Early Lung Branching Morphogenesis Research Articles

The Axonal Guidance Cue Semaphorin 3C Contributes to Alveolar Growth and Repair

Lung diseases characterized by alveolar damage such as bronchopulmonary dysplasia (BPD) in premature infants and emphysema lack efficient treatments. Understanding the mechanisms contributing to normal and impaired alveolar growth and repair may identify new therapeutic targets for these lung diseases. Axonal guidance cues are molecules that guide the outgrowth of axons. Amongst these axonal guidance cues, members of the Semaphorin family, in particular Semaphorin 3C (Sema3C), contribute to early lung branching morphogenesis. The role of Sema3C during alveolar growth and repair is unknown. We hypothesized that Sema3C promotes alveolar development and repair. In vivo Sema3C knock down using intranasal siRNA during the postnatal stage of alveolar development in rats caused significant air space enlargement reminiscent of BPD. Sema3C knock down was associated with increased TLR3 expression and lung inflammatory cells influx. In a model of O2-induced arrested alveolar growth in newborn rats mimicking BPD, air space enlargement was associated with decreased lung Sema3C mRNA expression. In vitro, Sema3C treatment preserved alveolar epithelial cell viability in hyperoxia and accelerated alveolar epithelial cell wound healing. Sema3C preserved lung microvascular endothelial cell vascular network formation in vitro under hyperoxic conditions. In vivo, Sema3C treatment of hyperoxic rats decreased lung neutrophil influx and preserved alveolar and lung vascular growth. Sema3C also preserved lung plexinA2 and Sema3C expression, alveolar epithelial cell proliferation and decreased lung apoptosis. In conclusion, the axonal guidance cue Sema3C promotes normal alveolar growth and may be worthwhile further investigating as a potential therapeutic target for lung repair.

Rewiring of the Lung: Axonal Guidance Cues Promote Alveolar Growth and Repair

Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, is the main complication after premature birth. Perinatal lung injury in premature infants disrupts the normal sequence of lung development and results in the histological pattern of alveolar “simplification” (larger but fewer alveoli) and decreased capillary density. Structural abnormalities mimicking human BPD are recapitulated in newborn rodents by exposure to hyperoxia. Little is known about the mechanisms of normal alveolar development and its anatomical substratum, the outgrowth of secondary crests. While even less is known about how these pathways are disordered in severe BPD. Interactions between airways and blood vessels are critical for normal lung development. Axonal guidance cues (AGC) are molecules that regulate neural network formation in the nervous system and the outgrowth of axons by acting as attractants, guiding neurons to their target or as repellents, creating exclusion zones that neurons avoid. Recent data suggest that AGC are involved in angiogenesis, cell migration and early lung branching morphogenesis. Thus, AGC are appealing candidates in guiding also the outgrowth of secondary crests during alveolar development and repair. Here we present novel data suggesting that the AGC Ephrin B2 These findings may lead to the development of new treatments for neonatal lung injury.

Prenatal Lung Epithelial Cell-Specific Abrogation of Alk3-Bone Morphogenetic Protein Signaling Causes Neonatal Respiratory Distress by Disrupting Distal Airway Formation

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

Molecular Mechanisms of Early Lung Specification and Branching Morphogenesis

The "hard wiring" encoded within the genome that determines the emergence of the laryngotracheal groove and subsequently early lung branching morphogenesis is mediated by finely regulated, interactive growth factor signaling mechanisms that determine the automaticity of branching, interbranch length, stereotypy of branching, left-right asymmetry, and finally gas diffusion surface area. The extracellular matrix is an important regulator as well as a target for growth factor signaling in lung branching morphogenesis and alveolarization. Coordination not only of epithelial but also endothelial branching morphogenesis determines bronchial branching and the eventual alveolar-capillary interface. Improved prospects for lung protection, repair, regeneration, and engineering will depend on more detailed understanding of these processes. Herein, we concisely review the functionally integrated morphogenetic signaling network comprising the critical bone morphogenetic protein, fibroblast growth factor, Sonic hedgehog, transforming growth factor-beta, vascular endothelial growth factor, and Wnt signaling pathways that specify and drive early embryonic lung morphogenesis.

Smad1 expression and function during mouse embryonic lung branching morphogenesis

Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells (alpha-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing approximately 20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.

Lgl1, a mesenchymal modulator of early lung branching morphogenesis, is a secreted glycoprotein imported by late gestation lung epithelial cells

Secreted glycoproteins serve a variety of functions related to cell-cell communication in developmental systems. We cloned LGL1, a novel glucocorticoid-inducible gene in foetal lung, and described its temporal and spatial localization in the rat. Disruption of foetal mesenchyme-specific LGL1 expression using antisense oligodeoxynucleotides, which was associated with a 50% decrease in lgl1 protein levels, inhibited airway epithelial branching in foetal rat gestational day 13 lung buds in explant culture. These findings suggested that lgl1 functions as a secreted signalling molecule. We now provide evidence supporting a role for lgl1 in mesenchymal-epithelial interactions that govern lung organogenesis. Lgl1 is a secreted glycoprotein with a conserved N-terminal secretory signal peptide. Using dual immunofluorescence, intracellular lgl1 was found to co-localize with markers of the Golgi apparatus and endoplasmic reticulum, consistent with its association with secretory vesicles. Using pulse-chase studies, we show that lgl1 is a stable protein with a half-life of 11.5 h. Furthermore, at gestational days 20 and 21 (term=22), foetal distal lung epithelial cells import lgl1 protein. Taken together, our findings support distinct roles for lgl1 as a mediator of glucocorticoid-induced mesenchymal-epithelial interactions in early and late foetal lung organogenesis.

PDGF‐a/PDGF alpha‐receptor signaling is required for lung growth and the formation of alveoli but not for early lung branching morphogenesis

PDGF‐a/PDGF alpha‐receptor signaling is required for lung growth and the formation of alveoli but not for early lung branching morphogenesis

PDGF-A/PDGF alpha-receptor signaling is required for lung growth and the formation of alveoli but not for early lung branching morphogenesis.

Platelet-derived growth factors (PDGF) constitute a family of four gene products (PDGF-A-D) acting by means of two receptor tyrosine kinases, PDGFR alpha and beta. Three of the ligands (PDGF-A, -B, and -C) bind to PDGFR alpha with high affinity. Knockout of pdgf-a in mice has demonstrated a role for PDGF-A in the recruitment of smooth muscle cells to the alveolar sacs and their further compartmentalization into alveoli. Although this is a late, postnatal step in lung development, pdgf-a antisense oligonucleotides were previously shown to inhibit epithelial branching in rat lung explants in vitro, which reflects an early embryonic process. These conflicting results may be explained by substitution of genetic loss of pdgf-a by maternal transfer of PDGF-A to the knockout embryo or the presence of other PDGFR alpha agonists (PDGF-B and -C) in vivo, potentially masking an effect of PDGF-A on branching morphogenesis. Alternatively, the administration of pdgf-a antisense oligonucleotides affected other processes than the intended. To discriminate between these opposing possibilities, we have analyzed lung development in pdgfr alpha -/- embryos and lung primordia grown in vitro. Our analysis shows that, while the pdgfr alpha -/- lungs and explanted lung rudiments were smaller than normal, branching morphogenesis appears qualitatively intact and proceeds until at least embryonic day 15.5, generating both prospective conducting and respiratory airways. We conclude that, although PDGF-AA signaling over PDGFR alpha may have direct or indirect roles in overall lung growth, it does not specifically control early branching of the lung epithelium.

Transforming growth factor beta2, but not beta1 and beta3, is critical for early rat lung branching.

Mesenchymal-epithelial tissue interactions are critical for lung branching morphogenesis, and polypeptide growth factors are likely involved in these tissue interactions. Transforming growth factorbetas (TGFbetas) have been implicated in lung development, but their involvement in early lung branching morphogenesis is unclear. In the present study, we investigated the role of the three mammalian TGFbeta subtypes (beta1, beta2, and beta3) and their receptors (type III (TbetaR-III), type II (TbetaR-II), and two types I (TbetaR-I), ALK-1 and ALK-5) in early rat lung organogenesis by using an embryonic rat lung explant culture. Transcripts and proteins for all three TGFbetas and their receptors were detected during the embryonic period of fetal rat lung development. Inhibition of TGFbeta2, but not beta1 and beta3, with antisense oligonucleotides and neutralizing antibodies resulted in significant inhibition of early lung branching in culture. Addition of minute amounts (</=1 ng/ml) of exogenous TGFbeta2, but not beta1 and beta3, restored the branching of TGFbeta2 antisense-treated explants. Higher concentrations of TGFbeta2 were inhibitory. BrdU labeling of lung explants was not altered by antisense TGFbeta2 treatment, but low concentrations of TGFbeta2 increased thymidine uptake by isolated epithelial cells. Fibronectin and metallogelatinase activities of embryonic lung cells were not affected by any TGFbeta isoform but TGFbeta2 specifically decreased mesenchymal hyaluronan synthesis. Antisense inhibition of ALK-5 and TbetaR-II showed a similar reduction in early lung branching as observed with antisense TGFbeta2. Incubation of lung explants with soluble TbetaR-II receptors also abrogated lung branching. ALK-1 antisense treatment did not affect early branching. Administration of neither activin A, which can act via ALK-1, nor follistatin, the natural inhibitor of activin, to the explants cultures had any significant effect on lung branching. Antisense inhibition of the activin receptor-II (Act-RII) also did not affect lung branching. These results are consistent with TGFbeta2, but not beta1 and beta3, regulating pattern formation during early rat lung organogenesis. This TGFbeta signaling in rat lung branching in vitro appears to be predominantly mediated via the TbetaR-I(ALK-5)/TbetaR-II heteromeric complex.

PDGF-AA and its receptor influence early lung branching via an epithelial–mesenchymal interaction

The biological role of platelet-derived growth factor (PDGF)-AA in lung morphogenesis was investigated by incubating embryonic lung explants with phosphorothioate antisense PDGF-A oligonucleotides, which decreased PDGF-AA but not PDGF-BB protein content. Antisense PDGF-A oligonucleotides inhibited DNA synthesis. This inhibitory effect of antisense PDGF-A was reversed by the addition of exogenous PDGF-AA but not PDGF-BB. Morphometric analyses of antisense-treated cultures showed a significant reduction in lung size. The number of terminal buds of the lung explants was significantly decreased by antisense PDGF-A oligonucleotides. PDGF-AA but not PDGF-BB attenuated the inhibitory effect of antisense PDGF-A on early lung branching. Sense PDGF-A had no effect on DNA synthesis and early lung branching. Reverse transcriptase-polymerase chain reaction analysis revealed PDGF-A mRNA expression in the epithelial component of the embryonic lung, while message for PDGF alpha-receptor was expressed in the mesenchyme. Incubation of explants with neutralizing PDGF-AA antibodies also reduced DNA synthesis and early branching morphogenesis. We conclude that PDGF-AA and its receptor represent an important epithelial-mesenchymal interaction which plays a critical role in early lung branching morphogenesis.