Role of the extracellular matrix in morphogenesis
Introduction
Most cells in multicellular organisms are in contact with an intricate meshwork of interacting extracellular collagens, proteoglycans and adhesion proteins, as well as growth factors, chemokines and cytokines. Together, these components constitute the extracellular matrix (ECM) 1., 2.. Many ECM proteins form large families with some 30 genes identified for collagens and 12 identified for laminins. Additional events, such as alternative splicing, proteolytic processing and glycosylation, increase the number of unique structures and expand the functions of these large, multifunctional molecules. Major recent advances have been made in the identification of new ECM molecules and in the determination of the domain structures and molecular organization of many components. The role of these components in development has also been defined and progress has been made in the identification of structurally and biologically active sites. The amount and type of these components vary considerably in different tissues and usually differ within the same tissue depending on the developmental stage.
ECMs not only provide support, tensile strength and scaffolding for tissues and cells, but also serve as three-dimensional substructures for cell adhesion and movement, as a storage depot for growth factors, chemokines and cytokines, and as signals for morphogenesis and differentiation (Box 1). Cartilage ECM, which is highly enriched in large proteoglycans and collagen II, has an additional unique function in resisting compression. By contrast, basement membrane matrices, which are enriched in the glycoproteins laminin and entactin/nidogen and collagen IV with lesser amounts of proteoglycans and growth factors, regulate cell polarity, separate different tissue types, and have the specialized function of acting as a molecular filter in the kidney.
With the identification of many new ECM family members and elucidation of the atomic structures by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, advances have been made in defining the domain structure and higher order architecture of extracellular components and matrices [3••]. Such approaches have begun to specify the intermolecular interactions important in ECM assembly and biological activity. For example, agrin was found to be important in acetylcholine receptor clustering in the neuromuscular junction and requires laminin-binding to localize to the synaptic basement membrane [4]. The surface-exposed residues on the γ1 laminin chain directly interact with an N-terminal agrin domain.
In general, the biological response involves multiple cellular interactions with individual ECM molecules and with multiple sites within the same molecule; the response is also influenced by the biomechanical properties of the ECM. Multiple ECM signals transmitted via diverse surface receptors are integrated by intracellular signaling pathways to affect the cellular response. The biological activity of the ECM has been studied in vitro using either complex three-dimensional matrices (either cell- or tissue-derived), gels of individual components (collagen or laminin), proteolytic or recombinant fragments of ECM components or peptides. Three-dimensional matrices appear to mimic the in vivo functions of the ECM, and the function of individual components has been further studied with synthetic peptides and recombinant fragments to identify targets for tissue engineering and repair 5.••, 6.•, 7..
Laminin has been the most vigorously studied ECM molecule using this approach and has been analyzed with much success; more than 40 active sites for specific functions have been defined. In addition, laminin and other ECM molecules contain cryptic active sites that are released or become available after proteolytic processing. For example, cleavage of laminin during mammary gland involution releases a fragment that binds to the epidermal growth factor (EGF) receptor and increases cell migration. Similarly, the anti-angiogenic molecules endostatin and tumstatin are degraded products of collagen XVIII and collagen IV, respectively 8.•, 9.. These cryptic sites, as well as the active sites, relate to in vivo functions in development and during remodeling of tissues, as evidenced by the mammary gland.
The biological responses to the ECM are regulated by specific cell-surface receptors [10•]. Many different receptors have been identified that transduce signals to the cytoskeleton and nucleus. These include members of the heterodimeric integrins, receptor tyrosine kinases and phosphatases, immunogloblulin superfamily receptors, dystroglycan, and cell-surface proteoglycans. It is important to note that most of the biologically active ECM molecules, including laminins, collagens, thrombospondin and fibronectin, contain multiple active sites, often for different activities, and interact with different receptors. For example, some 40 active sites have been identified on laminin-1 and 20 different receptors have been characterized.
The importance of ECM molecules in development has been proven by in vivo studies using gene targeting (Table 1) [11]. Mice lacking certain ECM component genes die before birth, whereas others survive and exhibit unique tissue phenotypes. The basement membrane, a critical ECM component during early development, is composed of the trimeric glycoprotein laminin, collagen IV, perlecan, entactin/nidogen, and various growth factors 12., 13., 14.. At the two-cell stage, the first ECM molecule, the laminin γ1 chain, is synthesized and serves as an initial matrix scaffolding (organizer of the ECM). Mice that lack the gene encoding laminin γ1 fail to organize a basement membrane matrix and die at embryonic day 5.5 and stem cells that lack this gene cannot form an epiblast 11., 15.•. It is thought that laminin self-polymerization preceeds and is required for proper basement membrane formation. Mice lacking the laminin α2 chain have a muscular-dystrophy-like phenotype. More than half of the human congenital muscular dystrophies are associated with mutations or loss of the laminin α2 chain. Mice lacking the laminin α3 chain have skin blisters similar to certain bullous diseases. The comparison of perlecan knockout mouse cartilage defects with those of the human genetic disorders Schwartz–Jampel syndrome and Dyssegmental dysplasia (Silverman–Handmaker type), led to the discovery that these disorders were caused by mutations in perlecan [16•]. It is likely that additional genetic diseases will be found to result from mutations in ECM molecules.
The large number of ECM molecules and their interactions with each other define unique biological matrices important in morphogenesis [17]. There is considerable variation in the amount and type of specific components present in ECMs in different tissues and at different stages of development 1., 2.. For example, the basement membrane of the kidney glomerulus has different proportions and types of collagens and laminins from that of the skin basement membrane. Also, the specific laminin isoform in each of these tissues varies during development. In addition, there are different growth factors in each of these tissue ECMs. This review will focus on the role of the ECM in a few organ systems, but it should be noted that all cells are influenced by the ECM. Emphasis will be placed on the role of the basement membrane and its components, as this is an area of active discovery.
Section snippets
Stem cell differentiation
Embryonic stem cells have the capacity to proliferate and to differentiate. In vivo, these cells are contacted by various soluble and insoluble ECM components that influence their differentiation [17]. In vitro studies have shown that ECM components and growth factors regulate the differentiation of stem cells. For example, a feeder layer of fibroblasts is not required if a basement membrane ECM (Matrigel™, containing laminin-1, collagen IV, perlecan, entactin and growth factors) and
Vascular morphogenesis
The ECM is important in both angiogenesis (blood vessel formation from pre-existing vessels) and vasculogenesis (blood vessel formation de novo). During vasculogenesis, endothelial cells interact with ECM components to migrate, proliferate and to form three-dimensional tubular structures 6.•, 20., 21., 22.•. This tubular morphogenesis requires integrin receptors for the ECM components, signaling processes that regulate cell shape through changes in the cytoskeleton, and cell–cell interactions
Organ morphogenesis
Morphogenesis of the salivary gland and other organs, including lung, breast, prostate, pancreas and kidney, is dependent on the multiple activities of the ECM as well as on soluble factors 33.•, 34.••. The embryonic salivary gland is a classic model to study organ morphogenesis ex vivo (Figure 2). The signals from the ECM to the salivary gland epithelium are integrated with signals from growth factors, particularly FGFs. Inhibition of FGF signaling decreases epithelial cell proliferation and
Conclusions and future directions
The ECM has profound structural and biological effects on developmental processes. Studies with in vitro cell and organ culture models include the use of ECM proteins, simple gels (laminin and collagen), complex gels (Matrigel™), and complex three-dimensional matrices (matrices laid down by cells in culture or derived from tissues). Based on the success of the collagen I and basement membrane matrices in vitro and in vivo, it is likely that additional ECMs will be developed that are tissue and
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
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