Early Lung Development Research Articles

Expression of Iroquois genes is up-regulated during early lung development in the nitrofen-induced pulmonary hypoplasia

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

PLoS Genetics Issue Image | Vol. 6(10) October 2010

The lung and trachea originate in the ventral foregut, but their separation from the future oesophagus remains poorly understood. In this issue of PLoS Genetics, Jurado et al report that ASCIZ, a Zn2+-finger protein previously linked to the repair of methylating and oxidative DNA damage, also has an unexpected function during early lung development. Although embryos lacking Asciz are able to generate respiratory precursors, they never form lung buds, and separation of the trachea from the foregut stalls at an early stage. The authors propose that ASCIZ may function as a new transcriptional regulator of foregut separation. Image Credit: Ian Smyth

Embryonic Wnt gene expression in the nitrofen-induced hypoplastic lung using 3-dimensional imaging

Purpose Wnts have been reported to play a key role in the lung morphogenesis. We have previously reported that pulmonary gene expression of Wnt2 and Wnt7b is downregulated on day 15 of gestation in the nitrofen-induced congenital diaphragmatic hernia (CDH) model. However, the distribution pattern of gene expression of Wnts in the very early lung development remains unclear. Optical projection tomography (OPT) is a new technique for 3-dimensional imaging of small developing organs and gene distribution combined with whole-mount in situ hybridization. We designed this study to investigate the distribution pattern of Wnts gene expression in lung buds of nitrofen-induced CDH model using OPT. Methods Embryos from normal and nitrofen-treated dams were harvested on embryonic day 10 (E10), and divided into controls and nitrofen group, respectively. Whole-mount in situ hybridization to detect transcripts of Wnt2 and Wnt7b was performed, analyzed, and reconstructed using OPT. Results The expression of Wnt2 transcripts was detected in the lung bud mesenchyme and markedly diminished in nitrofen group compared to controls, whereas Wnt7b transcripts were expressed in the mesoderm of bronchi and the lung bud with no detectable difference between 2 groups. Conclusion We provide evidence for the first time that Wnt2 expression is downregulated at lung bud stage in the nitrofen model. Optical projection tomography is potentially a useful approach to visualize both gene expression and morphology during very early stages of lung development.

A neonate with left pulmonary artery thrombosis and left lung hypoplasia: a case report

IntroductionSpontaneous intrauterine arterial thrombosis and congenital pulmonary hypoplasia are rare conditions and have not been reported to occur together. The literature rather includes two reports of babies with neonatal pulmonary artery occlusion and post-infarction cysts of the lungs.Case presentationWe report a case of a live Caucasian male newborn with left lung hypoplasia that occurred in association with left pulmonary artery thrombosis. Despite a critical neonatal course, including extracorporeal membrane oxygenation, this infant is alive and well at 18 months of age without any neurodevelopmental sequelae or reactive airway disease.ConclusionThis association suggests the possibility of an intrauterine vascular event between the fifth and eighth weeks of gestation during early pulmonary artery and lung development.

Expression of components of Wnt and Hedgehog pathways in different tissue layers during lung development in Xenopus laevis

Although Wnt and Hedgehog (Hh) signaling pathways play important roles in mouse lung development, these have not been explored in the development of Xenopus lung. This may be due to the lack of specific molecular markers for different layers of tissue in Xenopus lung and/or insufficient knowledge on expression patterns of Wnt and Hh signaling components in Xenopus lung. In this study, we first described the early morphogenesis of Xenopus laevis lung by using surfactant protein C ( sftpc) as a marker of lung epithelium and compared it with the expression patterns of several genes of Wnt and Hh pathways in Xenopus lungs. Our data showed that wnt7b was expressed in the entire lung epithelium from stage 37 to stage 45, while two other Wnt signaling components, wnt5a and wif1 ( wnt inhibitory factor 1), were expressed in the mesenchyme layer of the entire lungs through stages 39–41. We also found that sonic hedgehog ( shh) was expressed at stage 41 only in the anterior, but not in the posterior part of the lungs. These results show the expression of wnt5a, wnt7b, wif1 and shh in different layers of tissue of Xenopus lungs at early developmental stages, which implies different roles of these genes in the early development of Xenopus lungs. Our study for the first time defined specific molecular markers for description of early lung development in Xenopus, as well as provided information about expression of components of Wnt and Hh pathways in early Xenopus lungs, which should be useful for future functional studies.

Lung tissue regeneration after induced injury in Runx3 KO mice

Runx3 is essential for normal murine lung development, and Runx3 knockout (KO) mice, which die soon after birth, exhibit alveolar hyperplasia. Wound healing, tissue repair, and regeneration mechanisms are necessary in humans for proper early lung development. Previous studies have reported that various signaling molecules, such as pErk, Tgf-beta1, CCSP, pJnk, Smad3, and HSP70 are closely related to wound healing. In order to confirm the relationship between lung defects caused by the loss of function of Runx3 and wound healing, we have localized various wound-healing markers after laser irradiation in wild-type and in Runx3 KO mouse lungs at post-natal day 1. Our results indicate that pERK, Tgf-beta1, CCSP, pJnk, and HSP70 are dramatically down-regulated by loss of Runx3 during lung wound healing. However, Smad3 is up-regulated in the Runx3 KO laser-irradiated lung region. Therefore, the lung wound-healing mechanism is inhibited in the Runx3 KO mouse, which shows abnormal lung architecture, by reduced pErk, Tgf-beta1, CCSP, pJnk, and HSP70 and by induced Smad3.

Maternal Vitamin A Supplementation and Lung Function in Offspring

Vitamin A is important in regulating early lung development and alveolar formation. Maternal vitamin A status may be an important determinant of embryonic alveolar formation, and vitamin A deficiency in a mother during pregnancy could have lasting adverse effects on the lung health of her offspring. We tested this hypothesis by examining the long-term effects of supplementation with vitamin A or beta carotene in women before, during, and after pregnancy on the lung function of their offspring, in a population with chronic vitamin A deficiency. We examined a cohort of rural Nepali children 9 to 13 years of age whose mothers had participated in a placebo-controlled, double-blind, cluster-randomized trial of vitamin A or beta-carotene supplementation between 1994 and 1997. Of 1894 children who were alive at the end of the original trial, 1658 (88%) were eligible to participate in the follow-up trial. We performed spirometry in 1371 of the children (83% of those eligible) between October 2006 and March 2008. Children whose mothers had received vitamin A had a forced expiratory volume in 1 second (FEV(1)) and a forced vital capacity (FVC) that were significantly higher than those of children whose mothers had received placebo (FEV(1), 46 ml higher with vitamin A; 95% confidence interval [CI], 6 to 86; FVC, 46 ml higher with vitamin A; 95% CI, 8 to 84), after adjustment for height, age, sex, body-mass index, calendar month, caste, and individual spirometer used. Children whose mothers had received beta carotene had adjusted FEV(1) and FVC values that were similar to those of children whose mothers had received placebo (FEV(1), 14 ml higher with beta carotene; 95% CI, -24 to 54; FVC, 17 ml higher with beta carotene, 95% CI, -21 to 55). In a chronically undernourished population, maternal repletion with vitamin A at recommended dietary levels before, during, and after pregnancy improved lung function in offspring. This public health benefit was apparent in the preadolescent years.

Epithelial N-cadherin and nuclear β-catenin are up-regulated during early development of human lung

BackgroundThe aim of this study was to analyze the cell-specific expression of E- and N-cadherin and β-catenin in developing human lung tissues from 12 to 40 weeks of gestation.MethodsFortyseven cases of developing human lung including pseudoglandular, canalicular, saccular and alveolar periods were analyzed by immunohistochemisty for E- and N-cadherin and β-catenin and twentyone cases were also investigated by RT-PCR for E- and N-cadherin and β-catenin. For identifying the lung cells, the sections were also stained with antibodies against thyroid transcription factor-1 (TTF-1) and caveolin-1. Normal adult lung tissue was used as a control.E-cadherin was strongly expressed in epithelium of bronchi and large bronchioles from week 12 onwards and it was also positive in alveoli in pretype II cells and type II cells. N-cadherin was present in most of the epithelial cells of bronchi and the largest bronchioles during the pseudo-glandular and canalicular periods. N-cadherin was not detected in epithelium of developing alveoli. β-catenin was strongly membrane-bound and positively expressed in bronchial epithelium from week 12 to week 40; it showed nuclear positivity in both developing airway epithelium and in the cells underneath the epithelium during pseudo-glandular period and to a lesser degree also in the canalicular period. β-catenin was positive in pretype II cells as well as in type I and type II pneumocytes within alveoli.RT-PCR analyses revealed detectable amounts of RNAs of E- and N-cadherin and β-catenin in all cases studied. The amounts of RNAs were higher in early stages of gestation.ConclusionsE-cadherin is widely expressed in bronchial and alveolar epithelial cells. N-cadherin exhibit extensive epithelial positivity in bronchial epithelial cells during early lung development. The presence of β-catenin was observed in several cell types with a distinct location in tissue and cells in various gestational stages, indicating that it possesses several roles during lung development. The expressions of protein and mRNAs of E- and N-cadherin and β-catenin were higher in early gestation compared to of the end. Moreover, the expressions of these factors were higher during the lung development than in the adult human lung.

Surfactant biology and pulmonary fibrosis: A ChILD shall lead them

ELF5, an Ets family transcription factor found exclusively in epithelial cells, is expressed in the distal lung epithelium during embryogenesis, then becomes restricted to proximal airways at the end of gestation and postnatally. To test the hypothesis that ELF5 represses distal epithelial differentiation, we generated a transgenic mouse model in which a doxycycline inducible HA-tagged mouse Elf5 transgene was placed under the control of the lung epithelium-specific human SFTPC promoter. We found that expressing high levels of ELF5 during early lung development disrupted branching morphogenesis and produced a dilated epithelium. The effects of ELF5 on morphogenesis were stage-dependent, since inducing the transgene on E16.5 had no effect on branching. ELF5 reduced expression of the distal lung epithelial differentiation markers Erm, Napsa and Sftpc, and type II cell ultrastructural differentiation was immature. ELF5 overexpression did not induce the proximal airway epithelial markers Ccsp and Foxj1, but did induce expression of p63, a marker of basal cells in the trachea and esophagus. High ELF5 levels also induced the expression of genes found in other endodermal epithelia but not normally associated with the lung. These results suggest that precise levels of ELF5 regulate the specification and differentiation of epithelial cells in the lung.

Up-regulation of Wnt5a gene expression in the nitrofen-induced hypoplastic lung

Purpose The pathogenesis of pulmonary hypoplasia in nitrofen-induced congenital diaphragmatic hernia (CDH) still remains unclear. Wnt signaling pathways play a critical role in lung development. Whereas canonical Wnt signaling regulates branching morphogenesis during early lung development, the noncanonical Wnt5a controls late lung morphogenesis, including patterning of distal airway and vascular tubulogenesis (alveolarization). Overexpression of Wnt5a in transgenic mice and in the chick has been reported to result in severe pulmonary hypoplasia. We designed this study to test the hypothesis that the pulmonary Wnt5a gene expression is up-regulated in late stages of lung morphogenesis in CDH. Methods Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, D18, and D21 and divided into 3 groups: control; nitrofen without CDH, CDH(−); and nitrofen with CDH, CDH(+) (n = 8 at each time-point, respectively). Wnt5a pulmonary gene expression was analyzed by real-time reverse transcription polymerase chain reaction. Immunohistochemistry was performed to evaluate Wnt5a protein expression at each time-point. Results Pulmonary relative mRNA expression levels of Wnt5a were significantly increased in CDH(−) and CDH(+) at D18 (1.61 ± 0.92 and 1.81 ± 1.20, respectively) and D21 (2.40 ± 0.74* and 2.65 ± 0.35*, respectively) compared to controls at D18 and D21 (0.90 ± 0.17* and 1.69 ± 0.53**, respectively) (* P < .05, ** P < .001 vs control ). Strong Wnt5a immunoreactivity was seen in the distal epithelium at D18 and D21 in nitrofen-induced hypoplastic lung compared to controls. Conclusion Up-regulation of pulmonary Wnt5a gene expression in the late lung morphogenesis may interfere with patterning of alveolarization, causing pulmonary hypoplasia in the nitrofen-induced CDH.

Mechanisms for split localization of Fgf10 expression in early lung development

In early lung development, epithelial tubes (lung buds) intrude into mesenchyme covered with pleural cells (lung border), and form tree-like networks, by means of repeated use of morphogenetic processes: "elongation," "terminal bifurcation," and "lateral budding." When a bud is elongating, a peak of Fgf10 expression is formed in the mesenchyme near the tip; whereas when terminal bifurcation and lateral budding occur, two separate peaks are formed instead. To explain the spatial pattern of Fgf10 expression, we developed a mathematical model for the regulation of Fgf10 expression with geometrical conditions including shapes of the lung buds and the lung border. Different localization patterns of Fgf10 expression can be explained by the geometrical conditions. Fgf10 expression has a single peak when a length between the tip of lung bud and the lung border is large. When the length is small, Fgf10 expression has two peaks, whose location depends on the curvature of lung border.

Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers

The swimbladder is a hydrostatic organ in fish postulated as a homolog of the tetrapod lung. While lung development has been well studied, the molecular mechanism of swimbladder development is essentially uncharacterized. In the present study, swimbladder development in zebrafish was analyzed by using several molecular markers: hb9 (epithelium), fgf10a and acta2 (mesenchyme), and anxa5 (mesothelium), as well as in vivo through enhancer trap transgenic lines Et(krt4:EGFP)sq33-2 and Et(krt4:EGFP)sqet3 that showed strong EGFP expression in the swimbladder epithelium and outer mesothelium respectively. We defined three phases of swimbladder development: epithelial budding between 36 and 48 hpf, growth with the formation of two additional mesodermal layers up to 4.5 dpf, and inflation of posterior and anterior chambers at 4.5 and 21 dpf respectively. Similar to those in early lung development, conserved expression of Hedgehog (Hh) genes, shha and ihha, in the epithelia, and Hh receptor genes, ptc1 and ptc2, as well as fgf10a in mesenchyme was observed. By analyzing several mutants affecting Hh signaling and Ihha morphants, we demonstrated an essential role of Hh signaling in swimbladder development. Furthermore, time-specific Hh inhibition by cyclopamine revealed different requirements of Hh signaling in the formation and organization of all three tissue layers of swimbladder.

Chronological expression of Ciliated Bronchial Epithelium 1 during pulmonary development

Ciliated Bronchial Epithelium (CBE) 1 is a novel gene, which is expressed in ciliated cells. As cilia are important during embryogenesis, the present authors characterised the murine homologue of CBE1 (Cbe1) and compared its temporal expression during murine and human lung development. Cbe1 cDNA was cloned and characterised using sequencing, standard PCR and Western blotting. Mouse and human embryonic/fetal lungs (HELs) were harvested for mRNA analysis and protein localisation in vivo and in vitro using RT-PCR and immunohistochemistry. The Cbe1 amino acid sequence was >75% identical with CBE1 and its alternative splicing and tissue distribution were highly conserved. Pulmonary expression of Cbe1 mRNA was increased at embryonic day (E)16, 1 day later than Foxj1, which is consistent with a role in ciliogenesis. In HELs, CBE1 mRNA was detectable at 8-9 weeks post-conception and increased in explant culture. CBE1 protein expression was weak at 10 weeks post-conception but strong at 12.3 weeks post-conception, in parallel with cilia formation. Additionally, Cbe1 mRNA was expressed at E11 (4-5 weeks post-conception in HELs) in the absence of Foxj1, implying a distinct role in early development. Chronological regulation of CBE1/Cbe1 expression during pulmonary differentiation suggests involvement in ciliogenesis, with an additional role during early lung development.

MicroRNAs Expressed during Lung Cancer Development Are Expressed in Human Pseudoglandular Lung Embryogenesis

Background/Aims: MicroRNAs (miRNAs) play a role during mouse embryonic development and are also important in carcinogenesis. In order to investigate whether there are similar patterns of miRNA expression levels in pseudoglandular human embryonic lung and in human lung tumors, we have analyzed 18 miRNAs (the let-7 family, the miR-17–92 cluster, miR-221 and miR-222) in human embryonic lung samples and in paired lung tumor and normal lung tissue samples and correlated the results with clinicopathological characteristics. Methods: RNA was obtained from 12 human embryonic lung samples, 33 lung tumor samples and 33 paired normal lung samples. miRNAs were assessed by quantitative real-time PCR. Results: Members of the let-7 family were downregulated and members of the miR-17–92 cluster and miR-221 were overexpressed both in embryonic lung tissue and in lung tumors. Low levels of let-7c were associated with absence of metastases (p = 0.015), early-stage non-small cell lung cancer (NSCLC, p = 0.05), and smokers (p = 0.009). High levels of miR-106a were associated with small-cell lung cancer (p = 0.031), and high levels of miR-19a with advanced NSCLC (p = 0.008). Conclusion: Our study lends support to the model of cancer as an alteration of normal development, as many miRNAs were similarly expressed in early human lung development and stage I-II of lung cancer development.

Air pollution during pregnancy and lung function in newborns: a birth cohort study

Post-natal exposure to air pollution is associated with diminished lung growth during school age. The current authors aimed to determine whether pre-natal exposure to air pollution is associated with lung function changes in the newborn. In a prospective birth cohort of 241 healthy term-born neonates, tidal breathing, lung volume, ventilation inhomogeneity and exhaled nitric oxide (eNO) were measured during unsedated sleep at age 5 weeks. Maternal exposure to particles with a 50% cut-off aerodynamic diameter of 10 microm (PM(10)), nitrogen dioxide (NO(2)) and ozone (O(3)), and distance to major roads were estimated during pregnancy. The association between these exposures and lung function was assessed using linear regression. Minute ventilation was higher in infants with higher pre-natal PM(10) exposure (24.9 mL x min(-1) per microg x m(-3) PM(10)). The eNO was increased in infants with higher pre-natal NO(2) exposure (0.98 ppb per microg x m(-3) NO(2)). Post-natal exposure to air pollution did not modify these findings. No association was found for pre-natal exposure to O(3) and lung function parameters. The present results suggest that pre-natal exposure to air pollution might be associated with higher respiratory need and airway inflammation in newborns. Such alterations during early lung development may be important regarding long-term respiratory morbidity.

Role of Secretoglobin 3A2 in Lung Development

Secretoglobin 3A2 (SCGB3A2) was originally identified as a downstream target in lung for the homeodomain transcription factor NKX2-1, whose null mutation resulted in severely hypoplastic lungs. A very low level of SCGB3A2 is expressed in lungs at Embryonic Day (E) 11.5 during mouse development, which markedly increases by E16.5, the time when lung undergoes dramatic morphologic changes, suggesting that SCGB3A2 may be involved in lung development in addition to a known role in lung inflammation. To determine whether SCGB3A2 plays a role in lung development. To assess a potential role for SCGB3A2 during early lung development, wild-type and Nkx2-1-null fetal lungs of early developmental stages were subjected to ex vivo organ culture in the presence of SCGB3A2. Nkx2-1-null fetuses were exposed to SCGB3A2 during early organogenesis period through intravenous administration of this protein to Nkx2-1-heterozygous pregnant females carrying these null fetuses. Cultured lungs and fetal lungs were subjected to histologic and immunohistochemical analyses. To assess a role for SCGB3A2 in late lung development, SCGB3A2 was administered to pregnant wild-type females during mid- to late organogenesis stages, and the preterm pups and/or their lungs were evaluated for extent of maturity using breathing motion, gross morphology and histology of lungs, expression of gestational stage-specific genes, and phospholipid profiles. SCGB3A2 significantly promoted both early and late stages of lung development. SCGB3A2 is a novel growth factor in lung.

Misexpression of ELF5 disrupts lung branching and inhibits epithelial differentiation

ELF5, an Ets family transcription factor found exclusively in epithelial cells, is expressed in the distal lung epithelium during embryogenesis, then becomes restricted to proximal airways at the end of gestation and postnatally. To test the hypothesis that ELF5 represses distal epithelial differentiation, we generated a transgenic mouse model in which a doxycycline inducible HA-tagged mouse Elf5 transgene was placed under the control of the lung epithelium-specific human SFTPC promoter. We found that expressing high levels of ELF5 during early lung development disrupted branching morphogenesis and produced a dilated epithelium. The effects of ELF5 on morphogenesis were stage-dependent, since inducing the transgene on E16.5 had no effect on branching. ELF5 reduced expression of the distal lung epithelial differentiation markers Erm, Napsa and Sftpc, and type II cell ultrastructural differentiation was immature. ELF5 overexpression did not induce the proximal airway epithelial markers Ccsp and Foxj1, but did induce expression of p63, a marker of basal cells in the trachea and esophagus. High ELF5 levels also induced the expression of genes found in other endodermal epithelia but not normally associated with the lung. These results suggest that precise levels of ELF5 regulate the specification and differentiation of epithelial cells in the lung.

Misexpression of MIA disrupts lung morphogenesis and causes neonatal death

Microarray experiments designed to identify genes differentially expressed in the E11.5 lung and trachea showed that melanoma inhibitory activity (Mia1) was expressed only in the lung. Mia1 was abundantly expressed during early lung development, but was virtually absent by the end of gestation. Distal embryonic lung epithelium showed high levels of Mia1 expression, which was suppressed by treatment with either retinoic acid or the FGF signaling antagonist SU5402. Late-gestation fetuses in which lung epithelial hyperplasia was induced by misexpression of FGF7 or FGF10 showed continued expression of Mia1 in areas of aberrant morphogenesis. Mia1 expression was also significantly increased in urethane-induced lung adenomas. Treatment of E18.5 lung explants with exogenous MIA caused significant reductions in the expression of the lung differentiation markers Sftpa, Sftpb, Sftpc, and Abca3. Bitransgenic mice expressing MIA under the control of the SFTPC promoter after E16.5, the age when Mia1 is normally silenced, died from respiratory failure at birth with morphologically immature lungs associated with reduced levels of saturated phosphatidylcholine and mature SP-B. Microarray analysis showed significant reductions in the expression of Sftpa, Sftpb, Abca3, Aqp5, Lzp-s, Scd2, and Aytl2 in lungs misexpressing MIA. These results suggest that the silencing of Mia1 that occurs in late gestation may be required for maturation of the surfactant system.

TGF‐β signaling is dynamically regulated during the alveolarization of rodent and human lungs

Although transforming growth factor-beta (TGF-beta) signaling negatively regulates branching morphogenesis in early lung development, few studies to date have addressed the role of this family of growth factors during late lung development. We describe here that the expression, tissue localization, and activity of components of the TGF-beta signaling machinery are dynamically regulated during late lung development in the mouse and human. Pronounced changes in the expression and localization of the TGF-beta receptors Acvrl1, Tgfbr1, Tgfbr2, Tgfbr3, and endoglin, and the intracellular messengers Smad2, Smad3, Smad4, Smad6, and Smad7 were noted as mouse and human lungs progressed through the canalicular, saccular, and alveolar stages of development. TGF-beta signaling, assessed by phosphorylation of Smad2, was detected in the vascular and airway smooth muscle, as well as the alveolar and airway epithelium throughout late lung development. These data suggest that active TGF-beta signaling is required for normal late lung development.

Tracheal occlusion increases the rate of epithelial branching of embryonic mouse lung via the FGF10-FGFR2b-Sprouty2 pathway

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 Fgf10LacZ/− 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.