Article |
Address correspondence to Peter D. Yurchenco, Dept. of Pathology and Laboratory Medicine, UMDNJ, Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854. Tel.: (732) 235-5166. Fax: (732) 235-4825. E-mail: yurchenc{at}umdnj.edu
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Abstract |
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Key Words: basement membrane; gastrulation; integrin; dystroglycan; apoptosis
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Introduction |
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The first basement membranes to form during mouse embryonic development are those located between visceral endoderm and developing epiblast, and underneath the parietal endoderm (Reichert's membrane), which extends over the trophectoderm (Leivo et al., 1980). Although primitive endodermal cell differentiation precedes basement membrane assembly, epiblast differentiation and proamniotic cavitation require and follow it (Murray and Edgar, 2000; Murray and Edgar, 2001a, b). In this study, we examined wild-type, 1-lamininnull, ß1-integrinnull, and dystroglycan-null differentiating EBs. We report that the integrin- and laminin-deficient cells are unable to form basement membranes or undergo epiblast differentiation and cavitation because, in both states, they fail to express heterotrimeric laminin. Exogenous laminin bypasses the defect in each null embryoid body, restoring basement membrane along with epiblast differentiation and cavitation. This activity requires participation of long arm laminin LG modules that include a critical heparin-binding sequence as well as polymerization mediated by the three short arms. Strikingly, neither integrin nor dystroglycan is uniquely required for basement membrane assembly. Instead, they are necessary for regulation of their own expression, that of major basement membrane components, and cell differentiation. Finally, dystroglycan and integrin promote epiblast survival.
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Results |
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Wild-type and ß1-integrinnull EBs treated with exogenous laminin-1 were examined for ß1- and 6-integrin expression (Fig. 1, E and F). These integrin chains were localized in differentiated wild-type EBs in the epiblast and in the basement membrane zone. In contrast, the integrin-null EBs, regardless of treatment, failed to express and accumulate
6-integrin. ß1-integrin levels in dystroglycan-null EBs were similar to those detected by immunoblotting in the wild-type state (and were absent in the integrin-null state), suggesting that integrin does not compensate for loss of dystroglycan by up-regulation.
Contributions of laminin polymerization and LG modules
Incubation of 1-lamininnull EBs with exogenous laminin-1 results in basement membrane formation, epiblast differentiation, and cavitation (Murray and Edgar, 2000), providing a basis to analyze laminin domain contributions. We evaluated (a) the three short arms that mediate laminin polymerization as well as
1ß1,
2ß1 integrin, and heparin binding and (b) G-domain that mediates
6ß1,
7ß1 integrin-, heparin/heparan sulfate, and
-dystroglycan binding (Colognato and Yurchenco, 2000). To assess polymerization and discriminate it from other short arm functions, we determined the ability of nonpolymerizing laminin-1, prepared by treatment with the selective polymer-inactivating agent aminoethyl benzene sulfonyl fluoride (AEBSF; Colognato et al., 1999), to assemble a basement membrane in
1-lamininnull EBs (Fig. 2 and Table I) and evaluated the activity of laminin-1 maintained in the presence of fragments that selectively inhibit polymerization through either the
1/
1 (E1', AEBSF-treated E1' as noninhibiting control) or the ß1 (E4) short arms. Neither a basement membrane immunostaining pattern of laminin and type IV collagen nor epiblast formation was detected after inhibition of laminin polymerization, regardless of reagent used. This inhibition was considered specific because the only activity of E4 is polymerization inhibition, and the cell adhesion, heparin-binding, and nidogen-binding properties of laminin and E1' are not found affected by AEBSF treatment (Colognato et al., 1999; unpublished data).
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After treatment with either laminin-4 or laminin-2/4, laminin, type IV collagen, nidogen, and perlecan were detected in a subendodermal linear pattern accompanied by epiblast differentiation and cavitation (Table I). In contrast, laminin-8 (4ß1
1) failed to induce basement membranetype immunostaining, epiblast differentiation, or central cavitation. Laminin-5 (
3Aß3
2) adhered to the outer surface of the EBs (image not depicted); however, laminin-5,
1-laminin,
3-laminin, and nidogen did not accumulate within the basement membrane zone. Although a weaker and discontinuous linear subendodermal pattern of type IV collagen and perlecan was noted, no ECM was detected by electron microscopy. Thus, of the laminins tested, only
1- and
2-laminins, both polymerizing laminins, were capable of inducing basement membrane.
Site of activity in an LG module
To determine whether heparin/dystroglycan binding within LG module 4 is required for basement membrane, we inactivated the KRK (residues 27912793) sequence common to both by alanine substitution (Andac et al., 1999) in recombinant LG45 and determined its ability to block laminin-1 rescue of Lm-1null EBs (Fig. 3). The mutant LG45 showed substantially reduced binding by heparin affinity chromatography (Fig. 3 A), eluting at 0.16 M NaCl compared with 0.26 M for wild-type protein. In contrast to its recombinant control, the KRK mutant protein was largely unable to block laminin rescue of the Lm-
1null phenotype (Fig. 3, B and C). In keeping with this result, 0.1 mg/ml heparin completely prevented laminin-1 induction of basement membrane. We concluded that this particular surface-exposed triplet basic sequence (Fig. 3 D) plays a critical role in basement membrane assembly, likely contributing to anchorage.
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Dystroglycan expression
Examination of -dystroglycan immunofluorescence of early and differentiated wild-type EBs revealed that
-dystroglycan was initially diffusely distributed throughout the ICM in a pericellular pattern, but then redistributed to the basement membrane zone after basement membrane formation (Fig. 8). The latter pattern appeared to be largely confined to the epiblast aspect of the zone (this was particularly evident if the epiblast layer became detached from basement membrane during sectioning). No staining, as expected, was observed in dystroglycan-null EBs. The pericellular dystroglycan immunostaining intensity was greater in ß1-integrin and
1-lamininnull EBs compared with wild-type EBs (Fig. 8 A). After laminin-1 treatment of the integrin-null EBs, dystroglycan became redistributed to the basement membrane zone, whereas the staining intensity remained high. Laminin-1 treatment of the
1-lamininnull EBs similarly caused redistribution of dystroglycan to the basement membrane zone; however, the staining intensity was now decreased.
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Alterations of basement membrane component synthesis and accumulation
The expression and accumulation of basement membrane components were evaluated (Fig. 9). Loads of all fractions for analysis were normalized to total protein present in each EB extract. Conditioned medium protein contained the ECM components that accumulated into a final "pool" in transit from the EB, resulting either from turnover or cell death. The EB cell lysate, containing 10% of total endogenous basement membrane proteins present in each culture, represented the material that accumulated in basement membrane and cell (most epitopes were present within the basement membrane zone as determined by microscopy). When EB extracts or conditioned media were immunoprecipitated with laminin
1, ß1, or
1 chainspecific antibodies, no heterotrimeric laminin-1 was detected in the
1-lamininnull state. In addition to the expected absent
1 chain, the
1 chain was absent, possibly a consequence of degradation. In ß1-integrinnull EBs, laminin chains were not detected with EHS-lamininspecific antibody after precipitation with
1 subunitspecific antibody from either medium or cell lysates. However, immunoprecipitation of ß1-integrinnull cell extracts with either laminin ß1 or laminin
1specific antibody revealed an incompletely resolved ß/
doublet. We concluded that these chains, present in low amount, are present within the endodermal cell cytoplasm because no basement membrane formed in these EBs, whereas weak diffuse intracellular endodermal staining could be detected. These data support and extend the conclusions of Aumailley et al. (2000) but do not support those of Lohikangas et al. (2001) i.e., laminin
1 expression is selectively absent in ß1-integrinnull EBs. It follows that the block to assembly is due to the laminin expression defect rather than to a role of ß1-integrin in the polymerization of laminin or in its cell surface receptormediated assembly.
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Discussion |
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ß1-Integrin functions
The ability of exogenous laminin-1 to rescue the integrin-defect with restoration of basement membrane formation and epiblast development indicates that the early differentiation block in ß1-integrinnull EBs is due to the failure of laminin 1chain expression (either transcriptional or posttranscriptional) and is not at the level of basement membrane anchorage and assembly. This contribution may be specific for laminin-
1 because similar regulation has not been observed for Schwann cell basement membranes lacking this integrin subunit (Feltri et al., 2002). ß1-Integrin was not required for the integration of type IV collagen, nidogen, or perlecan into the basement membrane, suggesting that their incorporation into a laminin scaffold is mediated either directly through laminin interactions or through novel cell surface molecules. Furthermore, the data show that ß1-integrin, once the laminin synthesis block is bypassed, is not required for epiblast differentiation and cavitation, although it is essential for mesodermal differentiation. Although other ß-integrins might compensate for the missing ß1 subunit, there is no obvious candidate. None of the known laminin-interacting integrins (
6 and therefore ß4) were detected in ß1-integrinnull ES cells and/or EBs.
Although integrin compensation was not detected, dystroglycan was substantially overexpressed in ß1-integrinnull EBs. Laminin induced a topographical redistribution of dystroglycan such that it localized to sites in the basement membrane zone; however, it did not restore normal dystroglycan levels. Dystroglycan was similarly overexpressed in the 1-lamininnull EBs, suggesting that laminin is required to maintain normal dystroglycan expression. The hypothesis was supported by the finding that exogenous laminin mediated both the correct basement membrane zone localization and normalization of dystroglycan expression. Furthermore, treatment of
1-lamininnull EBs with E8 (ligand for
6ß1 integrin) abrogated laminin-mediated dystroglycan down-regulation. These data not only show that dystroglycan expression and localization is regulated by laminin and ß1-integrin, but also suggest that this regulation requires the direct ligation of laminin G-domain within a polymer to the integrin.
Role of a heparin-binding site in LG4
6ß1,
7ß1, and
6ß4 integrinbinding sites are located in LG modules 13, whereas heparin/heparan sulfate, sulfatide, and
-dystroglycanbinding sites are located within LG module-4. A third cell-interactive domain, capable of binding to heparin and
1ß1 and
2ß1 integrins, and located within the LN domain of the
1 subunit (Colognato-Pyke et al., 1995; Colognato et al., 1997), was not found to participate in embryonic basement membrane assembly. In EBs, LG module 4 was found to be required for basement membrane assembly, which is consistent with the concept that it provides for the key anchorage to the cell surface. We examined this further by alanine mutagenesis of an important heparin/dystroglycan-binding sequence, EYIKRKAF, located between inter-ß strand loops H and I of LG4 (Tisi et al., 2000). We found that the mutation, which substantially decreased heparin-binding, inactivated LG45 inhibition of assembly. The possibility that dystroglycan was an essential receptor for assembly, mediated through this site, was ruled out because dystroglycan-null EBs spontaneously formed basement membrane. This in turn suggests that the critical LG4 interaction is mediated by a heparan sulfate proteoglycan, or possibly by a sulfatide. However, an important remaining question is whether laminin anchorage and basement membrane assembly can occur in the absence of both integrin and dystroglycan, requiring only LG4 heparin-type binding and polymerization. Because integrin and dystroglycan may provide some of the anchorage activity themselves, it is possible that a minimum of two of the three binding sites in G-domain are required. Alternatively, the heparin-site may provide sufficient anchorage in the absence of either receptor. Resolution of this question will require further experimentation.
Role of dystroglycan
Our study has shown that dystroglycan is not required for formation of the developmentally critical basement membrane between endoderm and epiblast. This conclusion isseemingly in disagreement with the Henry and Campbell (1998) article. In that analysis, it was reported that basement membranes failed to form in dystroglycan-null EBs, and it was therefore suggested that dystroglycan is essential for basement membrane assembly. However, this could not represent a general receptor requirement as was implied because knockout of the dystroglycan gene in mice is characterized by a loss of Reichert's membrane, but not a loss of the embryonal basement membrane adjacent to epiblast (Williamson et al., 1997). Furthermore, the skeletal muscle of dystroglycan-deficient chimeric mice has been found to possess basement membrane (Cote et al., 1999). Of note, neither the wild-type nor dystroglycan-null EBs used in the study of Henry and Campbell developed epiblast and only 1% of EBs cavitated (both central attributes of embryonic differentiation), making unclear what step of differentiation was modeled. Together, we conclude that dystroglycan is not a fundamental requirement for basement membrane assembly in tissues.
A striking finding in our analysis was the loss of the epiblast layer through apoptosis. It has previously been observed that only those cells that adhere to basement membrane survive to differentiate with the nonadherent cells undergoing anoikis (Coucouvanis and Martin, 1995). However, continued survival of the epiblast was clearly dependent upon a dystroglycan interaction. This receptor dependency was significantly greater than that which we observed in the laminin-rescued integrin null, and a general survival role for dystroglycan is supported by in vitro studies conducted on muscle cells (Montanaro et al., 1999). The survival deficit seen in both receptor nulls raises the possibility that cell adhesion strength determines survival regardless of the specific receptor involved, and that the observed difference in survivability is due to asymmetric compensation in which only the integrin-null loss of receptor binding is largely replaced by high cell surface expression of dystroglycan.
EBs lacking 1-laminin, ß1-integrin, or functional FGF receptors fail to express essential laminin subunits, fail to form a basement membrane, and fail to differentiate (Li et al., 2001, and this study). In each case, assembly and differentiation could be rescued with exogenous laminin-1, strongly suggesting that lack of extracellular laminin, rather than a problem with cell surface ability to mediate assembly, caused the defect. During development, laminin expression became restricted to the zone underneath the endodermal layer, the major source of laminin synthesis and secretion (Murray and Edgar, 2001a). This step requires FGF signaling and ß1-integrin. Interestingly, the findings of Li et al. (2001) argue that laminin is both necessary and sufficient to mediate epiblast differentiation in the absence of endoderm. Our data provide evidence for a mechanism in which laminin must both polymerize through its LN domains (Yurchenco and Cheng, 1993) and interact with the cells of the ICM through a heparin-binding sequence in LG4 to initiate site-specific basement membrane assembly and to trigger differentiation. The new findings also argue that the laminin polymer creates the initial architectural scaffolding that must assemble before other components can accumulate into the ECM, and that is crucial for cellular differentiation.
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Materials and methods |
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Proteins and antibodies
Laminin-1 (DEAE-unbound fraction) and laminin fragments E1' (short arm complex), E3 (1-LG modules 45), E4 (ß1-domains VI and V), E8 (lower coiled-coil with LG13), and C14 (polymerizing
1ß1
1 short-arm complex) were prepared from the mouse EHS tumor as described previously (Yurchenco and Cheng, 1993; Yurchenco and O'Rear, 1994). Nonpolymerizing laminin-1 was prepared by treatment with 5 mM AEBSF in 50 mM Tris-HCl and 90 mM NaCl, pH 7.4, in the cold overnight (Colognato et al., 1999). AEBSF-E1' (nonpolymerization inhibition control) was prepared by incubation of E1' under the same conditions followed by dialysis to remove AEBSF. Laminin-2/4 and laminin-4 were prepared from collagenase-treated human placenta as described previously (Cheng et al., 1997). Recombinant laminin-5 (
3Aß3
2), produced in transfected HEK-293 cells, was a gift of Dr. Ariel Boutaud (BioStratum Incorporated, Research Triangle Park, NC). Reducing SDS-PAGE revealed 150-kD (
3), 140-kD (ß2), and 105-kD (
2) bands. Recombinant laminin-8 (
4ß1
1) was prepared as described previously (Kortesmaa et al., 2000).
Rat monoclonal antilaminin 1 (clone A5; Upstate Biotechnology), rabbit antimouse type IV collagen antibody (Rockland Immunochemicals), rat antimouse perlecan mAb, and rabbit antimouse type I collagen antibody (CHEMICON International, Inc.) were used for immunostaining at 1, 2.5, 2, and 2.5 µg/ml respectively. Rabbit pAbs specific for laminin-1, E4 (ß1 subunit), mouse laminin-1 RG50 (
1 LG 45) fractionated from recombinant G-domain were prepared and characterized as described previously (Yurchenco and Ruben, 1987; Handler et al., 1997; Yurchenco et al., 1997). E4 and RG50 antibodies were used for immunoprecipitation at 10 µg/ml and EHSlaminin-1 antibody was applied on immunoblots at 3 µg/ml. Rabbit polyclonal nidogen-specific antibody was generated with purified EHS-nidogen, affinity-purified with immobilized nidogen and cross-absorbed with laminin, and used at 3 (immunoprecipitation) and 1 µg/ml (immunoblotting, immunofluorescence). Mouse monoclonal IgM antibody IIH6 hybridoma medium specific for
-dystroglycan (Ervasti and Campbell, 1991), a gift of Kevin Campbell (Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa), was used as conditioned hybridoma medium at 1:2 dilution. Mouse mAb specific for ß-dystroglycan (Novocastra Laboratories Ltd) was used at a dilution of 1:100. Rat antimouse integrin ß1-chain mAb (2 µg/ml for immunoblotting and 5 µg/ml for immunofluorescence), and hamster antimouse integrin ß3-chain mAb (5 µg/ml for immunofluorescence) were obtained from BD Biosciences.
FITC- and Cy5-conjugated antibodies specific for mouse IgG, mouse IgM, and rabbit IgG (Jackson ImmunoResearch Laboratories) were used at 1:100 dilutions. HRP-linked antibodies specific for mouse IgG, rat IgG, and rabbit IgG (Amersham Pharmacia Biotech) were used as secondary antibodies for immunoblotting at a dilution of 1:3,000.
Sample preparation
EBs were collected into 10-ml tubes and allowed to sediment by gravity. After washing in PBS with 0.5% BSA, the EBs were fixed with 3% paraformaldehyde in PBS and followed by incubation in 7.5% sucrose-PBS for 3 h at room temperature and then in 15% sucrose-PBS at 4°C overnight. The EBs were embedded in Tissue-Tek OCT (Miles, Inc.) and 4-µm-thick frozen sections were prepared. Nonspecific binding sites were blocked with 5% goat serum. FITC- and/or Cy5-conjugated antibodies were used as secondary reagents and nuclei were counterstained with DAPI.
Microscopy
Slides were viewed by indirect immunofluorescence using an inverted microscope (model IX70; Olympus) fitted with an IX-FLA fluorescence observation attachment and a MicroMax 5-mHz CCD camera (Princeton Instruments) controlled by IP Lab 3.0 (Scanalytics). EBs were allowed to settle in 15-ml conical tubes, and then washed with PBS by resuspension/settling. The cell pellet was fixed in 0.5% glutaraldehyde and 0.2% tannic acid in PBS for 1 h (room temperature), washed with 0.1 M sodium cacodylate buffer, transferred to modified Karnovsky's fixative, post-fixed in 1% osmium tetroxide for 1 h, and then dehydrated and embedded in Epon/SPURR resin (EM Science). Thick (1 µm) and thin sections (90 nm) were cut with a diamond knife on an ultramicrotome. Thick sections were stained with 1% methylene blue in 1% sodium borate for light microscopy, and thin sections were stained with saturated uranyl acetate followed by 0.2% lead citrate. Images were photographed with an electron microscope (model JEM-1200EX; JEOL USA, Inc.).
TUNEL staining.
Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling (Promega). EB cryosections were washed in PBS, fixed with 3% paraformaldehyde in PBS for 30 min, and permeabilized with 0.2% Triton X-100 in PBS. DNA fragments were end-labeled with 0.5 U/ml terminal transferase and 5 mM fluorescein 12-dUTP for 1 h at 37°C. Slides were washed twice in 2x SSC followed by three washes in PBS. EBs were immunostained for laminin and counterstained with DAPI.
Protein assays
Protein in solution was determined either by absorbance at 280 nm or the Bradford assay (Bio-Rad Laboratories). SDS-PAGE was performed in 3.512% linear gradient gels and electrophoretic transfer of proteins onto PVDF membranes was performed as described previously (Yurchenco and Cheng, 1993; Cheng et al., 1997). Blots were blocked with 5% nonfat dried milk and 0.2% Tween 20 in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, and then incubated with primary antibody followed by antibody-HRP. Reacting bands were detected by ECL (Amersham Biosciences). Immunoprecipitation (IP) was performed at 4°C with the addition of protease inhibitor cocktails (Sigma-Aldrich) to all the protein samples and buffers. EB-conditioned medium or lysates were precleared with 20 µl of 50% protein Aagarose (pAb IP) or protein GSepharose bead slurry (mAb IP). Samples were incubated with antibody overnight and precipitated with 40 µl protein Aagarose or protein GSepharose beads for 2 h and followed by washing in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, and 0.1% SDS. After an additional wash, the supernatant was removed and the immunoprecipitates were analyzed by SDS-PAGE. Duplicates of type IV collagen antibody immunoprecipitates were incubated with 5 U bacterial collagenase (CLSPA; Worthington Biochemical Corporation) at 37°C for 1 h. After collagenase digestion, the immunoprecipitates were washed twice in PBS and analyzed.
Semi-quantitative RT-PCR
Total RNA was isolated with TRIzol® reagent (Life Technologies) and reverse-transcribed to cDNA using SuperScriptTM II reverse transcriptase (Life Technologies). The primers and PCR annealing conditions for brachyury, BMP4, low molecular weight NFL, -globulin, and hypoxanthine guanine phosphoribosyl transferase (HPRT) were described previously (Levinson-Dushnik and Benvenisty, 1997; Rohwedel et al., 1998; Weinhold et al., 2000). PCR products were electrophoretically resolved on 2% agarose gels.
Production of recombinant E3 and its mutant
Laminin-1LG45 was amplified by PCR from a mouse laminin
1 chain cDNA. BM40 signal sequence and a FLAG epitope were introduced into the 5'-end of the cDNA fragment. The heparin/dystroglycan-binding site KRK in LG4 was replaced with AAA via PCR-based mutagenesis as described previously (Andac et al., 1999). Both wild-type and KRK mutant were cloned into mammalian expression vector pcDNA3.1/Zeo+ (Invitrogen) and the sequence of the inserts was confirmed by automated sequencing. The constructs were expressed in HEK 293 cells and stable clones expressing wild-type or the mutant E3 were selected with ZeocinTM. Recombinant LG45 proteins were purified to homogeneity by FLAG affinity chromatography (Yurchenco et al., 1997).
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Footnotes |
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Acknowledgments |
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This study was supported by National Institutes of Health grant DK36425 (P.D. Yurchenco) and grants from the Center for Molecular Medicine Cologne and DFG Basement Membrane Study Program (to N. Smyth).
Submitted: 15 March 2002
Revised: 6 May 2002
Accepted: 7 May 2002
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References |
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