IN-SITU HYBRIDIZATION REVEALS TRANSIENT LAMININ B-CHAIN EXPRESSION BYINDIVIDUAL GLIAL AND MUSCLE-CELLS IN EMBRYONIC LEECH CENTRAL-NERVOUS-SYSTEM

Laminin, which strongly stimulates axon outgrowth in vitro, appears tr ansiently within the central nervous system (CNS) in embryos. After CN S injury, laminin reportedly reappears along axonal pathways only in a nimal species in which central axon regeneration is successful, includ ing the leech Hirudo medicinalis. Although glia have been suspected of making CNS laminin, in adult leeches glia are not required for lamini n synthesis and evidently microglia, not present in the early embryo, produce laminin. To determine which embryonic cells make laminin, a 1. 2 kb DNA fragment of leech laminin B1 chain, with homology to Drosophi la, human, and mouse B1 laminins and rat S laminin, was isolated using reverse-transcription and degenerate polymerase chain reaction (PCR) cloning. In situ hybridization revealed that laminin expression began before embryonic day 8, and by days 8 and 9 it was seen in paired CNS muscle cells. By late day 9, the two neuropil glial cells began to exp ress laminin. Lucifer Yellow dye was injected intracellularly and musc le cells stimulated to contract, confirming the identities of muscle a nd glial cells. Packet glial cells began to express B1 laminin by embr yonic day 12. By day 15, the cells of the perineurial sheath expressed B1 laminin, whereas it was no longer detectable in CNS muscle and gli a. The results agree with published immunohistochemistry showing lamin in within the CNS among growing axons by day 8, and only later in the perineurial sheath, by which time laminin disappears from within the C NS. Therefore, different cells synthesize laminin in the embryo and du ring repair in adults. (C) 1995 John Wiley & Sons, Inc.