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1.  Coordinate control of axon defasciculation and myelination by laminin-2 and -8 
The Journal of Cell Biology  2005;168(4):655-666.
Schwann cells form basal laminae (BLs) containing laminin-2 (Ln-2; heterotrimer α2β1γ1) and Ln-8 (α4β1γ1). Loss of Ln-2 in humans and mice carrying α2-chain mutations prevents developing Schwann cells from fully defasciculating axons, resulting in partial amyelination. The principal pathogenic mechanism is thought to derive from structural defects in Schwann cell BLs, which Ln-2 scaffolds. However, we found loss of Ln-8 caused partial amyelination in mice without affecting BL structure or Ln-2 levels. Combined Ln-2/Ln-8 deficiency caused nearly complete amyelination, revealing Ln-2 and -8 together have a dominant role in defasciculation, and that Ln-8 promotes myelination without BLs. Transgenic Ln-10 (α5β1γ1) expression also promoted myelination without BL formation. Rather than BL structure, we found Ln-2 and -8 were specifically required for the increased perinatal Schwann cell proliferation that attends myelination. Purified Ln-2 and -8 directly enhanced in vitro Schwann cell proliferation in collaboration with autocrine factors, suggesting Lns control the onset of myelination by modulating responses to mitogens in vivo.
PMCID: PMC2171752  PMID: 15699217
2.  Developmental and Pathogenic Mechanisms of Basement Membrane Assembly 
Current pharmaceutical design  2009;15(12):1277-1294.
Basement membranes are sheet-like cell-adherent extracellular matrices that serve as cell substrata and solid-phase agonists, contributing to tissue organization, stability and differentiation. These matrices are assembled as polymers of laminins and type IV collagens that are tethered to nidogens and proteoglycans. They bind to cell surface molecules that include signal-transducing receptors such as the integrins and dystroglycan and form attachments to adjacent connective tissues. The cell receptors, in turn, provide links between the matrix and underlying cytoskeleton. Genetic diseases of basement membrane and associated components, collectively the basement membrane zone, disrupt the extracellular matrix and/or its linkages to affect nerve, muscle, skin, kidney and other tissues. These diseases can arise due to a loss of matrix integrity, adhesion strength and/or receptor-mediated signaling. An understanding of the mechanisms of basement membrane zone assembly and resulting structure can provide insights into the development of normal tissues and the pathogenic mechanisms that underlie diverse disorders.
PMCID: PMC2978668  PMID: 19355968
Extracellular matrix; laminin; collagen; nidogen; perlecan; integrin; dystroglycan; agrin; muscular dystrophy; peripheral neuropathy; Alport’s syndrome; Goodpasture syndrome; epidermolysis bullosa; Pierson syndrome
3.  A Laminin-2, Dystroglycan, Utrophin Axis is Required for Compartmentalization and Elongation of Myelin Segments 
Animal and plant cells compartmentalize to perform morphogenetic functions. Compartmentalization of myelin-forming Schwann cells may favor elongation of myelin segments to the size required for efficient conduction of nerve impulses. Compartments in myelinated fibers were described by Ramon-y-Cajal and depend on periaxin, mutated in the hereditary neuropathy Charcot-Marie-Tooth 4F. Lack of periaxin in mice causes loss of compartments, formation of short myelin segments (internodes) and reduced nerve conduction velocity. How compartments are formed and maintained, and their relevance to human neuropathies is largely unknown. Here we show that formation of compartments around myelin is driven by the actin cytoskeleton, and maintained by actin and tubulin fences through linkage to the dystroglycan complex. Compartmentalization and establishment of correct internodal length requires the presence of glycosylated dystroglycan, utrophin and extracellular laminin-2/211. A neuropathic patient with reduced internodal length and nerve conduction velocity due to absence of laminin-2/211 (congenital muscular dystrophy 1A) also shows abnormal compartmentalization. These data link formation of compartments through a laminin2-dystroglycan-utrophin-actin axis to internodal length, and provide a common pathogenetic mechanism for two inherited human neuropathies. Other cell types may exploit dystroglycan complexes in similar fashions to create barriers and compartments.
PMCID: PMC2940832  PMID: 19321787
Cajal-bands; Schwann cell compartments; Dystroglycan complex; internodal length; myelin; inherited peripheral neuropathies
4.  Compositional Differences between Infant and Adult Human Corneal Basement Membranes 
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. In the infant DM, type IV collagen α1-α6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
The distribution of laminin γ3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
PMCID: PMC2151758  PMID: 17962449
5.  A Hypomorphic Mutation in the Mouse Laminin α5 Gene (Lama5) Causes Polycystic Kidney Disease 
Extracellular matrix abnormalities have been found in both human and animal models of polycystic kidney disease (PKD). We have produced a new mouse PKD model through insertion of a PGKneo cassette in an intron of the gene encoding laminin α5, a major tubular and glomerular basement membrane component important for glomerulogenesis and ureteric bud branching. Lama5neo represents a hypomorphic allele due to aberrant splicing. Lama5neo/neo mice exhibit PKD, proteinuria, and death from renal failure by 4 weeks of age. This contrasts with mice totally lacking laminin α5, which die in utero with multiple developmental defects. At 2 days of age, Lama5neo/neo mice exhibited mild proteinuria and microscopic cystic transformation. By 2 weeks, cysts were grossly apparent in cortex and medulla, involving both nephron and collecting duct segments. Tubular basement membranes appeared to form normally, and early cyst basement membranes showed normal ultrastructure but developed marked thickening as cysts enlarged. Overall, laminin α5 protein levels were severely reduced due to mRNA frameshift caused by exon skipping. This was accompanied by aberrant accumulation of laminin-332 (α3β3γ2; formerly called laminin-5) in some cysts, as also observed in human PKD. This constitutes the first evidence that a primary defect in an extracellular matrix component can cause PKD.
PMCID: PMC1482806  PMID: 16790509
BM, basement membrane; E, embryonic day; GBM, glomerular BM; P, postnatal day; PKD, polycystic kidney disease; PC1, polycystin-1
6.  Transgenic isolation of skeletal muscle and kidney defects in laminin beta2 mutant mice: Implications for Pierson syndrome 
Development (Cambridge, England)  2006;133(5):967-975.
Pierson syndrome is a recently defined disease usually lethal within the first postnatal months and caused by mutations in the gene encoding laminin β2 (LAMB2). The hallmarks of Pierson syndrome are congenital nephrotic syndrome accompanied by ocular abnormalities, including microcoria (small pupils), with muscular and neurological developmental defects also present. Lamb2−/− mice are a model for Pierson syndrome; they exhibit defects in the kidney glomerular barrier, in the development and organization of the neuromuscular junction, and in the retina. Lamb2−/− mice fail to thrive and die very small at 3 weeks of age, but to what extent the kidney and neuromuscular defects each contribute to this severe phenotype has been obscure, though highly relevant to understanding Pierson syndrome. To investigate this, we generated transgenic mouse lines expressing rat laminin β2 either in muscle or in glomerular epithelial cells (podocytes) and crossed them onto the Lamb2−/− background. Rat β2 was confined in skeletal muscle to synapses and myotendinous junctions, and in kidney to the glomerular basement membrane. In transgenic Lamb2−/− mice, β2 deposition only in glomeruli prevented proteinuria but did not ameliorate the severe phenotype. In contrast, β2 expression only in muscle restored synaptic architecture and led to greatly improved health, but the mice died from kidney disease at 1 month. Rescue of both glomeruli and synapses was associated with normal weight gain, fertility, and lifespan. We conclude that muscle defects in Lamb2−/− mice are responsible for the severe failure to thrive phenotype, and that renal replacement therapy alone will be an inadequate treatment for Pierson syndrome.
PMCID: PMC1363729  PMID: 16452099
7.  Distribution and Function of Laminins in the Neuromuscular System of Developing, Adult, and Mutant Mice  
The Journal of Cell Biology  1997;139(6):1507-1521.
Laminins, heterotrimers of α, β, and γ 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 α chains (α1, α2, α4, and α5), 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 α chains and two β chains, but each is regulated differently. Initially, the α2, α4, α5, and β1 chains are present both extrasynaptically and synaptically, whereas β2 is restricted to synaptic BL from its first appearance. As development proceeds, α2 remains broadly distributed, whereas α4 and α5 are lost from extrasynaptic BL and β1 from synaptic BL. In adults, α4 is restricted to primary synaptic clefts whereas α5 is present in both primary and secondary clefts. Thus, adult extrasynaptic BL is rich in laminin 2 (α2β1γ1), and synaptic BL contains laminins 4 (α2β2γ1), 9 (α4β2γ1), and 11 (α5β2γ1). Likewise, in cultured muscle cells, α2 and β1 are broadly distributed but α5 and β2 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: α2, β1, γ1, and α4, α5, β2, γ1, respectively. Mutation of the laminin α2 or β2 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 β2 from synaptic BL in β2−/− “knockout” mice is accompanied by loss of α5, and decreased levels of α2 in dystrophic α2dy/dy mice are accompanied by compensatory retention of α4. Finally, we show that motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (α1β1γ1) 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.
PMCID: PMC2132624  PMID: 9396756
8.  The Laminin α Chains: Expression, Developmental Transitions, and Chromosomal Locations of α1-5, Identification of Heterotrimeric Laminins 8–11, and Cloning of a Novel α3 Isoform 
The Journal of Cell Biology  1997;137(3):685-701.
Laminin trimers composed of α, β, and γ chains are major components of basal laminae (BLs) throughout the body. To date, three α chains (α1–3) have been shown to assemble into at least seven heterotrimers (called laminins 1–7). Genes encoding two additional α chains (α4 and α5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant α4 and α5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that α4 and α5 assemble into four novel laminin heterotrimers (laminins 8–11: α4β1γ1, α4β2γ1, α5β1γ1, and α5β2γ1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of α1-5 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, α4 and α5 exhibited the broadest patterns of expression, while expression of α1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the α chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one α chain, all α chains were present in multiple BLs, and some BLs contained two or three α 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 α chains and two different β chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five α chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length α3 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.
PMCID: PMC2139892  PMID: 9151674

Results 1-8 (8)