The Mechanism Of Sonic Hedgehog Signaling Pathway In Regulating Murine Lung Development

BackgroundEmbryo development is regulated by the interactions between different signaling pathways,and influenced by epithelial-mesenchymal interactions.Abnormal activation or deletion of gene will lead to a series of consequences,including pulmonary dysplasia diseases or even death.It is helpful to understand the relationship and mechanisms between different signal pathways in the process of lung development.In spite of slight differences,the considerable similarities of gene expression in human and mice suggest that conserved molecular networks regulate mammalian lung development.Hedgehog(Hh)signaling pathway plays an important role in cell differentiation,tissue and organ development during embryonic period.Canonical Hh signaling pathway is mainly composed by three ligands:Sonic Hedgehog(Shh)?Indian Hedgehog(Ihh)and Desert Hedgehog(Dhh),requiring the presence of patched(Ptch)and smoothened(Smo)transmembrane receptors in order to induce the activation of glioblastoma(Gli)zinc finger transcription factors include Gli1?Gli2?Gli3 and the downstream target gene that are the true effectors of the pathway.The Shh pathway performs an variety of control functions in the development of mouse embryonic lung.In the presence of Shh combined with Ptchl,Shh relieves the inhibition of Ptchl to Smo.Then,Smo moving into the cell releases micro tubule activated Glil and Gli2,and activates the expression of downstream target genes.ObjectIn this study,the role of classical Shh signaling pathway,as the breakthrough point,was investigated in the epithelial-to-mesenchymal(EMT)transition in the process of mouse embryonic lung development using immunohistochemistry and immunofluorescence technique.Firstly,we use immunohistochemistry to detecte the tissue interactions,molecular crosstalk that underlie the stereotypic patterning,and spatial and temporal variations of Smo and platelet-derived growth factor receptor alpha,which is one of a important molecule in Shh pathway in the developing lung.Secondly,established a transgenic mouse model.Lastly,we use the interstitial specific knockdown of Smo transgenic mouse model to explore the effect and regulation mechanism of Shh signaling pathway in mouse lung development.Methods and resultsFirstly,we used immunohistochemical technique to detecte the expression of Shh signaling pathways receptor,Smo and Pdgfr-alpha,in murine fetal lung.The results show that in the whole pseudoglandular stage,there was Smo expression in the airway epithelium and pulmonary mesenchyme;but in the canalicular stage,the expression of the Smo declined rapidly.In the pseudoglandular stage,Pdgfr-alpha expressed in proximal airway and at E12.5,in the distal lung epithelial cells and some of the stroma cells to surround the trachea,bronchi and bronchioles.At E14.5,the expression of Pdgfr-alpha was strictly localized around the large bronchi,the bronchial smooth muscle cells adjoining to mesenchymal cells,but it was not observed in the distal lung.In the canalicular stage,Pdgfr-alpha appeared in the farther pulmonary mesenchyme.Next,we used the Pdgfr-alpha Cre to knockdown Smo gene and established a transgenic mouse model which was a kind gift provide by the University of Southern California/Losangeles children's hospital.Finally,to determine the potential role of Smo-mediated Shh signaling pathway on lung development in mice,we used Pdgfr-alpha Cre mouse lines to generate Smo deletions in the trachea.The results showed that compared with the control group,during E12.5-E15.5,the volume of transgenic mouse lung and bronchial branching decreased,as well as the expression of TTF-1 by immunofluorescence assay was decreased,while there were no significant difference in the level of TTF-1 expression and bronchial branching at E16.5.During E12.5-E15.5 quantity of cartilaginous matrix chondroitin sulfate from bronchial mesenchyme was lower while formation of tracheal ring lagged with tracheal stenosis,but at E16.5,there were no significant difference.During E12.5-E13.5,compared with the control group,the expression of a-Sma in the proximal and distal trachea increased.At E14.5,increased ?-Sma expression was observed in proximal lung,but no significant difference was detected in distal lung.During E15.5-E16.5,there was no significant difference of the expression of ?-Sma between the proximal and distal lung.During E12.5-E15.5,the proximal epithelial marker P63 was decreased and had no obvious changes at E16.5.During E12.5-E14.5,distal epithelial marker p4-tublin was not detected and the expression was reduced at E15.5,but it showed no clear difference at E16.5.ConclusionIn summary,this study showed that the temporospatial accurate expression of Shh signaling pathway plays an important regulatory role in embryonic bronchial branching morphology,bronchial cartilage matrix and cartilage ring development,proximal and distal smooth muscle and epithelial development in mouse.In addition to the influence of the development of cartilage and smooth muscle,Shh pathway is essential for the development of epithelial and alveolar cells,and participates in the process of epithelial mesenchymal transition.That Shh pathway may be involved in the pathogenesis of bronchial alveolar dysplasia will probably illuminate some molecular basis for this congenital defects associated with pulmonary dysplasia diseases,and can provide a reference for future molecular target therapy which has a certain clinical significance.