Zebrafish mutants identify an essential role for laminins in notochord formation

Development ◽  
2002 ◽  
Vol 129 (13) ◽  
pp. 3137-3146 ◽  
Author(s):  
Michael J. Parsons ◽  
Steven M. Pollard ◽  
Leonor Saúde ◽  
Benjamin Feldman ◽  
Pedro Coutinho ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Positional Cloning ◽  
Essential Role ◽  
Basement Membranes ◽  
Form Complex ◽  
Organ Formation ◽  
Notochord Cells ◽  
Laminin 1

Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin β1 and laminin γ1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin β1 and laminin γ1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord.

Altered synthesis of laminin 1 and absence of basement membrane component deposition in (beta)1 integrin-deficient embryoid bodies

2000 ◽  
Vol 113 (2) ◽  
pp. 259-268 ◽  
Author(s):  
M. Aumailley ◽  
M. Pesch ◽  
L. Tunggal ◽  
F. Gaill ◽  
R. Fassler
Keyword(s):  
Basement Membrane ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Mouse Genetics ◽  
Embryoid Bodies ◽  
Membrane Formation ◽  
Beta 1 Integrin ◽  
Laminin 1

Basement membranes are the earliest extracellular matrices produced during embryogenesis. They result from synthesis and assembly into a defined supramolecular architecture of several components, including laminins, collagen IV, nidogen, and proteoglycans. In vitro studies have allowed us to propose an assembly model based on the polymerisation of laminin and collagen IV in two separate networks associated together by nidogen. How nucleation of polymers and insolubilisation of the different components into a basement membrane proceed in vivo is, however, unknown. A most important property of several basement membrane components is to provide signals controling the activity of adjacent cells. The transfer of information is mediated by interactions with cell surface receptors, among them integrins. Mouse genetics has demonstrated that the absence of these interactions is not compatible with development as deletion of either laminin (gamma)1 chain or integrin (beta)1 chain lead to lethality of mouse embryos at the peri-implantation stage. We have used embyoid bodies as a model system recapitulating the early steps of embryogenesis to unravel the respective roles of laminin and (beta)1 integrins in basement membrane formation. Our data show that there is formation of a basal lamina in wild-type, but not in (beta)1-integrin deficient, embryoid bodies. Surprisingly, in the absence of (beta)1 integrins, laminin 1 was not secreted in the extracellular space due to a rapid switch off of laminin (alpha)1 chain synthesis which normally drives the secretion of laminin heterotrimers. These results indicate that (beta)1 integrins are required for the initiation of basement membrane formation, presumably by applying a feed-back regulation on the expression of laminin (alpha)1 chain and other components of basement membranes.

In vivo biosynthesis and turnover of glomerular basement membrane in diabetic rats

1982 ◽  
Vol 242 (4) ◽  
pp. F385-F389
Author(s):  
M. P. Cohen ◽  
M. L. Surma ◽  
V. Y. Wu
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Diabetic Rats ◽  
Basement Membranes ◽  
Membrane Turnover ◽  
Proline Incorporation ◽  
Basement Membrane Collagen ◽  
Osmotic Lysis ◽  
Specific Activities

Glomerular basement membrane (GBM) was labeled in vivo by the injection of tracer amounts of tritiated proline into normal and streptozotocin-diabetic rats. Basement membrane biosynthesis and turnover were determined from the specific activities of proline and hydroxyproline in samples purified following osmotic lysis of glomeruli isolated 4 h to 12 days after injection. Peak radiolabeling of normal and diabetic GBM occurred within 24-48 h and 48-72 h, respectively, and, when corrected for differences in the serum proline specific activities, [3H]proline incorporation was greater in diabetic than in normal samples. In contrast to the subsequent time-dependent progressive decline in radiolabeling in basement membranes from normal animals, specific activities of proline and hydroxyproline in diabetic glomerular basement membrane did not change significantly over the same period of observation. Renal cortical mass and glomerular basement membrane collagen content were preserved in diabetic animals despite loss of body weight. The findings are compatible with prolongation of glomerular basement membrane turnover in experimental diabetes, and suggest that diminished degradation contributes to the accumulation of glomerular basement membrane that is characteristic of chronic diabetes.

"BM180": a novel basement membrane protein with a role in stimulus-secretion coupling by lacrimal acinar cells

1996 ◽  
Vol 270 (6) ◽  
pp. C1743-C1750 ◽  
Author(s):  
G. W. Laurie ◽  
J. D. Glass ◽  
R. A. Ogle ◽  
C. M. Stone ◽  
J. R. Sluss ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Membrane Protein ◽  
Acinar Cells ◽  
Basement Membranes ◽  
Enzyme Linked Immunosorbent Assay ◽  
Regulated Secretion ◽  
Basement Membrane Protein ◽  
Stimulus Secretion Coupling ◽  
Lacrimal Acinar Cells ◽  
Laminin 1

Regulated secretion requires the developmental coupling of neuronal or hormonal stimuli to an exocytotic response, a multistep pathway whose appearance may be linked with cellular adhesion to the newly formed exocrine cell basement membrane. We screened for adhesion-associated coupling activity using lacrimal acinar cells and have identified “BM180”, a novel basement membrane protein enriched in guanidine HCl extracts of lacrimal and parotid exocrine secretory glands. BM180 resides primarily in a previously inexamined lower molecular-mass basement membrane peak (peak 2) that contains cell adhesion activity inhibitable with the anti-BM180 monoclonal antibody 3E12. Removal of peak 2 by gel filtration or preincubation of basement membrane with 3E12 decreased regulated peroxidase secretion by one-half without affecting constitutive secretion or the amount of cellular peroxidase available for release. Adding back peak 2 restored regulated secretion in a dose-dependent and 3E12-inhibitable manner and suggested a synergistic relationship between BM180 and laminin 1. BM180 has a mobility of 180 and 60 kDa in the absence or presence of dithiothreitol, respectively, and shows no immunological identity by competitive enzyme-linked immunosorbent assay with laminin 1, collagen IV, entactin, fibronectin, BM-40, perlecan, or vitronectin. We propose that BM180 is an important resident of certain glandular basement membranes where it interacts with the cell surface, thereby possibly signaling the appearance of a transducing element in the stimulus-secretion coupling pathway.

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

1996 ◽  
Vol 133 (2) ◽  
pp. 417-430 ◽  
Author(s):  
A De Arcangelis ◽  
P Neuville ◽  
R Boukamel ◽  
O Lefebvre ◽  
M Kedinger ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Basement Membrane ◽  
Regulatory Element ◽  
Digestive Enzyme ◽  
Eukaryotic Expression ◽  
Basement Membranes ◽  
Chain Gene ◽  
Membrane Assembly ◽  
Development And Differentiation ◽  
Laminin 1

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.

Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo

1995 ◽  
Vol 108 (12) ◽  
pp. 3795-3805 ◽  
Author(s):  
F. Schuler ◽  
L.M. Sorokin
Keyword(s):  
In Situ Hybridization ◽  
Basement Membranes ◽  
Chain Gene ◽  
Muscle Fibres ◽  
Myogenic Cells ◽  
Mouse Tissues ◽  
Laminin 1

The expression of laminin-1 (previously EHS laminin) and laminin-2 (previously merosin) isoforms by myogenic cells was examined in vitro and in vivo. No laminin alpha 2 chainspecific antibodies react with mouse tissues, 50 rat monoclonal antibodies were raised against the mouse laminin alpha 2 chain: their characterization is described here. Myoblasts and myotubes from myogenic cell lines and primary myogenic cultures express laminin beta 1 and gamma 1 chains and form a complex with a 380 kDa alpha chain identified as laminin alpha 2 by immunofluorescence, immunoprecipitation and PCR. PCR from C2C12 myoblasts and myotubes for the laminin alpha 2 chain gene (LamA2) provided cDNA sequences which were used to investigate the in vivo expression of mouse LamA2 mRNA in embryonic tissues by in situ hybridization. Comparisons were made with specific probes for the laminin alpha 1 chain gene (LamA1). LamA2 but not LamA1 mRNA was expressed in myogenic tissues of 14- and 17-day-old mouse embryos, while the laminin alpha 2 polypeptide was localized in adjacent basement membranes in the muscle fibres. In situ hybridization also revealed strong expression of the LamA2 mRNA in the dermis, indicating that laminin alpha 2 is expressed other than by myogenic cells in vivo. Immunofluorescence studies localized laminin alpha 2 in basement membranes of basal keratinocytes and the epithelial cells of hair follicles, providing new insight into basement membrane assembly during embryogenesis. In vitro cell attachment assays revealed that C2C12 and primary myoblasts adhere to laminin-1 and -2 isoforms in a similar manner except that myoblast spreading was significantly faster on laminin-2. Taken together, the data suggest that laminins 1 and 2 play distinct roles in myogenesis.

scholarly journals The netrin receptor DCC focuses invadopodia-driven basement membrane transmigration in vivo

2013 ◽  
Vol 201 (6) ◽  
pp. 903-913 ◽  
Author(s):  
Elliott J. Hagedorn ◽  
Joshua W. Ziel ◽  
Meghan A. Morrissey ◽  
Lara M. Linden ◽  
Zheng Wang ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Cancer Metastasis ◽  
Normal Development ◽  
Basement Membranes ◽  
Membrane Interface ◽  
Anchor Cell ◽  
Membrane Components ◽  
Invasive Cell ◽  
Basement Membrane Components

Though critical to normal development and cancer metastasis, how cells traverse basement membranes is poorly understood. A central impediment has been the challenge of visualizing invasive cell interactions with basement membrane in vivo. By developing live-cell imaging methods to follow anchor cell (AC) invasion in Caenorhabditis elegans, we identify F-actin–based invadopodia that breach basement membrane. When an invadopodium penetrates basement membrane, it rapidly transitions into a stable invasive process that expands the breach and crosses into the vulval tissue. We find that the netrin receptor UNC-40 (DCC) specifically enriches at the site of basement membrane breach and that activation by UNC-6 (netrin) directs focused F-actin formation, generating the invasive protrusion and the cessation of invadopodia. Using optical highlighting of basement membrane components, we further demonstrate that rather than relying solely on proteolytic dissolution, the AC’s protrusion physically displaces basement membrane. These studies reveal an UNC-40–mediated morphogenetic transition at the cell–basement membrane interface that directs invading cells across basement membrane barriers.

scholarly journals Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

2012 ◽  
Vol 197 (5) ◽  
pp. 677-689 ◽  
Author(s):  
Daiji Kiyozumi ◽  
Makiko Takeichi ◽  
Itsuko Nakano ◽  
Yuya Sato ◽  
Tomohiko Fukuda ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Direct Consequence ◽  
Basement Membranes ◽  
Dystrophic Epidermolysis Bullosa ◽  
Membrane Assembly ◽  
Fraser Syndrome ◽  
Basement Membrane Protein ◽  
Integrin Binding Site ◽  
Associated Proteins

Dysfunction of the basement membrane protein QBRICK provokes Fraser syndrome, which results in renal dysmorphogenesis, cryptophthalmos, syndactyly, and dystrophic epidermolysis bullosa through unknown mechanisms. Here, we show that integrin α8β1 binding to basement membranes was significantly impaired in Qbrick-null mice. This impaired integrin α8β1 binding was not a direct consequence of the loss of QBRICK, which itself is a ligand of integrin α8β1, because knock-in mice with a mutation in the integrin-binding site of QBRICK developed normally and do not exhibit any defects in integrin α8β1 binding. Instead, the loss of QBRICK significantly diminished the expression of nephronectin, an integrin α8β1 ligand necessary for renal development. In vivo, nephronectin associated with QBRICK and localized at the sublamina densa region, where QBRICK was also located. Collectively, these findings indicate that QBRICK facilitates the integrin α8β1–dependent interactions of cells with basement membranes by regulating the basement membrane assembly of nephronectin and explain why renal defects occur in Fraser syndrome.

scholarly journals Light and electron microscopic localization of the alpha 1-chain and the E1 and E8 domains of laminin-1 in mouse kidney using monoclonal antibodies to establish the orientation of laminin-1 within basement membranes.

1995 ◽  
Vol 43 (7) ◽  
pp. 675-680 ◽  
Author(s):  
N Miosge ◽  
E Günther ◽  
E Heyder ◽  
B Manshausen ◽  
R Herken
Keyword(s):  
Monoclonal Antibodies ◽  
Proximal Tubule ◽  
Distal Tubule ◽  
Distinct Pattern ◽  
Mouse Kidney ◽  
Basement Membranes ◽  
Mouse Tissue ◽  
Electron Microscopic ◽  
Laminin 1

To localize the different domains of the laminin-1 molecule in tissues and gain insight into their in vivo relevance, we raised rat anti-mouse monoclonal antibodies (MAbs) against the entire molecule. Then we tested eight of the 20 clones producing anti-laminin-1 MAbs to specify their reactivity towards the alpha 1-, beta 1-, and gamma 1-chains and the elastase-cleaved fragments of the laminin-1 molecule. We found three MAbs with high titers in ELISA that showed good reactivity in embedded tissue. One of these reacted specifically against the E1 fragment, one against the E8 fragment, and one MAb detected the alpha 1-chain of laminin-1 but not the beta 1- or gamma 1-chain. All three MAbs are useful for light immunohistochemical investigations on cryosections and on paraffin-embedded material, and for ultrastructural localization of laminin-1 in LR Gold-embedded mouse tissue. Antibody staining of the E1 and E8 domains of laminin-1 revealed distinct localization of the molecule in the proximal tubule basement membranes of mouse kidney. The short arms (E1) of the laminin-1 molecule are predominantly located in the lamina lucida and the long arms (E8) are oriented towards the lamina fibroreticularis. Therefore, both MAbs are useful for studies of the orientation of the laminin-1 molecule in basement membranes. The distal tubule basement membranes did not show any distinct pattern of laminin-1 distribution. In general, the distal tubules showed the strongest reactions over the entire width of the basement membrane for all three MAbs. In contrast, the proximal tubule basement membranes showed somewhat weaker reactivity but a distinct pattern of laminin-1 distribution, with the E1 fragments oriented towards the adjacent epithelial cell surface.

scholarly journals Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition

2002 ◽  
Vol 115 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Thorarinn Gudjonsson ◽  
Lone Rønnov-Jessen ◽  
René Villadsen ◽  
Fritz Rank ◽  
Mina J. Bissell ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Specific Antigen ◽  
Myoepithelial Cells ◽  
Normal Breast ◽  
Breast Epithelial ◽  
Luminal Epithelial Cells ◽  
Laminin 1

The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. We have shown previously that luminal epithelial cells can be polarized when cultured within a reconstituted basement membrane gel. We reasoned that such cues in vivo may be given by myoepithelial cells. Accordingly, we used an assay where luminal epithelial cells are incorrectly polarized to test this hypothesis. We show that culturing human primary luminal epithelial cells within collagen-I gels leads to formation of structures with no lumina and with reverse polarity as judged by dual stainings for sialomucin, epithelial specific antigen or occludin. No basement membrane is deposited, and β4-integrin staining is negative. Addition of purified human myoepithelial cells isolated from normal glands corrects the inverse polarity, and leads to formation of double-layered acini with central lumina. Among the laminins present in the human breast basement membrane (laminin-1, -5 and -10/11), laminin-1 was unique in its ability to substitute for myoepithelial cells in polarity reversal.Myoepithelial cells were purified also from four different breast cancer sources including a biphasic cell line. Three out of four samples either totally lacked the ability to interact with luminal epithelial cells, or conveyed only correction of polarity in a fraction of acini. This behavior was directly related to the ability of the tumor myoepithelial cells to produce α-1 chain of laminin. In vivo, breast carcinomas were either negative for laminin-1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Dual staining with myoepithelial markers revealed that tumor-associated myoepithelial cells were either negative or weakly positive for expression of laminin-1, establishing a strong correlation between loss of laminin-1 and breast cancer. We conclude that the double-layered breast acinus may be recapitulated in culture and that one reason for the ability of myoepithelial cells to induce polarity is because they are the only source of laminin-1 in the breast in vivo. A further conclusion is that a majority of tumor-derived/-associated myoepithelial cells are deficient in their ability to impart polarity because they have lost their ability to synthesize sufficient or functional laminin-1. These results have important implications for the role of myoepithelial cells in maintenance of polarity in normal breast and how they may function as structural tumor suppressors.

scholarly journals Gene Structure and Functional Analysis of the Mouse Nidogen-2 Gene: Nidogen-2 Is Not Essential for Basement Membrane Formation in Mice

2002 ◽  
Vol 22 (19) ◽  
pp. 6820-6830 ◽  
Author(s):  
Jürgen Schymeinsky ◽  
Sabine Nedbal ◽  
Nicolai Miosge ◽  
Ernst Pöschl ◽  
Cherie Rao ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Biochemical Characterization ◽  
Gene Knockout ◽  
Basement Membranes ◽  
Membrane Formation ◽  
Gene Knockout Mice ◽  
Almost All ◽  
Nidogen 1 ◽  
Deficient Mice

ABSTRACT Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.

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