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Integrins and Basal Lamina Formation 261 Journal of Cell Science 113, 259-268 (2000) 259 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0774 Altered synthesis of laminin 1 and absence of basement membrane component deposition in β1 integrin-deficient embryoid bodies Monique Aumailley1,*, Monika Pesch1, Lucy Tunggal1, Françoise Gaill2 and Reinhard Fässler3 1Institute II for Biochemistry, University of Cologne, 50931 Cologne, Germany 2Laboratoire de Biologie Moléculaire et Cellulaire du Dévelopement, UPMC/CNRS, 75252 Paris, France 3Department of Experimental Pathology, Lund University, 22185 Lund, Sweden *Author for correspondence (e-mail: [email protected]) Accepted 15 November 1999; published on WWW 13 January 2000 SUMMARY Basement membranes are the earliest extracellular embryos at the peri-implantation stage. We have used matrices produced during embryogenesis. They result from embyoid bodies as a model system recapitulating the early synthesis and assembly into a defined supramolecular steps of embryogenesis to unravel the respective roles of architecture of several components, including laminins, laminin and β1 integrins in basement membrane collagen IV, nidogen, and proteoglycans. In vitro studies formation. Our data show that there is formation of a basal have allowed us to propose an assembly model based on the lamina in wild-type, but not in β1-integrin deficient, polymerisation of laminin and collagen IV in two separate embryoid bodies. Surprisingly, in the absence of β1 networks associated together by nidogen. How nucleation integrins, laminin 1 was not secreted in the extracellular of polymers and insolubilisation of the different space due to a rapid switch off of laminin α1 chain components into a basement membrane proceed in vivo is, synthesis which normally drives the secretion of laminin however, unknown. A most important property of several heterotrimers. These results indicate that β1 integrins are basement membrane components is to provide signals required for the initiation of basement membrane controling the activity of adjacent cells. The transfer of formation, presumably by applying a feed-back regulation information is mediated by interactions with cell surface on the expression of laminin α1 chain and other receptors, among them integrins. Mouse genetics has components of basement membranes. demonstrated that the absence of these interactions is not compatible with development as deletion of either laminin γ1 chain or integrin β1 chain lead to lethality of mouse Key words: Laminin, Integrin, ES cell, Basement membrane INTRODUCTION into two independent networks (Yurchenco and Schittny, 1990; Timpl and Brown, 1996) that are connected by nidogen (Fox Basement membranes are thin layers of specialised et al., 1991; Aumailley and Smyth, 1998). In the presence of extracellular matrices surrounding cells or separating layer of Ca2+, laminin 1, as well as laminin variants with full-length cells of different lineages. During embryogenesis they are the chains, self-assembles by a cooperative heat-gelation process earliest extracellular matrix synthesised and they appear in the involving interactions between amino-terminal LN motifs blastocyts between the primitive endoderm and the inner cell (domain VI) of the short arms (Paulsson, 1988; Yurchenco et mass (Graham and Lehtonen, 1979). At this stage they result al., 1992; Yurchenco and Cheng, 1993; Cheng et al., 1997). from the sequential synthesis of several components, including The process is reversible which allows the extraction of at least laminin, nidogen, proteoglycans, and collagen IV. The laminin from tissues with physiological buffers containing laminin β1 and γ1 chains are detected at the two-cell stage, the chelating agents (Paulsson et al., 1987). High affinity laminin α1 chain and nidogen are present at the 8-16 cell stage interactions between the carboxy- and amino-terminal domains (Cooper and Mac Queen, 1983; Dziadek and Timpl, 1985), of nidogen with an LE motif in the laminin γ1 chain and while collagen IV appears later in the inner cell mass of 3- collagen IV, respectively, lead to the formation of ternary to 4-day-old blastocysts (Leivo et al., 1980). At further complexes in vitro (Fox et al., 1991; Mayer et al., 1993; developmental stages, basement membranes become more Aumailley et al., 1989). Altogether these data strongly suggest versatile in composition but still contain at least one that in vivo the polymeric networks of laminin and collagen IV isoform of each laminin, collagen IV and nidogen families, become connected by nidogen and that these multiple proteoglycans, and other glycoproteins. interactions are crucial for basement membrane formation and In vitro studies have shown that basement membrane stability. The fact that laminin 1 is the first network-forming formation involves self assembly of laminin and of collagen IV component expressed during development makes it a good 260 M. Aumailley and others candidate for a role in the initiation of basement membrane maintained in culture for up to 3 weeks with medium renewal once a assembly. This is supported by the observation that embryos week. The EB were routinely inspected and counted under phase with a null mutation in the gene coding for γ1 chain develop contrast microscopy and photographed at regular intervals. At various to the blastocyst stage but no further, and fail to form proper times EB were collected with a pasteur pipet and processed for basement membranes (Smyth et al., 1999). However, how the analysis. minimal concentration of 70-140 nM required for nucleation Rescue of integrin β1 deficient G201 cells of laminin polymers (Yurchenco and Cheng, 1993) is reached To obtain G201 cells that express β1A integrin splice variant the in vivo is not known. mouse β1A integrin cDNA was cloned after a phosphoglycerol kinase Besides being a key component of basement membrane (PGK) or Simian Virus 40 (SV-40) promotor. Both expression vectors architecture, laminins bind to cell surface components, in contained a puromycin expression cassette for selecting transfected particular to integrins and α-dystroglycan, and in doing so they G201 cells with puromycin. The plasmids were linearised and 15 µg control cellular activities by providing adjacent cells with electroporated into 1×107 G201 cells. Transfected cells were cultured multiple informations (Ekblom, 1996; Aumailley and Smyth, in ES medium (see above) on embryonic fibroblasts and selected with 1998). Interestingly, deletion of β1 integrins or of dystroglycan either 1, 2, 4 or 8 µg puromycin per ml ES medium. Twenty four ES in mice results in peri-implantation lethality and the embryos cell clones that survived the puromycin selection and tightly adhered show a defective morphogenesis of the endoderm or of the to the fibroblasts were picked, expanded in 24-well plates in the presence of feeders and analysed by flow cytometry using a polyclonal Reichert’s membrane, respectively (Fässler and Meyer, 1995; antiserum against rat β1 integrin (a gift from Dr S. Johansson, Stephens et al., 1995; Williamson et al., 1997). Thus both University of Uppsala, Sweeden). Thirteen clones electroporated with laminin 1, β1 integrins and dystroglycan are required at the PGK-β1 integrin cassette and 4 clones electroporated with the SV-40- same development stage, i.e. at the time when the first β1 integrin cassette showed β1 integrin expression on the cell surface. basement membrane appears. To identify a possible synergy of Two weeks and 8 passages later the expression level of the β1 integrin laminin and β1 integrins in basement membrane formation we remained high in three PGK-driven clones but not in the clones have used as model system embryoid bodies (EB) which transfected with the SV-40-β1 integrin cassette. One of the PGK- sequentially reproduce several stages of early embryonic driven clones (G201β1/9) was used in the studies. development (Keller, 1995). We show here by indirect Antibodies immunofluorescence and by transmission electron microscopy Rabbit antisera raised against mouse laminin fragment P1 recognising that EB derived from wild-type embryonic stem (ES) cells the α1, β1 and γ1 chains and referred to here as anti-laminin develop a basal lamina at the basis of the outer cell layer. This antiserum, nidogen, fibulin-1, collagen IV, and BM-40, and against is supported by biochemical analyses showing that laminin 1 human fibronectin were kindly provided by Dr R. Timpl (Max-Planck and nidogen are present in the extracellular space as EDTA- Institute for Biochemistry, Martinsried, Germany). A rabbit antiserum soluble material. By contrast, in EB derived from β1-null against a recombinant polypeptide of the mouse laminin α1 chain ES cells, there are no immunological, ultrastructural, or (LG4-5 domains) was kindly provided by Dr L. Sorokin (University biochemical clues for the presence of any basement membrane of Erlangen, Erlangen, Germany). Monoclonal antibodies (mAb) material. Moreover, while in wild-type EB laminin 1 against integrin subunits β1 (9GE7), α6 (GoH3), and α7 (CA5) were accumulates extracellularly as a function of time and of provided by Dr A. Sonnenberg (The Netherlands Cancer Institute, laminin α1 chain synthesis, the later is rapidly switched off in Amsterdam, Netherlands), Dr A. Sutherlands (University of Virginia, β Charlottesville, VA), and Dr D. Vestweber (University of Münster, EB derived from 1-null ES cells. These results clearly Münster, Germany), respectively. Rabbit antiserum against the demonstrate that initiation of basement membrane
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