Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice

Laminins, heterotrimers of alpha, beta, and gamma chains, are prominent constituents of basal laminae (BLs) throughout the body. Previous studies have shown that laminins affect both myogenesis and synaptogenesis in skeletal muscle. Here we have studied the distribution of the 10 known laminin chains in muscle and peripheral nerve, and assayed the ability of several heterotrimers to affect the outgrowth of motor axons, We show that cultured muscle cells express four different alpha chains (alpha 1, alpha 2, alpha 4, and alpha 5), and that developing muscles incorporate all four into BLs, The portion of the muscle's BL that occupies the synaptic cleft contains at least three alpha chains and two beta chains, but each is regulated differently. Initially, the alpha 2, alpha 4, alpha 5, and beta 1 chains are present both extrasynaptically and synaptically, whereas beta 2 is restricted to synaptic BL from its first appearance. As development proceeds, alpha 2 remains broadly distributed, whereas alpha 4 and alpha 5 are lost from extrasynaptic BL and beta 1 from synaptic BL. In adults, alpha 4 is restricted to primary synaptic clefts whereas alpha 5 is present in both primary and secondary clefts. Thus, adult extrasynaptic BL is rich in laminin 2 (alpha 2 beta 1 gamma 1), and synaptic BL contains laminins 4 (alpha 2 beta 2 gamma 1), 9 (alpha 4 beta 2 gamma 1), and 11 (alpha 5 beta 2 gamma 1). Likewise, in cultured muscle cells, alpha 2 and beta 1 are broadly distributed but alpha 5 and beta 2 are concentrated at acetylcholine receptor-rich "hot spots," even in the absence of nerves, The endoneurial and perineurial BLs of peripheral nerve also contain distinct laminin chains: alpha 2, beta 1, gamma 1, and alpha 4, alpha 5, beta 2, gamma 1, respectively. Mutation of the laminin alpha 2 or beta 2 genes in mice not only leads to loss of the respective chains in both nerve and muscle, but also to coordinate loss and compensatory upregulation of other chains, Notably, loss of beta 2 from synaptic BL in beta 2(-/-) "knockout" mice is accompanied by loss of alpha 5, and decreased levels of alpha 2 in dystrophic alpha 2(dy/dy) mice are accompanied by compensatory retention of alpha 4. Finally, we show that motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (alpha 1 beta 1 gamma 1) and laminin 2, fail to cross from laminin 4 to laminin 1, and stop upon contacting laminin 11, The ability of laminin 11 to serve as a stop signal for growing axons explains, in part, axonal behaviors observed at developing and regenerating synapses in vivo.