The renal glomerulus of mice lacking s-laminin/laminin beta 2: nephrosis despite molecular compensation by laminin beta 1.

S-laminin/laminin beta 2, a homologue of the widely distributed laminin B1/beta 1 chain, is a major component of adult renal glomerular basement membrane (GBM). Immature GBM bears beta 1, which is replaced by beta 2 as development proceeds. In mutant mice that lack beta 2, the GBM remains rich in beta 1, suggesting that a feedback mechanism normally regulates GBM maturation. The beta 2-deficient GBM is structurally intact and contains normal complements of several collagenous and noncollagenous glycoproteins. However, mutant mice develop massive proteinuria due to failure of the glomerular filtration barrier. These results support the idea that laminin beta chains are functionally distinct although they assemble to form similar structures. Laminin beta 2-deficient mice may provide a model for human congenital or idiopathic nephrotic syndromes.

Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome.

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

Role of fatty acid transport protein 4 in oleic acid-induced glucagon-like peptide-1 secretion from murine intestinal L cells.

The antidiabetic intestinal L cell hormone glucagon-like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion and inhibits gastric emptying. GLP-1 secretion is stimulated by luminal oleic acid (OA), which crosses the cell membrane by an unknown mechanism. We hypothesized that L cell fatty acid transport proteins (FATPs) are essential for OA-induced GLP-1 release. Therefore, the murine GLUTag L cell model was used for immunoblotting, [(3)H]OA uptake assay, and GLP-1 secretion assay as determined by radioimmunoassay following treatment with OA ± phloretin, sulfo-N-succinimidyl oleate, or siRNA against FATP4. FATP4(-/-) and cluster-of-differentiation 36 (CD36)(-/-) mice received intraileal OA, and plasma GLP-1 was measured by sandwich immunoassay. GLUTag cells were found to express CD36, FATP1, FATP3, and FATP4. The cells demonstrated specific (3)H[OA] uptake that was dose-dependently inhibited by 500 and 1,000 µM unlabeled OA (P < 0.001). Cell viability was not altered by treatment with OA. Phloretin and sulfo-N-succinimidyl oleate, inhibitors of protein-mediated transport and CD36, respectively, also decreased [(3)H]OA uptake, as did knockdown of FATP4 by siRNA transfection (P < 0.05-0.001). OA dose-dependently increased GLP-1 secretion at 500 and 1,000 µM (P < 0.001), whereas phloretin, sulfo-N-succinimidyl oleate, and FATP4 knockdown decreased this response (P < 0.05-0.01). FATP4(-/-) mice displayed lower plasma GLP-1 at 60 min in response to intraileal OA (P < 0.05), whereas, unexpectedly, CD36(-/-) mice displayed higher basal GLP-1 levels (P < 0.01) but a normal response to intraileal OA. Together, these findings demonstrate a key role for FATP4 in OA-induced GLP-1 secretion from the murine L cell in vitro and in vivo, whereas the precise role of CD36 remains unclear.

Mystery solved: discovery of a novel integrin ligand in the developing kidney.

Mutant mice lacking the integrin alpha8 subunit exhibit variable defects in kidney development with most mutants missing both kidneys. Several lines of evidence indicate that the known extracellular matrix ligands for integrin alpha8beta1 are either dispensable for or not involved in alpha8beta1 signaling during kidney development. This suggests the presence of an unknown ligand. A novel alpha8beta1 ligand, nephronectin, has now been identified. Nephronectin is a new extracellular matrix protein associated with the Wolffian duct and the ureteric bud, epithelial structures with well-defined roles in kidney development.

Molecular and functional defects in kidneys of mice lacking collagen alpha 3(IV): implications for Alport syndrome.

Collagen IV is a major structural component of all basal laminae (BLs). Six collagen IV alpha chains are present in mammals; alpha 1 and alpha 2(IV) are broadly expressed in embryos and adults, whereas alpha 3-6(IV) are restricted to a defined subset of BLs. In the glomerular BL of the kidney, the alpha 1 and alpha 2(IV) chains are replaced by the alpha 3-5(IV) chains as development proceeds. In humans, mutation of the collagen alpha 3, alpha 4, or alpha 5(IV) chain genes results in a delayed onset renal disease called Alport syndrome. We show here that mice lacking collagen alpha 3(IV) display a renal phenotype strikingly similar to Alport syndrome: decreased glomerular filtration (leading to uremia), compromised glomerular integrity (leading to proteinuria), structural changes in glomerular BL, and glomerulonephritis. Interestingly, numerous changes in the molecular composition of glomerular BL precede the onset of renal dysfunction; these include loss of collagens alpha 4 and alpha 5(IV), retention of collagen alpha 1/2(IV), appearance of fibronectin and collagen VI, and increased levels of perlecan. We suggest that these alterations contribute, along with loss of collagen IV isoforms per se, to renal pathology.

Roles for laminin in embryogenesis: exencephaly, syndactyly, and placentopathy in mice lacking the laminin alpha5 chain.

Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are alpha/beta/gamma heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development. Here, we show that the laminin alpha5 chain is required during embryogenesis. The alpha5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose alpha5 retain or acquire other alpha chains. Embryos lacking laminin alpha5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are alpha5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin alpha chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.

Collagen IV alpha 3, alpha 4, and alpha 5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches.

Collagen IV is a major component of vertebrate basal laminae (BLs). Studies in humans have revealed a family of genes encoding alpha 1-alpha 6 collagen IV chains and implicated alpha 3-alpha 6 in disease processes (Goodpasture and Alport syndromes and diffuse leiomyomatosis). To extend studies of these components to an experimentally accessible animal, we cloned cDNAs encoding partial collagen alpha 3, alpha 4, and alpha 5(IV) chains from the mouse. Ribonuclease protection assays showed that all three genes were expressed at highest levels in kidney and lung; alpha 5(IV) was also expressed at high levels in heart. We then made antibodies specific for each collagen IV chain. Immunohistochemical studies of several tissues revealed many combinations of collagen IV chains; however, alpha 3 and alpha 4 (IV) were always coexpressed, and only appeared in BLs that were alpha 5(IV) positive. The alpha 3-alpha 5(IV) chains were frequently but not exclusively associated with the S (beta 2) chain of laminin, as were the alpha 1, 2 (IV) collagen chains with laminin B1 (beta 1). An analysis of developing rat kidney BLs showed that newly formed (S-shaped) nephrons harbored collagen alpha 1 and alpha 2(IV) and laminin B1; maturing (capillary loop stage) BLs contained collagen alpha 1-alpha 5(IV) and laminin B1 and S-laminin; and mature glomerular BLs contained mainly collagen alpha 3-alpha 5(IV) and S-laminin. Thus, collagen alpha 1 and alpha 2(IV) and laminin B1 appear to be fetal components of the glomerular BL, and there is a developmental switch to collagen alpha 3-alpha 5(IV) and S-laminin expression.

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 (alpha1, alpha2, alpha4, and alpha5), 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 alpha2, alpha4, alpha5, and beta1 chains are present both extrasynaptically and synaptically, whereas beta2 is restricted to synaptic BL from its first appearance. As development proceeds, alpha2 remains broadly distributed, whereas alpha4 and alpha5 are lost from extrasynaptic BL and beta1 from synaptic BL. In adults, alpha4 is restricted to primary synaptic clefts whereas alpha5 is present in both primary and secondary clefts. Thus, adult extrasynaptic BL is rich in laminin 2 (alpha2beta1gamma1), and synaptic BL contains laminins 4 (alpha2beta2gamma1), 9 (alpha4beta2gamma1), and 11 (alpha5beta2gamma1). Likewise, in cultured muscle cells, alpha2 and beta1 are broadly distributed but alpha5 and beta2 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: alpha2, beta1, gamma1, and alpha4, alpha5, beta2, gamma1, respectively. Mutation of the laminin alpha2 or beta2 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 beta2 from synaptic BL in beta2(-/-) "knockout" mice is accompanied by loss of alpha5, and decreased levels of alpha2 in dystrophic alpha2(dy/dy) mice are accompanied by compensatory retention of alpha4. Finally, we show that motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (alpha1beta1gamma1) 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.

Cell elongation induces laminin alpha2 chain expression in mouse embryonic mesenchymal cells: role in visceral myogenesis.

Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) alpha2 chain not present in round mesenchymal cells. LM alpha2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM beta1 and gamma1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM alpha2. Dy/dy mice express very low levels of LM alpha2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM alpha-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM alpha2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.

The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Congenital nephrotic syndrome in mice lacking CD2-associated protein.

CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associated with nephrin, the primary component of the slit diaphragm.

c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation.

In vertebrate development, a prominent feature of several cell lineages is the coupling of cell cycle regulation with terminal differentiation. We have investigated the basis of this relationship in the skeletal muscle lineage by studying the effects of the proliferation-associated regulator, c-myc, on the differentiation of MyoD-initiated myoblasts. Transient cotransfection assays in NIH 3T3 cells using MyoD and c-myc expression vectors demonstrated c-myc suppression of MyoD-initiated differentiation. A stable cell system was also developed in which MyoD expression was constitutive, while myc levels could be elevated conditionally. Induction of this conditional c-myc suppressed myogenesis effectively, even in the presence of MyoD. c-myc suppression also prevented up-regulation of a relative of MyoD, myogenin, which is normally expressed at the onset of differentiation in all muscle cell lines examined and may be essential for differentiation. Additional experiments tested whether failure to differentiate in the presence of myc could be overcome by providing myogenin ectopically. Cotransfection of c-myc with myogenin, MyoD, or a mixture of myogenin and MyoD showed that neither myogenin alone nor myogenin plus MyoD together could bypass the c-myc block. The effects of c-myc were further dissected by showing that c-myc can inhibit differentiation independently of Id, a negative regulator of muscle differentiation. These results lead us to propose that c-myc and Id constitute independent negative regulators of muscle differentiation, while myogenin and any of the other three related myogenic factors (MyoD, Myf-5, and MRF4/herculin/Myf-6) act as positive regulators.

Overexpression of alpha4 chain-containing laminins in human glial tumors identified by gene microarray analysis.

Differential gene expression in tumors often involves growth factors and extracellular matrix/basement membrane components. Here, 11,000- gene microarray was used to identify gene expression profiles in brain tumors including high-grade gliomas [glioblastoma multiforme (GBM) and anaplastic astrocytoma], low-grade astrocytomas, or benign extra-axial brain tumors (meningioma) in comparison with normal brain tissue. Histologically normal tissues adjacent to GBMs were also studied. All GBMs studied overexpressed 14 known genes compared with normal human brain tissue. Overexpressed genes belonged to two broad groups: (a) growth factor-related genes; and (b) structural/extracellular matrix-related genes. For most of these 14 genes, expression levels were lower in low-grade astrocytoma than in GBM and were barely detectable in normal brain. Despite normal-appearing histology, gene expression patterns of tissues immediately adjacent to GBM were similar to those of their respective primary GBMs. Two genes were consistently up-regulated in both high-grade and low-grade gliomas, as well as in histologically normal tissues adjacent to GBMs. These genes coded for the epidermal growth factor receptor (previously reported to be overexpressed in gliomas) and for the alpha4 chain of laminin, a major blood vessel basement membrane component. Changes in expression of this laminin chain have not been previously associated with malignant tumors. Overexpression of laminin alpha4 chain in GBM and astrocytoma grade II by gene microarray analysis was confirmed by semiquantitive reverse transcription-PCR and immunohistochemistry. Importantly, an alpha4 chain-containing laminin isoform, laminin-8 (alpha4beta1gamma1), was expressed mainly in blood vessel walls of GBMs and histologically normal tissues adjacent to GBMs, whereas another alpha4 chain-containing laminin isoform, laminin-9 (alpha4beta2gamma1), was expressed mainly in blood vessel walls of low-grade tumors and normal brain. GBMs that overexpressed laminin-8 had a shorter mean time to tumor recurrence (4.3 months) than GBMs with overexpression of laminin-9 (9.7 months, P = 0.0007). Up-regulation of alpha4 chain-containing laminins could be important for the development of glioma-induced neovascularization and glial tumor progression. Overexpression of laminin-8 may be predictive of glioma recurrence.

The presynaptic calcium channel is part of a transmembrane complex linking a synaptic laminin (alpha4beta2gamma1) with non-erythroid spectrin.

Nerve regeneration studies at the neuromuscular junction (NMJ) suggest that synaptic basal lamina components tell the returning axon where to locate neurotransmitter release machinery, including synaptic vesicle clusters and active zones. Good candidates for these components are the synaptic laminins (LNs) containing alpha4, alpha5, or beta2 chains. Results from a beta2 laminin knockout mouse have suggested a linkage of this extracellular laminin to cytosolic synaptic vesicle clusters. Here we report such a transmembrane link at the electric organ synapse, which is homologous to the NMJ. We immunopurified electric organ synaptosomes and found on their surface two laminins of 740 and 900 kDa. The 740 kDa laminin has a composition of alpha4beta2gamma1 (laminin-9). Immunostaining reveals that as in the NMJ, alpha4 and beta2 chains are concentrated at the electric organ synapse. Using detergent-solubilized synaptosomes, we immunoprecipitated a complex containing alpha4beta2gamma1 laminin, the voltage-gated calcium channel, and the cytoskeletal protein spectrin. Other presynaptic proteins such as 900 kDa laminin are not found in this complex. We hypothesize that alpha4beta2gamma1 laminin in the synaptic basal lamina attaches to calcium channel, which in turn is attached to cytosolic spectrin. Spectrin could then organize synaptic vesicle clusters by binding vesicle-associated proteins.

Identification and expression of mouse netrin-4.

Netrins are secreted proteins that serve as potent axon guidance molecules in vertebrates and invertebrates. We report the identification of a novel mammalian member of this family. Netrin-4 is similar in predicted size and secondary structure to the other three netrins; all contain, in order, an amino-terminal signal sequence, a laminin-type globular domain of the 'VI' type, three laminin-type epidermal growth factor (EGF) repeats, and a carboxyl-terminal 'netrin module'. In terms of primary sequence, however, netrin-4 is a distant relative of netrins-1-3, and its globular domain is more closely related to those of laminins than to those of other netrins. Netrin-4 is broadly expressed in both neural and non-neural tissues of embryonic and adult mice. In embryonic spinal cord, it is selectively expressed by cells at the lateral margins of the floor plate. In postnatal brain, it is selectively expressed in subsets of neurons, including cerebellar granule and hippocampal pyramidal cells.

Laminin-11.

Laminins are a family of glycoproteins which are ubiquitous components of basement membranes and play key structural and functional roles. Eleven isoforms have been identified to date; each is an alpha beta gamma heterotrimer assembled from a repertoire of five alpha, three beta and two gamma chains. Studies of laminin-11 (alpha 5 beta 2 gamma 1) illustrate the unique expression patterns and distinct functions that can be displayed by laminin isoforms. Laminin-11 is found in the glomerular basement membrane in kidney, in the neuromuscular synaptic cleft in skeletal muscle and in other tissues such as placenta and lung. Mice lacking laminin-11 exhibit defective glomerular filtration and developmental defects in neuromuscular synapse formation, with Schwann cells invading the synaptic cleft. Consistent with these observations, both motoneurons and Schwann cells distinguish laminin-11 from other isoforms in vitro. These results suggest that laminin-11 is a structural component of the basement membrane which influences cell behavior in physiologically relevant ways. A greater understanding of laminin-11 assembly and basement membrane incorporation could provide a logical basis for therapy in merosin-deficient congenital muscular dystrophy.

The functions of laminins: lessons from in vivo studies.

This series of three short reviews is an attempt to summarize our current knowledge of the in vivo tests of hypotheses of laminin functions. The structures of the laminins have been thoroughly reviewed recently (P. Ekblom and R. Timpl, in press), and I will not attempt to repeat this information here. Instead, I will focus on the recent evidence gathered from gene knock out experiments in mice and from naturally occurring human and mouse gene mutations. The most obvious lesson from the above studies--other than demonstrating the importance of laminins in general--is that the structural diversity of the laminin family members makes highly specialized functions possible. While all laminins may share many functional properties, the individual chains are involved in interactions which cannot be substituted for by other laminins or by other basement membrane components. While this concept is not new, it is very satisfying to see its validity so dramatically confirmed. It is therefore predictable that additional gene ablation experiments using other known and yet undescribed laminin genes will be equally interesting and informative. To me, one of the most striking lessons from these studies is how strongly the induced mouse mutations mimic human disease. With all the concerns with genetic background differences and species specific effects, manipulation of the laminin genes appears to be a particularly good first approach to identifying the causes of human disease. There is an abundant literature accumulated from biochemical and, more recently, molecular structural analyses, and from in vitro systems, suggesting a role of laminins contributing directly to the stability of the basement membrane. There is an equally vast literature supporting an indirect role in mediating cellular behavior, through interactions with various receptors. It is interesting that the in vivo studies summarized above support both activities. In the case of laminin 5 mutations, the phenotypic consequence appears to be due primarily to the loss of an important structural link between the epithelial cytokeratins and the dermal anchoring fibrils. The ultrastructure of the epithelium appears normal, as does the architecture of the papillary dermis. Only the anchoring complex itself is aberrant. The absence of laminin 5 appears not to compromise the development or viability of the epidermis. The basement membrane appears normal-other than the anchoring complex itself. The pathology observed in the newborn is believed to be due to the frictional trauma of birth, with the expectation that the function of the fetal skin is normal in utero. The Herlitz epidermolysis bullosa phenotype is obvious immediately at birth, and it does not progress postnatally beyond the extent to which the affected individual experiences additional frictional trauma or secondary consequences such as infection or fluid loss. Since laminin 5 is only one of a series of structural links within the anchoring complex, one would predict that a loss of any of these links would result in the same phenotype. Current evidence supports this view, as the absence of integrin alpha 6 beta 4 (Vidal et al., 1995; Dowling et al., 1996; Georges-Labouesse et al., 1996; van der Neut et al., 1996) or of collagen VII (A. M. Christiano and J. Uitto, in press) also results in dramatic neonatal dermal-epidermal fragility. The differences in phenotype, such as the pyloric atresia in the case of loss of integrin alpha 6 beta 4, are presumably due to additional functions of the integrin in other tissues or in other developmental processes. Therefore, the laminin 5 mutations may be unique, in that the in vivo studies suggest that the primary role of the molecule is in the elaboration and stability of the anchoring complex, but not in the basement membrane itself. Of course, since the in vivo phenotype reflects only losses that cannot be compensated, this interpretation may be much too narrow. (ABSTRACT TRUNCATED)

Distribution of the ten known laminin chains in the pathways and targets of developing sensory axons.

Laminins are heterotrimers of alpha, beta, and gamma chains. At present, five alpha, three beta, and two gamma chains have been described. The best characterized laminin (laminin 1 = alpha 1, beta 1, gamma 1) promotes neurite outgrowth from virtually all classes of developing neurons, implying that laminins may serve as axon guidance molecules in vivo. Moreover, different laminin trimers exert distinct effects on subsets of laminin-1-responsive cells, suggesting that isoform diversity may underlie some axonal choices in vivo. As a first step toward evaluating these hypotheses, we have documented the expression patterns of all 10-known laminin chains in the peripheral nervous system and spinal cord of the murine embryo. The alpha 2, alpha 4, beta 1, and gamma 1 chains are expressed in peripheral axonal pathways by embryonic day (E) 11.5, when sensory and motor axonal outgrowth is underway. Thus, laminins (but not laminin 1) may promote peripheral axonal outgrowth. By E 13.5, laminin chains are differentially expressed in the limb-bud, with prominent expression of alpha 2 and alpha 4 in muscle and of alpha 3 and alpha 5 in skin. This pattern raises the possibility that laminin isoform diversity contributes to the ability of cutaneous and muscle sensory axons to distinguish their targets. Later in development, some chains (e.g., alpha 2, alpha 4, and beta 1) are downregulated in peripheral nerve while others (e.g., gamma 1), continue to be expressed by Schwann cells into adulthood. In contrast to peripheral nerves and ganglia, laminin chains are expressed at low levels, if at all, in the developing spinal cord gray matter.

Distribution of ten laminin chains in dystrophic and regenerating muscles.

Using immunohistochemical methods, we assessed the distribution of all 10 known laminin chains (alpha1-5, beta1-3, gamma1 and gamma2) in skeletal muscles from patients with Duchenne, congenital, limb girdle, or Emery-Dreifuss muscular dystrophies. The alpha2, beta1 and gamma1 chains were abundant in the basal lamina surrounding muscle fibers in normal controls; alpha1, alpha3-alpha5, beta3, and gamma2 were undetectable; and beta2 was present at a low level. Compared to controls, levels of the alpha5 chain were increased in muscles from many dystrophic patients; levels of beta1 were reduced and/or levels of beta2 were increased in a minority. However, these changes were neither specific for, nor consistent within, diagnostic categories. In contrast, levels of alpha4 were increased in muscles from all patients with alpha2 laminin (merosin)-deficient congenital muscular dystrophy. Loss of alpha2 laminin in congenital dystrophy is disease-specific but some other changes in laminin isoform expression in dystrophic muscles could be secondary consequences of myopathy, denervation, regeneration or immaturity. To distinguish among these possibilities, we compared the laminins of embryonic, denervated, regenerating, and mutant mouse muscles with those in normal adult muscle. Embryonic muscle basal lamina contained alpha4 and alpha5 along with alpha2, and regenerating muscle re-expressed alpha5 but not alpha4. Levels of alpha5 but not alpha4 were increased in dystrophin (mdx) mutants and in dystrophin/utrophin double mutants (mdx:utrn -/-), models for Duchenne dystrophy. In contrast, laminin alpha4 was upregulated more than alpha5 in muscles of laminin alpha2 mutant mice (dy/dy; a model for alpha2-deficient congenital dystrophy). Based on these results, we hypothesize that the expression of alpha5 in many dystrophies reflects the regenerative process, whereas the selective expression of alpha4 in alpha2-deficient muscle is a specific compensatory response to loss of alpha2.

Human corneal epithelial basement membrane and integrin alterations in diabetes and diabetic retinopathy.

Corneas of diabetic patients have abnormal healing and epithelial adhesion, which may be due to alterations of the corneal extracellular matrix (ECM) and basement membrane (BM). To identify such alterations, various ECM and BM components and integrin receptors were studied by immunofluorescence on sections of normal and diabetic human corneas. Age-matched corneas from 15 normal subjects, 10 diabetics without diabetic retinopathy (DR), and 12 diabetics with DR were used. In DR corneas, the composition of the central epithelial BM was markedly altered, compared to normal or non-DR diabetic corneas. In most cases the staining for entactin/nidogen and for chains of laminin-1 (alpha1beta1gamma1) and laminin-10 (alpha5beta1gamma1 was very weak, discontinuous, or absent over large areas. Other BM components displayed less frequent changes. The staining for alpha3beta1 (VLA-3) laminin binding integrin was also weak and discontinuous in DR corneal epithelium. Components of stromal ECM remained unchanged even in DR corneas. Therefore, distinct changes were identified in the composition of the epithelial BM in DR corneas. They may be due to increased degradation or decreased synthesis of BM components and related integrins. These alterations may directly contribute to the epithelial adhesion and wound healing abnormalities found in diabetic corneas.