Mini-Review |
2 Department of Biological Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
Address correspondence to Erhard Hohenester, Department of Biological Sciences, Biophysics Group, Blackett Laboratory, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. Tel.: 44-20-7594-7701. Fax: 44-20-7589-0191. email: e.hohenester{at}imperial.ac.uk
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Abstract |
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Key Words: extracellular matrix; embryo development; mutagenesis; structure determination
Basement membranes (BMs) are cell-associated sheet-like extracellular matrices covering the basal aspect of all epithelia and endothelia and surrounding muscle, fat, and peripheral nerve cells. BMs are essential for tissue formation in all animals. They provide mechanical stability and barriers between different cell types and are critically involved in cell differentiation, survival, and migration. Initially described as a distinct ultrastructure in electron micrographs, BMs are now defined as much by their molecular composition as by their intimate association with cell surfaces (Timpl and Brown, 1996; Erickson and Couchman, 2000; Kalluri, 2003). The major BM proteins and receptors are conserved in Caenorhabditis elegans, Drosophila, and man, although many more isoforms exist in vertebrates (Hutter et al., 2000; Hynes and Zhao, 2000). The first BM protein to be analyzed biochemically was collagen IV, soon followed by the discovery 25 years ago of the major noncollageneous BM glycoprotein, laminin (Chung et al., 1979; Timpl et al., 1979). Today we probably know the identity of most BM proteins (Table I), but many of their activities still remain to be elucidated.
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BM structure and assembly |
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FGF signaling is required for primitive endoderm differentiation and laminin expression (Li et al., 2001). EBs expressing a dominant-negative FGF receptor mutant fail to form BM, epiblast, and cavity. These defects can be partially rescued by the addition of exogeneous laminin, confirming that the primitive endoderm is required for the secretion of laminin, which, in turn, is both necessary and sufficient for subsequent formation of BM, epiblast, and cavity. This interpretation is largely consistent with observations on EBs lacking laminin 1 expression (Murray and Edgar, 2000, 2001). These EBs form primitive endoderm, which continues to differentiate into visceral and parietal endoderm, but they also fail to deposit a BM and do not develop epiblast and cavity.
Although not necessary for BM formation and stability in early development, the other major BM components, including all other laminin isoforms, nevertheless are essential at later stages. Genetic ablation in mice of the collagen 1(IV) and
2(IV) chains (Pöschl et al., 2004), the laminin
5 chain (Miner et al., 1998), and perlecan (Costell et al., 1999; Arikawa-Hirasawa et al., 1999) results in lethality due to multiple severe defects. Lack of the laminin
2 and
3 chains causes, respectively, severe muscular dystrophy (Kuang et al., 1998) and skin blistering (Ryan et al., 1999), both in knock-out mice and in humans afflicted by hereditary diseases. Finally, mice lacking the laminin
4 chain have defective microvessels (Thyboll et al., 2002). In all cases, the phenotypes are largely consistent with the specific expression pattern of the respective BM component and likely result from both compromised BM integrity and the absence of specific instructive cues.
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Lamininreceptor interactions |
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Mice and EBs lacking ß1 integrin gene expression resemble those deficient in the laminin 1 chain (Fässler and Meyer, 1995; Stephens et al., 1995). Mutant mice die at peri-implantation and mutant EBs develop primitive endoderm, but no BM, epiblast, or cavity. However, addition of laminin-1 to ß1 integrin-null EBs restores BM formation, showing that ß1 integrins are not the critical receptors in primary BM assembly (Li et al., 2002). The phenotype of ß1 integrin-null EBs appears to be due to the low level of laminin
1 chain expression by the primitive endoderm (Aumailley et al., 2000; Li et al., 2002). Mice deficient in DG die post-implantation due to impaired development of Reichert's membrane (Williamson et al., 1997), and abnormalities have been described in the BM and cavitation of DG-null EBs (Li et al., 2002).
If neither integrins nor DG are essential, what is the receptor that recruits laminin to nascent BM at the cell surface? The available EB data do not rule out that the functions of ß1 integrins and DG are redundant at this stage of development. However, preliminary studies of mice expressing a truncated laminin 1 chain, lacking the LG4LG5 portion, have shown that these embryos die before gastrulation, i.e., at an earlier stage than the DG-null embryos (Ekblom et al., 2003). This result implies the existence of a critical receptor for the LG4LG5 portion other than DG or integrins (which bind to the LG1LG3 region). Indeed, Li et al. (2002) have shown that a heparin-binding sequence in LG4 is required for laminin-mediated BM assembly in laminin
1-null EBs; this sequence is located at the distal tip of the G domain (Fig. 1). The binding sites for heparin and DG overlap in LG4 of the laminin
1 chain, however, and further experiments with mutant laminins are therefore required to resolve this issue. A recent study of the closely related laminin
2 chain has shown that it is possible to produce mutant laminins that specifically lack only one type of receptor binding site (Wizemann et al., 2003).
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Lamininnidogen interaction |
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The importance of the lamininnidogen interaction for epithelial morphogenesis was first demonstrated by antibody perturbation studies in organ culture (Ekblom et al., 1994). Therefore, it was somewhat surprising that mice lacking nidogen-1 displayed only subtle abnormalities (Murshed et al., 2000; Dong et al., 2002). However, it has now been demonstrated that the more recently discovered nidogen-2 can compensate for the lack of nidogen-1 by the appearance of a severe phenotype of mice null for both nidogens (Nischt, R., personal communication).
The lamininnidogen interaction was addressed directly in an elegant genetic study. Building on previous biochemical mapping studies, Willem et al. (2002) deleted the nidogen-binding LE domain from the laminin 1 chain. Because this deletion did not affect laminin heterotrimer formation and mutant laminin
1 chain was present in BMs of heterozygous and homozygous mice, it reasonably was assumed that the mutant laminins retained their ability to polymerize and bind cellular receptors. Nidogen-1, although expressed normally and present in intact form, was not retained in BMs of most tissues, indicating that its interaction with laminins is indeed important for BM localization. Homozygous mice exhibit multiple defects and die at birth due to incomplete maturation of the lungs, as well as impaired kidney and urinary tract development. The lung defects are characterized by a discontinuous BM between alveolar and endothelial cells, as well as by thickening of the connective tissue surrounding the alveoli. Interestingly, however, branching morphogenesis is not affected. Around 90% of mutant embryos lack one or both kidneys; detailed analysis revealed that the renal agenesis is due to a failure of the Wolffian duct (WD) to elongate. Proper growth of the WD is a prerequisite for the outgrowth of the ureteric bud, which in turn induces the metanephric mesenchymalepithelial conversion, resulting in kidney formation. The mechanism by which the lamininnidogen interaction influences WD growth is unknown, but it is noteworthy that the BM surrounding the tips of the faulty WDs shows subtle discontinuities in mutant embryos.
In addition to the lung and kidney abnormalities, the brain cortex of mutant mice is abnormally laminated and shows multiple ectopias on the surface (Halfter et al., 2002). The brain defects arise from the disruption of the pial BM. This BM covers the brain and serves as the attachment site for radial glia cells, which, in turn, provide a scaffold for neuroblasts migrating toward the pial surface. Because the pial BM is fragile, radial glia cells retract; Cajal-Retzius cells are misplaced or lost; and cortical plate neurons migrate abnormally, either passing through the meninges to form ectopias or terminating their migration prematurely. Similar defects have been observed in mice lacking perlecan (Costell et al., 1999), collagen IV (Pöschl et al., 2004), ß1 integrins (Graus-Porta et al., 2001), or DG (Moore et al., 2002). Thus, multiple cellmatrix interactions appear to be essential for the pial BM to resist mechanical forces, as they occur during brain vesicle expansion in development.
The specific deletion of the nidogen binding site on the laminin 1 chain impressively demonstrates how molecular structurefunction analyses can guide genetic studies, leading to much sharper experimental tools compared with the blunt instrument of complete gene knock-outs. Although nidogen binding is the only known activity of this region of the laminin
1 chain, the remote possibility remains that the observed defects are due to the disruption of other, currently unidentified activities. Such problems could be circumvented by the introduction of suitable point mutations, rather than domain deletions. The pioneering work of Rupert Timpl's laboratory in mapping BM protein interactions at the atomic level should prove to be a treasure chest for such future genetic studies of BM function.
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Acknowledgments |
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E. Hohenester is a Wellcome Senior Research Fellow.
Submitted: 13 January 2004
Accepted: 13 February 2004
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