 transgenic mouse strain functions as a reporter for Fgf10 expression and displays decreased endogenous Fgf10 expression. In this paper, we have generated an allelic series to determine the impact of Fgf10 dosage on lung development. We report that 80% of the newborn Fgf10 hypomorphic mice die within 24 h of birth due to respiratory failure. These mutant mouse lungs display severe hypoplasia, dilation of the distal airways and large hemorrhagic areas. Epithelial differentiation and proliferation studies indicate a specific decrease in TTF1 and SP-B expressing cells correlating with reduced epithelial cell proliferation and associated with a decrease in activation of the canonical Wnt signaling in the epithelium. Analysis of vascular development shows a reduction in PECAM expression at E14.5, which is associated with a simplification of the vascular tree at E18.5. We also show a decrease in alpha-SMA expression in the respiratory airway suggesting defective smooth muscle cell formation. At the molecular level, these defects are associated with decrease in Vegfa and Pdgfa expression likely resulting from the decrease of the epithelial/mesenchymal ratio in the Fgf10 hypomorphic lungs. Thus, our results indicate that FGF10 plays a pivotal role in maintaining epithelial progenitor cell proliferation as well as coordinating alveolar smooth muscle cell formation and vascular development.

Epithelial-mesenchymal interactions that govern the development of the colon from the primitive gastrointestinal tract are still unclear. In this study, we determine the temporal-spatial expression pattern of Fibroblast growth factor 10 (Fgf10), a key developmental gene, in the colon at different developmental stages. We found that Fgf10 is expressed in the mesenchyme of the distal colon, while its main receptor Fgfr2-IIIb is expressed throughout the entire intestinal epithelium. We demonstrate that Fgf10 inactivation leads to decreased proliferation and increased cell apoptosis in the colonic epithelium at E10.5, therefore resulting in distal colonic atresia. Using newly described Fgf10 hypomorphic mice, we show that high levels of FGF10 are dispensable for the differentiation of the colonic epithelium. Our work unravels for the first time the pivotal role of FGF10 in the survival and proliferation of the colonic epithelium, biological activities which are essential for colonic crypt formation.

Fibroblast growth factor (FGF) signaling has been shown to regulate lung epithelial development but its influence on mesenchymal differentiation has been poorly investigated. To study the role of mesenchymal FGF signaling in the differentiation of the mesenchyme and its impact on epithelial morphogenesis, we took advantage of Fgfr2c(+/Delta) mice, which due to a splicing switch express Fgfr2b in mesenchymal tissues and manifest Apert syndrome-like phenotypes. Using a set of in vivo and in vitro studies, we show that an autocrine FGF10-FGFR2b signaling loop is established in the mutant lung mesenchyme, which has several consequences. It prevents the entry of the smooth muscle progenitors into the smooth muscle cell (SMC) lineage and results in reduced fibronectin and elastin deposition. Levels of Fgf10 expression are raised within the mutant mesenchyme itself. Epithelial branching as well as epithelial levels of FGF and canonical Wnt signaling is dramatically reduced. These defects result in arrested development of terminal airways and an "emphysema like" phenotype in postnatal lungs. Our work unravels part of the complex interactions that govern normal lung development and may be pertinent to understanding the basis of respiratory defects in Apert syndrome.

Mesothelial Fibroblast Growth Factor 9 (Fgf9) has been demonstrated by inactivation studies in mouse to be critical for the proliferation of the mesenchyme. We now show that Fgf9 is also expressed at significant levels in the distal epithelium from the mid-pseudoglandular stages. Using mesenchymal-free lung endoderm culture, we show that FGF9 triggers the proliferation of the distal epithelium leading to the formation of a cyst-like structure. On embryonic Fgfr2b-/- lungs, FGF9 induces proliferation of the mesenchyme but fails to trigger a similar effect on the epithelium, therefore involving the FGFR2b receptor in the proliferative response of the epithelium to FGF9. While FGF9 inhibits the differentiation of the mesenchyme, the epithelium appears to differentiate normally. At the molecular level, FGF9 up-regulates Fgf10 expression in the mesenchyme likely via increased expression of Tbx4 and 5 and controls the transcription of Hedgehog targets Ptc and Gli-1 in a Hedgehog-independent manner. We also show that FGF9 inhibits the activation of the canonical Wnt pathway in the epithelium by increasing Dkk1 expression, a canonical Wnt antagonist. Our work shows for the first time that FGF9 acts on the epithelium involving FGFR2b to control its proliferation but not its differentiation and contributes to the regulation of canonical Wnt signaling in the epithelium.

Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial-mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis.

Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must be tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. Herein, we demonstrate for the first time an important function for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. We show that Shp2 is specifically expressed in embryonic lung epithelial buds, and that loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, we provide direct evidence that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. We demonstrate that Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung was associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation was not affected. Our results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis.

Members of the Dickkopf (Dkk) family of secreted proteins are potent inhibitors of Wnt/beta-catenin signaling. In this study we show that Dkk1, -2, and -3 are expressed distally in the epithelium, while Kremen1, the needed co-receptor, is expressed throughout the epithelium of the developing lung. Using TOPGAL mice [DasGupta, R., Fuchs, E., 1999. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126, 4557-4568] to monitor the Wnt pathway, we show that canonical Wnt signaling is dynamic in the developing lung and is active throughout the epithelium and in the proximal smooth muscle cells (SMC) until E12.5. However, from E13.5 onwards, TOPGAL activity is absent in the SMC and is markedly reduced in the distal epithelium coinciding with the onset of Dkk-1 expression in the distal epithelium. To determine the role of Wnt signaling in early lung development, E11.5 organ cultures were treated with recombinant DKK1. Treated lungs display impaired branching, characterized by failed cleft formation and enlarged terminal buds, and show decreased alpha-smooth muscle actin (alpha-SMA) expression as well as defects in the formation of the pulmonary vasculature. These defects coincide with a pattern of decreased fibronectin (FN) deposition. DKK1-induced morphogenetic defects can be mimicked by inhibition of FN and overcome by addition of exogenous FN, suggesting an involvement of FN in Wnt-regulated morphogenetic processes.

Herein we report the knock-on cytotoxic effect of lethal toxin (LeTx) on human umbilical vascular endothelial cells (HUVECs). HUVECs were treated either directly with LeTx or indirectly with LeTx conditioned medium (LeTxCM) prepared from RAW264.7 macrophage cells. Cytotoxicity assays were done on HUVECs and A549 cells using LeTx. HUVECs were more susceptible to LeTx (61-74% survivals) as compared to A549 cells (83-94% survivals, P < 0.005). However, LeTxCM from RAW264.7 further potentiated killing of HUVECs (37% survival) compared to the LeTxCM from A549 cells (up to 70-100% survivals). LeTxCM challenge induced an apoptotic cell death in HUVECs, and this was confirmed by reduction of BCL-2 levels to 54%. Protective antigen (PA) binding to macrophage cell line RAW264.7 > HUVECs >> A549 cells. Thus, we postulate that after the initial prodormal phase of pulmonary entry, LeTx causes not only significant direct damage to macrophages and endothelial cells, but also mediates additional indirect damage to endothelial cells mediated by a knock-on effect of LeTx on macrophages that causes apoptotic cell death in endothelial cells.

Bronchopulmonary dysplasia is a chronic lung disease of premature human infancy that shows pathological features comprising varying sized areas of interstitial fibrosis in association with distorted large alveolar spaces. We have previously shown that transfer of active transforming growth factor (TGF)-beta 1 (AdTGF beta 1(223/225)) genes by adenovirus vector to embryonic lungs results in inhibition of branching morphogenesis and primitive peripheral lung development, whereas transfer to adult lungs results in progressive interstitial fibrosis. Herein we show that transfer of TGF-beta1 to newborn rat pups results in patchy areas of interstitial fibrosis developing throughout a period of 28 days after transfer. These areas of fibrosis appear alongside areas of enlarged alveolar spaces similar to the prealveoli seen at birth, suggesting that postnatal lung development and alveolarization has been inhibited. In rats treated with AdTGF beta 1(223/225), enlarged alveolar spaces were evident by day 21, and by 28 days, the mean alveolar cord length was nearly twice that in control vector or untreated rats. Hydroxyproline measurements confirmed the presence of fibrosis. These data suggest that overexpression of TGF-beta 1 during the critical period of postnatal rat lung alveolarization gives rise to pathological, biochemical, and morphological changes consistent with those seen in human bronchopulmonary dysplasia, thus inferring a pathogenic role for TGF-beta in this disorder.

Tumor necrosis factor-alpha converting enzyme (TACE) is a membrane-anchored, Zn-dependent metalloprotease, which belongs to the ADAM (a disintegrin and metalloprotease) family. TACE functions as a membrane sheddase to release the ectodomain portions of many transmembrane proteins, including the precursors of TNFalpha, TGFalpha, several other cytokines, as well as the receptors for TNFalpha, and neuregulin (ErbB4). Mice with TACE(DeltaZn/DeltaZn) null mutation die at birth with phenotypic changes, including failure of eyelid fusion, hair and skin defects, and abnormalities of lung development. Abnormal fetal heart development was not previously described. Herein, we report that TACE(DeltaZn/DeltaZn) null mutant mice by late gestation exhibit markedly enlarged fetal hearts with increased myocardial trabeculation and reduced cell compaction, mimicking the pathological changes of noncompaction of ventricular myocardium. In addition, larger cardiomyocyte cell size and increased cell proliferation were observed in ventricles of TACE(DeltaZn/DeltaZn) knockout mouse hearts. At the molecular level, reduced expression of epidermal growth factor receptor, attenuated protein cleavage of ErbB4, and changes in MAPK activation were also detected in TACE(DeltaZn/DeltaZn) knockout heart tissues. The data suggest that TACE-mediated cell surface protein ectodomain shedding plays an essential and a novel regulatory role during cardiac development and modeling.