LAMININ ALPHA 2 IN DEVELOPMENT

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The long term goal of this research is to determine the molecular mechanisms behind the observed importance of basement membranes for tissue development and function. The present application is focused on laminin, specifically the laminin alpha2 chain. The alpha2 chain is a homologue of the A or alpha1 chain of the classical laminin from the Engelbreth-Holm- Swarm (EHS) tumor and is expressed most prominently in mature striated muscle and peripheral nerve. The laminin alpha2 chain is affected in certain muscle diseases in humans and animals. It is reduced in patients with the Fukuyama congenital muscular dystrophy and missing in patients with certain other congenital muscular dystrophies. Two allelic mouse mutants with muscular dystrophy exist: the dy, with severe phenotype and greatly reduced laminin alpha2 chain, and dy2J with milder phenotype and a truncated laminin alpha2 chain. The dy2J mouse has a mutation in an RNA splice consensus sequence causing abnormal splicing of mRNA and deletions in the domain VI of the alpha2 chain. Aim l: We will use the dy and dy2J mutants to study structure and function of laminin. The tissue localization of the laminin in the mutant mice will be studied by light and electron microscopy in relation to other basement membrane proteins. Electron microscopy will be used to study the structure of isolated, mutated laminin. The interaction of mutated laminin with itself and other basement membrane proteins and with cells will be studied in polymerization-, affinity-, and cell adhesion assays. Aim 2: We will generate a laminin alpha2 chain null mutant mouse to determine the effect of complete absence of this laminin chain on development. A portion of the coding sequence of the laminin alpha2 chain gene will be replaced with the LacZ gene by homologous recombination in embryonic stem cells. The phenotype of the homozygous null mutant mouse will be analyzed and compared to that of the dy and dy2J mice. Heterozygous mice will be analyzed for expression of beta-galactosidase, particularly in early embryogenesis, to obtain information on the normal expression of the laminin alpha2 chain. Aim 3: The defect in spontaneous and experimental laminin alpha2-chain deficient mice will be partially corrected by expression of wild type alpha2 chain. Transgenic mice overexpressing laminin alpha2 chain will be generated. These mice will be bred to alpha2 mutant mice to produce mice with a more restricted phenotype. Results with spontaneous and experimentally mutated mice will result in better knowledge of the function and activities of laminin in different tissues and may lead to improved understanding, diagnosis, and treatment of muscular dystrophies and other neuromuscular diseases.