Essential and overlapping roles for laminin alpha chains in notochord and blood vessel formation

Laminins are major constituents of basement membranes and have wide ranging functions during development and in the adult. They are a family of heterotrimeric molecules created through association of an alpha, beta and gamma chain. We previously reported that two zebrafish loci, grumpy (gup) and sleepy (sly), encode laminin beta1 and gamma1, which are important both for notochord differentiation and for proper intersegmental blood vessel (ISV) formation. In this study we show that bashful (bal) encodes laminin alpha1 (lama1). Although the strongest allele, bal(m190), is fully penetrant, when compared to gup or sly mutant embryos, bal mutants are not as severely affected, as only anterior notochord fails to differentiate and ISVs are unaffected. This suggests that other alpha chains, and hence other isoforms, act redundantly to laminin 1 in posterior notochord and ISV development. We identified cDNA sequences for lama2, lama4 and lama5 and disrupted the expression of each alone or in mutant embryos also lacking laminin alpha1. When expression of laminin alpha4 and laminin alpha1 are simultaneously disrupted, notochord differentiation and ISVs are as severely affected as sly or gup mutants. Moreover, live imaging of transgenic embryos expressing enhanced green fluorescent protein in forming ISVs reveals that the vascular defects in these embryos are due to an inability of ISV sprouts to migrate correctly along the intersegmental, normally laminin-rich regions.     

Differential expression of laminin chains in the human major salivary gland

Laminin represents a macromolecule family. The heterotrimeric isoforms of laminin can be determined by immunohistochemical demonstration of the single chains. The laminin chain heterogeneity of the basement membrane in adult human major salivary glands was evaluated in relation to cellular differentiation of the epithelia and the stromal compartment. Monoclonal antibodies to the laminin alpha1, alpha3 (BM165) chains and epiligrin reacted with the basement membranes of serous and mucous acini and of intercalated, striated and excretory ducts. As evidenced by a double-labelling technique, the alpha2 chain showed a spatial association with the myoepithelium of the acini, whereas the ductal basement membranes containing no myoepithelial cells were negative. Almost exclusively, beta1 chain was detected in acinar basement membrane, beta2 chain whereas in ductal basement membrane. gamma2 chain is a unique chain belonging to the laminin-5 isoform. It was restricted to the ductal basement membrane. alpha1, alpha2, beta1, beta2 and gamma1 chains were detected in nerves of salivary tissue and alpha1, alpha3, beta1, beta2 and gamma1 chains and epiligrin in blood vessels. Our results indicate that the acinar ductal unit contains basement membranes with different isoforms, which relate to cell differentiation and cell function. © 1998 Chapman & Hall     

Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

Laminins are components of all basement membranes and have well demonstrated roles in diverse developmental processes, from the peri-implantation period onwards. Laminin 1 (alpha1beta1gamma1) is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes. The laminin gamma1 chain has been shown by targeted mutation to be required for endodermal differentiation and formation of basement membranes; Lamc1(-/-) embryos die within a day of implantation. We report the generation of mice lacking laminin alpha1 and laminin beta1, the remaining two laminin 1 chains. Mutagenic insertions in both Lama1 and Lamb1 were obtained in a secretory gene trap screen. Lamb1(-/-) embryos are similar to Lamc1(-/-) embryos in that they lack basement membranes and do not survive beyond embryonic day (E) 5.5. However, in Lama1(-/-) embryos, the embryonic basement membrane forms, the embryonic ectoderm cavitates and the parietal endoderm differentiates, apparently because laminin 10 (alpha5beta1gamma1) partially compensates for the absent laminin 1. However, such compensation did not occur for Reichert's membrane, which was absent, and the embryos died by E7. Overexpression of laminin alpha5 from a transgene improved the phenotype of Lama1(-/-) embryos to the point that they initiated gastrulation, but this overexpression did not rescue Reichert's membrane, and trophoblast cells did not form blood sinuses. These data suggest that both the molecular composition and the integrity of basement membranes are crucial for early developmental events.     

Laminin alpha5 chain is required for intestinal smooth muscle development

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.     

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.     

Laminin alpha5-chain is expressed early and widely during the development of the anterior segment of rat eye

The distribution of a novel laminin alpha5-chain in the basement membranes of the anterior segment of rat eye was studied. Frozen sections of embryonic day (E)16--17, post-natal day (P)2, 5, 10, 15 and 30 and adult rat eyes were immunostained for laminin chains alpha2, alpha5, beta1, beta2 and gamma1 and for laminin-5, as well as for EHS-laminin, to visualize all basement membranes. Laminin alpha5-, beta1- and gamma1-chain immunoreactivities were found in the basement membranes of the inner and outer layers of optic cup, lens epithelium, further corneal epithelium and skin of the eyelids in E16--17 rat eyes. In P2 and older rat eyes, laminin alpha5-, beta1- and gamma1-chains were all seen in the basement membranes of the corneal and conjunctival epithelium, Descemet's membrane, lens epithelium, ciliary processes, blood vessels and skin of the eyelids. There was a change in the expression pattern of laminin alpha5, beta1- and gamma1-chains in Descemet's membrane from the endothelial side of the membrane (P2--P15 eyes) to both sides of the membrane after P30. Immunoreactivity for laminin-5 was weak in the basement membrane of E16--17 epidermis, but strong in the basement membrane of corneal, conjunctival and eyelid epithelium in P2 and older rat eyes. Laminin alpha2- and beta2-chains were seen in conjunctival and uveal blood vessels in P15 and older rat eyes. The laminin beta2-chain emerged into the basement membrane of conjunctival epithelium in P30 and older rat eyes, suggesting a role for the laminin beta2-chain in the maturation of conjunctiva. The results suggest that laminin alpha5-chain, possibly in laminin-10 (alpha5beta1gamma1), is early and widely expressed in the basement membranes of developing and adult rat eye and, further, that laminin alpha5-chain is a major laminin alpha-chain, partly in coexpression with the alpha3-chain of laminin-5 in the basement membranes of the anterior segment of the eye in developing and adult rats. © 1998 Chapman & Hall     

Matrix assembly,regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation

Laminin-1 is essential for early embryonic basement membrane assembly and differentiation. Several steps can be distinguished, i.e., the expression of laminin and companion matrix components, their accumulation on the cell surface and assembly into basement membrane between endoderm and inner cell mass, and the ensuing differentiation of epiblast. In this study, we used differentiating embryoid bodies derived from mouse embryonic stem cells null for gamma1-laminin, beta1-integrin and alpha/beta-dystroglycan to dissect the contributions of laminin domains and interacting receptors to this process. We found that (a) laminin enables beta1-integrin-null embryoid bodies to assemble basement membrane and achieve epiblast with beta1-integrin enabling expression of the laminin alpha1 subunit; (b) basement membrane assembly and differentiation require laminin polymerization in conjunction with cell anchorage, the latter critically dependent upon a heparin-binding locus within LG module-4; (c) dystroglycan is not uniquely required for basement membrane assembly or initial differentiation; (d) dystroglycan and integrin cooperate to sustain survival of the epiblast and regulate laminin expression; and (e) laminin, acting via beta1-integrin through LG1-3 and requiring polymerization, can regulate dystroglycan expression.     

Molecular analysis of laminin N-terminal domains mediating self-interactions

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 alpha, beta, and gamma chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five alpha, three beta, and three gamma 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 alpha chains and from the beta2 and beta3 subunits. The gamma chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the gamma3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca2+ 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.     

Immunohistochemical distribution of laminin-5 gamma2 chain and its developmental change in human embryonic and foetal tissues

Immunohistochemical distribution of laminin gamma2 chain, a subunit of the basement membrane protein laminin-5, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin gamma2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5-6 weeks of gestation. Between 6-7 and 12-13 weeks, laminin gamma2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin gamma2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin gamma2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin gamma2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin-5 and some specific roles of its gamma2 chain in the morphogenesis of human tissues.     

Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens

Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin gamma1 chain (gamma1III4) is important for kidney development and for nerve guidance. Other proteins may also bind gamma1III4, although human nidogen-2 binds poorly to the mouse laminin gamma1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and gamma1III3-5, which both contain the gamma1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial-mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin gamma1 chain is dependent on epithelial-mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by alpha3beta1 and alpha6beta1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized.     

Laminin isoforms and lung development: all isoforms are not equal

Laminins are a major component of basement membranes. Each laminin molecule is a heterotrimeric glycoprotein composed of one alpha, one beta, and one gamma chain. Fifteen laminin isoforms exist, assembled from various combinations of 5alpha, 3beta, and 3gamma chains. The embryonic lung has abundant laminin isoforms. Increasing evidence suggests that different laminin isoforms have unique functions in lung development. Studies of embryonic lung explants and organotypic co-cultures show that laminin alpha1 and laminin 111 are important for epithelial branching morphogenesis and that laminin alpha2 and laminin 211 have a role in smooth muscle cell differentiation. In vivo studies of laminin alpha5-deficient mice indicate that this laminin chain, found in laminins 511 and 521, is essential for normal lobar septation in early lung development and normal alveolization and distal epithelial cell differentiation and maturation in late lung development. However, not all of the laminin chains present in the developing lung appear to be necessary for normal lung development since laminin alpha4 null mice do not have obvious lung abnormalities and laminin gamma2 null mice have only minimal changes in lung development. The mechanisms responsible for the lung phenotypes in mice with laminin mutations are unknown, but it is clear that multiple laminin isoforms are crucial for lung development and that different laminin isoforms exhibit specific, non-overlapping functions.     

Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes

Branching epithelial morphogenesis requires interactions between the surrounding mesenchyme and the epithelium, as well as interactions between basement membrane components and the epithelium. Embryonic submandibular gland was used to study the roles of two mesenchymal proteins, epimorphin and tenascin-C, as well as the epithelial protein laminin-1 and one of its integrin receptors on branching morphogenesis. Laminin-1 is a heterotrimer composed of an alpha 1 chain and two smaller chains (beta 1 and gamma 1). Immunofluorescence revealed a transient expression of laminin alpha 1 chain in the epithelial basement membrane during early stages of branching morphogenesis. Other laminin-1 chains and alpha 6, beta 1, and beta 4 integrin subunits seemed to be expressed constitutively. Expression of epimorphin, but not tenascin-C, was seen in the mesenchyme during early developmental stages, but a mAb against epimorphin did not perturb branching morphogenesis of this early epithelium. In contrast, inhibition of branching morphogenesis was seen with a mAb against the carboxy terminus of laminin alpha 1 chain, the E3 domain. An inhibition of branching was also seen with a mAb against the integrin alpha 6 subunit. The antibodies against laminin alpha 1 chain and integrin alpha 6 subunit perturbed development in distinct fashions. Whereas treatment with the anti-E3 resulted in discontinuities of the basement membrane at the tips of the branching epithelium, treatment with the mAb against alpha 6 integrin subunit seemed to leave the basement membrane intact. We suggest that the laminin E3 domain is involved in basement membrane formation, whereas alpha 6 beta 1 integrin binding to laminin-1 may elicit differentiation signals to the epithelial cells.     

Laminin gamma3 chain binds to nidogen and is located in murine basement membranes

Recently a novel laminin gamma3 chain was identified in mouse and human and shown to have the same modular structure as the laminin gamma1 chain. We expressed two fragments of the gamma3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3-5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The gamma3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the beta2 VI/V fragment. The gamma3 III3-5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the gamma1 III3-5 fragment. These data suggested that laminins containing the gamma3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against gamma3 VI/V and gamma3 III3-5 showed no cross-reaction with homologous fragments from the gamma1 and gamma2 chains of laminin and allowed the establishment of gamma chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20-100-fold lower content of the gamma3 chain compared with the gamma1 chain in various tissue extracts of adult mice. The expression of gamma3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the gamma3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the gamma3 chain to basement membranes of these tissues.     

Defective glomerulogenesis in the absence of laminin alpha5 demonstrates a developmental role for the kidney glomerular basement membrane

Laminins are major components of all basement membranes. They are a diverse group of alpha/beta/gamma heterotrimers formed from five alpha, three beta, and three gamma chains. Laminin alpha5 is a widely expressed chain found in many embryonic and adult basement membranes. During embryogenesis, alpha5 has a role in disparate developmental processes, including neural tube closure, digit septation, and placentation. Here, we analyzed kidney development in Lama5 mutant embryos and found a striking defect in glomerulogenesis associated with an abnormal glomerular basement membrane (GBM). This correlates with failure of the developmental switch in laminin alpha chain deposition in which alpha5 replaces alpha1 in the GBM at the capillary loop stage of glomerulogenesis. In the absence of a normal GBM, glomerular epithelial cells were in disarray, and endothelial and mesangial cells were extruded from within the constricting glomerulus, leading to a complete absence of vascularized glomeruli. In addition, a minority of Lama5 mutant mice lacked one or both kidneys, indicating that laminin alpha5 is also important in earlier kidney development. Our results demonstrate a dual role for laminin alpha5 in kidney development, illustrate a novel defect in glomerulogenesis, and indicate a heretofore unappreciated developmental role for the GBM in influencing the behavior of epithelial and endothelial cells.     

Bone morphogenetic protein 1 is an extracellular processing enzyme of the laminin 5 gamma 2 chain

Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (alpha3beta3gamma2) within the alpha3 and gamma2 chains (). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first alpha3 chain cleavage (200-l65 kDa alpha3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa alpha3) occurs within the lIla domain, 11 residues N-terminal to the start of domain II. The gamma chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the gamma2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (). Recombinant BMP-1 cleaves gamma2 in vitro, both within intact laminin 5 and at the predicted site of a recombinant gamma2 short arm. alpha3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin alpha3 and gamma2 chains to be substrates for BMP-1 in vitro. We speculate that gamma2 cleavage is required for formation of the laminin 5-6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermal-epidermal junction basement membrane in vivo.     

Expression and biological role of laminin-1.

Of the approximately 15 laminin trimers described in mammals, laminin-1 expression seems to be largely limited to epithelial basement membranes. It appears early during epithelial morphogenesis in most tissues of the embryo, and remains present as a major epithelial laminin in some adult tissues. Previous organ culture studies with embryonic tissues have suggested that laminin-1 is important for epithelial development. Recent data using genetically manipulated embryonic stem (ES) cells grown as embryoid bodies provide strong support for the view of a specific role of laminin-1 in epithelial morphogenesis. One common consequence of genetic ablation of FGF signaling, beta1-integrin or laminin gamma1 chain expression in ES cells is the absence of laminin-1, which correlates with failure of BM assembly and epiblast differentiation. Partial but distinct rescue of epiblast differentiation has been achieved in all three mutants by exogenously added laminin-1. Laminin-1 contains several biologically active modules, but several are found in beta1 or gamma1 chains shared by at least 11 laminins. However, the carboxytermini of the alpha chains contain five laminin globular (LG) modules, distinct for each alpha chain. There is increasing evidence for a particular role of alpha1LG4 binding to its receptors for epithelial tubulogenesis. The biological roles of this and other domains of laminin-1 are currently being explored by genetic means. The pathways controlling laminin-1 synthesis have remained largely unknown, but recent advances raise the possibility that laminin-1 and collagen IV synthesis can be regulated by pro-survival kinases of the protein kinase B/Akt family.     

Generation of a conditionally null allele of the laminin alpha1 gene

Laminins are heterotrimeric glycoproteins of the basement membranes. Laminin 1 (alpha1, beta1, gamma1) is the major laminin expressed during early mouse embryogenesis. To gain access to the physiological function of laminin alpha1 chain, we developed a conditionally null allele of its encoding gene (Lama1) using the cre/loxP system. Floxed-allele-carrying mice (Lama1(flox/flox)) display no overt phenotype. Lama1(flox/flox) mice were crossed with transgenic deleter mice (CMV-Cre) to generate Lama1-deficient mice (Lama1(Delta/Delta)). Lama1(Delta/Delta) embryos die during the early postimplantation period after embryonic day 6.5. They lack Reichert's membrane, an extraembryonic basement membrane in which laminin alpha1 is normally highly expressed. In parallel, Lama1(Delta/Delta) embryos display 1) parietal and visceral endoderm differentiation defects with altered expression of cytokeratin 19 and GATA4, respectively, and 2) an induction of apoptosis. This new mouse model is of particular interest as it will allow time- and tissue-specific inactivation of the Lama1 gene in various organs.     

Laminins in normal,keratoconus, bullous keratopathy and scarred human corneas

The laminin composition (LMalpha1-alpha5, beta1-beta3, gamma1 and gamma2 chains) of normal corneas and corneal buttons from keratoconus, bullous keratopathy (BKP), Fuchs' dystrophy + BKP, Fuchs' dystrophy without BKP and scar after deep lamellar keratoplasty (DLKP) was investigated with immunohistochemistry. The epithelial basement membranes (BMs) of both normal and diseased corneas contained LMalpha3, alpha5, beta1, beta3, gamma1 and gamma2 chains. The epithelial BM morphology was altered in the different diseases. Scarring was associated with irregular BM and ectopic stromal localization of different laminin chains. The Descemet's membrane (DM) contained LMalpha5, beta1 and gamma1 chains in all cases and additionally LMbeta3 and gamma2 chains in the majority of keratoconus corneas. The interface in the DLKP cornea had patches of LMalpha3, alpha4, alpha5, beta1 and beta2 chains, and an extra BM-like structure under the Bowman's membrane. These results suggest that laminin chains participate in the process of corneal scarring and in the pathogenesis of some corneal diseases. The novel finding of LMalpha3, beta3 and gamma2 in the DM of keratoconus buttons indicates that this membrane is also involved in the disease and that some cases of keratoconus may have a congenital origin, without normal downregulation of the LMbeta3 chain.     

Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood-brain barrier in experimental autoimmune encephalomyelitis

An active involvement of blood-brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE).Endothelial basement membranes contained laminin 8 (alpha4beta1gamma1) and/or 10 (alpha5beta1gamma1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin alpha6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells.