Journal of Cell Science 113, 259-268 (2000) 259 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0774

Altered synthesis of 1 and absence of component deposition in β1 integrin-deficient embryoid bodies

Monique Aumailley1,*, Monika Pesch1, Lucy Tunggal1, Françoise Gaill2 and Reinhard Fässler3 1Institute II for , 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 , laminin and β1 integrins in basement membrane 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 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 formation cytoplasmic domain of the integrin α3 subunit was from commercial requires synthesis, secretion, and insolubilisation of laminin 1 source (Chemicon International). and that the process is driven by integrins of the β1 family. Immunofluorescence staining EB were collected in Eppendorf tubes (10-20 EB/tube), centrifuged MATERIALS AND METHODS at low speed and resuspended in 1% paraformaldehyde, 0.1 M phosphate buffer, pH 7.4. After fixation (30 minutes, room ES cell and embryoid body cultures temperature), the EB were washed and incubated in 0.1 M phosphate Wild-type D3 (Doetschman et al., 1985), β1-deficient G201 (Fässler buffer containing, successively, 0%, 7.5% and 15% sucrose. The last et al., 1995) and β1 integrin-rescued G201β1/9 (see below) ES cells suspension of EB was mixed 1:1 (v:v) with Tissue Tek O.C.T. were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with Compound (Sakura distributed by Vogel, Giessen, Germany) and 2 mM glycyl L-glutamine, supplemented with 15% fetal calf serum, finally resuspended in Tissue Tek O.C.T. Compound. After instant- 0.1 mM non-essential amino acids, penicillin/streptomycin, 0.1 mM freeze in liquid nitrogen, the bottoms of the Eppendorf tubes were cut β-mercaptoethanol, and leukemia inhibitory factor (LIF; 0.02 µg/ml). and the EB were embedded in freezing Tissue Tek. Cryosections (7 All reagents were from Gibco BRL (Life Technologies, Eggenstein, µm, 2800 Frigocut; E. Reichert; Jung) were blocked with 1% bovine Germany). The D3 and G201β1/9 cells were cultivated on feeder serum albumin (Fraction V, Serva, Boehringer-Ingelheim, Germany) layers of mitomycin-treated mouse embryonic fibroblasts and the and incubated with primary antibodies followed by Cy3-conjugated G201 cells were directly cultivated on tissue culture plates (Falcon, antibodies against mouse, rabbit, or rat immunoglobulins (Jackson, Faust, Cologne, Germany). distributed through Immunotech). The histoslides were mounted in Nearly confluent ES cells were resuspended with 0.05% trypsin, 9:1 (v/v) glycerol/PBS and observed with an Axiophot microscope 0.02% EDTA in phosphate-buffered saline (PBS) and used to initiate (Zeiss, Oberkochen, Germany) equiped with epifluorescence optics. embryoid body culture by the hanging drop technique (600 cells/20 Photomicrographs were taken on Kodak T-Max 400 film. µl/drop; 50 drops/plate) on the lid of bacteriological culture plates (Greiner, Fastnacht, Cologne, Germany) in ES medium with 20% fetal Histology and transmission electron microscopy calf serum and without LIF. After 48 hours, embryoid bodies were EB (10/batch) were fixed by 2% glutaraldehyde in PBS (60 minutes) dislodged from the lid by gentle washing with culture medium and followed by 2% glutaraldehyde in 0.1 M Na cacodylate/HCl, pH 7.4 Integrins and basal lamina formation 261

Fig. 1. Overall morphology of wild-type and β1- deficient embryoid bodies. Live EB were photographed under phase contrast microscopy at 14 days (A,B) or after fixation and under magnifying glass at 21 days (C,D). Note aggregation, irregular contours, and out-growing processes of β1-deficient EB and the regular rounded shape of wild-type EB. Bar, 200 µm (A and B); 330 µm (C and D).

(20 minutes). After three washes with 0.1 M Na cacodylate/HCl, pH 7.4, the EB were post-fixed (30 minutes) with 1% OsO4 in 0.15 M Na cacodylate/HCl, pH 7.4, dehydrated in graded ethanol, and individually embedded in either Epon or Araldite. Semi-thin (1 µm) or ultrathin sections (60-80 nm) were prepared with an Ultracut (Reichert). The semi-thin sections were stained with toluidine blue and observed with a photonic microscope (Nikon). Ultra-thin sections were contrasted with uranyl acetate and lead citrate, and observed with a transmission electron microscope (LEO 912). These studies were performed at the Centre de Microscopie Electronique de Jussieu (Paris, France). BM extraction, SDS-PAGE and immunoblotting EB were resuspended (20 EB/100 µl) in 150 mM NaCl, 50 mM Tris-HCl, pH 7.4 (TBS) containing 20 mM EDTA and staining was observed on the whole sections with antibodies proteases inhibitors and extracted in this buffer for 2 hours at 4¡C. specific for laminin, nidogen, collagen IV or fibronectin (not The extracts were collected after centrifugation and the pellets were shown). In 7-day-old wild-type EB, antibodies against laminin resuspended and homogenised in the same buffer (100 µl). The or nidogen showed an intense labeling preferentially restricted extracts (EDTA-soluble fraction) or the homogenised pellets (EDTA- at the periphery of the specimen, while cells with a more insoluble fraction) were diluted 1:1 (v/v) with Laemmli buffer and central position were faintly stained. A distinct linear staining submitted to sodium dodecyl sulfate polyacrylamide (4-10%) gel electrophoresis (SDS-PAGE) in the presence of 5% β- was seen at some location between the positively and mercaptoethanol. The polypeptides were then electro-transfered negatively labeled zones (Fig. 2a,b). Most cells of the sections overnight under 200 mA current at 4¡C to nitrocellulose membranes were stained with antibodies raised against collagen IV or (Schleicher and Schuell, Dassel, Germany) in 50 mM borate buffer, fibronectin (Fig. 2c,d). In 14-day-old wild-type EB, a clearly pH 8.5, containing 5% methanol. For immunodetection, the linear labeling was seen for laminin, nidogen, and to some nitrocellulose membranes were saturated with 5% milk powder and extent for collagen IV (Fig. 2e-g). In 21-day-old wild-type EB, 0.1% Tween-20, and successively incubated with primary antibodies all three antisera specific for basement membrane components and peroxidase-conjugated secondary antibodies (Dako, distributed gave a strong and continous staining at the basis of the outer by Dianova, Hamburg, Germany). Bound antibodies were revealed cell layer which was not seen with anti-fibronectin (Fig. 2i-l). using the ECL chemiluminiscence kit (Amersham, Braunschweig, In all cases, there was no immunofluorescence reaction with Germany). peripheral cells. By contrast, at any time point of the culture, none of the antisera revealed a linear staining in EB derived RESULTS from β1-null ES, including after 21 days (Fig. 2m-p). These results suggested that laminin and nidogen, starting at day 7, The development of wild-type and β1-deficient EB was and later collagen IV, were progressively secreted and monitored under phase contrast microscopy up to 21 days of extracellularly deposited in order to form a basal lamina at the culture. By size measurement on photographs taken at regular basis of the peripheral cell layer of wild-type EB, and that these intervals, the β1-deficient EB were found to be ~20% smaller events were perturbed in EB derived from β1-null ES cells. than the wild-type counterparts, in agreement with a A closer histological observation of semi-thin sections from significantly lower proliferation rate of the mutant EB (not 21-day-old EB revealed that an outer layer of polarised cells shown). Both types of EB started to pulsate at day 8-9. Wild- had formed in the wild type but not in β1-deficient EB (Fig. type EB were round and with regular contours while β1- 3). The overall organisation of the latter was anarchic, cells deficient EB were irregular in shape, developed out-growing were loosely packed, heterogeneous in shape and fusiform processes and were frequently aggregated (Fig. 1). cells, absent in wild-type EB, constituted the occasionally seen Batches of 10-20 EB were successively collected at day out-growing processes (Fig. 3). Compared to their wild-type 3, 4, 5, 6, 7, 14 and 21 and processed for indirect counterparts, the cells forming β1-deficient EB had larger immunofluorescence labeling of cryosections. For 3- to 6-day- nucleus, less cytoplasm and less perinuclear granules (Fig. 3). old wild-type or β1-deficient EB a punctuate and intracellular At an ultrastructural level, electron dense extracellular material 262 M. Aumailley and others

Fig. 2. Immunofluorescence staining of wild-type and β1-deficient EB with antibodies against laminin 1, nidogen, collagen IV, or fibronectin. EB were cultivated for 7, 14, or 21 days and processed for immunofluorescence staining of sections (7 µm) with rabbit antiserum against laminin 1 (a,e,i,m), nidogen (b,f,j,n), collagen IV (c,g,k,o), and fibronectin (d,h,l,p), followed by Cy3-conjugated antibodies against rabbit immunoglobulins. a-l, wild-type EB; m-p, β1-deficient EB. Bar, 55 µm. was seen in the immediate vicinity of the basal aspect of cells laminin material in the EDTA-insoluble fractions. Those forming the outer layer of wild-type EB (Fig. 4A). At higher contained a faint doublet and two major bands migrating as the magnification, these structures resembled a patchy basal α1 and β1/γ1 chains of EHS laminin, respectively (Fig. 5A). lamina (Fig. 4C). By contrast, distinct extracellular deposits By contrast, a major difference was observed at day 14 (not were absent in β1-deficient EB, and the zone localised below shown) and 21 (Fig. 5A). Distinct bands migrating at the the outer cell layer was electron-clear (Fig. 4B,D). Moreover, position of laminin α1 and β1/γ1 chains were seen for the β1-deficient cells were apparently not polarised, contained less EDTA-soluble fraction from wild-type EB but not for that of secretory vesicles and had poorly developed Golgi apparatus β1-deficient EB. Similar bands were observed in the EDTA- and endoplasmic reticulum than their wild-type counterparts insoluble material of wild-type EB, which contained additional (Fig. 4). bands of ~180 and 120 kDa possibly corresponding to To unravel the molecular mechanism underlying these intracellularly degraded polypeptides and of ~150 kDa observations, 7-, 14-, and 21-day-old wild-type and β1-null EB corresponding to nidogen (see below).The EDTA-insoluble were extracted with 10 mM EDTA-containing physiological fraction of β1-null EB contained polypeptides migrating at the buffer according to a protocol routinely used to extract laminin- position of the laminin β1/γ1 chains, but not at the position of nidogen complexes from tissues (Paulsson et al., 1987). The the laminin α1 chain (Fig. 5A). Immunoblotting with an extracts and the insoluble material were fractionated by SDS- antiserum specific for the laminin α1 chain confirmed the PAGE and analysed by immunoblotting with antibodies raised presence of that chain as a faint doublet in the insoluble against fragment P1 of laminin 1. At day 7, analysis of wild- fractions of 7-day-old wild-type or β1-deficient EB, and as a type or mutant EB gave similar results with no detection of strong band in the EDTA-soluble and -insoluble fractions of laminin in the EDTA-soluble fractions and the presence of wild-type EB, but not in those from β1-deficient EB (Fig. 5B). Integrins and basal lamina formation 263

Fig. 3. Histological observation of wild-type and β1-deficient EB. Semi-thin (1 µm) sections of 21-day-old wild-type (A, D) or β1-deficient (B,C, E-G) EB were stained with toluidine blue and observed with a photonic microscope at low (A-C) and high (D-G) magnification. Bar, 80 µm (A and B); 160 µm (C); 16 µm (D-G).

As seen on some lanes in Fig. 5A the anti-laminin antiserum, degradation, or release into the culture medium. Immunoblotting known to react with nidogen, detected a band at ~150 kDa. analysis of the latter showed that intact nidogen was indeed Immunoblotting with a nidogen specific antiserum confirmed the released into the medium of both types of EB, in amounts, presence of the latter in all the EDTA-soluble and -insoluble however, higher for β1-null than for wild-type EB (Fig. 6B). By fractions (Fig. 5C). However, as judged by band intensity there comparison, intact or degraded laminin chains were not detected was less nidogen in β1-deficient than in wild-type EB. This in the supernatants collected after 7, 14 or 21 days of culture for could have been caused by either reduced synthesis, increased wild-type or β1-deficient EB (Fig. 6A). 264 M. Aumailley and others

Fig. 4. Ultrastructure of wild-type and β1- deficient embryoid bodies. 21-day-old EB were processed for transmission electron microscopy as indicated in Materials and Methods. The specimens were observed at magnifications of ×6300 (A,B) and ×31500 (C,D). Note the presence of electron-dense deposits (arrowheads) and filaments in the vicinity of the cell plasma membrane in wild-type EB (A,C) and the absence of such material in β1-deficient EB (B,D). Bar, 1.36 µm (A and B); 262 nm (C and D).

In order to see whether other basement membrane proteins were affected, EDTA extractable or insoluble material from 21-day- old EB were immunoblotted with antibodies specific for BM-40 or fibulin 1. Both proteins were found in the cellular as well as in the extracellular compartments of wild-type EB, but were below detection levels in G201 EB (Fig. 7). Altogether these results indicated that in the β1-null EB either laminin α1 chain synthesis was perturbed per se or that formation and deposition of a basal lamina are β1 integrin-dependent and submitted to an integrin-mediated feed-back regulation on laminin α1 chain synthesis. To confirm that formation of a basal lamina was dependent on functional integrins and to rule out the remote possibility that lack of laminin α1 chain up- regulation was not an intrinsic defect of the G201 cells, EB derived from ES cells in which the integrin β1 integrin has been rescued (clone G201β1/9) were analysed by immunofluorescence staining. The results showed that the cell capacity to deposit laminin (not shown) or nidogen (Fig. 8) in a polarised manner was restored upon re-expression of the β1 laminin, nidogen, and collagen IV, are synthesised, secreted, integrins. To determine which integrin α subunit(s) was and deposited in a timely and polarised manner at the basal associated with the β1 subunit, wild-type EB were aspect of the peripheral layer of cells. In the 21-day-old EB, immunostained with antibodies directed against integrin α the ultrastructure of the extracellular matrix accumulated at this subunits usually involved in laminin binding. It revealed a location appeared as filaments and a patchy basal lamina. At linear staining for the β1 and α6 integrin subunits, but not for the biochemical level, the laminin α1, β1, and γ1 chains, the α3 or α7 subunits (Fig. 9). This suggested that the integrin nidogen and other extracellular matrix polypeptides were interacting with laminin 1 in wild-type EB was α6β1. synthesised and secreted into the extracellular space. With the exception of nidogen secretion, all other events were absent in β1-deficient EB raising the possibility that secretion and DISCUSSION deposition of basement membrane components require functional integrins of the β1 family. Embryoid bodies that result in vitro from the aggregation of A detailed analysis by immunoblotting revealed that embryonic stem cells contain differentiated cells from all germ secretion of correctly assembled laminin heterotrimers was layers (Doetschman et al., 1985; Abe et al., 1996) and can regulated by the expression level of the laminin α1 chain. At reproduce several morphogenetic events (Keller, 1995). This a low expression level of the latter, as observed in 7-day-old model has been instrumental to investigate the role of proteins wild-type EB and at any time for mutant EB, laminin was not in early development, including β1 integrins. For example, detected in the extracellular compartment, although the β1 and formation of sarcomeres occurs normally in β1-null EB γ1 were synthesised in substantial amounts. By contrast, in 14- (Hirsch et al., 1998), while myogenic and neuronal and 21-day-old wild-type EB, the high expression level of the differentiation are retarded or accelerated, respectively (Fässler laminin α1 chain was associated with secretion of et al., 1996; Rohwedel et al., 1998). Here we report that in heterotrimers. This indicated that at a low level of laminin α1 wild-type EB, basement membrane components, including chain synthesis (7-day-old wild and all mutant EB), either Integrins and basal lamina formation 265 A

Fig. 5. Immunoblotting detection of laminin chains and nidogen in wild-type (+/+) and β1- deficient (−/−) EB. 7- and 21-day-old EB were extracted with 20 mM EDTA in Tris buffer (20 EB /100 µl). The pellets were resuspended and homogenised in 100 µl of the same buffer. EDTA-soluble (extract) and -insoluble (pellet) fractions were mixed 1:1 with Laemmli buffer and analysed by SDS-PAGE on 4-10% acrylamide gels (50 µl/lane) After transfer to nitrocellulose membranes, the blots were incubated with rabbit antisera against laminin 1 (A), laminin α1 chain (B), or nidogen (C) followed by horseradish-conjugated secondary antibodies. Reactive bands were revealed by ECL. The positions of the laminin α1, β1 and γ1 chains from EHS laminin 1 (lane 5) are indicated by arrows at right in A. B and C show only the relevant portion of the blots. laminin βγ dimers were not secreted and the β1 and γ1 chains hypothesis which agrees with a recent report showing that were retained in the intracellular compartment or they were secretion of laminin heterotrimers is driven by the α1 chain secreted and lost into the culture medium. The absence of (Yurchenco et al., 1997). By contrast, although nidogen laminin immunoreactive material in the latter favors the first expression was slightly reduced in β1-deficient EB, its expression and secretion was independent from that of laminin and of integrin expression. However, substantial amounts of nidogen were lost in the culture medium of β1-deficient EB. This agrees with the current model of basement membrane formation which assumes nidogen insolubilisation by the formation of stable equimolar complexes with laminin (Paulsson et al., 1987; Fox et al., 1991; Aumailley et al., 1993). Alternative approaches have led to envision a role for integrins in basement membrane formation, but no definitive conclusions were drawn. By targeted deletion of β1 integrins in F9 embryonal carcinomas cells, it was shown that loss of β1 integrins did not interfere with laminin expression but prevented deposition of the protein in a polarised manner (Stephens et al., 1993). Abnormal basement membranes were also observed in teratomas induced by injection of β1-null ES cells into syngeneic mice. This was attributed to reduced amounts of laminin 1 and nidogen in β1-null tumours compared to normal teratomas, due to decreased synthesis and increased proteolysis, respectively (Bloch et al., 1997; Sasaki et al., 1997). It could not be concluded, however, whether these alterations were contributed by the β1-null cells or the host wild-type cells. Association of the β1 integrin chain with

Fig. 6. Nidogen, but not laminin 1, is released in higher quantities in the culture medium of β1-deficient (−/−) EB than in that of wild-type EB (+/+). The culture medium of 7-, 14- and 21-day-old EB was collected, fractionated by SDS-PAGE and immunoblotted with antibodies against laminin 1 (A) or nidogen (B). The EHS laminin- nidogen complex (EHS) as well as non-conditioned culture medium (M) were loaded on separate lanes as indicated on the figure. 266 M. Aumailley and others

integrins may play a role in their organisation or stability. In α6-null mice (George-Labouesse et al., 1996) and in patients afflicted with a mutation in the α6 integrin gene (Ruzzi et al., 1997; Pulkkinen et al., 1997), basement membranes appear normal but detach from the basal surface of cells, while neuro- muscular junctions are progressively altered in α7-null mice (Mayer et al., 1997). Whether each of these laminin binding integrins play a dispensable or indispensable role in the formation, assembly and/or the stability of basement membranes cannot be decided since genetic ablation of one of the integrin α chains in totipotent ES cells may have activated a compensatory pathway by the others. Alternatively, α- dystroglycan, also a laminin receptor (Henry and Campbell, Fig. 7. Immunoblot detection of BM-40 and fibulin 1 in 21-day-old 1996; Ekblom, 1996), plays a crucial role in the early steps of wild-type (+/+) and β1-deficient (−/−) EB. EDTA soluble and morphogenesis and ablation of its gene leads to peri- insoluble material were fractionated by SDS-PAGE as indicated Fig. implantation lethality of mouse embryos (Williamson et al., 3. After electro-transfer the nitrocellulose membranes were probed 1997) and the absence of a basal lamina (Henry and Campbell, with antibodies against BM-40 or fibulin 1 as indicated on the figure. 1998). These authors proposed that α-dystroglycan induces the clustering of laminin molecules in order to initiate different α chains results in the formation of different receptors polymerisation, an hypothesis which is supported by other for laminins, including α3β1, α6β1 and α7β1 (Aumailley and experiments showing aggregation of exogenous laminin at the Smyth, 1998). Extinction of the ITGA3, ITGA6, or ITGA7 surface of cultivated myotubes (Colognato et al., 1999). genes is compatible with mouse embryo development. In α3- Here we show that functional β1 integrins are required for null mice the basement membranes are disorganised Kreidberg basement membrane formation by being involved in the feed- et al., 1996; DiPersio et al., 1997), suggesting that α3β1 back regulation of laminin α1 chain expression. At this point,

Fig. 8. Immunofluorescence staining of EB after rescue of the integrin β1 subunit in ES G201 cells. EB derived from D3 (wild type, +/+), G201β1/9 (β1 integrin rescue, rescue) or G201 (β1 integrin-deficient, −/−) ES cells were cultivated for 14 days. After fixation EB cryosections were stained with a rabbit polyclonal antibody against nidogen.

Fig. 9. Immunofluorescence staining of integrin subunits in 21-day-old wild-type EB. Wild-type EB were cultivated for 21 days and then processed for immunofluorescence staining of sections (7 µm) with antibodies against integrin subunits as indicated on the figure. Bar, 25 µm Integrins and basal lamina formation 267 the issue of which integrin heterodimer is involved in the Yurchenco, P. D. (1997). Self-assembly of laminin isoforms. J. Biol. Chem. regulation is still open. Our immunofluorescence data show 272, 31525-31532. that both the β1 and α6 integrin subunits, and not the α3 or Colognato, H., Winkelmann, D. A. and Yurchenco, P. D. (1999). Laminin α polymerization induces a receptor-cytoskeletal network. J. Cell Biol. 145, 7, are polarised at the basal side of the outer cell layer in wild- 619-631. type EB. Consequently, the α6β1 integrin appears to be the Cooper, A. R. and MacQueen, H. A. (1983). Subunits of laminin are best candidate for regulating basement membrane formation. differentially synthesized in mouse eggs and early embryos. Dev. Biol. 96, It would agree with previous reports showing that synchronised 467-471. α β α DiPersio, C. M., Hodivala-Dilke, K. M., Jaenisch, R., Kreidberg, J. A. and expression of 6 1 integrin and laminin 1 chain is required Hynes, R. O. (1997). α3β1 integrin is required for normal development of for epithelial cell polarisation and basal lamina development the epidermal basement membrane. J. Cell Biol. 137, 729-742. during kidney tubulogenesis (Klein et al., 1988; Sorokin et al., Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W. and Kemler, R. 1990; Falk et al., 1996). In addition, our data disclose a novel (1985). The in vitro development of blastocyst-derived embryonic stem cell regulatory loop between β1 integrin and laminin α1 chain. lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87, 27-45. Long ago, feed-back regulation was observed for collagen Dziadek, M. and Timpl, R. (1985). Expression of nidogen and laminin in synthesis and fibrillogenesis (Wiestner et al., 1979), and it is basement membranes during mouse embryogenesis and in teratocarcinoma just recently that the α1β1 and α2β1 integrins were shown to cells. Dev. Biol. 111, 372-382. be involved in this regulatory loop (Klein et al., 1991; Schiro Ekblom, P. (1996). Receptors for laminins during epithelial morphogenesis. Curr. Opin. Cell Biol. 8, 700-706. et al., 1991; Langholz et al., 1995). Similarly, fibronectin Falk, M., Salmivirta, K., Durbeej, M., Larsson, E., Ekblom, M., polymerisation is cell-driven and requires active integrins Vestweber, D. and Ekblom, P. (1996). Integrin α6β1 is involved in kidney (Ruoslahti, 1996). The feed-back regulation of matrix protein tubulogenesis in vitro. J. 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