CLONING AND BIOLOGICAL FUNCTION OF LAMININ IN HYDRA-VULGARIS

The Cnidarian, hydra, lends itself to studies related to the role of e xtracellular matrix (ECM) components in development because of its hig h regenerative capacity and its simple structure, which is organized a s an epithelial bilayer with an intervening ECM termed the mesoglea. P revious immunocytochemical and biochemical studies have established th at hydra mesoglea contains many of the major matrix components (e.g., fibronectin, laminin, type IV collagen, and heparan sulfate proteoglyc an) associated with the ECM of vertebrate and more complex invertebrat e species. Additional studies have also established that ECM component s have a critical role in hydra development as monitored during head r egeneration and morphogenesis of hydra cell aggregates. In the present study a monoclonal antibody (mAb52) raised to isolated hydra mesoglea was used as a probe in additional functional studies and to screen a cDNA expression library made from poly(A)(+) RNA isolated from Hydra v ulgaris. Immunofluorescent analysis indicated that mAb52 was localized along the entire longitudinal axis of adult polyps in what is termed the subepithelial zones of hydra mesoglea. Cytochemical studies found these subepithelial zones to be rich in anionic sites. Previous studie s have shown that mAb52 blocks hydra cell aggregate development and ex periments in the current study have shown that mAb52 also blocks in vi vo interstitial cell (I-cell) migration in hydra grafts. Sequence anal ysis of cDNA clones isolated using mAb52 indicated that the protein en coded by these clones had structural homology with mammalian and Droso phila laminin B1 chain and hybridized to a single 6.75-kb band on Nort hern blots of total hydra RNA. One interesting difference in hydra lam inin B1 was the presence of a FTGTQ amino acid sequence in place of th e vertebrate YIGSR cell binding domain. Under nonreducing conditions, polyclonal antibodies against FTGTQ bound to the same >200-kDa band on Western blots of mesoglea as mAb52 and also immunolocalized to the su bepithelial zones. Under reducing conditions, anti-FTGTQ antibodies bo und to a single band with a mass of approximately 200 kDa. In addition , FTGTQ peptide inhibited adhesion of dissociated hydra cells to mesog lea and anti-FTCTQ antibodies inhibited hydra cell binding to substrat es coated with mesoglea or FTGTQ peptide. Anti-FTGTQ antibodies also i nhibited in vivo I-cell migration in hydra grafts. Given the early div ergence of Cnidarians during evolution, these studies indicate the hig hly conserved nature of laminin and provide additional information reg arding the critical role of ECM components during hydra development. ( C) 1994 Academic Press, Inc.