Spatial-specific TGF-β1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung

Abstract

The precise spatial-temporal role that expression and activation of transforming growth factor (TGF)-β plays in mammalian organ morphogenesis remains incompletely understood. Using replication deficient adenoviral vectors containing engineered TGF-β1 cDNAs, we studied the spatial effects of locally over-expressing either latent or mutated, constitutively active TGF-β1 protein during embryonic mouse lung branching morphogenesis in culture. Transfer of exogenous genes into lung epithelium was achieved by intra-tracheal microinjection of recombinant adenovirus, while submerging lungs in virus resulted in gene transfer into the pleura and subjacent mesenchymal cells, as revealed by cytochemical staining for β-galactosidase. Only lungs transfected with active, but not latent TGF-β1 gene, showed elevated levels of active TGF-β. Epithelial over-expression of active, but not latent TGF-β1, via intra-tracheal micro-injection inhibited lung branching morphogenesis by 36%. In contrast, lungs submerged with either active or latent TGF-β1 recombinant virus did not demonstrate an inhibitory effect upon branching. Pulmonary gene regulation was assayed by competitive polymerase chain reaction coupled with reverse transcription. Direct respiratory tract micro-injection of adenovirus over-expressing active TGF-β1 resulted in a dose-dependent inhibition of epithelial surfactant protein (SP)-C and SP-B mRNA levels by up to 76% and 70%, respectively, while in contrast, fibronectin and matrix Gla protein (MGP) mRNA levels remained stable. However, lungs that had been submerged in adenovirus expressing active TGF-β1 demonstrated a concentration-dependent induction of both fibronectin and MGP mRNA levels up to 4.3- and 4.7-fold respectively in the presence of 1 X 10 11 pfu/ml active TGF-β1 virus. On the other hand, lungs treated with adenovirus expressing latent TGF-β1 either by micro-injection or submerging failed to demonstrate any regulatory effect either upon epithelial or mesenchymal gene expression. We conclude that adenovector-mediated overexpression of activated TGF-β1 in specific spatial compartments results respectively in either inhibition of branching morphogenesis and epithelium-specific gene expression, or in induction of matrix gene expression without affecting morphogenesis or epithelium-specific gene expression, depending on the route of administration. Also, the lack of effect of latent TGF-β1 over-expression strongly suggests that TGF-β activation per se provides an important locus of fine regulation ofthe spatial effects of TGF-β signaling during embryonic lung branching morphogenesis.