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Receptors for laminins during epithelial morphogenesis
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Receptors for laminins during epithelial morphogenesis Peter Ekblom Laminin-1 is expressed by many embryonic epithelial cell types. It binds to receptors on the epithelial cell surface. The integrin a6131 is a well known laminin-1 receptor that is expressed on many embryonic epithelial cells. More recently, dystroglycan was discovered as a high-affinity receptor for laminin-1 and laminin-2. It is expressed not only by muscle cells but also by embryonic epithelial cells. In embryonic epithelia, dystroglycan may act by binding to the E3 fragment of laminin-l. Integrins and the dystroglycan complex seem to be important for epithelial morphogenesis, but the relative roles of these two receptor systems for epithelial cells are still unclear.
Addresses Department of Animal Physiology, Uppsala University, Biomedical Center, Box 596, Husargatan3, S-751 R4 Uppsala, Sweden; e-maih [email protected] Current Opinion in Cell Biology 1996, 8:700-706 © Current Biology Lid ISSN 0955-0674
Introduction Epithelial cells are firmly attached to the basement membrane in the adult. Long before individual basement membrane components had been identified, embryonic studies using electron microscopy revealed that basement membranes form very early during epithelial morphogenesis [1,2]. These and many other studies led to the widely held belief that basement membranes are required for the initial stages of epithelial development and for the maintenance of the morphology of the epithelial sheets [1]. Laminin-1 is expressed by many epithelial cell types, and binds with high affinity to mesenchyme-derived nidogen. This interaction may be crucial for basement membrane assembly. Laminin-1 also binds to epithelial cell surface receptors such as ~6131 integrin. This review will describe current knowledge about the nature of the laminin isoforms that are expressed by embryonic epithelial cells. In addition, recent results suggesting that laminin-1 is important for embryonic development of epithelial cells will be covered. Finally, the importance of different laminin receptors for epithelial morphogenesis will be discussed. The potential importance of a recently described laminin receptor, dystroglycan, for epithelial morphogenesis is emphasized. E p i t h e l i a l b a s e m e n t m e m b r a n e a s s e m b l y in the extracellular space Although different basement membranes are fairly heterogeneous molecularly, several features are common to most basement membranes. All basement membranes are composed of two fairly independent protein sheets,
the collagen IV and the laminin networks, which are bound together by nidogen (also called entactin) and possibly by other not yet identified components. The nature of the collagen IV and laminin isoforms varies from tissue to tissue. T h e assembly during embryogenesis of basement membranes, which are huge protein networks, is extremely complex. Nevertheless, some basic principles of the assembly process are emerging [2,3"]. One principle, discovered largely by immunolocalization and localization of mRNAs in tissue sections, is that epithelial basement membranes form as a result of epithelial-mesenchymal interactions [4",5"]. Some components, such as the laminin 131 chain [6], are produced by both cells during epithelial morphogenesis, whereas some basement membrane components are produced exclusively by the mesenchyme. A more strict mesenchymal origin of nidogen seems to hold for most tissues in which epithelial morphogenesis occurs [6-8] and is of particular interest as there is some evidence that the binding of nidogen to the laminin y1 chain [3"] is required for epithelial morphogenesis [8]. Expression of laminin isoforms during epithelial development More than ten individual laminin chains have already been discovered and they can form a large number of different heterotrimers [9,10,11", 12]. Many of the newly discovered laminin isoforms have, so far, been characterized mainly at the primary sequence level, and it remains to be studied where they are expressed during embryogenesis [9,10,11°]. For many of the first described chains, much more is known, but a comprehensive catalogue of their expression patterns can nevertheless not be provided [12]. In many cases, only a few tissues and a few developmental stages have been analyzed. Thus, the simple rules of expression provided here will be refined as more data become available. It is important to note that cells can probably produce many laminin heterotrimers simultaneously. T h e laminin 131 and y1 chains probably have the broadest tissue distribution of the laminin chains (being found in mesenchyme, epithelium, endothelium and muscle) and it is likely that most embryonic epithelial cells produce these chains [6,13,14]. Of the laminin ~ chains, the ¢tl, ~3 and c~5 chains seem to be prominently expressed in epithelium, whereas the c~2 and c~4 chains are not [9,10,15]. The a2 chain is found in mesenchyme, muscle and peripheral nerves [15], and the ~4 chain mRNA is restricted to certain mesenchymal cells such as smooth muscle and dermis [10]. Of the 'epithelial' ~ chains, the ~,3 chain is prominently expressed in skin basement membranes, as part of laminin-5. T h e ~3 chain mRNA is also expressed in the epithelium of salivary gland and tooth, but only at later developmental stages [9]. For a more comprehensive discussion of the o~3 laminin chain,
Receptors for laminins during epithelial morphogenesis Ekblom
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the reader is referred to Borradori and Sonnenberg, this issue, pp 647-656.
shown to alter epithelial cell polarity in an epithelioid cell line.
Curiously, there are still some controversies concerning the expression pattern of the laminin otl chain although it was the first described ct chain. Most investigators agree that the laminin c~l chain is found in many epithelial basement membranes [12], but it is debatable whether it is also present in other basement membranes. By immunofluorescence, using well characterized polyclonal and monoclonal antibodies against the E3 fragment of laminin-1 (which contains the laminin o~1 chain), the (x1 chain was localized to epithelial basement membranes of several mouse embryonic tissues [13,16-18]. These studies agree well with in situ hybridization results which revealed a limited expression of the c~l chain mRNA in the epithelium or in cells closely associated with the epithelium during mouse [6,7,18,19 °] and human [15] embryogenesis. A further expression site was found to be the human embryonic central nervous system [15]. In contrast, a broader expression pattern of the laminin ~1 chain, with expression also occurring in endothelial basement membranes, has been noted by immunofluorescence by a large number of investigators, who were mainly working with adult human tissues and some other species [20,21°,22,23°]. These differences could be due to species differences, different stages being analyzed, or the fact that the different reagents react with different molecules. It will be important to clarify this issue, and here attention should be paid to a further characterization of the reagents used.
Among the organs forming by means of epithelialmesenchymal interactions, the developing kidney is unique in the sense that there is a conversion of mesenchyme to epithelium [24]. T h e conversion is accompanied by the appearance of a new basement membrane, and it is thus a good model in which to study the development of basement membranes. In developing kidneys, the Ill and ~'1 chains are fairly widely expressed, as they are in mesenchyme. In contrast, laminin a l chain expression is specific for epithelial cells [16-18]. Antibodies reacting with the E8 or E3 fragment of laminin-1 have been shown to inhibit the development of mesenchyme into epithelium in organ cultures of embryonic kidneys, suggesting that these domains are important for epithelial cell polarization [16,17].
T h e recently discovered laminin c~5 chain of mouse has a broad tissue distribution in adult tissues, based on Northern blot analysis [11"]. In situ hybridization of mouse embryonic tissues has revealed that the c~5 chain is found prominently in embryonic epithelia in addition to in some muscle tissues (M Durbeej et al., unpublished data). Only a few embryonic endothelial cell types seemed to produce this chain. Interestingly, the (~5 chain was found prominently in those epithelial cell types that lacked the ecl chain, and vice versa, and only a few locations with an overlapping expression pattern were noted in the embryo (M Durbeej et al., unpublished data).
Laminin-1 and organ and cell culture s t u d i e s Although it is still unclear how broadly the c~l chain is expressed during embryogenesis or in adulthood, most investigators agree that the c~l chain is expressed by embryonic epithelial cells, suggesting that it is important for epithelial morphogencsis (see above). This view is supported by several in vitro studies. First, by adding antibodies against different domains of laminin-1 in organ cultures of embryonic tissues, epithelial morphogenesis has been perturbed in many culture systems. Second, by culturing embryonic epithelial cells on laminin-1, differentiation has been enhanced. Third, stable transfection of antisense constructs for the laminin c~l chain has been
In the developing salivary gland, the expression pattern of the col laminin chain is somewhat different from in the developing kidney. T h e c~l chain mRNA is transiently expressed by the salivary gland mesenchyme adjacent to the epithelial stalk at early stages of development [25]. A faint expression is also seen transiently at the tips of the branching epithelium. At later stages, no laminin col chain mRNA can be seen in salivary glands. Instead, the ~c5 chain can be detected in the epithelium at later stages (M Durbeej et al., unpublished data). The E3 fragment of laminin-1 might be important at the very early stages of branching morphogenesis of the salivary gland; monoclonal antibodies against the E3 fragment were found to perturb branching morphogenesis in organ cultures of embryonic salivary glands [25]. In the embryonic lung, laminin c~l chain mRNA is also synthesized by a thin layer of mesenchyme close to the stalk of the branching epithelium, and by the branching epithelial tips during early stages of embryonic lung morphogenesis [6,7]. So far, there have been no reports of using monoclonal antibodies against the E3 fragments in organ cultures of embryonic lungs. T h e E8 fragment, however, has been suggested to be involved in later developmental stages of lung alveolar morphogenesis [26]. Furthermore, fragment P1 of laminin-1, consisting of the inner part of the three disulphide-bonded short arms, has been suggested to be required for the early stages of branching epithelial morphogenesis of the lung [27]. Another approach was used to study the role of laminins in the development of retinal neuroepithelium [28]. Dissociated neuroepithelial cells from chicken embryos were cultured on different types of laminin substrata. The cells were found to survive, proliferate and differentiate into neurons when cultured on laminin-1, whereas they did not survive or differentiate well on Schwannoma-derived laminin, which has an oc2 chain and is devoid of c~l chain. The neuron-stimulating activity of laminin-1 was found to
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reside on the E8 fragment and the authors stressed the importance of the carboxy-terminal end of the laminin a l chain for neuroepithelial differentiation [28]. The antisense approach has been used to study the role of the laminin a l chain in gut development [29°]. A cDNA fragment of the cxl chain in the antisense orientation was stably transfected into a human colon cancer cell line. These cells normally form epithelial structures when cocultured with fibroblast cells. Absence of c~l chain expression caused by the antisense construct led to an incorrect secretion of the 131 and T1 chains, to a lack of basement membrane formation, and to alterations in the epithelial cell polarity [29°].
Receptors for laminin-1 on embryonic epithelial cells If laminin-1 is important for epithelial cell development, cell surface receptors for it should exist on embryonic epithelial cells. Initial efforts to find such epithelial receptors some ten years ago concentrated on a 68 kDa protein [30]. However, this protein might be an intracellular protein and it is apparently not particularly well enriched in epithelial cells [30,31]. It is thus possible that this protein is not a major laminin receptor for epithelial cells in vivo. Subsequently, integrins emerged as more likely laminin receptors [32]. Two epithelial laminin-1 receptors, integrins ot6151 and oc6154, have been well characterized [32-34], and a role for integrin ot6134 in the attachment of epidermal cells to basement membrane is supported by many different types of studies (see Borradori and Sonnenberg, this issue, pp 647-656). Integrin ct6151 might be important for other epithelial cells. Nevertheless, recent studies raise the possibility that a protein first discovered from muscle membranes, dystroglycan, could act as a major laminin-binding receptor for epithelial cells also. Integrin (x6131during branching epithelial morphogenesis Several cultured cell lines use the ot6151 integrin to bind to the E8 fragment of laminin-1 in vitro. The ~6 integrin subunit was initially identified by use of a monoclonal antibody that reacts with the cell surface of epithelium and endothelium in tissue sections [32]. The integrin a6 subunit is detected on the surface of embryonic kidney epithelium in vivo [35,36]. In the embryonic kidney, immunofluorescence staining has demonstrated a coappearance of the integrin a6 subunit and laminin ~1 chain during conversion of mesenchyme to epithelium [36]. The immunolocalization studies of both embryonic and adult tissues are compatible with the view that integrins containing the cx6 subunit act as receptors for laminins in vivo also (see Fig. 1). The integrin cx6 subunit is known to associate with either the 151 or the 134 integrin subunit to form the laminin-binding integrins a6151 and cx6154[32-34]. In embryonic kidney tubules, the a6 integrin subunit associates with the 151
subunit rather than with the 134 subunit [37]. Antibodies against the a6 subunit partially inhibit kidney tubulogenesis in organ culture [36]. Antibodies against the integrin or6 subunit and against the integrin 1~1 subunit have also been reported to inhibit epithelial morphogenesis [25,36]. These studies can be considered to provide strong evidence that the a6151 integrin is important for branching epithelial morphogenesis. However, mice lacking the o~6 integrin subunit undergo normal embryogenesis and defects are seen in skin at birth [38°°]. This striking finding suggests either that other receptors for laminins are more important than integrin ot6131 during embryonic epithelial morphogenesis, or that other laminin-binding integrins take over the role of integrin a6 subunit during embryogenesis when integrin a6 is missing. T h e integrin 151 subunit is probably associated with many other integrin o~ chains in developing epithelia, and some might bind iaminin-1.
Dystroglycan during branching epithelial morphogenesis Although integrins have long been considered to be the main receptors for basement membrane components, other laminin receptors may exist. In muscle, dystroglycan links the cytoskeleton to the basement membrane [39]. T h e dystroglycan complex includes the extracellular Qt-dystroglycan, which binds to E3-1ike G domains of laminin-2 in muscle, and five transmembrane proteins, namely 15-dystroglycan, oc-sarcoglycan, 15-sarcoglycan, T-sarcoglycan and a 25 kDa protein [39]. The 15-dystroglycan binds to the carboxyl terminus of intracellular dystrophin [40]. T h e syntrophin triplet is also directly associated with the carboxy-terminal domain of dystrophin, and the amino-terminal domain of dystrophin binds F-actin [41]. Genetic studies suggest that the linkage between laminin-2 and dystrophin is required for muscle integrity. Absence of dystrophin, a reduction of dystrophin-associated glycoproteins, or deficiency of laminin-2 leads to muscular dystrophy [39]. Mutations in the individual components of the sarcoglycan complex also lead to muscular dystrophies [40]. Although a-dystroglycan is extracellular and 15-dystroglycan is a transmembrane protein, both proteins are encoded by a single mRNA. Post-translational modifications of a 97kDa precursor protein give rise to two proteins with molecular masses of 43kDa (for 15-dystroglycan) and 156kDa (for cx-dystroglycan) [42]. Dystroglycan is covered in more detail by Henry and Campbell, this issue, pp 625-631. Interestingly, cx-dystroglycan also binds to the E3 fragment of laminin-1 [43,44,45°], which is not found in muscle [13,19°]. T h e precise binding sites on the two proteins have not yet been determined. It is notable that ~-dystroglycan binds laminin-1 with high affinity and does not bind nidogen, fibronectin or type IV collagen [39,43]. T h e E3 fragment of laminin-1 can support adhesion of some cell types in vitro [17]. As mentioned previously, kidney tubule development can be partially inhibited by
Receptors for laminins during epithelial morphogenesis Ekblom
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Figure 1 Diagram of laminin-1 interactions in the embryonic epithelium. Laminin-1 is made up of an eel, a 61 and a 71 chain. Laminin epidermal growth factor like (LE) modules are represented by rectangles within the laminin chains. The 71 chain is shown to bind to nidogen. Note that nidogen is derived from the adjacent mesenchyme. Dystroglycan (composed of oc- and ~-dystroglycan) and the ot6~1 integrin are shown here to bind to the E3 and the E8 fragments of laminin-1, respectively. These interactions are thought to attach the basement membrane to the epithelial cell surface. The proteins associated with ~-dystroglycan in the cell surface and the intracellular space are not well known for epithelial cells. Intracellular proteins which potentially bind to [~-dystroglycan in epithelial cells may include Dp 140, a truncated form of dystrophin. The straight lines around a-dystroglycan indicate that epithelial dystroglycan might be heavily glycosylated. The integrin receptor and dystroglycan might be involved in signal transduction, but this possibility has not yet been studied. The model of laminin-1 is to some extent based on data from [2,3°]. The model of dystroglycan has been modified from references in [39,45°]. Figure adapted, and reproduced with the permission of Karger, Basel, from [58].
col
senchymal
Laminin-1
"- Nidogen
pl
~
'y1
Integrin \ (
Basement membrane
E8
I
E3-[ ~]
. Dystroglycan
Epithelial cell
p 140 Signal transduction?
antibodies to the E3 fragment [16,17], suggesting that epithelial receptors for the E3 fragment should exist in the embryonic kidney. Recently, it was shown that the dystroglycan and laminin (~1 chain mRNAs are coexpressed in epithelial cells during kidney development; both appear at the onset of epithelial cell polarization [46°°]. Moreover, the polypeptides could be colocalized in the basement membrane region of the developing epithelial cells in the embryonic kidney. Finally, development of epithelial cells in organ culture of embryonic kidneys could be inhibited by the anti-oc-dystroglycan antibody IIH6; this antibody blocks the interaction between laminin-1 and a-dystroglycan [46"']. These findings suggest that dystroglycan is important for epithelial cell development. It might act as a receptor for the E3 fragment of laminin-1 (Fig. 1). During embryogenesis, dystroglycan mRNA is expressed in the epithelium of several nonmuscle organs, suggesting that dystroglycan is important for epithelial
branching morphogenesis in many tissues. A remarkable finding is that dystroglycan-/- mice die soon before gastrulation, well before overt muscle morphogenesis (K Campbell, R Williamson, personal communication). It is still unclear why the embryos die, but the phenotype of the dystroglycan null mice is compatible with the view that dystroglycan could be important for epithelial morphogenesis. As death occurs so early, however, the mutant embryos are difficult to study.
Conclusions and perspectives Laminin-1 (all31y1) and laminins containing the laminin (x5 chain seem to be major epithelial laminins during embryonic development. Although receptors for the (x5 chain have not yet been identified, several receptors for laminin-1 are known. In the kidney model system for epithelial morphogenesis, c(6131 integrin and dystroglycan have been suggested to act as two independent laminin-1
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Cell-to-cell contact and extracellular matrix
receptors (see Fig. 1). Genetic studies in mice have raised some doubt about the role of integrin a6131 for embryonic development of epithelial cells, and emphasize the importance of dystroglycan.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: =
of special interest
ee of outstanding interest
T h e suggestion that dystroglycan has a role in epithelial morphogenesis relies much on the antibody perturbation studies performed in the in vitro assay for kidney development, using one blocking antibody. It would be important to generate other blocking antibodies and to test these in many systems. Other methods to test the suggestion should also be performed. In particular, it should be possible to create mice which lack dystroglycan in only some epithelial cells. An important aim is to clarify the nature of the binding sites of c~-dystroglycan to epithelial basement membrane components. Binding of dystroglycan to laminin-1 can be more easily perturbed by heparin than can binding to laminin-2 [47]. The binding affinities may be strongly dependent on the glycosylation states of the interacting proteins. Brain oc-dystroglycan is smaller than muscle ~-dystroglycan, probably because of a different degree of glycosylation [44]. T h e glycosylation of epithelial dystroglycans and laminins should be studied. Binding to laminin-1 could be the major function of epithelial dystroglycan, but it should be kept in mind that dystroglycan also binds to some forms of agrin, another basement membrane component. Several splice variants of agrin are known, and some are expressed in non-neural tissues [48,49].
The proteins associated with 13-dystroglycan in the epithelial membrane and in the intracellular space also remain to be identified. Organ-specific differences in the associated proteins may exist. Dystrophin is not found in epithelial cells [50], but l~-dystroglycan might instead bind to utrophin, a homologue of dystrophin known to bind dystroglycan [51] that is enriched in focal adhesions in cultured cell lines [52]. Additional intracellular proteins that bind to epithelial dystroglycan might include smaller proteins also encoded by the dystrophin gene. Many such shorter transcripts containing the dystroglycan-binding site exist [53-56]. Their presence during epithelial morphogenesis should be studied. Preliminary studies suggest that one short product of the dystrophin gene, Dp 140, is selectively expressed during kidney tubulogenesis (M Durbeej et al., unpublished data).
There is some evidence that dystroglycan in muscle does not only act as a structural protein. 13-dystroglycan was found to be directly associated with Grb2, an adapter protein involved in signal transduction and cytoskeletal organization, suggesting that the dystroglycan complex could also be involved in signal transduction and cytoskeletal organization [57]. It should be an interesting task to study Grb2 during epithelial morphogenesis.
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Receptors for laminins during epithelial morphogenesis Peter Ekblom Laminin-1 is expressed by many embryonic epithelial cell types. It binds to receptors on the epithelial cell surface. The integrin a6131 is a well known laminin-1 receptor that is expressed on many embryonic epithelial cells. More recently, dystroglycan was discovered as a high-affinity receptor for laminin-1 and laminin-2. It is expressed not only by muscle cells but also by embryonic epithelial cells. In embryonic epithelia, dystroglycan may act by binding to the E3 fragment of laminin-l. Integrins and the dystroglycan complex seem to be important for epithelial morphogenesis, but the relative roles of these two receptor systems for epithelial cells are still unclear.
Addresses Department of Animal Physiology, Uppsala University, Biomedical Center, Box 596, Husargatan3, S-751 R4 Uppsala, Sweden; e-maih [email protected] Current Opinion in Cell Biology 1996, 8:700-706 © Current Biology Lid ISSN 0955-0674
Introduction Epithelial cells are firmly attached to the basement membrane in the adult. Long before individual basement membrane components had been identified, embryonic studies using electron microscopy revealed that basement membranes form very early during epithelial morphogenesis [1,2]. These and many other studies led to the widely held belief that basement membranes are required for the initial stages of epithelial development and for the maintenance of the morphology of the epithelial sheets [1]. Laminin-1 is expressed by many epithelial cell types, and binds with high affinity to mesenchyme-derived nidogen. This interaction may be crucial for basement membrane assembly. Laminin-1 also binds to epithelial cell surface receptors such as ~6131 integrin. This review will describe current knowledge about the nature of the laminin isoforms that are expressed by embryonic epithelial cells. In addition, recent results suggesting that laminin-1 is important for embryonic development of epithelial cells will be covered. Finally, the importance of different laminin receptors for epithelial morphogenesis will be discussed. The potential importance of a recently described laminin receptor, dystroglycan, for epithelial morphogenesis is emphasized. E p i t h e l i a l b a s e m e n t m e m b r a n e a s s e m b l y in the extracellular space Although different basement membranes are fairly heterogeneous molecularly, several features are common to most basement membranes. All basement membranes are composed of two fairly independent protein sheets,
the collagen IV and the laminin networks, which are bound together by nidogen (also called entactin) and possibly by other not yet identified components. The nature of the collagen IV and laminin isoforms varies from tissue to tissue. T h e assembly during embryogenesis of basement membranes, which are huge protein networks, is extremely complex. Nevertheless, some basic principles of the assembly process are emerging [2,3"]. One principle, discovered largely by immunolocalization and localization of mRNAs in tissue sections, is that epithelial basement membranes form as a result of epithelial-mesenchymal interactions [4",5"]. Some components, such as the laminin 131 chain [6], are produced by both cells during epithelial morphogenesis, whereas some basement membrane components are produced exclusively by the mesenchyme. A more strict mesenchymal origin of nidogen seems to hold for most tissues in which epithelial morphogenesis occurs [6-8] and is of particular interest as there is some evidence that the binding of nidogen to the laminin y1 chain [3"] is required for epithelial morphogenesis [8]. Expression of laminin isoforms during epithelial development More than ten individual laminin chains have already been discovered and they can form a large number of different heterotrimers [9,10,11", 12]. Many of the newly discovered laminin isoforms have, so far, been characterized mainly at the primary sequence level, and it remains to be studied where they are expressed during embryogenesis [9,10,11°]. For many of the first described chains, much more is known, but a comprehensive catalogue of their expression patterns can nevertheless not be provided [12]. In many cases, only a few tissues and a few developmental stages have been analyzed. Thus, the simple rules of expression provided here will be refined as more data become available. It is important to note that cells can probably produce many laminin heterotrimers simultaneously. T h e laminin 131 and y1 chains probably have the broadest tissue distribution of the laminin chains (being found in mesenchyme, epithelium, endothelium and muscle) and it is likely that most embryonic epithelial cells produce these chains [6,13,14]. Of the laminin ~ chains, the ¢tl, ~3 and c~5 chains seem to be prominently expressed in epithelium, whereas the c~2 and c~4 chains are not [9,10,15]. The a2 chain is found in mesenchyme, muscle and peripheral nerves [15], and the ~4 chain mRNA is restricted to certain mesenchymal cells such as smooth muscle and dermis [10]. Of the 'epithelial' ~ chains, the ~,3 chain is prominently expressed in skin basement membranes, as part of laminin-5. T h e ~3 chain mRNA is also expressed in the epithelium of salivary gland and tooth, but only at later developmental stages [9]. For a more comprehensive discussion of the o~3 laminin chain,
Receptors for laminins during epithelial morphogenesis Ekblom
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the reader is referred to Borradori and Sonnenberg, this issue, pp 647-656.
shown to alter epithelial cell polarity in an epithelioid cell line.
Curiously, there are still some controversies concerning the expression pattern of the laminin otl chain although it was the first described ct chain. Most investigators agree that the laminin c~l chain is found in many epithelial basement membranes [12], but it is debatable whether it is also present in other basement membranes. By immunofluorescence, using well characterized polyclonal and monoclonal antibodies against the E3 fragment of laminin-1 (which contains the laminin o~1 chain), the (x1 chain was localized to epithelial basement membranes of several mouse embryonic tissues [13,16-18]. These studies agree well with in situ hybridization results which revealed a limited expression of the c~l chain mRNA in the epithelium or in cells closely associated with the epithelium during mouse [6,7,18,19 °] and human [15] embryogenesis. A further expression site was found to be the human embryonic central nervous system [15]. In contrast, a broader expression pattern of the laminin ~1 chain, with expression also occurring in endothelial basement membranes, has been noted by immunofluorescence by a large number of investigators, who were mainly working with adult human tissues and some other species [20,21°,22,23°]. These differences could be due to species differences, different stages being analyzed, or the fact that the different reagents react with different molecules. It will be important to clarify this issue, and here attention should be paid to a further characterization of the reagents used.
Among the organs forming by means of epithelialmesenchymal interactions, the developing kidney is unique in the sense that there is a conversion of mesenchyme to epithelium [24]. T h e conversion is accompanied by the appearance of a new basement membrane, and it is thus a good model in which to study the development of basement membranes. In developing kidneys, the Ill and ~'1 chains are fairly widely expressed, as they are in mesenchyme. In contrast, laminin a l chain expression is specific for epithelial cells [16-18]. Antibodies reacting with the E8 or E3 fragment of laminin-1 have been shown to inhibit the development of mesenchyme into epithelium in organ cultures of embryonic kidneys, suggesting that these domains are important for epithelial cell polarization [16,17].
T h e recently discovered laminin c~5 chain of mouse has a broad tissue distribution in adult tissues, based on Northern blot analysis [11"]. In situ hybridization of mouse embryonic tissues has revealed that the c~5 chain is found prominently in embryonic epithelia in addition to in some muscle tissues (M Durbeej et al., unpublished data). Only a few embryonic endothelial cell types seemed to produce this chain. Interestingly, the (~5 chain was found prominently in those epithelial cell types that lacked the ecl chain, and vice versa, and only a few locations with an overlapping expression pattern were noted in the embryo (M Durbeej et al., unpublished data).
Laminin-1 and organ and cell culture s t u d i e s Although it is still unclear how broadly the c~l chain is expressed during embryogenesis or in adulthood, most investigators agree that the c~l chain is expressed by embryonic epithelial cells, suggesting that it is important for epithelial morphogencsis (see above). This view is supported by several in vitro studies. First, by adding antibodies against different domains of laminin-1 in organ cultures of embryonic tissues, epithelial morphogenesis has been perturbed in many culture systems. Second, by culturing embryonic epithelial cells on laminin-1, differentiation has been enhanced. Third, stable transfection of antisense constructs for the laminin c~l chain has been
In the developing salivary gland, the expression pattern of the col laminin chain is somewhat different from in the developing kidney. T h e c~l chain mRNA is transiently expressed by the salivary gland mesenchyme adjacent to the epithelial stalk at early stages of development [25]. A faint expression is also seen transiently at the tips of the branching epithelium. At later stages, no laminin col chain mRNA can be seen in salivary glands. Instead, the ~c5 chain can be detected in the epithelium at later stages (M Durbeej et al., unpublished data). The E3 fragment of laminin-1 might be important at the very early stages of branching morphogenesis of the salivary gland; monoclonal antibodies against the E3 fragment were found to perturb branching morphogenesis in organ cultures of embryonic salivary glands [25]. In the embryonic lung, laminin c~l chain mRNA is also synthesized by a thin layer of mesenchyme close to the stalk of the branching epithelium, and by the branching epithelial tips during early stages of embryonic lung morphogenesis [6,7]. So far, there have been no reports of using monoclonal antibodies against the E3 fragments in organ cultures of embryonic lungs. T h e E8 fragment, however, has been suggested to be involved in later developmental stages of lung alveolar morphogenesis [26]. Furthermore, fragment P1 of laminin-1, consisting of the inner part of the three disulphide-bonded short arms, has been suggested to be required for the early stages of branching epithelial morphogenesis of the lung [27]. Another approach was used to study the role of laminins in the development of retinal neuroepithelium [28]. Dissociated neuroepithelial cells from chicken embryos were cultured on different types of laminin substrata. The cells were found to survive, proliferate and differentiate into neurons when cultured on laminin-1, whereas they did not survive or differentiate well on Schwannoma-derived laminin, which has an oc2 chain and is devoid of c~l chain. The neuron-stimulating activity of laminin-1 was found to
702
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reside on the E8 fragment and the authors stressed the importance of the carboxy-terminal end of the laminin a l chain for neuroepithelial differentiation [28]. The antisense approach has been used to study the role of the laminin a l chain in gut development [29°]. A cDNA fragment of the cxl chain in the antisense orientation was stably transfected into a human colon cancer cell line. These cells normally form epithelial structures when cocultured with fibroblast cells. Absence of c~l chain expression caused by the antisense construct led to an incorrect secretion of the 131 and T1 chains, to a lack of basement membrane formation, and to alterations in the epithelial cell polarity [29°].
Receptors for laminin-1 on embryonic epithelial cells If laminin-1 is important for epithelial cell development, cell surface receptors for it should exist on embryonic epithelial cells. Initial efforts to find such epithelial receptors some ten years ago concentrated on a 68 kDa protein [30]. However, this protein might be an intracellular protein and it is apparently not particularly well enriched in epithelial cells [30,31]. It is thus possible that this protein is not a major laminin receptor for epithelial cells in vivo. Subsequently, integrins emerged as more likely laminin receptors [32]. Two epithelial laminin-1 receptors, integrins ot6151 and oc6154, have been well characterized [32-34], and a role for integrin ot6134 in the attachment of epidermal cells to basement membrane is supported by many different types of studies (see Borradori and Sonnenberg, this issue, pp 647-656). Integrin ct6151 might be important for other epithelial cells. Nevertheless, recent studies raise the possibility that a protein first discovered from muscle membranes, dystroglycan, could act as a major laminin-binding receptor for epithelial cells also. Integrin (x6131during branching epithelial morphogenesis Several cultured cell lines use the ot6151 integrin to bind to the E8 fragment of laminin-1 in vitro. The ~6 integrin subunit was initially identified by use of a monoclonal antibody that reacts with the cell surface of epithelium and endothelium in tissue sections [32]. The integrin a6 subunit is detected on the surface of embryonic kidney epithelium in vivo [35,36]. In the embryonic kidney, immunofluorescence staining has demonstrated a coappearance of the integrin a6 subunit and laminin ~1 chain during conversion of mesenchyme to epithelium [36]. The immunolocalization studies of both embryonic and adult tissues are compatible with the view that integrins containing the cx6 subunit act as receptors for laminins in vivo also (see Fig. 1). The integrin cx6 subunit is known to associate with either the 151 or the 134 integrin subunit to form the laminin-binding integrins a6151 and cx6154[32-34]. In embryonic kidney tubules, the a6 integrin subunit associates with the 151
subunit rather than with the 134 subunit [37]. Antibodies against the a6 subunit partially inhibit kidney tubulogenesis in organ culture [36]. Antibodies against the integrin or6 subunit and against the integrin 1~1 subunit have also been reported to inhibit epithelial morphogenesis [25,36]. These studies can be considered to provide strong evidence that the a6151 integrin is important for branching epithelial morphogenesis. However, mice lacking the o~6 integrin subunit undergo normal embryogenesis and defects are seen in skin at birth [38°°]. This striking finding suggests either that other receptors for laminins are more important than integrin ot6131 during embryonic epithelial morphogenesis, or that other laminin-binding integrins take over the role of integrin a6 subunit during embryogenesis when integrin a6 is missing. T h e integrin 151 subunit is probably associated with many other integrin o~ chains in developing epithelia, and some might bind iaminin-1.
Dystroglycan during branching epithelial morphogenesis Although integrins have long been considered to be the main receptors for basement membrane components, other laminin receptors may exist. In muscle, dystroglycan links the cytoskeleton to the basement membrane [39]. T h e dystroglycan complex includes the extracellular Qt-dystroglycan, which binds to E3-1ike G domains of laminin-2 in muscle, and five transmembrane proteins, namely 15-dystroglycan, oc-sarcoglycan, 15-sarcoglycan, T-sarcoglycan and a 25 kDa protein [39]. The 15-dystroglycan binds to the carboxyl terminus of intracellular dystrophin [40]. T h e syntrophin triplet is also directly associated with the carboxy-terminal domain of dystrophin, and the amino-terminal domain of dystrophin binds F-actin [41]. Genetic studies suggest that the linkage between laminin-2 and dystrophin is required for muscle integrity. Absence of dystrophin, a reduction of dystrophin-associated glycoproteins, or deficiency of laminin-2 leads to muscular dystrophy [39]. Mutations in the individual components of the sarcoglycan complex also lead to muscular dystrophies [40]. Although a-dystroglycan is extracellular and 15-dystroglycan is a transmembrane protein, both proteins are encoded by a single mRNA. Post-translational modifications of a 97kDa precursor protein give rise to two proteins with molecular masses of 43kDa (for 15-dystroglycan) and 156kDa (for cx-dystroglycan) [42]. Dystroglycan is covered in more detail by Henry and Campbell, this issue, pp 625-631. Interestingly, cx-dystroglycan also binds to the E3 fragment of laminin-1 [43,44,45°], which is not found in muscle [13,19°]. T h e precise binding sites on the two proteins have not yet been determined. It is notable that ~-dystroglycan binds laminin-1 with high affinity and does not bind nidogen, fibronectin or type IV collagen [39,43]. T h e E3 fragment of laminin-1 can support adhesion of some cell types in vitro [17]. As mentioned previously, kidney tubule development can be partially inhibited by
Receptors for laminins during epithelial morphogenesis Ekblom
703
Figure 1 Diagram of laminin-1 interactions in the embryonic epithelium. Laminin-1 is made up of an eel, a 61 and a 71 chain. Laminin epidermal growth factor like (LE) modules are represented by rectangles within the laminin chains. The 71 chain is shown to bind to nidogen. Note that nidogen is derived from the adjacent mesenchyme. Dystroglycan (composed of oc- and ~-dystroglycan) and the ot6~1 integrin are shown here to bind to the E3 and the E8 fragments of laminin-1, respectively. These interactions are thought to attach the basement membrane to the epithelial cell surface. The proteins associated with ~-dystroglycan in the cell surface and the intracellular space are not well known for epithelial cells. Intracellular proteins which potentially bind to [~-dystroglycan in epithelial cells may include Dp 140, a truncated form of dystrophin. The straight lines around a-dystroglycan indicate that epithelial dystroglycan might be heavily glycosylated. The integrin receptor and dystroglycan might be involved in signal transduction, but this possibility has not yet been studied. The model of laminin-1 is to some extent based on data from [2,3°]. The model of dystroglycan has been modified from references in [39,45°]. Figure adapted, and reproduced with the permission of Karger, Basel, from [58].
col
senchymal
Laminin-1
"- Nidogen
pl
~
'y1
Integrin \ (
Basement membrane
E8
I
E3-[ ~]
. Dystroglycan
Epithelial cell
p 140 Signal transduction?
antibodies to the E3 fragment [16,17], suggesting that epithelial receptors for the E3 fragment should exist in the embryonic kidney. Recently, it was shown that the dystroglycan and laminin (~1 chain mRNAs are coexpressed in epithelial cells during kidney development; both appear at the onset of epithelial cell polarization [46°°]. Moreover, the polypeptides could be colocalized in the basement membrane region of the developing epithelial cells in the embryonic kidney. Finally, development of epithelial cells in organ culture of embryonic kidneys could be inhibited by the anti-oc-dystroglycan antibody IIH6; this antibody blocks the interaction between laminin-1 and a-dystroglycan [46"']. These findings suggest that dystroglycan is important for epithelial cell development. It might act as a receptor for the E3 fragment of laminin-1 (Fig. 1). During embryogenesis, dystroglycan mRNA is expressed in the epithelium of several nonmuscle organs, suggesting that dystroglycan is important for epithelial
branching morphogenesis in many tissues. A remarkable finding is that dystroglycan-/- mice die soon before gastrulation, well before overt muscle morphogenesis (K Campbell, R Williamson, personal communication). It is still unclear why the embryos die, but the phenotype of the dystroglycan null mice is compatible with the view that dystroglycan could be important for epithelial morphogenesis. As death occurs so early, however, the mutant embryos are difficult to study.
Conclusions and perspectives Laminin-1 (all31y1) and laminins containing the laminin (x5 chain seem to be major epithelial laminins during embryonic development. Although receptors for the (x5 chain have not yet been identified, several receptors for laminin-1 are known. In the kidney model system for epithelial morphogenesis, c(6131 integrin and dystroglycan have been suggested to act as two independent laminin-1
704
Cell-to-cell contact and extracellular matrix
receptors (see Fig. 1). Genetic studies in mice have raised some doubt about the role of integrin a6131 for embryonic development of epithelial cells, and emphasize the importance of dystroglycan.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: =
of special interest
ee of outstanding interest
T h e suggestion that dystroglycan has a role in epithelial morphogenesis relies much on the antibody perturbation studies performed in the in vitro assay for kidney development, using one blocking antibody. It would be important to generate other blocking antibodies and to test these in many systems. Other methods to test the suggestion should also be performed. In particular, it should be possible to create mice which lack dystroglycan in only some epithelial cells. An important aim is to clarify the nature of the binding sites of c~-dystroglycan to epithelial basement membrane components. Binding of dystroglycan to laminin-1 can be more easily perturbed by heparin than can binding to laminin-2 [47]. The binding affinities may be strongly dependent on the glycosylation states of the interacting proteins. Brain oc-dystroglycan is smaller than muscle ~-dystroglycan, probably because of a different degree of glycosylation [44]. T h e glycosylation of epithelial dystroglycans and laminins should be studied. Binding to laminin-1 could be the major function of epithelial dystroglycan, but it should be kept in mind that dystroglycan also binds to some forms of agrin, another basement membrane component. Several splice variants of agrin are known, and some are expressed in non-neural tissues [48,49].
The proteins associated with 13-dystroglycan in the epithelial membrane and in the intracellular space also remain to be identified. Organ-specific differences in the associated proteins may exist. Dystrophin is not found in epithelial cells [50], but l~-dystroglycan might instead bind to utrophin, a homologue of dystrophin known to bind dystroglycan [51] that is enriched in focal adhesions in cultured cell lines [52]. Additional intracellular proteins that bind to epithelial dystroglycan might include smaller proteins also encoded by the dystrophin gene. Many such shorter transcripts containing the dystroglycan-binding site exist [53-56]. Their presence during epithelial morphogenesis should be studied. Preliminary studies suggest that one short product of the dystrophin gene, Dp 140, is selectively expressed during kidney tubulogenesis (M Durbeej et al., unpublished data).
There is some evidence that dystroglycan in muscle does not only act as a structural protein. 13-dystroglycan was found to be directly associated with Grb2, an adapter protein involved in signal transduction and cytoskeletal organization, suggesting that the dystroglycan complex could also be involved in signal transduction and cytoskeletal organization [57]. It should be an interesting task to study Grb2 during epithelial morphogenesis.
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