Defective formation of the inner limiting membrane in laminin beta2- and gamma3-null mice produces retinal dysplasia.
ABSTRACT: Retinal basement membranes (BMs) serve as attachment sites for retinal pigment epithelial cells on Bruch's membrane and Müller cells (MCs) on the inner limiting membrane (ILM), providing polarity cues to adherent cells. The beta2 and gamma3 chains of laminin are key components of retinal BMs throughout development, suggesting that they play key roles in retinal histogenesis. This study was conducted to analyze how the absence of both beta2- and gamma3-containing laminins affects retinal development. Methods. The function of the beta2- and gamma3-containing laminins was tested by producing a compound deletion of both the beta2 and the gamma3 laminin genes in the mouse and assaying the effect on postnatal retinal development by using anatomic and electrophysiological techniques. Results. Despite the widespread expression of beta2 and gamma3 laminin chains in wild-type (WT) retinal BMs, the development of only one, the ILM, was disrupted. The postnatal consequence of the ILM disruption was an alteration of MC attachment and a resultant disruption in MC apical-basal polarity, which culminated in retinal dysplasia. Of importance, although their density was altered, retinal cell fates were unaffected. The laminin mutants have a markedly decreased visual function, resulting in part from photoreceptor dysgenesis. Conclusions. These data suggest that beta2 and gamma3 laminin isoforms are critical for the formation and stability of the ILM. These data also suggest that attachment of the MC to the ILM provides important polarity cues to the MC and for postnatal retinal histogenesis.
Project description:Laminins are heterotrimeric glycoproteins of the extracellular matrix. Eleven different laminin chains have been identified in vertebrates. They are ubiquitously expressed in the human body, with a distinct tissue distribution. Laminin expression in neural retina and their functional role during human retinogenesis is still unknown. This study investigated the laminin expression in human developing and adult retina, showing laminin ?1, ?5, ?1, ?2 and ?1 to be predominantly expressed in Bruch's membrane and the inner limiting membrane. Laminin-332 and laminin ?3 expression were mainly observed in the neural retina during retinal histogenesis. These expression patterns were largely conserved in pluripotent stem cell-derived retinal organoids. Blocking of laminin ?3 function in retinal organoids resulted in the disruption of laminar organisation and synapse formation, the loss of photoreceptor organisation and retinal ganglion cells. Our data demonstrate a unique temporal and spatial expression for laminins and reveal a novel role for laminin ?3 during human retinogenesis.
Project description:Laminins are the major cell adhesive proteins in basement membranes, and consist of three subunits termed alpha, beta, and gamma. Recently, we found that the Glu residue at the third position from the C termini of the gamma1 and gamma2 chains is critically involved in integrin binding by laminins. However, the gamma3 chain lacks this Glu residue, suggesting that laminin isoforms containing the gamma3 chain may be unable to bind to integrins. To address this possibility, we expressed the E8 fragment of laminin-213 and found that it was incapable of binding to integrins. Similarly, the E8 fragment of laminin-113 was expressed and also found to be inactive in binding to integrins, confirming the distinction between the integrin binding activities of gamma3 chain-containing isoforms and those containing the gamma1 or gamma2 chain. To further address the importance of the Glu residue, we swapped the C-terminal four amino acids of the gamma3 chain with the C-terminal nine amino acids of the gamma1 chain, which contain the Glu residue. The resulting chimeric E8 fragment of laminin-213 became fully active in integrin binding, whereas replacement with the nine amino acids of the gamma1 chain after substitution of Gln for the conserved Glu residue failed to restore the integrin binding activity. These results provide both loss-of-function and gain-of-function evidence that laminin isoforms containing the gamma3 chain are unable to bind to integrins due to the absence of the conserved Glu residue, which should play a critical role in integrin binding by laminins.
Project description:Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.
Project description:Laminins are heterotrimeric molecules composed of an alpha, a beta, and a gamma chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known alpha and beta chains but containing a novel gamma chain, gamma3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that gamma3 contains all the expected domains of a gamma chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that gamma3-containing laminins are likely to exist in a stable matrix. Studies of the tissue distribution of gamma3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, gamma3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The gamma3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of gamma3-containing laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells.
Project description: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 alpha1-alpha2 to alpha3-alpha4 chains after 3 years of life; in the adult, alpha1-alpha2 chains were retained only in the limbal BM. Laminin alpha2 and beta2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma3 chain. In the infant DM, type IV collagen alpha1-alpha6 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 gamma3 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.
Project description:Proper arteriovenous morphogenesis is crucial for maintaining normal tissue perfusion. However, our understanding of how arterial morphogenesis is regulated in the CNS is incomplete. In this study, we asked whether vascular basement membrane (BM) laminins, specifically the γ3-containing isoforms, regulate retinal arterial morphogenesis. We provide evidence that Laminin-γ3 is deposited at both arterial and venous BMs during arteriogenesis. Vascular BM Laminin-γ3 bound dystroglycan (DG), a laminin receptor preferentially expressed by arterial endothelial cells (ECs) during arteriogenesis. Blockade of laminin-DG binding in vitro led to decreased Delta-like ligand (DLL)-4 expression in ECs. Moreover, genetic deletion of the Laminin-γ3- and EC-specific deletion of DG led to similar defects in retinal arteriogenesis, including reduced Dll4 expression, hyperbranching and reduced smooth muscle coverage. These results implicate a newly identified Laminin-γ3-DG signaling cascade that regulates arterial Dll4/Notch signaling to specify and stabilize retinal arteries.-Biswas, S., Watters, J., Bachay, G., Varshney, S., Hunter, D. D., Hu, H., Brunken, W. J. Laminin-dystroglycan signaling regulates retinal arteriogenesis.
Project description:Laminins promote early stages of peripheral nerve myelination by assembling basement membranes (BMs) on Schwann cell surfaces, leading to activation of ?1 integrins and other receptors. The BM composition, structural bonds and ligands needed to mediate this process, however, are not well understood. Mice hypomorphic for laminin ?1-subunit expression that assembled endoneurial BMs with reduced component density exhibited an axonal sorting defect with amyelination but normal Schwann cell proliferation, the latter unlike the null. To identify the basis for this, and to dissect participating laminin interactions, LAMC1 gene-inactivated dorsal root ganglia were treated with recombinant laminin-211 and -111 lacking different architecture-forming and receptor-binding activities, to induce myelination. Myelin-wrapping of axons by Schwann cells was found to require higher laminin concentrations than either proliferation or axonal ensheathment. Laminins that were unable to polymerize through deletions that removed critical N-terminal (LN) domains, or that lacked cell-adhesive globular (LG) domains, caused reduced BMs and almost no myelination. Laminins engineered to bind weakly to ?6?1 and/or ?7?1 integrins through their LG domains, even though they could effectively assemble BMs, decreased myelination. Proliferation depended upon both integrin binding to LG domains and polymerization. Collectively these findings reveal that laminins integrate scaffold-forming and cell-adhesion activities to assemble an endoneurial BM, with myelination and proliferation requiring additional ?6?1/?7?1-laminin LG domain interactions, and that a high BM ligand/structural density is needed for efficient myelination.
Project description:Pathologies of retinal blood vessels are among the major causes of blindness worldwide. A key cell type that regulates retinal vascular development is the astrocyte. Generated extrinsically to the retina, astrocytes migrate into the retina through the optic nerve head. Even though there is a strong correlation between astrocyte distribution and retinal vascular development, the factors that guide astrocytes into the retina remain unclear. In this study, we show that astrocytes migrate within a laminin-containing basement membrane - the inner limiting membrane. Genetic deletion of the laminin ?2 and ?3 chains affects astrocyte migration and spatial distribution. We show that laminins act as haptotactic factors in vitro in an isoform-specific manner, inducing astrocyte migration and promoting astrocyte differentiation. The addition of exogenous laminins to laminin-null retinal explants rescues astrocyte migration and spatial patterning. Furthermore, we show that the loss of laminins reduces ?1 integrin expression in astrocytes. Culturing laminin-null retinal astrocytes on laminin substrates restores focal localization of ?1 integrin. Finally, we show that laminins containing ?2 and ?3 chains regulate subsequent retinal blood vessel growth and maintain vascular integrity. These in vivo and in vitro studies demonstrate clearly that laminins containing ?2 and ?3 chains are indispensable for migration and spatial organization of astrocytes and that they play a crucial role during retinal angiogenesis in vivo.
Project description:A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the alpha4, alpha5, and beta2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin alpha5 and arrested in mutants lacking both alpha4 and alpha5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.
Project description:Laminins are heterotrimeric extracellular glycoproteins found in, but not confined to, basement membranes (BMs). They are important components in formation of the molecular networks of BMs as well as in cell polarity, cell differentiation and tissue morphogenesis. Each laminin is composed by an ?, a ? and a ? chain. Previous studies have shown that the ?3 chain is partnered with either the ?1 chain (in placenta) or ?2 chain (in the CNS) (Libby et al., 2000). Several studies, including our own, suggested that the ?3 chain is expressed in both apical and basal compartments (Koch et al., 1999; Gersdorff et al., 2005; Yan and Cheng, 2006). This study investigates the expression pattern of the ?3 chain in mouse. We developed three new ?3-reactive antibodies, and we show that the ?3 chain is present in BMs. The distribution pattern is considerably more restricted than that of the ?1 chain and within any tissue there is differential deposition into BM compartments. This is particularly true in the retina and brain, where ?3 is uniquely expressed in a subset of the vascular basement membranes and the pial surface. We used conventional genetic ablation techniques to remove the ?3 chain in mice; unlike other laminin null mice (?5, ?2, ?1 nulls), these mice live a normal lifespan and have only minor abnormalities, the most striking of which are ectopic granule cells in the cerebellum and an apparent increase in capillary branching in the outer retina. These data support the suggestion that the ?3 chain is deposited in BMs and contributes some unique properties to their function, particularly in the nervous system.