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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.03.032 Byline: Nguyet M. Nguyen (a), Robert M. Senior (a)(b) Keywords: Basement membrane; Laminin; Lung development; Lung epithelial cell differentiation Abbreviations: BM, basement membrane; Lama, laminin [alpha]; Lamb, laminin [beta]; Lamc, laminin [gamma] Abstract: Laminins are a major component of basement membranes. Each laminin molecule is a heterotrimeric glycoprotein composed of one [alpha], one [beta], and one [gamma] chain. Fifteen laminin isoforms exist, assembled from various combinations of 5[alpha], 3[beta], and 3[gamma] chains. The embryonic lung has abundant laminin isoforms. Increasing evidence suggests that different laminin isoforms have unique functions in lung development. Studies of embryonic lung explants and organotypic co-cultures show that laminin [alpha]1 and laminin 111 are important for epithelial branching morphogenesis and that laminin [alpha]2 and laminin 211 have a role in smooth muscle cell differentiation. In vivo studies of laminin [alpha]5-deficient mice indicate that this laminin chain, found in laminins 511 and 521, is essential for normal lobar septation in early lung development and normal alveolization and distal epithelial cell differentiation and maturation in late lung development. However, not all of the laminin chains present in the developing lung appear to be necessary for normal lung development since laminin [alpha]4 null mice do not have obvious lung abnormalities and laminin [gamma]2 null mice have only minimal changes in lung development. The mechanisms responsible for the lung phenotypes in mice with laminin mutations are unknown, but it is clear that multiple laminin isoforms are crucial for lung development and that different laminin isoforms exhibit specific, non-overlapping functions. Author Affiliation: (a) Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA (b) Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA Article History: Received 21 January 2006; Revised 16 March 2006; Accepted 27 March 2006

Laminin [alpha]5 is prominent in the basement membrane of alveolar walls, airways, and pleura in developing and adult lung. Targeted deletion of laminin [alpha]5 in mice causes developmental defects in multiple organs, but embryonic lethality has precluded examination of the latter stages of lung development. To identify roles for laminin [alpha]5 in lung development, we have generated an inducible lung epithelial cell-specific Lama5 null (SP-Clama[5.sup.fl/-]) mouse through use of the Cre/loxP system, the human surfactant protein C promoter, and the reverse tetracycline transactivator. SP-Clama[5.sup.fl/-] embryos exposed to doxycycline from E6.5 died a few hours after birth. Compared to control littermates, SP-Clama[5.sup.fl/-] lungs had dilated, enlarged distal airspaces, but basement membrane ultrastructure was preserved. Distal epithelial cell differentiation was perturbed, with a marked reduction of alveolar type II cells and a virtual absence of type I cells. Cell proliferation was reduced and apoptosis was increased. Capillary density was diminished, and this was associated with a decrease in total lung VEGF production. Overall, these findings indicate that epithelial laminin [alpha]5, independent of its structural function, is necessary for murine lung development, and suggest a role for laminin [alpha]5 in signaling pathways that promote alveolar epithelial cell differentiation and VEGF expression. Keywords: Laminin; Lung development; Basement membrane; Alveolar epithelial cell; Differentiation; VEGF

Laminin [alpha/beta/gamma] heterotrimers are the major noncollagenous components of all basement membranes. To date, five [alpha], three [beta], and three [gamma] chains have been identified. Laminin [alpha]5 is expressed early in lung development and colocalizes with laminin [alpha]l. While laminin [alpha]1 expression in the lung is restricted to the embryonic period, laminin [alpha]5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin [alpha]5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama[5.sup.-/-] mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin [alpha]4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin [alpha]5, which was found to be the only laminin [alpha] chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked. Key Words: laminin; lung development; basement membrane; visceral pleura; lobar septation.

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.