WHERE AM I? HOW A CELL RECOGNIZES ITS POSITIONAL INFORMATION DURING MORPHOGENESIS

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WHERE AM I? HOW A CELL RECOGNIZES ITS POSITIONAL INFORMATION DURING MORPHOGENESIS RYU-ICHIRO HATA Department of Tissue Physiology, Division of Adult Diseases, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 101, Japan

Morphogenesis is an old, and one of the latest, fascinating fields in biological science and a huge number of papers on molecular mechanisms underlying it have been published. But most of the works and reviews on these mechanisms pertain to molecules of, as it were, the planning or design of morphogenesis, such as morphogens and homeodomain proteins. In this review, I will describe the function of extracellular matrix (ECM) and other cell adhesion molecules in morphogenesis as that of actual morpho-creating molecules, morphocreators, and discuss their ? 1996 Academic Press Limited roles as positional information-pertaining molecules. K: positional information; morphogenesis; extracellular matrix; morphocreator

INTRODUCTION Advances in understanding pattern formation and the regulation of morphogenesis have revealed the existence of diffusible factors such as bicoid and activin, which play roles as morphogens (Johnston and Nusslein-Volhard, 1992; Jessell and Melton, 1992; Gurdon et al., 1995) and transcription factors such as homeodomain proteins (Gehring, 1992) and nuclear retinoic acid receptors (De Luca, 1991; McGinnis and Krumlauf, 1992), which control the development of form. These molecules provide information or signals of morphogenesis but none of them actually performs morphogenesis; they function, instead, to control patterns of expression of genes involved in the cellular processes underlying morphogenesis. Candidate molecules regulating actual morphogenesis, morphocreators, have to be: (i) molecules essential for morphogenesis; (ii) molecules that give positional information on the cells producing them or on neighbouring cells; and (iii) molecules whose genes may be targets of the morphological signal molecules described above. Molecules pertaining to cell-to-cell and cell-to-matrix interactions satisfy requirements for the candidate molecules. In this mini-review I will describe the function of ECM components and cell adhesion molecules in morphogenesis and discuss the intracellular transduction pathway of adhesion signals in comparison with that of other signals such as those of growth factors. 1065–6995/96/010059+07 $12.00/0

FUNCTIONS OF EXTRACELLULAR MATRIX IN TISSUE FORMATION Owing to the progress made in culture methods, we are now able to maintain and proliferate various kinds of normal cells. But still there are two major differences between cells in culture and those in vivo. Firstly, isolated cells cannot form three-dimensional tissue or organ in culture, and, secondly, cells cannot retain their differentiated state for a long time under traditional culture conditions. For example, fibroblasts obtained from dermis show density-dependent growth characteristics and almost cease to proliferate once they have formed a two-dimensional monolayer, even though the cells in vivo in the dermis, for example, construct a three-dimensional structure surrounded by ECM components such as various type of collagens, proteoglycans, and adhesive glycoproteins produced by the cells themselves. These phenomena are commonly observed when normal, non-transformed cells are cultured. This cessation of proliferation is not dependent on merely a deficiency of nutrients or accumulation of waste materials in the culture medium, because proliferation of the cells does not resume by replacing the culture medium with serumsupplemented fresh medium. Several years ago, we found that the cause of the density-dependent growth of fibroblasts mentioned above is attributable to incomplete synthesis and ? 1996 Academic Press Limited

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maturation of collagen, thus malformation of the ECM, due to deficiency of vitamin C (-ascorbic acid). In fact, daily addition of the vitamin, 0.1 m, stimulated collagen synthesis and cell growth (Hata et al., 1988). But vitamin C itself is very labile under normal culture conditions, i.e. at neutral pH and in aqueous solution, and it has toxic effects on the cells by the production of H2O2 when a higher concentration, e.g. 1 m, is employed (Hata et al., 1988). So we further sought for a stable and bioactive vitamin C derivative and found that a phosphate derivative, ascorbic acid 2-phosphate (Asc 2-P), had co-factor activity for collagen biosynthesis in the cells and was very stable in culture (Hata and Senoo, 1989). Thus we called it long-acting vitamin C. The addition of 0.1 or 0.2 m Asc 2-P to cultures of dermal fibroblasts stimulated cell growth, transcription of type I collagen genes (Kurata and Hata, 1991, Kurata et al., 1993), processing of procollagen to collagen, collagen fibre formation, and construction of a three-dimensional tissue, similar to the dermis from which the cells had been isolated (Fig. 1, Hata and Senoo, 1989). Asc 2-P also stimulated the differentiation and tissue formation of various other kinds of cells. The presence of Asc 2-P in the co-culture system of hepatic parenchymal cells and fibroblasts resulted in the formation of three-dimensional lobular structures and the normal production level of albumin by the hepatic parenchymal cells was much better preserved (Senoo et al., 1989). Supplementation of the medium with Asc 2-P also stimulated the differentiation of myoblastic cells. Investigation of the molecular mechanisms of the process clarified that the long-acting vitamin C stimulated collagen production of the myoblasts, and adhesion of the cells to collagen, which in turn stimulated the synthesis of myogenin, a myogenic transcription factor that binds to genes for myoblast-specific proteins and stimulates transcription of the genes (Mitsumoto et al., 1994). Basement membrane or an adhesive glycoprotein component of it, laminin, and prolactin both bound to their receptors (LAMR, PRLR), and stimulated differentiation of mammary epithelial cells and activate transcription of a gene for a milk protein, â-casein (Schmidhauser et al., 1992; Streuli et al., 1995; Table 1). The co-presence of inhibitors of collagen synthesis with Asc 2-P attenuated its stimulative effects on the cell differentiation and tissue formation (Hata and Senoo, 1989; Mitsumoto et al., 1994). These results indicate that collagen is essential for morphogenesis and formation of collagen matrix and that adhesion

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Fig. 1. Electron photomicrographs of dermal fibroblasts cultured in the absence (a and c) or presence (b and d) of 0.1 m ascorbic acid 2-phosphate, a long-acting vitamin C derivative. The micrographs are cultures sectioned perpendicular to the plane of growth. C, Collagen fibres; G, Golgi apparatus; M, Mitochondrion; rER, rough endoplasmic reticulum. Bars indicate 1 ìm. Arrows and arrowheads indicate, respectively, the basal layer and top layer of the sheet. Reproduced from Hata and Senoo, 1989.

of cells to it gives stimulative signals for differentiation and morphogenesis of various kinds of cells. FEEDBACK REGULATION OF GENE EXPRESSION BY COLLAGEN MATRIX During and after differentiation, cells involved in tissue formation recognize their position in the tissue and attenuate the rates of their growth and expression of their differentiation specific genes. Actual morphocreators may be responsible for this feedback regulation. Collagen is also a good candidate molecule in this respect. Morphological heterogeneity of the cells was observed when the three-dimensional structure produced by fibroblasts cultured in the presence of Asc 2-P was analysed by electron microscopy. Well-developed rough endoplasmic reticulum (rEM) and Golgi apparatus (G) were notable in the

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Fig. 2. Transduction of adhesion signals. PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-biphosphate; IP3, inositol 1,4,5-triphosphate; DAG, diacylglycerol; PKC, protein kinase C; smgGDS, small molecular G protein GDP dissociation stimulator; Rac, Ras and Rho, small molecular GTP-binding proteins; RhoGDI, RhoGDP dissociation inhibitor; RhoGAP, Rho GTPase activating protein; Na + H + , Na + /H + antiporter; AF, actin filaments; áA, á actinin; V, vinculin; Te, tensin; Ta, talin; Pa, paxillin; TyrK, tyrosine kinase; c-Src, cellular src tyrosine kinase; v-Src, viral src tyrosine kinase; Fadk, focal adhesion kinase; Grb2, growth factor bound protein 2; Sos, son of sevenless; MAPK, MAP kinase; MAPKK, MAP kinase kinase; á5â1, fibronectin receptor; FN, fibronectin; HSPG, heparansulfate proteoglycan; LPA, lysophosphatidic acid; LPAR, LPA receptor; C3, C3 transferase; +, activation, and ", suppression, of activity. Letters in rectangles indicate molecules with enzyme activity.

upper-layer cells compared with the appearance of these organelles in the cells present in the middle of the tissue where the cells were surrounded by extracellular matrix (Fig. 1(b and d)). These observations suggest that cells on the ECM and cells in the ECM are metabolically different. When fibroblasts are cultured on type I collagencoated dishes, growth and collagen synthesis are greater than when the cells are cultured directly on uncoated polystyrene dishes. On the other hand, when the cells are cultured in a threedimensional collagen matrix, growth and collagen synthesis are inhibited (Senoo and Hata, 1994a,b; and references cited in the papers and Hata et al., in press). These results indicate that the cells recognize whether they are on the twodimensional collagen or in the three-dimensional collagen matrix and respond by regulating growth

and expression of type I collagen genes. They further suggest type I collagen is a suitable candidate molecule for the morphocreator. REGULATION OF MORPHOGENESIS BY CELL-TO-CELL INTERACTIONS Cell-to-cell interactions through cell surface molecules also regulate differentiation of cells. One typical example is differentiation of the compound eye of Drosophila. It is composed of 800 unit eyes or ommatidia that each contain eight photoreceptor neurons (R1–R8) and 12 accessory cells. The fate of each cell within the developing ommatidium is not determined by lineage but by communication. The development of one ommatidial cell, the R7 photoreceptor cell, has been particularly

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Table 1 Intracellular signal transduction pathways of differentiation factors and growth factors*

*See text and legend to Fig. 2 for abbreviations.

amenable to analysis. Investigation of the homeotic mutation of R7 cells (sevenless) indicated that the product of the gene, Sevenless, is a transmembrane tyrosine kinase-containing receptor, and the ligand of it, Boss, bride of sevenless, is a transmembrane protein present in the R8 cell. Further analysis of the signal transduction pathway by use of various kinds of cells, including temperaturesensitive mutant flies, clarified the presence of the signal transduction pathway of Drk (downstream of receptor tyrosine kinase), Sos (son of sevenless), Ras1 (small molecular G protein), Gap1, (GTPase activating protein), Draf, Dsor1 (downstream of Draf1), a serine/threonine/tyrosine kinase), rolled, a serine/threonine kinase, and target genes pnt (pointed) and yan (Carthew and Rubin, 1990; Dickson et al., 1992; Dickson and Hafen, 1994; Table 1). In vertebrate development as well as in invertebrate, for example, in avian wing buds, differentiating cells also express position- or differentiation stage-dependent specific cell adhesion molecules (Ide et al., 1994). These results indicate that cell-tocell adhesion molecules can determine the fate of cell differentiation.

CELL–MATRIX ADHESION SIGNAL One characteristic of the ECM is its insolubility which is due to the formation of a supramolecular complex resulting from inter-molecular interaction of specific domains present in each ECM molecule. What is the significance of this insolubility? Cell adhesion to a fibronectin matrix is one of the best characterized examples of signal transduction. One of the early responses commonly observed in cells treated with peptide growth factors is an increase in intracellular pH due to activation of the Na + /H + antiporter. Similar activation of the antiporter and increase in pH are observed when cells attach themselves to an insoluble fibronectin matrix, but this does not occur if the cells are incubated with soluble fibronectin instead. Insolubility of the fibronectin matrix may be essential for clustering of the fibronectin receptor (FNR), integrin á5â1 (VLA-5), because clustering of the integrin subunit by antibodies against each subunit also stimulated the Na + /H + antiporter and an increase in intracellular pH (Schwartz et al., 1991; Hata and Senoo, 1993; Fig. 2). Sites where cells

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Fig. 3. Regulation of tissue formation by extracellular matrix (ECM) system.

adhere to ECM form a characteristic structure called a focal adhesion, at which an accumulation of molecules such as vinculin, talin, paxillin and tensin is observed. A specific tyrosine kinase called focal adhesion kinase is also accumulated in the focal adhesion and activation of the enzyme is observed as well as activation of c-Src tyrosine kinase. In addition to recognition of the RGD (Arg-Gly-Asp) sequence in the fibronectin molecule by á5â1, the occupation of a heparansulfate binding site on fibronectin is essential for emission of the adhesion signal; and this second signal can be substituted by protein kinase C (Woods and Couchman, 1992), suggesting that the serine/ threonine kinase pathway is important for the formation of focal adhesion and signal transduction. Formation of actin stress fibres is also observed intracellularly next to the site of adhesion. Rho, a small molecular weight G protein, plays an important role in the stress fibre formation from globular actin molecules (Ridley and Hall, 1992, 1994). Crosstalk between the signal transduction pathway of the adhesion signals and that of growth factors is observed at this level (Ridley et al., 1992). Adhesion to ECM is essential for the growth of normal cells but not for transformed cells. This may be because products of oncogenes such as v-Src bypass the adhesion signal pathway and

activate the MAP kinase cascade as well as normal cells (Schlaepfer et al., 1994); thus adhesion would not be necessary for the growth of transformed cells (Fig. 2). For the activation of T lymphocytes, adhesion to ECM is not necessary; but presentation of MHC bound-antigen to T cell receptor (TCR) is essential for activation in addition to a cytokine signal. And the latter signal is substitutable by adhesion to fibronectin (Yamada, 1991). The intracellular signal transduction pathway of the adhesion signal is quite similar to the activation pathway of T lymphocytes as well as to the transduction pathway of growth factors (Kazlauskas, 1994; Table 1, Fig. 2). These data indicate the universality of intracellular signal transduction pathways of different signalling molecules and suggest the possibility of presence of crosstalk between different signals. CONCLUSION I have described in this review ECM components such as type I collagen and fibronectin as being actual morphocreative molecules. These molecules are essential for growth, differentiation, and morphogenesis in culture system.

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Recent studies on molecular pathogenesis of osteogenesis imperfecta revealed that type I collagen is essential for development and morphogenesis in vivo (Hata, 1993). Targeted destruction of genes for fibronectin or its receptor or defective expression of type I collagen genes in mice induced mortality (Hata, 1993; Hynes, 1994), indicating product proteins of these genes are indispensable to development and morphogenesis. These molecules also retain positional information and give it back as signals to the cells that produced them or to neighbouring cells, as well as regulate their growth and metabolism. Deficiency of a proteoglycan, type III collagen, or fibronectin, or a defect in processing of type I collagen, all give rise to Ehlers–Danlos syndrome, even though some phenotypic variations exist. These data support the idea that ECM components construct an insoluble supramolecular complex by using specific interacting domains that are contained in each component, and also indicate that the mature ECM thus formed as a whole regulates cell growth, metabolism and morphogenesis through cell surface receptors such as integrins. Therefore the ECM can be viewed as a kind of system that regulates multicellular organization much like the nerve system, endocrine system, and immune system (Fig. 3; Hata and Senoo, 1992). Type I collagen is the most abundant component of the ECM and forms the back-bone structure of our body. It is not clear at present that type I collagen genes, encoding proá1(I) and proá2(I) chains, are direct targets of morphogenesisregulating transcription factors such as homeodomain proteins. But genes for some ECM components, tenascin (cytotactin, Jones et al., 1992), or cell adhesion molecules, neural cell adhesion molecule (Jones et al., 1993) and L-CAM (Goomer et al., 1994) have been shown to be direct targets of homeodomain proteins. Further investigation of the regulatory mechanism of gene expression of ECM components and of the molecular mechanism of cell regulation by the ECM system will intensify our understanding of the mechanisms responsible for the development, morphogenesis, and homeostasis of our body. ACKNOWLEDGEMENTS I thank Dr Haruki Senoo for valuable discussion and critical reading of the manuscript. Original works of ours cited in this review were supported in part by Grants-in-Aid for Scientific Research and Co-operative Research from the Ministry of Education, Science, and Culture of

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