Development and Disorders of the Airway in Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), a disorder characterized by arrested lung development, is a frequent cause of morbidity and mortality in premature infants. Parenchymal lung changes in BPD are relatively well-characterized and highly studied; however, there has been less emphasis placed on the role that airways disease plays in the pathophysiology of BPD. In preterm infants born between 22 and 32 weeks gestation, the conducting airways are fully formed but still immature and therefore susceptible to injury and further disruption of development. The arrest of maturation results in more compliant airways that are more susceptible to deformation and damage. Consequently, neonates with BPD are prone to developing airway pathology, particularly for patients who require intubation and positive-pressure ventilation. Airway pathology, which can be divided into large and small airways disease, results in increased respiratory morbidity in neonates with chronic lung disease of prematurity.

Is Lactoferrin Supplementation Beneficial for All Preterm Infants?

OBJECTIVE: Human milk (HM) has antibacterial properties due to the presence of immune-modulators, including lactoferrin (LF). This study will determine effect(s) of HM maturation, fortification, and storage conditions on LF levels and its antibacterial properties. STUDY DESIGN: HM samples (n = 30) were obtained from preterm and term mothers. The LF levels were analyzed by ELISA, and the antibacterial activity was measured after inoculation with Escherichia coli. RESULTS: The highest level of LF in preterm HM was observed in the first week of lactation. However, storage of preterm HM at 4°C decreased LF levels significantly. Both LF levels and antibacterial activity in preterm HM was lower compared with term HM, but significantly higher than donor HM even after HM-based fortification. LF supplementation of donor HM improved its antibacterial activity. CONCLUSION: Preterm infants fed donor HM, formula, or stored HM at 4°C may benefits from LF supplementation to improve HM antibacterial properties. KEY POINTS: · Milk LF levels vary with storage and maturity.. · Donor milk is deficient in LF even after adding HM-based fortification.. · Donor HM and formula fed infants may benefit from LF..

Light protection of parenteral nutrition, cholestasis, and other prematurity-related morbidities in premature infants.

Introduction: Parenteral Nutrition (PN) can lead to intestinal failure associated liver disease (IFALD). There are no human studies to date studying specifically the benefits of light-protection on neonatal IFALD. Recently, the European Medicines Agency and the American Society for Parenteral and Enteral Nutrition (ASPEN) both recommended full light protection of PN to reduce the risk of adverse clinical outcomes. Objective: The primary objective of this study was to evaluate the impact of light-protecting PN on the incidence of cholestasis and peak direct bilirubin levels in premature infants. Study design: Retrospective chart review of preterm infants requiring PN for a minimum of 2 weeks with or without light-protection. After light protection of the PN solution, primary outcomes (including cholestasis and direct bilirubin levels) of both groups were compared. Secondary outcomes include evaluation of bronchopulmonary dysplasia (BPD), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP), sepsis and mortality. Results: A total of 50 preterm infants <37 weeks gestation were included, 25 infants in each group. There was a statistically significant decrease in the rate of cholestasis (12 vs. 3, p = 0.005), median peak direct bilirubin levels (1.7 vs. 0.9 mg/dL, p = 0.02) and total bilirubin levels (4.1 vs. 3.4, p = 0.05) in the light-protection group compared to no light-protection group. There was a decrease in the incidence of severe BPD (with an increase of mild BPD, resulting in the same overall BPD rate) in the light-protection compared to no light-protection group (7 vs. 15, p = 0.0223). There was no difference in NEC, ROP, sepsis or mortality. Conclusion: Our study supports that the practice of light-protecting PN may reduce the incidence of IFALD in premature infants. Moreover, there was a trend toward decreased incidence of severe BPD in the light-protection group. Further light protection studies are needed to confirm these findings.

Noninfectious influencers of early-onset sepsis biomarkers.

Diagnostic tests for sepsis aim to either detect the infectious agent (such as microbiological cultures) or detect host markers that commonly change in response to an infection (such as C-reactive protein). The latter category of tests has advantages compared to culture-based methods, including a quick turnaround time and in some cases lower requirements for blood samples. They also provide information on the immune response of the host, a critical determinant of clinical outcome. However, they do not always differentiate nonspecific host inflammation from true infection and can inadvertently lead to antibiotic overuse. Multiple noninfectious conditions unique to neonates in the first days after birth can lead to inflammatory marker profiles that mimic those seen among infected infants. Our goal was to review noninfectious conditions and patient characteristics that alter host inflammatory markers commonly used for the diagnosis of early-onset sepsis. Recognizing these conditions can focus the use of biomarkers on patients most likely to benefit while avoiding scenarios that promote false positives. We highlight approaches that may improve biomarker performance and emphasize the need to use patient outcomes, in addition to conventional diagnostic performance analysis, to establish clinical utility.

Placental extracellular vesicles-associated microRNA-519c mediates endotoxin adaptation in pregnancy.

BACKGROUND: Pregnancy represents a unique challenge for the maternal-fetal immune interface, requiring a balance between immunosuppression, which is essential for the maintenance of a semiallogeneic fetus, and proinflammatory host defense to protect the maternal-fetal interface from invading organisms. Adaptation to repeated inflammatory stimuli (endotoxin tolerance) may be critical in preventing inflammation-induced preterm birth caused by exaggerated maternal inflammatory responses to mild or moderate infections that are common during pregnancy. However, the exact mechanisms contributing to the maintenance of tolerance to repeated infections are not completely understood. MicroRNAs play important roles in pregnancy with several microRNAs implicated in gestational tissue function and in pathologic pregnancy conditions. MicroRNA-519c, a member of the chromosome 19 microRNA cluster, is a human-specific microRNA mainly expressed in the placenta. However, its role in pregnancy is largely unknown. OBJECTIVE: This study aimed to explore the role of "endotoxin tolerance" failure in the pathogenesis of an exaggerated inflammatory response often seen in inflammation-mediated preterm birth. In this study, we investigated the role of microRNA-519c, a placenta-specific microRNA, as a key regulator of endotoxin tolerance at the maternal-fetal interface. STUDY DESIGN: Using a placental explant culture system, samples from term and second-trimester placentas were treated with lipopolysaccharide. After 24 hours, the conditioned media were collected for analysis, and the placental explants were re-exposed to repeated doses of lipopolysaccharide for 3 days. The supernatant was analyzed for inflammatory markers, the presence of extracellular vesicles, and microRNAs. To study the possible mechanism of action of the microRNAs, we evaluated the phosphodiesterase 3B pathway involved in tumor necrosis factor alpha production using a microRNA mimic and phosphodiesterase 3B small interfering RNA transfection. Finally, we analyzed human placental samples from different gestational ages and from women affected by inflammation-associated pregnancies. RESULTS: Our data showed that repeated exposure of the human placenta to endotoxin challenges induced a tolerant phenotype characterized by decreased tumor necrosis factor alpha and up-regulated interleukin-10 levels. This reaction was mediated by the placenta-specific microRNA-519c packaged within placental extracellular vesicles. Lipopolysaccharide treatment increased the extracellular vesicles that were positive for the exosome tetraspanin markers, namely CD9, CD63, and CD81, and secreted primarily by trophoblasts. Primary human trophoblast cells transfected with a microRNA-519c mimic decreased phosphodiesterase 3B, whereas a lack of phosphodiesterase 3B, achieved by small interfering RNA transfection, led to decreased tumor necrosis factor alpha production. These data support the hypothesis that the anti-inflammatory action of microRNA-519c was mediated by a down-regulation of the phosphodiesterase 3B pathway, leading to inhibition of tumor necrosis factor alpha production. Furthermore, human placentas from normal and inflammation-associated pregnancies demonstrated that a decreased placental microRNA-519c level was linked to infection-induced inflammatory pathologies during pregnancy. CONCLUSION: We identified microRNA-519c, a human placenta-specific microRNA, as a novel regulator of immune adaptation associated with infection-induced preterm birth at the maternal-fetal interface. Our study serves as a basis for future experiments to explore the potential use of microRNA-519c as a biomarker for infection-induced preterm birth.

First Case of Ewingella americana Meningitis in a Term Newborn: A Rare but Real Pathogen.

Ewingella americana is a Gram-negative, catalase positive and anaerobic enterobacterium first described in 1983. Infections caused by this pathogen, such as bacteremia and pneumonia, are extremely rare and primarily occur in patients with underlying pathologies or immunosuppression. There is still a debate as to whether Ewingella americana is a real pathogen or if it can be considered an opportunistic infectious agent. We report the first documented case of Ewingella americana meningitis in literature and the first case of this pathogen causing infection in a newborn. Case presentation: A term newborn male was born via spontaneous vaginal delivery to a Gravida 2 Para 0, 28 year old woman with negative prenatal screening tests with a birth weight of 4.70 kilograms and Apgar scores of 9 and 9 at 1 and 5 minutes respectively. Rupture of membranes was 27 hours prior to delivery. Infant was noted to be febrile to 101°F at birth, so infant was admitted in the neonatal intensive care unit and started empirically on ampicillin and gentamycin. Cerebrospinal fluid (CSF) drawn due to irritability on day of life 1 presented normal cell and protein count but grew Gram negative rods after 2 days, identified subsequently as Ewingella americana; repeat CSF analysis done at 6 days of life showed pleocytosis. Brain MRI performed at 2 weeks of life showed leptomeningitis. The infant was treated with ceftazidime for 21 days from the first negative CSF culture. He has since followed up with the neurologist and infectious disease specialist. He had a normal electroencephalogram (EEG) and is meeting all developmental milestones at the 24 months of age follow up visit. Conclusion: Our case highlights that Ewingella americana can cause serious invasive infections such as meningitis in the neonatal period with minimal symptomatology. Antibiotic treatment in the neonatal period can present challenges due to the Ewingella americana's variable sensitivity. The role of these emerging low virulence organisms in causing infections has to be further elucidated, especially in vulnerable patients such as newborns.

Hydrocortisone and bronchopulmonary dysplasia: variables associated with response in premature infants.

OBJECTIVE: The primary objective was to evaluate hydrocortisone's efficacy for decreasing respiratory support in premature infants with developing bronchopulmonary dysplasia (BPD). Secondary objectives included assessment of the impact of intrauterine growth restriction (IUGR), maternal history of chorioamnionitis, side effects and route of administration associated with hydrocortisone's efficacy. Dexamethasone as second-line treatment to decrease respiratory support was reviewed. METHODS: Retrospective chart review of preterm infants requiring respiratory support receiving hydrocortisone. RESULTS: A total of 48 patients were included. Successful extubation was achieved in 50% of intubated patients after hydrocortisone treatment with no major complications. In our small study, history of maternal chorioamnionitis, IUGR or route of administration did not affect the response. Rescue dexamethasone after hydrocortisone therapy was ineffective in the ten patients who failed extubation following hydrocortisone. CONCLUSION: Hydrocortisone is effective in decreasing respiratory support in patients with developing BPD without major complications. Randomized studies are warranted to confirm our findings.

Fgf10 deficiency is causative for lethality in a mouse model of bronchopulmonary dysplasia.

Inflammation-induced FGF10 protein deficiency is associated with bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurely born infants characterized by arrested alveolar development. So far, experimental evidence for a direct role of FGF10 in lung disease is lacking. Using the hyperoxia-induced neonatal lung injury as a mouse model of BPD, the impact of Fgf10 deficiency in Fgf10+/- versus Fgf10+/+ pups was investigated. In normoxia, no lethality of Fgf10+/+ or Fgf10+/- pups was observed. By contrast, all Fgf10+/- pups died within 8 days of hyperoxic injury, with lethality starting at day 5, whereas Fgf10+/+ pups were all alive. Lungs of pups from the two genotypes were collected on postnatal day 3 following normoxia or hyperoxia exposure for further analysis. In hyperoxia, Fgf10+/- lungs exhibited increased hypoalveolarization. Analysis by FACS of the Fgf10+/- versus control lungs in normoxia revealed a decreased ratio of alveolar epithelial type II (AECII) cells over total Epcam-positive cells. In addition, gene array analysis indicated reduced AECII and increased AECI transcriptome signatures in isolated AECII cells from Fgf10+/- lungs. Such an imbalance in differentiation is also seen in hyperoxia and is associated with reduced mature surfactant protein B and C expression. Attenuation of the activity of Fgfr2b ligands postnatally in the context of hyperoxia also led to increased lethality with decreased surfactant expression. In summary, decreased Fgf10 mRNA levels lead to congenital lung defects, which are compatible with postnatal survival, but which compromise the ability of the lungs to cope with sub-lethal hyperoxic injury. Fgf10 deficiency affects quantitatively and qualitatively the formation of AECII cells. In addition, Fgfr2b ligands are also important for repair after hyperoxia exposure in neonates. Deficient AECII cells could be an additional complication for patients with BPD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development.

Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10(+) progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development.

A breath of fresh air on the mesenchyme: impact of impaired mesenchymal development on the pathogenesis of bronchopulmonary dysplasia.

The early mouse embryonic lung, with its robust and apparently reproducible branching pattern, has always fascinated developmental biologists. They have extensively used this embryonic organ to decipher the role of mammalian orthologs of Drosophila genes in controlling the process of branching morphogenesis. During the early pseudoglandular stage, the embryonic lung is formed mostly of tubes that keep on branching. As the branching takes place, progenitor cells located in niches are also amplified and progressively differentiate along the proximo-distal and dorso-ventral axes of the lung. Such elaborate processes require coordinated interactions between signaling molecules arising from and acting on four functional domains: the epithelium, the endothelium, the mesenchyme, and the mesothelium. These interactions, quite well characterized in a relatively simple lung tubular structure remain elusive in the successive developmental and postnatal phases of lung development. In particular, a better understanding of the process underlying the formation of secondary septa, key structural units characteristic of the alveologenesis phase, is still missing. This structure is critical for the formation of a mature lung as it allows the subdivision of saccules in the early neonatal lung into alveoli, thereby considerably expanding the respiratory surface. Interruption of alveologenesis in preterm neonates underlies the pathogenesis of chronic neonatal lung disease known as bronchopulmonary dysplasia. De novo formation of secondary septae appears also to be the limiting factor for lung regeneration in human patients with emphysema. In this review, we will therefore focus on what is known in terms of interactions between the different lung compartments and discuss the current understanding of mesenchymal cell lineage formation in the lung, focusing on secondary septae formation.

Transient Inhibition of FGFR2b-ligands signaling leads to irreversible loss of cellular ß-catenin organization and signaling in AER during mouse limb development.

The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤ 24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, ß1-integrin and E-cadherin, and a permanent loss of cellular ß-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development.

Mesodermal Pten inactivation leads to alveolar capillary dysplasia- like phenotype.

Alveolar capillary dysplasia (ACD) is a congenital, lethal disorder of the pulmonary vasculature. Phosphatase and tensin homologue deleted from chromosome 10 (Pten) encodes a lipid phosphatase controlling key cellular functions, including stem/progenitor cell proliferation and differentiation; however, the role of PTEN in mesodermal lung cell lineage formation remains unexamined. To determine the role of mesodermal PTEN in the ontogeny of various mesenchymal cell lineages during lung development, we specifically deleted Pten in early embryonic lung mesenchyme in mice. Pups lacking Pten died at birth, with evidence of failure in blood oxygenation. Analysis at the cellular level showed defects in angioblast differentiation to endothelial cells and an accompanying accumulation of the angioblast cell population that was associated with disorganized capillary beds. We also found decreased expression of Forkhead box protein F1 (Foxf1), a gene associated with the ACD human phenotype. Analysis of human samples for ACD revealed a significant decrease in PTEN and increased activated protein kinase B (AKT). These studies demonstrate that mesodermal PTEN has a key role in controlling the amplification of angioblasts as well as their differentiation into endothelial cells, thereby directing the establishment of a functional gas exchange interface. Additionally, these mice could serve as a murine model of ACD.

FGF9-Pitx2-FGF10 signaling controls cecal formation in mice.

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.

Embryonic epithelial Pten deletion through Nkx2.1-cre leads to thyroid tumorigenesis in a strain-dependent manner.

Even though the role of the tyrosine phosphatase Pten as a tumor suppressor gene has been well established in thyroid cancer, its role during thyroid development is still elusive. We therefore targeted Pten deletion in the thyroid epithelium by crossing Pten(flox/flox) with a newly developed Nkx2.1-cre driver line in the BALB/c and C57BL/6 genetic backgrounds. C57BL/6 homozygous Pten mutant mice died around 2 weeks of age due to tracheal and esophageal compression by a hyperplasic thyroid. By contrast, BALB/c homozygous Pten mutant mice survived up to 2 years, but with a slightly increased thyroid volume. Characterization of the thyroid glands from C57BL/6 homozygous Pten mutant mice at postnatal day 14 (PN14) showed abnormally enlarged tissue with areas of cellular hyperplasia, disruption of the normal architecture, and follicular degeneration. In addition, differing degrees of hypothyroidism, thyroxine (T(4)) decrease, and thyroid-stimulating hormone elevation between the strains in the mutants and the heterozygous mutant were detected at PN14. Finally, C57BL/6 heterozygous Pten mutant mice developed thyroid tumors after 2 years of age. Our results indicate that Pten has a pivotal role in thyroid development and its deletion results in thyroid tumor formation, with the timing and severity of the tumor depending on the particular genetic background.

Parabronchial smooth muscle constitutes an airway epithelial stem cell niche in the mouse lung after injury.

During lung development, parabronchial SMC (PSMC) progenitors in the distal mesenchyme secrete fibroblast growth factor 10 (Fgf10), which acts on distal epithelial progenitors to promote their proliferation. ß-catenin signaling within PSMC progenitors is essential for their maintenance, proliferation, and expression of Fgf10. Here, we report that this Wnt/Fgf10 embryonic signaling cascade is reactivated in mature PSMCs after naphthalene-induced injury to airway epithelium. Furthermore, we found that this paracrine Fgf10 action was essential for activating surviving variant Clara cells (the cells in the airway epithelium from which replacement epithelial cells originate) located at the bronchoalveolar duct junctions and adjacent to neuroendocrine bodies. After naphthalene injury, PSMCs secreted Fgf10 to activate Notch signaling and induce Snai1 expression in surviving variant Clara cells, which subsequently underwent a transient epithelial to mesenchymal transition to initiate the repair process. Epithelial Snai1 expression was important for regeneration after injury. We have therefore identified PSMCs as a stem cell niche for the variant Clara cells in the lung and established that paracrine Fgf10 signaling from the niche is critical for epithelial repair after naphthalene injury. These findings also have implications for understanding the misregulation of lung repair in asthma and cancer.

Contrasting expression of canonical Wnt signaling reporters TOPGAL, BATGAL and Axin2(LacZ) during murine lung development and repair.

Canonical WNT signaling plays multiple roles in lung organogenesis and repair by regulating early progenitor cell fates: investigation has been enhanced by canonical Wnt reporter mice, TOPGAL, BATGAL and Axin2(LacZ). Although widely used, it remains unclear whether these reporters convey the same information about canonical Wnt signaling. We therefore compared beta-galactosidase expression patterns in canonical Wnt signaling of these reporter mice in whole embryo versus isolated prenatal lungs. To determine if expression varied further during repair, we analyzed comparative pulmonary expression of beta-galactosidase after naphthalene injury. Our data show important differences between reporter mice. While TOPGAL and BATGAL lines demonstrate Wnt signaling well in early lung epithelium, BATGAL expression is markedly reduced in late embryonic and adult lungs. By contrast, Axin2(LacZ) expression is sustained in embryonic lung mesenchyme as well as epithelium. Three days into repair after naphthalene, BATGAL expression is induced in bronchial epithelium as well as TOPGAL expression (already strongly expressed without injury). Axin2(LacZ) expression is increased in bronchial epithelium of injured lungs. Interestingly, both TOPGAL and Axin2(LacZ) are up regulated in parabronchial smooth muscle cells during repair. Therefore the optimal choice of Wnt reporter line depends on whether up- or down-regulation of canonical Wnt signal reporting in either lung epithelium or mesenchyme is being compared.

FGF10 controls the patterning of the tracheal cartilage rings via Shh.

During embryonic development, appropriate dorsoventral patterning of the trachea leads to the formation of periodic cartilage rings from the ventral mesenchyme and continuous smooth muscle from the dorsal mesenchyme. In this work, we have investigated the role of two crucial morphogens, fibroblast growth factor 10 and sonic hedgehog, in the formation of periodically alternating cartilaginous and non-cartilaginous domains in the ventral mesenchyme. Using a combination of gain- and loss-of-function approaches for FGF10 and SHH, we demonstrate that precise spatio-temporal patterns and appropriate levels of expression of these two signaling molecules in the ventral area are crucial between embryonic day 11.5 and 13.5 for the proper patterning of the cartilage rings. We conclude that the expression level of FGF10 in the mesenchyme has to be within a critical range to allow for periodic expression of Shh in the ventral epithelium, and consequently for the correct patterning of the cartilage rings. We propose that disturbed balances of Fgf10 and Shh may explain a subset of human tracheomalacia without tracheo-esophageal fistula or tracheal atresia.

Lung organogenesis.

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.

Signaling via Alk5 controls the ontogeny of lung Clara cells.

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 (TGFbeta)is a multifunctional molecule with roles in normal development and disease pathogenesis. In this study, we deleted the TGFbeta 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 TGFbeta/ALK5 regulates Clara cell differentiation may entail inhibition of Pten expression, which in turn activates ERK and AKT phosphorylation.