Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung
Am J Physiol Lung Cell Mol Physiol doi:10.1152/ajplung.00185.2004 288:167-178, 2005.
Minke van Tuyl, Jason Liu, Jinxia Wang, Maciek Kuliszewski, Dick Tibboel and Martin Post
Fig. 1. Low oxygen enhances epithelial branching morphogenesis in vitro. C101-LacZ expression in embryonic day (E) 11.5 lung explants after 48 (A and B) and 96 (C and D) h in culture. Explants cultured at 3% oxygen (B and D) showed more complex branching compared with explants cultured at 20% oxygen (A and C). *Shows location of the trachea; arrowheads show the main bronchi, and the arrows show increased distal branch tips. Blue color is X-galactosidase (X-gal) staining in airway epithelial cells. Bars, 100 µm.
Recent investigations have suggested an active role for endothelial cells in organ development, including the lung. Herein, we investigated some of the molecular mechanisms underlying normal pulmonary vascular development and their influence on epithelial branching morphogenesis. Because the lung in utero develops in a relative hypoxic environment, we first investigated the influence of low oxygen on epithelial and vascular branching morphogenesis. Two transgenic mouse models, the C101-LacZ (epithelial-LacZ marker) and the Tie2-LacZ (endothelial-LacZ marker), were used. At embryonic day 11.5, primitive lung buds were dissected and cultured at either 20 or 3% oxygen. At 24-h intervals, epithelial and endothelial LacZ gene expression was visualized by X-galactosidase staining. The rate of branching of both tissue elements was increased in explants cultured at 3% oxygen compared with 20% oxygen. Low oxygen increased expression of VEGF, but not that of the VEGF receptor (Flk-1). Expression of two crucial epithelial branching factors, fibroblast growth factor-10 and bone morphogenetic protein-4, were not affected by low oxygen. Epithelial differentiation was maintained at low oxygen as shown by surfactant protein C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible factor-1 or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited. Collectively, the in vitro data show that a low-oxygen environment enhances branching of both distal lung epithelium and vascular tissue and that pulmonary vascular development appears to be rate limiting for epithelial branching morphogenesis.