The LAMC1 gene coding for the laminin γ1 subunit was targeted by homologous recombination in mouse embryonic stem cells. Mice heterozygous for the mutation had a normal phenotype and were fertile, whereas homozygous mutant embryos did not survive beyond day 5.5 post coitum. These embryos lacked basement membranes and although the blastocysts had expanded, primitive endoderm cells remained in the inner cell mass, and the parietal yolk sac did not develop. Cultured embryonic stem cells appeared normal after targeting both LAMC1 genes, but the embryoid bodies derived from them also lacked basement membranes, having disorganized extracellular deposits of the basement membrane proteins collagen IV and perlecan, and the cells failed to differentiate into stable myotubes. Secretion of the linking protein nidogen and a truncated laminin α1 subunit did occur, but these were not deposited in the extracellular matrix. These results show that the laminin γ1 subunit is necessary for laminin assembly and that laminin is in turn essential for the organization of other basement membrane components in vivo and in vitro. Surprisingly, basement membranes are not necessary for the formation of the first epithelium to develop during embryogenesis, but first become required for extra embryonic endoderm differentiation.

We have isolated the novel gene SMOC-1 that encodes a secreted modular protein containing an EF-hand calcium-binding domain homologous to that in BM-40. It further consists of two thyroglobulin-like domains, a follistatin-like domain and a novel domain. Recombinant expression in human cells showed that SMOC-1 is a glycoprotein with a calcium-dependent conformation. Results from Northern blots, reverse transcriptase-PCR, and immunoblots revealed a widespread expression in many tissues. Immunofluorescence studies with an antiserum directed against recombinant human SMOC-1 demonstrated a basement membrane localization of the protein and additionally its presence in other extracellular matrices. Immunogold electron microscopy confirmed the localization of SMOC-1 within basement membranes in kidney and skeletal muscle as well as its expression in the zona pellucida surrounding the oocyte.

The ability of laminins to self-polymerize is crucial for the formation of basement membranes. Development of this polymerized network has profound effects upon tissue architecture as well as on the intracellular organization and differentiation of neighboring cells. The laminin N-terminal (LN) domains have been shown to mediate this interaction and studies using proteolytic fragments derived from laminin-1 led to the theory that network assembly depends on the formation of a heterotrimeric complex between LN domains derived from ␣, ␤, and ␥ chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five ␣, three ␤, and three ␥ chains, of which some are truncated and lack the N-terminal LN domain. To address whether the model of heterotrimeric complex formation is applicable to laminin isoforms other than laminin-1, eight LN domains found in the laminin protein family were recombinantly expressed and tested in three different assays for homologous and heterologous interactions. The results showed that the lack of homologous interactions is an exception, with such interactions being seen for LN domains derived from all ␣ chains and from the ␤ 2 and ␤ 3 subunits. The ␥ chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the ␥ 3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca 2؉ ions, with EDTA reversibly abrogating binding, EDTA-induced conformational changes were not reversible. Together these results demonstrate that the assembly model proposed on the basis of laminin-1 may be a simplification, with the assembly of naturally occurring laminin networks being far more complex and highly dependent upon which laminin isoforms are present.Basement membranes are specialized extracellular matrices found underlying all epithelia and endothelia as well as surrounding many types of mesenchymal cells. Laminins constitute the major noncollagenous protein component within the basement membrane and are crucial for its formation (1, 2). Through their interactions with specific receptors, especially members of the ␤ 1 integrin family and ␣-dystroglycan, they induce many cellular effects, including differentiation and cellular and axonal migration. The prototype, laminin-1, isolated from embryonic carcinoma cells (3) or the Engelbreth-HolmSwarm tumor (4) has been shown to belong to a family consisting of 15 members (for nomenclature, see Refs. 5-7). Laminins are multidomain heterotrimers formed by the combination of one ␣, one ␤, and one ␥ chain. Laminin-1 is an 800-kDa glycoprotein composed of a 400-kDa ␣ 1 chain and ␤ 1 and ␥ 1 chains, each of 200 kDa. It has a cross structure with one long and three short arms, the latter being formed from the three free N-terminal ends of the ␣, ␤, and ␥ chains (8). These parts of the ␤ and ␥ chains each contain two globular domains, designated IV and VI, whereas there are three...