To determine whether epigenetic changes occurred during cyclophosphamide (CYP)-induced chronic bladder inflammation in mice. Epigenetics plays a role in the regulation of inflammatory genes in non-cancer diseases such as asthma and COPD. However, epigenetic (DNA methylation) changes during chronic bladder inflammation have not been described previously.
Chronic cystitis was induced in three groups of adult CD-1 male mice using multiple weight-based intraperitoneal cyclophosphamide (CYP) injections over a period of three months. Histopathologic and MethyLight assays were performed on chronic bladder inflammation specimens at multiple time points to monitor cystitis progression and DNA methylation changes in comparison to control specimens, respectively.
Histopathological analysis showed the most extensive edema and urothelial sloughing at the 1-month time point. MethyLight analyses revealed statistically significant changes in DNA methylation associated with the Calca, Timp3, Mmp2, and Igf2r genes in the chronic bladder injury model. The changes in DNA methylation associated with chronic cystitis were noted to be DNA hypomethylation of the Calca gene in the control tissue, and DNA hypermethylation for the Calca, Timp3, Mmp2, and Igf2r genes, in comparison to control tissue.
DNA methylation changes were noted in Calca, Timp3, Mmp2, and Igf2r genes during chronic cystitis in a murine model. Epigenetics appears to play a role in the regulation of inflammatory bladder genes during chronic cystitis; however, further studies are needed to elucidate the pathways associated with these genes.
Bladder Inflammation; Cystitis; Cyclophosphamide; Epigenetics; DNA; Methylation
Air pollution is a major health challenge worldwide and has previously been strongly associated with adverse reproductive health. This study aimed to examine the association between spontaneous abortion and seasonal variation of air pollutants in Ulaanbaatar, Mongolia.
Monthly average O3, SO2, NO2, CO, PM10 and PM2.5 levels were measured at Mongolian Government Air Quality Monitoring stations. The medical records of 1219 women admitted to the hospital due to spontaneous abortion between 2009–2011 were examined retrospectively. Fetal deaths per calendar month from January-December, 2011 were counted and correlated with mean monthly levels of various air pollutants by means of regression analysis.
Regression of ambient pollutants against fetal death as a dose–response toxicity curve revealed very strong dose–response correlations for SO2 r > 0.9 (p < 0.001) while similarly strongly significant correlation coefficients were found for NO2 (r > 0.8), CO (r > 0.9), PM10 (r > 0.9) and PM2.5 (r > 0.8), (p < 0.001), indicating a strong correlation between air pollution and decreased fetal wellbeing.
The present study identified alarmingly strong statistical correlations between ambient air pollutants and spontaneous abortion. Further studies need to be done to examine possible correlations between personal exposure to air pollutants and pregnancy loss.
Air pollution; Fetal death; Mongolia; Seasonal variation; Spontaneous abortion
Reciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. Although the molecular markers and related animal models that target lung epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme-specific manner are not available, which becomes a critical barrier to the study of lung mesenchymal biology and the related pulmonary diseases.
We have identified a mouse Tbx4 gene enhancer that contains conserved DNA sequences across many vertebrate species with lung or lung-like gas exchange organ. We then generate a mouse line to express rtTA/LacZ under the control of the Tbx4 lung enhancer, and therefore a Tet-On inducible transgenic system to target lung mesenchymal cells at different developmental stages. By combining a Tbx4-rtTA driven Tet-On inducible Cre expression mouse line with a Cre reporter mouse line, the spatial-temporal patterns of Tbx4 lung enhancer targeted lung mesenchymal cells were defined. Pulmonary endothelial cells and vascular smooth muscle cells were targeted by the Tbx4-rtTA driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be targeted during the entire developmental stage.
Developmental lung mesenchymal cells can be specifically marked by Tbx4 lung enhancer activity. With our newly created Tbx4 lung enhancer-driven Tet-On inducible system, lung mesenchymal cells can be specifically and differentially targeted in vivo for the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice.
Lung mesenchyme; Tbx4 lung enhancer; Tet-On system
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.
Pitx2; Fgf9; Fgf10; cecum; agenesis; development
The potential for amniotic fluid stem cell (AFSC) treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF), is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0) or chronic (day 14) intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL), but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.
Patients with Apert Syndrome (AS) display a wide range of congenital malformations including tracheal stenosis, which is a disease characterized by a uniform cartilaginous sleeve in place of a normally ribbed cartilagenous trachea. We have studied the cellular and molecular basis of this phenotype in a mouse model of Apert syndrome (Fgfr2c+/Δ mice), which shows ectopic expression of Fgfr2b in mesenchymal tissues. Here we report that tracheal stenosis is associated with increased proliferation of mesenchymal cells, where the expression of Fgf10 and its upstream regulators Tbx4 and Tbx5 are abnormally elevated. We show that Fgf10 has a critical inductive role in tracheal stenosis, as genetic knockdown of Fgf10 in Fgfr2c+/Δ mice rescues this phenotype. These novel findings demonstrate a regulatory role for Fgf10 in tracheal development and shed more light on the underlying cause of tracheal defects in Apert syndrome.
The key role played by of Fgf10 during early lung development is clearly illustrated in Fgf10 knockout mice, which exhibit complete 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 (Mailleux et al., 2005). 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 hours of respiratory failure. These mutant lungs display severe hypoplasia, dilation of the distal airways and large hemorrhagic areas. Epithelial differentiation and proliferation studies indicate a specific decrease in the percentile of 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 α-SMA expression in the respiratory airway suggesting defective formation of the alveolar smooth muscle cells. At the molecular level, these defects are associated with a decrease in Vegfa and Pdgfa expression likely resulting from the decrease of the epithelium/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.
Fgf10 hypomorph; mesenchymal differentiation; smooth muscle cells; lung emphysema; vascularization
The majority of epithelial cells in the distal lung of rodents and humans are quiescent in vivo, yet certain cell populations retain an intrinsic capacity to proliferate and differentiate in response to lung injury or in appropriate culture settings, thus giving them properties of stem/progenitor cells. Here, we describe the isolation of two such populations from adult mouse lung: alveolar epithelial type 2 cells (AEC2), which can generate alveolar epithelial type 1 cells, and bronchioalveolar stem cells (BASCs), which in culture can reproduce themselves, as well as generate a small number of other distal lung epithelial cell types. These primary epithelial cells are typically isolated using enzyme digestion, mechanical disruption, and serial filtration. AEC2 and BASCs are distinguished from other distal lung cells by expression of specific markers as detected by fluorescence-activated cell sorting, immunohistochemistry, or a combination of both of these techniques.
Mouse lung; Alveolus; Terminal airway; Alveolar epithelial type 2 cells; Bronchioalveolar stem cells; Epithelial cell culture; Fluorescence-activated cell sorting; Immunohistochemistry
Bronchodilator responses (BDR) are routinely used in the diagnosis and management of asthma; however, their acceptability and repeatability have not been evaluated using quality control criteria for preschool children.
To compare conventional spirometry with an impulse oscillometry system (IOS) in healthy and asthmatic preschool children.
Data from 30 asthmatic children and 29 controls (two to six years of age) who underwent IOS and spirometry before and after salbutamol administration were analyzed.
Stable asthmatic subjects significantly differed versus controls in their spirometry-assessed BDR (forced expiratory volume in 1 s [FEV1], forced vital capacity and forced expiratory flow at 25% to 75% of forced vital capacity) as well as their IOS-assessed BDR (respiratory resistance at 5 Hz [Rrs5], respiratory reactance at 5 Hz and area under the reactance curve). However, comparisons based on the area under the ROC curve for ΔFEV1 % initial versus ΔRrs5 % initial were 0.82 (95% CI 0.71 to 0.93) and 0.75 (95% CI 0.62 to 0.87), respectively. Moreover, the sensitivity and specificity for ΔFEV1 ≥9% were 0.53 and 0.93, respectively. Importantly, sensitivity increased to 0.63 when either ΔFEV1 ≥9% or ΔRrs5 ≥29% was considered as an additional criterion for the diagnosis of asthma.
The accuracy of asthma diagnosis in preschool children may be increased by combining spirometry with IOS when measuring BDR.
Asthma; Bronchodilator agents; Oscillometry; Preschool; Spirometry
Wound healing is the inherent ability of an organism to protect itself against injuries. Cumulative evidence indicates that the healing process patterns embryonic morphogenesis and may result in either organ regeneration or scarring – phenomena that are developmental stage-or age-dependent. Skin is the largest organ. Its morphogenesis and repair mechanisms have been studied extensively due not only to its anatomical location, which allows easy access and observation, but also its captivating structure and vital function. Thus, this review will focus on using skin as a model organ to illustrate new insights into the mechanisms of wound healing that are developmentally regulated in mammals, with special emphasis on the roles of Wnt signaling pathways and their crosstalk with TGF-β signaling. Relevant information from studies of other organs is discussed where it applies, and the clinical impact from such knowledge and emerging concepts on regenerative medicine are also discussed in perspective.
Wound healing; Development; Morphogenesis; Scarless; Scarring; Myofibroblasts; Wnt
Introduction or background
The adult lung is a complex organ whose large surface area interfaces extensively with both the environment and circulatory system. Yet, in spite of the high potential for exposure to environmental or systemic harm, epithelial cell turnover in adult lung is comparatively slow. Moreover, loss of lung function with advancing age is becoming an increasingly costly healthcare problem. Cell-based therapies stimulating endogenous stem/progenitor cells or supplying exogenous ones have therefore become a prime translational goal. Alternatively when lung repair becomes impossible, replacement with tissue-engineered lung is an attractive emerging alternative using a decellularized matrix or bioengineered scaffold.
Sources of data
Endogenous and exogenous stem cells for lung therapy are being characterized by defining developmental lineages, surface marker expression, functions within the lung and responses to injury and disease. Seeding decellularized lung tissue or bioengineered matrices with various stem and progenitor cells is an approach that has already been used to replace bronchus and trachea in human patients and awaits further development for whole lung tissue.
Areas of agreement
Cellular therapies have clear potential for respiratory disease. However, given the surface size and complexity of lung structure, the probability of a single cellular population sufficing to regenerate the entire organ, as in the bone marrow, remains low. Hence, lung regenerative medicine is currently focused around three aims: (i) to identify and stimulate resident cell populations that respond to injury or disease, (ii) to transplant exogenous cells which can ameliorate disease and (iii) to repopulate decellularized or bioengineered lung matrix creating a new implantable organ.
Areas of controversy
Lack of consensus on specific lineage markers for lung stem and progenitor cells in development and disease constrains transferability of research between laboratories and sources of cellular therapy. Furthermore, effectiveness of individual cellular therapies to correct gas exchange and provide other critical lung functions remains unproven. Finally, feasibility of autologous whole organ replacement has not been confirmed as a durable therapy.
Cellular therapies for lung regeneration would be enhanced by better lineage tracing within the lung, the ability to direct differentiation of exogenous stem or progenitor cells, and the development of functional assays for cellular viability and regenerative properties. Whether endogenous or exogeneous cells will ultimately play a greater therapeutic role remains to be seen. Reducing the need for lung replacement via endogenous cell-mediated repair is a key goal. Thereafter, improving the potential of donor lungs in transplant recipients is a further area where cell-based therapies may be beneficial. Ultimately, lung replacement with autologous tissue-engineered lungs is another goal for cell-based therapy.
Areas timely for developing research
Defining ‘lung stem or progenitor cell’ populations in both animal models and human tissue may help. Additionally, standardizing assays for assessing the potential of endogenous or exogenous cells within the lung is important. Understanding cell–matrix interactions in real time and with biomechanical insight will be central for lung engineering.
Communicating the real potential for cell-based lung therapy needs to remain realistic, given the keen expectations of patients with end-stage lung disease.
endogenous lung stem cells; exogenous stem cells; lung regeneration and repair; regenerative medicine; stem cell therapies
Alveolar epithelial integrity is dependent upon the alveolar milieu, yet the milieu of the damaged alveolar epithelial cell type 2 (AEC2) has been little studied. Characterization of its components may offer the potential for ex vivo manipulation of stem cells to optimize their therapeutic potential. We examined the cytokine profile of AEC2 damage milieu, hypothesizing that it would promote endogenous epithelial repair while recruiting cells from other locations and instructing their engraftment and differentiation. Bronchoalveolar lavage and lung extract from hyperoxic rats represented AEC2 in vivo damage milieu, and medium from a scratch-damaged AEC2 monolayer represented in vitro damage. CINC-2 and ICAM, the major cytokines detected by proteomic cytokine array in AEC2 damage milieu, were chemoattractive to normoxic AECs and expedited in vitro wound healing, which was blocked by their respective neutralizing antibodies. The AEC2 damage milieu was also chemotactic for exogenous uncommitted human amniotic fluid stem cells (hAFSCs), increasing migration greater than 20-fold. hAFSCs attached within an in vitro AEC2 wound and expedited wound repair by contributing cytokines migration inhibitory factor and plasminogen activator inhibitor 1 to the AEC2 damage milieu, which promoted wound healing. The AEC2 damage milieu also promoted differentiation of a subpopulation of hAFSCs to express SPC, TTF-1, and ABCA3, phenotypic markers of distal alveolar epithelium. Thus, the microenvironment created by AEC2 damage not only promotes autocrine repair but also can attract uncommitted stem cells, which further augment healing through cytokine secretion and differentiation.
AEC2; amniotic fluid stem cells; epithelial damage; CINC-2; ICAM
Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders. Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4+FoxP3+) and decrease of dendritic cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls. Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.
Interest regarding stem cell based therapies for the treatment of congenital or acquired craniofacial deformities is rapidly growing. Craniofacial problems such as periodontal disease, cleft lip and palate, ear microtia, craniofacial microsomia, and head and neck cancers are not only common but also some of the most burdensome surgical problems worldwide. Treatments often require a multi-staged multidisciplinary team approach. Current surgical therapies attempt to reduce the morbidity and social/emotional impact, yet outcomes can still be unpredictable and unsatisfactory. The concept of harvesting stem cells followed by expansion, differentiation, seeding onto a scaffold and re-transplanting them is likely to become a clinical reality. In this review, we will summarize the translational applications of stem cell therapy in tissue regeneration for craniofacial defects.
stem cell; craniofacial; regeneration
Lung development is a complex and finely balanced process. Yet the lung has a relatively limited repertoire of responses to injury, which, depending on severity of the injury and developmental stage and susceptibility of the lung, culminate in stopping development, followed by more or less successful repair or alternatively in fibrosis. Unlike fetal skin, which heals scarlessly early in gestation, but scars later in gestation and increasingly so postnatally, the damaged fetal lung does heal, but not very well. Thus lung injury appears to entrain a default developmental/repair mechanism involving increased amounts of activated TGF beta ligand signaling. When this occurs prior to or very early in the process of alveolarization, excessive TGF beta ligand inhibits further alveolarization, a disease process phenotype that has been termed Bronchopulmonary Dysplasia in extreme human prematurity. However, once alveolarization is sufficiently advanced as in mid to late gestation fetal monkey, late gestation human or adult mouse, rat or human lung, excessive TGF beta signaling results in pulmonary fibrosis. Recently we have further shown that FGF10 signaling, a process that is necessary for distal lung morphogenesis, can also antagonize bleomycin-induced lung fibrosis in adult mice by a mechanism involving inhibition of active TGF beta ligand bioavailability. We therefore suggest that lung development, repair and fibrosis have many fundamental mechanisms in common, that potentially can be manipulated using cells or soluble factors that optimize the alveolar milieu to prevent and possibly even to reverse lung fibrosis.
Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease of the lung parenchyma, without curative treatment. Gremlin is a bone morphogenic protein (BMP) antagonist, its expression being increased in IPF lungs. It has been implicated in promoting myofibroblast accumulation, likely through inhibited fibroblast apoptosis and epithelial-to-mesenchymal transition. In the current study, we examined the effects of selective adenovirus-mediated overexpression of Gremlin in rat lungs. We show that transient Gremlin overexpression results in activation of alveolar epithelial cells with proliferation and apoptosis, as well as partly reversible lung fibrosis. We found myofibroblasts arranged in fibroblastic foci. Fibroblast proliferation occurred delayed as compared with epithelial changes. Fibrotic pathology significantly declined after Day 14, the reversal being associated with an increase of the epithelium-protective element, fibroblast growth factor (FGF)–10. Our data indicate that Gremlin-mediated BMP inhibition results in activation of epithelial cells and transient fibrosis, but also induction of epithelium-protective FGF10. A Gremlin–BMP–FGF10 loop may explain these results, and demonstrate that the interactions between different factors are quite complex in fibrotic lung disease. Increased Gremlin expression in human IPF tissue may be an expression of continuing epithelial injury, and Gremlin may be part of activated repair mechanisms.
pulmonary fibrosis; gremlin; bone morphogenic protein; animal model; epithelial cell
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.
lung development; Six1; proliferation; differentiation; Shh; Fgf10
Murine lung development begins at embryonic day (E) 9.5. Normal lung structure and function depend on the patterns of localization of differentiated cells. Pulmonary mesenchymal cell lineages have been relatively unexplored. Importantly, there has been no prior evidence of clonality of any lung cells. Herein we use a definitive genetic approach to demonstrate a common origin for proximal and distal pulmonary mesenchymal cells. A retroviral library with 3,400 unique inserts was microinjected into the airway lumen of E11.5 lung buds. After 7–11 days of culture, buds were stained for placental alkaline phosphatase (PLAP). Most PLAP+ cells are peribronchial smooth muscle cells, initially localized laterally near the hilum, then migrating down airways to the subpleural region. Laser-capture microdissection and polymerase chain reaction confirm the clonal identities of PLAP+ cells proximally and distally. Our observation of this fundamental process during lung development opens new avenues for investigation of maladaptive mesenchymal responses in lung diseases.
retrovirus; microinjection; alkaline phosphatase; smooth muscle; clonal analysis
Developmental lung biology is a field that has the potential for significant human impact: lung disease at the extremes of age continues to cause major morbidity and mortality worldwide. Understanding how the lung develops holds the promise that investigators can use this knowledge to aid lung repair and regeneration. In the decade since the “molecular embryology” of the lung was first comprehensively reviewed, new challenges have emerged—and it is on these that we focus the current review. Firstly, there is a critical need to understand the progenitor cell biology of the lung in order to exploit the potential of stem cells for the treatment of lung disease. Secondly, the current familiar descriptions of lung morphogenesis governed by growth and transcription factors need to be elaborated upon with the reinclusion and reconsideration of other factors, such as mechanics, in lung growth. Thirdly, efforts to parse the finer detail of lung bud signaling may need to be combined with broader consideration of overarching mechanisms that may be therapeutically easier to target: in this arena, we advance the proposal that looking at the lung in general (and branching in particular) in terms of clocks may yield unexpected benefits.
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.
lung development; Eya1; proliferation; differentiation; Shh; Fgf10
A proper balance between self-renewal and differentiation of lung specific progenitors at the distal epithelial tips is absolutely required for normal lung morphogenesis. Cell polarity and mitotic spindle orientation play a critical role in the self-renewal/differentiation of epithelial cells and can impact normal physiological processes, including epithelial tissue branching and differentiation. Therefore, understanding the behavior of lung distal epithelial progenitors could identify innovative solutions to restoring normal lung morphogenesis. Yet little is known about cell polarity, spindle orientation and segregation of cell fate determinant in the embryonic lung epithelium, which contains progenitor cells. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized and highly mitotic with characteristic perpendicular cell divisions. Consistent with these findings, mInsc, LGN and NuMA polarity proteins, which control spindle orientation, are asymmetrically localized in mitotic distal epithelial progenitors of embryonic lungs. Furthermore, the cell fate determinant Numb is asymmetrically distributed at the apical side of distal epithelial progenitors and segregated to one daughter cell in most mitotic cells. These findings provide evidence for polarity in distal epithelial progenitors of embryonic lungs and provide a framework for future translationally oriented studies in this area.
lung progenitors; progenitor cell behavior; cell polarity; cell fate
Epithelial to mesenchymal transition (EMT) is a key process during embryonic development and disease development and progression. During EMT, epithelial cells lose epithelial features and express mesenchymal cell markers, which correlate with increased cell migration and invasion. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that induces EMT in multiple cell types. The TGF-β pathway is regulated by microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of specific messenger RNAs.
Herein, we identified mir-99a and mir-99b as two novel TGF-β target miRNA genes, the expression of which increased during TGF-β induced EMT of NMUMG cells. Mir-99a and mir-99b inhibition decreased TGF-β activity by inhibiting SMAD3 phosphorylation, resulting in decreased migration and increased proliferation in response to TGF-β. However, mir-99a and mir-99b inhibition was insufficient to block TGF-β induced EMT of NMUMG cells.
Mir-99a and mir-99b over-expression in epithelial NMUMG cells resulted in increased proliferation, migration and fibronectin expression, while E-cadherin and ZO-1 expression were negatively regulated.
In conclusion, we identified mir-99a and mir-99b as two novel modulators of TGF-β pathway that alter SMAD3 phosphorylation, in turn altering cell migration and adhesion of mesenchymal NMUMG cells. The effect of mir-99a and mir-99b over-expression on NMUMUG proliferation is dependent upon the epithelial or mesenchymal status of the cells. Our study suggests that mir-99a and mir-99b may function as modulators within a complex network of factors regulating TGF-β induced breast epithelial to mesenchymal transition, as well as proliferation and migration of breast cancer cells, providing a possible target for future translationally oriented studies in this area.