Cell Differentiation and Development, 32 (1990) 367-376 © 1990 Elsevier Scientific Publishers Ireland, Ltd. 0922-3371/90/$03.50
367
CELDIF 99920
Role of cell-matrix contacts in cell migration and epithelial-mesenchymal transformation Elizabeth D. H a y Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, U.S.A.
Epithelial cells make contact with extracellular matrix via receptors on the basal surface that interact with the basal actin cortex. In 3D matrix, the mesenchymal cell makes contact with matrix all around its circumference via similar receptors. When moving, the fibroblast is constantly constructing a new front end. We postulate in a 'fixed cortex' theory of cell motility that the circumferential actin cortex is firmly attached to matrix and that the myosin-rich endoplasm slides past it into the continually forming new front end. During epithelial-mesenchymal transformation, the presumptive mesenchymal cell seems to turn on the new front end mechanism as a way of emigrating from the epithelium into the underlying matrix with which it makes 'fixed' contacts. Master genes may exist that regulate the expression of epithelial genes on the one hand, and mesenchymal genes on the other.
Epithelium; Mesenchyme; Epithelial-mesenchymal transformation; Cell migration
Introduction
Cell-matrix contacts are of major importance in the development of the embryo. Epithelial cells (Fig. 1) sit on top of extracellular matrix (ECM) and exhibit apical-basal polarity. Contact with the underlying ECM organizes the basal actin cortex and promotes differentiation, as measured by increase in synthesis of tissue-specific proteins (Sugrue and Hay, 1981). The idea that the contact with matrix affects specific synthesis by its effect on F actin is supported by the fact that cytochalasin D abolishes ECM stimulation of collagen
Correspondence address: E.D. Hay, Department of Anatomy and Cellular Biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, U.S.A.
synthesis by corneal epithelium (Svoboda and Hay, 1987). ECM receptors characteristic of epithelia include syndecan (Bernfield and Sanderson, 1990), the 67 kDa laminin receptor (Liotta et al., 1984; Sugrne, 1988), and integrins (a3fll, a6fll, a6f14) that interact with laminin and/or type IV collagen (Hynes, 1987; Hemler, 1990). Mesenchymal cells invade ECM and give rise to fibroblasts, chondrocytes, osteocytes, and other residents of the matrix. The fibrobrast is the prototype of a mesenchymal cell (Fig. 1). In the embryo or in 3D collagen gels in vitro, the migrating mesenchymal cell is elongate in shape and has front end-back end polarity. On planar substrata (glass, plastic) in vitro, the fibroblast flattens, develops stress fibers, and stops moving (Herman et al., 1981). The presence of the actin-rich microfilament bundles allows colocalization of ECM, ECM
368 receptors, actin-binding proteins, and actin to be shown by immunohistochemistry (Burridge, 1986), but it must be remembered that stress fibers are artifacts of planar substrata. Fibroblasts in situ or in 3D ECM in vitro have ECM receptors and actin cortex all around the periphery of the cell body. They never have stress fibers along the substratum or an upper free surface that ruffles and moves back (Wang, 1985) toward the nucleus. Instead of ruffles, the front end is covered with filopodia. The fact that the receptors interact with the actin cortex is demonstrated by the inability of 3D ECM to elongate the mesenchymal cell in the presence of cytochalasin D (Tomasek and Hay, 1984). How the mesenchymal cell might use its cell matrix contacts to migrate It is generally agreed that the fibroblast must be attached to a substratum to move, but the mechanism of its motility is totally unknown. On a 2D matrix, like a fibronectin-coated dish, the
fibroblast is believed to attach and reattach to the matrix as it 'walks' along the substratum (see Bilozur and Hay, 1989, for review). Considering that the cell cortex is attached via stable receptors to matrix, constant attachment and reattachment to the dish seems unlikely. In 3D matrix, however, it would be impossible to synchronously attach and reattach to ECM all around the cell in a way that would permit forward movement. Observed by slow motion cinemaphotography, the fibroblast is seen to glide smoothly forward at 1 g m / m i n in 3D ECM, with periodic retraction of its entire rear end (Bard and Hay, 1975). In addition to the problem of seemingly stable receptor-mediated cell-matrix contact, one is confronted with the problem of how to invoke a Huxley-type sliding filament model to explain fibroblast movement. Huxley (1973) himself speculated that in fibroblasts, motility might be brought about by sliding of myosin along actin filaments attached to the plasmalemma. He stopped short of visualizing that the plasmalemma
Fig. 1. Diagram of a typical epithelial cell and a representative(migrating) mesenchymalcell. The epithelial cell shows apical-basal polarity with microvillion the apical surface and a well-developedactin cortex next to the basal surface that interacts with ECM. The Golgi complexis apical. The mesenchymalcell has a circumferentialactin cortex and, on its leading edge, actin-rich filopodia. The Golgi complexis in the front end. From Hay (1990).
369
would have to be anchored for sliding to move the myosin-rich endoplasm. Thinking about these two problems, a heretical solution occurred to this author: the cell surface or ectoplasm (plasmalemma and actin cortex) does not move during fibroblast locomotion (Hay, 1985). This 'fixed cortex' theory states that the myosin-rich endoplasm of the fibroblast slides forward along an actin cortex that is stably attached to matrix via actin-binding proteins and ECM receptors (Fig. 2). Obviously, the myosin, which occurs only in the endoplasm (Tomasek et al., 1982), cannot be soluble if it is to pull along the nucleus and other cell organelles. The mesenchymal cell in 3D ECM contains vimentin and tubulin filaments that fill the endoplasm and run the length of the cell in semicoiled fashion encompassing all the organelles (Tomasek and Hay, 1984; Greenburg and Hay, 1988). It would be simple enough for myosin to be attached to them. Of course, another problem immediately arises. What happens to the endoplasm when it runs out
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of actin cortex? It sounds as if it would be extruded out into the matrix, leaving behind an empty actin shell. Fortunately, a solution to this enigma has been provided recently by John Singer and his colleagues (Kupfer et al., 1987) and by Robert Singer's group (Lawrence and Singer, 1986): the moving fibroblast is constantly constructing a new front end. Newly synthesized membrane proteins insert into the front lamellipodium of the fibroblast on a 2D substratum and appear to 'move backward' (Kupfer et al., 1987). The fixed cortex theory states that the new membrane proteins polymerize in the front end and are fixed in the ectoplasm to substratum as the endoplasm moves forward past them. Lawrence and Singer (1988) localized actin mRNA in the front end of moving fibroblasts. Both ruffles and filopodia (Fig. 2) are very rich in F actin and contain no myosin (Tomasek et al., 1982). They could be the site of polymerization of F actin from G actin synthesized in the front end, and of binding of F actin to actin-binding proteins that attach to ma-
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Fig. 2. Diagram depicting the fixed cortex theory of mcsenchymal cell movement. The same actin cortcx-ECM receptor complex labeled 1,2 appears in all three drawings, where it remains in the same place because its interaction with matrix is stable. In A, the complex 1,2 is in front of the nuclens. The myosin-rich endoplasm containing cell orgaric]les, rnicrotubulcs, arid vimentin intermexiiat¢ filaments slides by at the rate of ] lira/rain (B). Fhially, part or all of the actin and plasmalcmma at 1,2 becomes locatex] in blobs in the ECM when the rear end of the cell recoils (C). From Hay (1989a).
370 trix via newly synthesized ECM receptors that are constantly inserting into the filopodial plasmalemma. The cell, moving in this manner, would be expected to stretch out its tall (Fig. 2B), which would gradually become myosin-poor and actinrich. The observed periodic recoil (Fig. 2C) would occur when the thinned rear end tears off the substratum, leaving pieces of cell cortex and plasmalemma behind as blobs. Such blobs on dishes have been analyzed by Lark and Culp (1984) and have been noted in 3D substrata by Bard and Hay (1975). Thus, the fixed cortex the-
Cell-matrix contacts during formation of mesenchymal cells from epithelia
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ory of fibroblast locomotion seems compatible with all the known facts about fibroblast morphology and metabolism during cell movement. We are labeling cell surface receptors currently to see where they go during fibroblast migration (Daniels and Hay, 1990) and we plan other experiments to test the fixed cortex theory. Preliminary evidence that the actin cortex is fixed during induced neural crest emigration into the neural tube lumen stems from our observations on the location of the zonula adherens during the process (Bilozur and Hay, 1989).
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Fig. 3. Camera lucida drawings of sections of a 16-somite chicken embryo at the level of somites 2, 7, and 12. Posteriorly, the mesoderm is epithelial (Epith.). Anteriorly, these epithelia give rise to connective tissue producing mesenchyme,such as sclerotome (Seler.), at the same time that neural crest (Cres0 forms. W.Duct, Wolffian Duct and other nephrogenic mesoderm. Cent. cells, central cells in the lumen of the somite. N.T., neural tube. Derm., dermatome. Myo, myotome. Lat. Mes., lateral mesoderm.From McCarthy and Hay (1990).
In the chordate phylum, primitive members such as amphioxus are entirely composed of epithelia, with an epithelial notochord for support and a dorsal neural tube. The embryos of all chordates express this epithelial pattern, but for the amniotes (reptiles, birds, mammals) to create an epithelial mesoderm, they first form mesenchymal cells from the primitive streak of their flat embryonic discs and then aggregate them into epithelial notochord, somites, nephrogenic and lateral mesoderm (lower right, Fig. 3). The formation of kidney epithelium from such mesenchyme (Saxen, 1987) is a well studied example of the phenomenon of mesenchymal-epithelial transformation. In addition to definitive epithelial cells, c o n n e c t i v e tissue a n d m u s c l e - p r o d u c i n g mesenchymal cells form from these epithelia. In the trunk of the embryo, the sclerotome (middle right, Fig. 3) is the first of the mesodermal mesenchymes to derive from mesodermal epithelia. Interestingly, neural crest starts to emigrate from the neural tube ectoderm at the same time that the first definitive mesodermal mesenchyme forms (middle and upper right, Fig. 3). These cells have the elongate shape and front end-back end polarity (Fig. 1) of mesoderm-derived mesenchymal cells and so their formation from neural tube can be considered an example of epithelialmesenchymal transformation, even though they give rise to nervous tissue and melanocytes. They produce little or no ECM, but neither does primitive streak mesenchyme. In the head, a second
371 wave of neural tube-derived m e s e n c h y m e emigrates that does produce connective tissue (Noden, 1983; Nichols, 1986). Bilozur and Hay (1988) isolated chicken neural tubes at the stage shown in Fig. 3 (middle to lower right) and suspended them inside type I collagen gels or basement membrane gels. After a day of culture, a healthy crop of neural crest cells has moved out into both kinds of 3D ECM. Neither gel contains fibronectin and the fibronectin peptide, RGD, does not inhibit crest migration in the gels (Bilozur and Hay, 1988), contrary to the
situation on planar substrata (Thiery et al., 1985). The laminin cell-binding peptide, Y I G S R (Kleinman et al., 1986), totally inhibits migration of trunk crest in basement membrane gel. This result (Bilozur and Hay, 1988) is very clear cut and simply cannot be explained away by investigators who wish to think that Y I G S R in laminin is not an important cell binding r e # o n of the molecule. Y I G S R binds to the 67 kDa laminin receptor (Kleinman et al., 1986) and it will be interesting to see if crest expresses this receptor when migrating in basement membrane gels.
Fig. 4. Light micrographs of embryonicrat palate immediatelyafter fusion of the shelves (A, 16.25 days) and an enlargementof the midline epithelial seam at the time it is breaking up into mesenchymalcells (arrows, B, 16.75 days). SC, sloughing periderm cell. A few periderm cells are caught in the seam (arrow, A). Bars: A, 100/Lm; B, 25 #m. From Fitchett and Hay (1989).
372 A completely new type of epithelial-mesenchymal transformation has recently been postulated to occur in older embryos during tissue remodeling. Evidence has been presented that removal of Mullerian duct epithelium in the male involves its transformation to mesenchyme (Trelstad et al., 1982). We asked whether or not epithelial-mesenchymal transformation could be involved in a dramatic case of epithelial removal during tissue remodeling: the disappearance of the medial edge epithelium (MEE) after fusion of the two secondary palatal shelves in the midline (Fitchett and Hay, 1989). During the 16th day of development of the rat fetus, the shelves increase in size, rotate, and move toward each other. The outer layer (periderm) of the two layered MEE sloughs, permitting the basal cells of the opposing epithelia to make contact (Fig. 4A). If the periderm is not allowed to slough before contact, as usually occurs in experiments in vitro, periderm is trapped in the seam and dies, giving the appearance that the seam is disappearing by cell death (Pourtois, 1966). Within a half day after contact is made in situ, the seam breaks up and gives rise to mesenchyme (Fig. 4B). A convincing series of micrographs can be assembled to show the steps in this epithelial-mesenchymal transformation (Fitchett and Hay, 1989). The MEE of the seam can be shown by immunohistochemistry to switch its intermediate filament profile from keratin to vimentin before the mesenchyme forms. Vimentin is found in mesenchymal cells and very rarely in epithelia in situ. The same process unites the nasal surface of the palate with the nasal septum and in both regions, epithelia lose syndecan before fusion (Fitchett et al., 1990). This heparan sulfate proteoglycan is a matrix receptor found in most epithelia in the basolateral plasmalemma, and it may stabilize the epithelial tissue phenotype (Bernfield and Sanderson, 1990). Mammary epithelial cells infected in vitro with antisense syndecan message, decrease syndecan expression and become mesenchymal in shape (see Bernfield, this volume). Currently, we are investigating whether or not epithelia-associated integrins also disappear prior to epithelial-mesenchymal transformation in the palate.
We have recently localized a3fll, a5fll, and
a6fll integrins during epithelial-mesenchymal transformation of the lens (Zuk and Hay, 1990). The anterior lens epithelium of the embryonic or adult chicken is one of a number of definitive (nonmesenchyme-forming) epithelia that can be induced to transform into mesenchyme by suspending them completely within 3D collagen gel (Greenburg and Hay, 1982, 1986, 1988). The process of transformation takes a relatively long time (3-5 days) to begin in vitro. Presumably, this is a period of cell proliferation during which the 3D collagen inactivates epithelial programs and turns on the mesenchymal genetic program in the cells facing the gel. The cells begin to accumulate mesenchymal type RER and other organelles at the time they elongate on the surface of the explant and start to move out into the ECM. They stop producing type IV collagen and laminin, and begin producing type I collagen. We found that they expressed at the time they were leaving the explant, all three of the fll integrins we tested, as did the parent epithelium. The et3 and a6 subunits were lost from the elongate, mesenchymal cells after a number of days in culture, whereas a5pl, the principal fibronectin receptor, persists and fibronectin continues to be synthesized. The persistence of the a3 and a6 subunits probably reflects their slow turnover. In these experiments with definitive epithelia, we found that keratin persists for a time in the mesenchyme-like cells that are expressing vimentin (Greenburg and Hay, 1988), whereas in epithelial-mesenchymal transformations that are normally programmed to occur in the embryo, keratin is not present or disappears before the transition takes place (Fitchett and Hay, 1989; Franke et al., 1982). A chicken syndecan is not currently available, but it is possible that it will also be more sluggish in disappearing from definitive epithelia than from epithelia already programmed to form mesenchyme in the embryo. In conclusion, we have described in this chapter a fixed cortex, new front end mechanism for movement of mesenchymal cells through ECM. The ability of mesenchymal cells to emigrate from embryonic and even adult epithelia attests to the validity of the theory, for the transforming cells
373
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Fig. 5. Diagram of a theory of epithelial-mesenchymal transformation based on the fixed cortex model of motility. The presumptive mesenchymal cell constructs a new front end on its base (A) into which its myosin rich endoplasm slides (B). The Golgi complex and actin mRNA will lie in this part of the cell. To get out of the epithelium, the cell recoils its rear end, leaving blobs of cytoplasm behind (C). From Hay (1989b).
clearly extend a new front end capped with filopodia into the adjacent ECM (Fig. 5). That mesenchymal cells, all of which derive from epithelia, might use the same motility mechanism to move through ECM as to depart from the epithelium seems reasonable. That both processes require synthesis of new actin and new plasmalemma in the front end does not seem surprising viewed in this new light. We have postulated (Hay, 1989a,b) that master regulatory gene(s) is/are turned on (Fig. 5) to create the new front end mechanisms. These same genes, or genes they activate down the line, could turn on vimentin mRNA and other components essential for mesenchymal cell elongation (Zuk et
al., 1989). We report in collaboration with Bernfield's group that syndecan disappears before epithelial-mesenchymal transformation in the embryonic palate. Some insight into the proposed function of the syndecan molecule itself in stabilizing the epithelial phenotype might be gained b y following the disappearance of syndecan during an ECM-induced transformation of a definitive epithelia, such as lens, to mesenchyme. In the latter case, we find that epithelial integrins and keratin intermediate filaments may persist in the mesenchyme-like cells without harm to the transformation as long as the fibronectin-integrin and vimentin are present. We hope in the future by in situ hybridization to be able to correlate more
374
exactly the activation of the genes involved in the mesenchymal program with the sequence of morphological changes observed.
References Bard, J.B.L. and E.D. Hay: The behavior of fibroblasts from the developing avian cornea: Morphology and movement in situ and in vitro. J. Cell Biol. 67, 400-418 (1975). Bernfield, M. and R.D. Sanderson: Syndecan, a morphogenetitally regulated cell surface proteoglycan that binds extracellular matrix and growth factors. Phil. Trans. R. Soc. Lond. 327, 171-186 (1990). Bilozur, M.E. and E.D. Hay: Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, and collagen. Dev. Biol. 125, 19-33 (1988). Bilozur, M.E. and E.D. Hay: Cell migration into neural tube lumen provides evidence for the 'fixed cortex' theory of cell motility. Cell Motil. Cytoskel. 14, 469-484 (1989). Burridge, K.: Substrate adhesions in normal and transformed fibroblasts: organiTation and regulation of cytoskeletal, membrane and extracellular matrix components at focal contacts. Cancer Rev. 4, 18-78 (1986). Daniels, K.J. and E.D. Hay: Distribution of fll integrins, matrix, actin, and actin-associated proteins during migration of elongated fibroblasts in 3D collagen gels. J. Cell Biol. 111, 1401 (1990). Fitchett, J. and E.D. Hay: Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse. Dev. Biol. 131, 455-474 (1989). Fitchett, J.E., K.R. McAlmon, E.D. Hay and M. Bernfield: Epithelial cells lose syndecan prior to epithelial-mesenchymal transformation in the developing rat palate. J. Cell Biol. (abstract, in press) (1990). Franke, W.W., C. Grund, C. Kuhn, B.W. Jackson and K. Illmensee: Formation of cytoskeletal elements during mouse embryogenesis. III. Primary mesenchymal cells and the first appearance of vimentin filaments. Differentiation 23, 43-59 (1982). Greenburg, G. and E.D. Hay: Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells. J. Cell Biol. 95, 333-339 (1982). Greenburg, G. and E.D. Hay: Cytodifferentiation and tissue phenotype change during transformation of embryonic lens epithelium to mesenchyme-like cells in vitro. Dev. Biol. 1~15, 363-379 (1986). Greenburg, G- and E.D. Hay: Cytoskeleton and thyroglobulin expression change during transformation of thyroid epithelium to mesenchyme-like cells. Development 102, 605622 (1988). Hay, E.D.: Interaction of migrating embryonic cells with extracellular matrix. Exp. Biol. Med. 10, 174-193 (1985). Hay, E.D.: Extracellular matrix, cell skeletons, and embryonic development. Am. J. Med. Genet. 34, 14-29 (19899). Hay, E.D.: Theory for epithelial-mesenchymal transformation
based on the 'fixed cortex' cell motility model. Cell Motil. Cytoskel. 14, 455-457 (1989b). Hay, E.D.: Cell Biology of Extracellular Matrix, 2nd edn. (Plenum Press, New York), in press (1990). Hemler, M.E.: VLA proteins in the integrin family: structures, functions, and their role on leucocytes. Annu. Rev. Immunol. 8, 365-400 (1990). Herman, I.M., N.J. Crisona and T.D. Pollard: Relation between cell activity and the distribution of actin and myosin. J. Cell Biol. 90, 84-91 (1981). Huxley, H.E.: Muscular contraction and cell motility. Nature (Lond.) 243, 445-449 (1973). Hynes, R.O.: Integrins: a family of cell surface receptors. Cell 48, 549-554 (1987). Kleinman, H.K., M.L. McGarvey, J.R. Hassell, V.L. Star, F.B. Cannon, G.W. Laurie and G.R. Martin: Basement membrane complexes with biological activity. Biochem. 25, 312318 (1986). Kupfer, A., P.J. Kronebusch, J.K. Rose and J. Singer: A critical role for the polarization of membrane recycling in cell motility. Cell Motil. Cytoskel. 8, 182-189 (1987). Lark, M.W. and L.A. Culp: Multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites. J. Biol. Chem. 259, 6773-6782 (1984). Lawrence, J.B. and R. Singer: Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45, 407-415 (1986). Liotta, L.A,, C.N. Rao and S.H. Barsky: Tumor cell interaction with the extracellular matrix. In: The Role of Extracellular Matrix in Development, ed. R.L. Trelstad (Alan R. Liss, New York) pp. 357-371 (1984). McCarthy, R.A. and E.D. Hay: Expression of type I collagen, laminin, and tenascin during early avian development: ultrastructure and correlation with neural crest and sclerotome pathways. Submitted to Am. J. Anat. (1990). Nichols, D.M.: Formation and distribution of neural crest mesenchyme to the first pharyngeal arch region of the mouse embryo. Am. J. Anat. 176, 221-231 (1986). Noden, D.M.: The embryonic origins of avian cephalic and cervical muscles and associated connective tissues. Am. J. Anat. 168, 257-276 (1983). Pourtois, M.: Onset of the acquired potentiality for fusion in the palatal shelves of rats. J. Embryol. Exp. Morphol. 16, 171-182 (1966). Saxen, L.: Organogenesis of the Kidney. Cambridge Univ., Cambridge (1987). Sugrue, S.: Identification and immunolocalization of the laminin binding protein from embryonic corneal epithelial cells. Differentiation 38, 169-176 (1988). Sugrue, S.P. and E.D. Hay: Response of basal epithelial cell surface and cytoskeleton to solubilized extracellular matrix molecules. J. Cell Biol. 91, 45-54 (1981). Svoboda, K.K.H. and E.D. Hay: Embryonic corneal epithelial interaction with exogenous laminin and basal lamina is F-actin dependent. Dev. Biol. 123, 455-469 (1987). Thiery, J.P., J.L. Duband and G.C. Tucker: Cell migration in the vertebrate embryo. Annu. Rev. Cell Biol. 1, 91-114 (1985).
375 Tomasek, J.J. and E.D. Hay: Analysis of the role of microfilaments and microtubules in the acquisition of bipolarity and the subsequent elongation of fibroblasts in hydrated collagen gels. J. Cell Biol. 99, 536-549 (1984). Tomasek, J.J., E.D. Hay and K. Fujiwara: Collagen modulates cell shape and cytoskeleton of embryonic corneal fibroblasts: distribution of actin, a-actinin and myosin. Dev. Biol. 92, 107-122 (1982). Trelstad, R.L., A. Hayashi, K. Hayashi and P.K. Donahoe: The epithelial-mesenchymal interface of the male rat Mullerian duct: loss of basement membrane integrity and ductal regression. Dev. Biol. 92, 27-40 (1982).
Wang, Y.-L.: Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J. Cell Biol. 101, 597-602 (1985). Zuk, A. and E.D. Hay: Changes in expression of the fll integrin family occur during transformation of lens epithelium to mesenchyme in 3D collagen gels. J. Cell Biol. 111, 2651 (1990). Zuk, A., K. Matlin and E.D. Hay: Type I collagen gel induces Madin-Darby canine kidney cells to become fusiform in shape and lose apical-basal polarity. J. Cell Biol. 108, 903-920 (1989).
367
CELDIF 99920
Role of cell-matrix contacts in cell migration and epithelial-mesenchymal transformation Elizabeth D. H a y Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, U.S.A.
Epithelial cells make contact with extracellular matrix via receptors on the basal surface that interact with the basal actin cortex. In 3D matrix, the mesenchymal cell makes contact with matrix all around its circumference via similar receptors. When moving, the fibroblast is constantly constructing a new front end. We postulate in a 'fixed cortex' theory of cell motility that the circumferential actin cortex is firmly attached to matrix and that the myosin-rich endoplasm slides past it into the continually forming new front end. During epithelial-mesenchymal transformation, the presumptive mesenchymal cell seems to turn on the new front end mechanism as a way of emigrating from the epithelium into the underlying matrix with which it makes 'fixed' contacts. Master genes may exist that regulate the expression of epithelial genes on the one hand, and mesenchymal genes on the other.
Epithelium; Mesenchyme; Epithelial-mesenchymal transformation; Cell migration
Introduction
Cell-matrix contacts are of major importance in the development of the embryo. Epithelial cells (Fig. 1) sit on top of extracellular matrix (ECM) and exhibit apical-basal polarity. Contact with the underlying ECM organizes the basal actin cortex and promotes differentiation, as measured by increase in synthesis of tissue-specific proteins (Sugrue and Hay, 1981). The idea that the contact with matrix affects specific synthesis by its effect on F actin is supported by the fact that cytochalasin D abolishes ECM stimulation of collagen
Correspondence address: E.D. Hay, Department of Anatomy and Cellular Biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, U.S.A.
synthesis by corneal epithelium (Svoboda and Hay, 1987). ECM receptors characteristic of epithelia include syndecan (Bernfield and Sanderson, 1990), the 67 kDa laminin receptor (Liotta et al., 1984; Sugrne, 1988), and integrins (a3fll, a6fll, a6f14) that interact with laminin and/or type IV collagen (Hynes, 1987; Hemler, 1990). Mesenchymal cells invade ECM and give rise to fibroblasts, chondrocytes, osteocytes, and other residents of the matrix. The fibrobrast is the prototype of a mesenchymal cell (Fig. 1). In the embryo or in 3D collagen gels in vitro, the migrating mesenchymal cell is elongate in shape and has front end-back end polarity. On planar substrata (glass, plastic) in vitro, the fibroblast flattens, develops stress fibers, and stops moving (Herman et al., 1981). The presence of the actin-rich microfilament bundles allows colocalization of ECM, ECM
368 receptors, actin-binding proteins, and actin to be shown by immunohistochemistry (Burridge, 1986), but it must be remembered that stress fibers are artifacts of planar substrata. Fibroblasts in situ or in 3D ECM in vitro have ECM receptors and actin cortex all around the periphery of the cell body. They never have stress fibers along the substratum or an upper free surface that ruffles and moves back (Wang, 1985) toward the nucleus. Instead of ruffles, the front end is covered with filopodia. The fact that the receptors interact with the actin cortex is demonstrated by the inability of 3D ECM to elongate the mesenchymal cell in the presence of cytochalasin D (Tomasek and Hay, 1984). How the mesenchymal cell might use its cell matrix contacts to migrate It is generally agreed that the fibroblast must be attached to a substratum to move, but the mechanism of its motility is totally unknown. On a 2D matrix, like a fibronectin-coated dish, the
fibroblast is believed to attach and reattach to the matrix as it 'walks' along the substratum (see Bilozur and Hay, 1989, for review). Considering that the cell cortex is attached via stable receptors to matrix, constant attachment and reattachment to the dish seems unlikely. In 3D matrix, however, it would be impossible to synchronously attach and reattach to ECM all around the cell in a way that would permit forward movement. Observed by slow motion cinemaphotography, the fibroblast is seen to glide smoothly forward at 1 g m / m i n in 3D ECM, with periodic retraction of its entire rear end (Bard and Hay, 1975). In addition to the problem of seemingly stable receptor-mediated cell-matrix contact, one is confronted with the problem of how to invoke a Huxley-type sliding filament model to explain fibroblast movement. Huxley (1973) himself speculated that in fibroblasts, motility might be brought about by sliding of myosin along actin filaments attached to the plasmalemma. He stopped short of visualizing that the plasmalemma
Fig. 1. Diagram of a typical epithelial cell and a representative(migrating) mesenchymalcell. The epithelial cell shows apical-basal polarity with microvillion the apical surface and a well-developedactin cortex next to the basal surface that interacts with ECM. The Golgi complexis apical. The mesenchymalcell has a circumferentialactin cortex and, on its leading edge, actin-rich filopodia. The Golgi complexis in the front end. From Hay (1990).
369
would have to be anchored for sliding to move the myosin-rich endoplasm. Thinking about these two problems, a heretical solution occurred to this author: the cell surface or ectoplasm (plasmalemma and actin cortex) does not move during fibroblast locomotion (Hay, 1985). This 'fixed cortex' theory states that the myosin-rich endoplasm of the fibroblast slides forward along an actin cortex that is stably attached to matrix via actin-binding proteins and ECM receptors (Fig. 2). Obviously, the myosin, which occurs only in the endoplasm (Tomasek et al., 1982), cannot be soluble if it is to pull along the nucleus and other cell organelles. The mesenchymal cell in 3D ECM contains vimentin and tubulin filaments that fill the endoplasm and run the length of the cell in semicoiled fashion encompassing all the organelles (Tomasek and Hay, 1984; Greenburg and Hay, 1988). It would be simple enough for myosin to be attached to them. Of course, another problem immediately arises. What happens to the endoplasm when it runs out
~ i-
~
---'--~ ~
of actin cortex? It sounds as if it would be extruded out into the matrix, leaving behind an empty actin shell. Fortunately, a solution to this enigma has been provided recently by John Singer and his colleagues (Kupfer et al., 1987) and by Robert Singer's group (Lawrence and Singer, 1986): the moving fibroblast is constantly constructing a new front end. Newly synthesized membrane proteins insert into the front lamellipodium of the fibroblast on a 2D substratum and appear to 'move backward' (Kupfer et al., 1987). The fixed cortex theory states that the new membrane proteins polymerize in the front end and are fixed in the ectoplasm to substratum as the endoplasm moves forward past them. Lawrence and Singer (1988) localized actin mRNA in the front end of moving fibroblasts. Both ruffles and filopodia (Fig. 2) are very rich in F actin and contain no myosin (Tomasek et al., 1982). They could be the site of polymerization of F actin from G actin synthesized in the front end, and of binding of F actin to actin-binding proteins that attach to ma-
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Fig. 2. Diagram depicting the fixed cortex theory of mcsenchymal cell movement. The same actin cortcx-ECM receptor complex labeled 1,2 appears in all three drawings, where it remains in the same place because its interaction with matrix is stable. In A, the complex 1,2 is in front of the nuclens. The myosin-rich endoplasm containing cell orgaric]les, rnicrotubulcs, arid vimentin intermexiiat¢ filaments slides by at the rate of ] lira/rain (B). Fhially, part or all of the actin and plasmalcmma at 1,2 becomes locatex] in blobs in the ECM when the rear end of the cell recoils (C). From Hay (1989a).
370 trix via newly synthesized ECM receptors that are constantly inserting into the filopodial plasmalemma. The cell, moving in this manner, would be expected to stretch out its tall (Fig. 2B), which would gradually become myosin-poor and actinrich. The observed periodic recoil (Fig. 2C) would occur when the thinned rear end tears off the substratum, leaving pieces of cell cortex and plasmalemma behind as blobs. Such blobs on dishes have been analyzed by Lark and Culp (1984) and have been noted in 3D substrata by Bard and Hay (1975). Thus, the fixed cortex the-
Cell-matrix contacts during formation of mesenchymal cells from epithelia
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ory of fibroblast locomotion seems compatible with all the known facts about fibroblast morphology and metabolism during cell movement. We are labeling cell surface receptors currently to see where they go during fibroblast migration (Daniels and Hay, 1990) and we plan other experiments to test the fixed cortex theory. Preliminary evidence that the actin cortex is fixed during induced neural crest emigration into the neural tube lumen stems from our observations on the location of the zonula adherens during the process (Bilozur and Hay, 1989).
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Fig. 3. Camera lucida drawings of sections of a 16-somite chicken embryo at the level of somites 2, 7, and 12. Posteriorly, the mesoderm is epithelial (Epith.). Anteriorly, these epithelia give rise to connective tissue producing mesenchyme,such as sclerotome (Seler.), at the same time that neural crest (Cres0 forms. W.Duct, Wolffian Duct and other nephrogenic mesoderm. Cent. cells, central cells in the lumen of the somite. N.T., neural tube. Derm., dermatome. Myo, myotome. Lat. Mes., lateral mesoderm.From McCarthy and Hay (1990).
In the chordate phylum, primitive members such as amphioxus are entirely composed of epithelia, with an epithelial notochord for support and a dorsal neural tube. The embryos of all chordates express this epithelial pattern, but for the amniotes (reptiles, birds, mammals) to create an epithelial mesoderm, they first form mesenchymal cells from the primitive streak of their flat embryonic discs and then aggregate them into epithelial notochord, somites, nephrogenic and lateral mesoderm (lower right, Fig. 3). The formation of kidney epithelium from such mesenchyme (Saxen, 1987) is a well studied example of the phenomenon of mesenchymal-epithelial transformation. In addition to definitive epithelial cells, c o n n e c t i v e tissue a n d m u s c l e - p r o d u c i n g mesenchymal cells form from these epithelia. In the trunk of the embryo, the sclerotome (middle right, Fig. 3) is the first of the mesodermal mesenchymes to derive from mesodermal epithelia. Interestingly, neural crest starts to emigrate from the neural tube ectoderm at the same time that the first definitive mesodermal mesenchyme forms (middle and upper right, Fig. 3). These cells have the elongate shape and front end-back end polarity (Fig. 1) of mesoderm-derived mesenchymal cells and so their formation from neural tube can be considered an example of epithelialmesenchymal transformation, even though they give rise to nervous tissue and melanocytes. They produce little or no ECM, but neither does primitive streak mesenchyme. In the head, a second
371 wave of neural tube-derived m e s e n c h y m e emigrates that does produce connective tissue (Noden, 1983; Nichols, 1986). Bilozur and Hay (1988) isolated chicken neural tubes at the stage shown in Fig. 3 (middle to lower right) and suspended them inside type I collagen gels or basement membrane gels. After a day of culture, a healthy crop of neural crest cells has moved out into both kinds of 3D ECM. Neither gel contains fibronectin and the fibronectin peptide, RGD, does not inhibit crest migration in the gels (Bilozur and Hay, 1988), contrary to the
situation on planar substrata (Thiery et al., 1985). The laminin cell-binding peptide, Y I G S R (Kleinman et al., 1986), totally inhibits migration of trunk crest in basement membrane gel. This result (Bilozur and Hay, 1988) is very clear cut and simply cannot be explained away by investigators who wish to think that Y I G S R in laminin is not an important cell binding r e # o n of the molecule. Y I G S R binds to the 67 kDa laminin receptor (Kleinman et al., 1986) and it will be interesting to see if crest expresses this receptor when migrating in basement membrane gels.
Fig. 4. Light micrographs of embryonicrat palate immediatelyafter fusion of the shelves (A, 16.25 days) and an enlargementof the midline epithelial seam at the time it is breaking up into mesenchymalcells (arrows, B, 16.75 days). SC, sloughing periderm cell. A few periderm cells are caught in the seam (arrow, A). Bars: A, 100/Lm; B, 25 #m. From Fitchett and Hay (1989).
372 A completely new type of epithelial-mesenchymal transformation has recently been postulated to occur in older embryos during tissue remodeling. Evidence has been presented that removal of Mullerian duct epithelium in the male involves its transformation to mesenchyme (Trelstad et al., 1982). We asked whether or not epithelial-mesenchymal transformation could be involved in a dramatic case of epithelial removal during tissue remodeling: the disappearance of the medial edge epithelium (MEE) after fusion of the two secondary palatal shelves in the midline (Fitchett and Hay, 1989). During the 16th day of development of the rat fetus, the shelves increase in size, rotate, and move toward each other. The outer layer (periderm) of the two layered MEE sloughs, permitting the basal cells of the opposing epithelia to make contact (Fig. 4A). If the periderm is not allowed to slough before contact, as usually occurs in experiments in vitro, periderm is trapped in the seam and dies, giving the appearance that the seam is disappearing by cell death (Pourtois, 1966). Within a half day after contact is made in situ, the seam breaks up and gives rise to mesenchyme (Fig. 4B). A convincing series of micrographs can be assembled to show the steps in this epithelial-mesenchymal transformation (Fitchett and Hay, 1989). The MEE of the seam can be shown by immunohistochemistry to switch its intermediate filament profile from keratin to vimentin before the mesenchyme forms. Vimentin is found in mesenchymal cells and very rarely in epithelia in situ. The same process unites the nasal surface of the palate with the nasal septum and in both regions, epithelia lose syndecan before fusion (Fitchett et al., 1990). This heparan sulfate proteoglycan is a matrix receptor found in most epithelia in the basolateral plasmalemma, and it may stabilize the epithelial tissue phenotype (Bernfield and Sanderson, 1990). Mammary epithelial cells infected in vitro with antisense syndecan message, decrease syndecan expression and become mesenchymal in shape (see Bernfield, this volume). Currently, we are investigating whether or not epithelia-associated integrins also disappear prior to epithelial-mesenchymal transformation in the palate.
We have recently localized a3fll, a5fll, and
a6fll integrins during epithelial-mesenchymal transformation of the lens (Zuk and Hay, 1990). The anterior lens epithelium of the embryonic or adult chicken is one of a number of definitive (nonmesenchyme-forming) epithelia that can be induced to transform into mesenchyme by suspending them completely within 3D collagen gel (Greenburg and Hay, 1982, 1986, 1988). The process of transformation takes a relatively long time (3-5 days) to begin in vitro. Presumably, this is a period of cell proliferation during which the 3D collagen inactivates epithelial programs and turns on the mesenchymal genetic program in the cells facing the gel. The cells begin to accumulate mesenchymal type RER and other organelles at the time they elongate on the surface of the explant and start to move out into the ECM. They stop producing type IV collagen and laminin, and begin producing type I collagen. We found that they expressed at the time they were leaving the explant, all three of the fll integrins we tested, as did the parent epithelium. The et3 and a6 subunits were lost from the elongate, mesenchymal cells after a number of days in culture, whereas a5pl, the principal fibronectin receptor, persists and fibronectin continues to be synthesized. The persistence of the a3 and a6 subunits probably reflects their slow turnover. In these experiments with definitive epithelia, we found that keratin persists for a time in the mesenchyme-like cells that are expressing vimentin (Greenburg and Hay, 1988), whereas in epithelial-mesenchymal transformations that are normally programmed to occur in the embryo, keratin is not present or disappears before the transition takes place (Fitchett and Hay, 1989; Franke et al., 1982). A chicken syndecan is not currently available, but it is possible that it will also be more sluggish in disappearing from definitive epithelia than from epithelia already programmed to form mesenchyme in the embryo. In conclusion, we have described in this chapter a fixed cortex, new front end mechanism for movement of mesenchymal cells through ECM. The ability of mesenchymal cells to emigrate from embryonic and even adult epithelia attests to the validity of the theory, for the transforming cells
373
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Fig. 5. Diagram of a theory of epithelial-mesenchymal transformation based on the fixed cortex model of motility. The presumptive mesenchymal cell constructs a new front end on its base (A) into which its myosin rich endoplasm slides (B). The Golgi complex and actin mRNA will lie in this part of the cell. To get out of the epithelium, the cell recoils its rear end, leaving blobs of cytoplasm behind (C). From Hay (1989b).
clearly extend a new front end capped with filopodia into the adjacent ECM (Fig. 5). That mesenchymal cells, all of which derive from epithelia, might use the same motility mechanism to move through ECM as to depart from the epithelium seems reasonable. That both processes require synthesis of new actin and new plasmalemma in the front end does not seem surprising viewed in this new light. We have postulated (Hay, 1989a,b) that master regulatory gene(s) is/are turned on (Fig. 5) to create the new front end mechanisms. These same genes, or genes they activate down the line, could turn on vimentin mRNA and other components essential for mesenchymal cell elongation (Zuk et
al., 1989). We report in collaboration with Bernfield's group that syndecan disappears before epithelial-mesenchymal transformation in the embryonic palate. Some insight into the proposed function of the syndecan molecule itself in stabilizing the epithelial phenotype might be gained b y following the disappearance of syndecan during an ECM-induced transformation of a definitive epithelia, such as lens, to mesenchyme. In the latter case, we find that epithelial integrins and keratin intermediate filaments may persist in the mesenchyme-like cells without harm to the transformation as long as the fibronectin-integrin and vimentin are present. We hope in the future by in situ hybridization to be able to correlate more
374
exactly the activation of the genes involved in the mesenchymal program with the sequence of morphological changes observed.
References Bard, J.B.L. and E.D. Hay: The behavior of fibroblasts from the developing avian cornea: Morphology and movement in situ and in vitro. J. Cell Biol. 67, 400-418 (1975). Bernfield, M. and R.D. Sanderson: Syndecan, a morphogenetitally regulated cell surface proteoglycan that binds extracellular matrix and growth factors. Phil. Trans. R. Soc. Lond. 327, 171-186 (1990). Bilozur, M.E. and E.D. Hay: Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, and collagen. Dev. Biol. 125, 19-33 (1988). Bilozur, M.E. and E.D. Hay: Cell migration into neural tube lumen provides evidence for the 'fixed cortex' theory of cell motility. Cell Motil. Cytoskel. 14, 469-484 (1989). Burridge, K.: Substrate adhesions in normal and transformed fibroblasts: organiTation and regulation of cytoskeletal, membrane and extracellular matrix components at focal contacts. Cancer Rev. 4, 18-78 (1986). Daniels, K.J. and E.D. Hay: Distribution of fll integrins, matrix, actin, and actin-associated proteins during migration of elongated fibroblasts in 3D collagen gels. J. Cell Biol. 111, 1401 (1990). Fitchett, J. and E.D. Hay: Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse. Dev. Biol. 131, 455-474 (1989). Fitchett, J.E., K.R. McAlmon, E.D. Hay and M. Bernfield: Epithelial cells lose syndecan prior to epithelial-mesenchymal transformation in the developing rat palate. J. Cell Biol. (abstract, in press) (1990). Franke, W.W., C. Grund, C. Kuhn, B.W. Jackson and K. Illmensee: Formation of cytoskeletal elements during mouse embryogenesis. III. Primary mesenchymal cells and the first appearance of vimentin filaments. Differentiation 23, 43-59 (1982). Greenburg, G. and E.D. Hay: Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells. J. Cell Biol. 95, 333-339 (1982). Greenburg, G. and E.D. Hay: Cytodifferentiation and tissue phenotype change during transformation of embryonic lens epithelium to mesenchyme-like cells in vitro. Dev. Biol. 1~15, 363-379 (1986). Greenburg, G- and E.D. Hay: Cytoskeleton and thyroglobulin expression change during transformation of thyroid epithelium to mesenchyme-like cells. Development 102, 605622 (1988). Hay, E.D.: Interaction of migrating embryonic cells with extracellular matrix. Exp. Biol. Med. 10, 174-193 (1985). Hay, E.D.: Extracellular matrix, cell skeletons, and embryonic development. Am. J. Med. Genet. 34, 14-29 (19899). Hay, E.D.: Theory for epithelial-mesenchymal transformation
based on the 'fixed cortex' cell motility model. Cell Motil. Cytoskel. 14, 455-457 (1989b). Hay, E.D.: Cell Biology of Extracellular Matrix, 2nd edn. (Plenum Press, New York), in press (1990). Hemler, M.E.: VLA proteins in the integrin family: structures, functions, and their role on leucocytes. Annu. Rev. Immunol. 8, 365-400 (1990). Herman, I.M., N.J. Crisona and T.D. Pollard: Relation between cell activity and the distribution of actin and myosin. J. Cell Biol. 90, 84-91 (1981). Huxley, H.E.: Muscular contraction and cell motility. Nature (Lond.) 243, 445-449 (1973). Hynes, R.O.: Integrins: a family of cell surface receptors. Cell 48, 549-554 (1987). Kleinman, H.K., M.L. McGarvey, J.R. Hassell, V.L. Star, F.B. Cannon, G.W. Laurie and G.R. Martin: Basement membrane complexes with biological activity. Biochem. 25, 312318 (1986). Kupfer, A., P.J. Kronebusch, J.K. Rose and J. Singer: A critical role for the polarization of membrane recycling in cell motility. Cell Motil. Cytoskel. 8, 182-189 (1987). Lark, M.W. and L.A. Culp: Multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites. J. Biol. Chem. 259, 6773-6782 (1984). Lawrence, J.B. and R. Singer: Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45, 407-415 (1986). Liotta, L.A,, C.N. Rao and S.H. Barsky: Tumor cell interaction with the extracellular matrix. In: The Role of Extracellular Matrix in Development, ed. R.L. Trelstad (Alan R. Liss, New York) pp. 357-371 (1984). McCarthy, R.A. and E.D. Hay: Expression of type I collagen, laminin, and tenascin during early avian development: ultrastructure and correlation with neural crest and sclerotome pathways. Submitted to Am. J. Anat. (1990). Nichols, D.M.: Formation and distribution of neural crest mesenchyme to the first pharyngeal arch region of the mouse embryo. Am. J. Anat. 176, 221-231 (1986). Noden, D.M.: The embryonic origins of avian cephalic and cervical muscles and associated connective tissues. Am. J. Anat. 168, 257-276 (1983). Pourtois, M.: Onset of the acquired potentiality for fusion in the palatal shelves of rats. J. Embryol. Exp. Morphol. 16, 171-182 (1966). Saxen, L.: Organogenesis of the Kidney. Cambridge Univ., Cambridge (1987). Sugrue, S.: Identification and immunolocalization of the laminin binding protein from embryonic corneal epithelial cells. Differentiation 38, 169-176 (1988). Sugrue, S.P. and E.D. Hay: Response of basal epithelial cell surface and cytoskeleton to solubilized extracellular matrix molecules. J. Cell Biol. 91, 45-54 (1981). Svoboda, K.K.H. and E.D. Hay: Embryonic corneal epithelial interaction with exogenous laminin and basal lamina is F-actin dependent. Dev. Biol. 123, 455-469 (1987). Thiery, J.P., J.L. Duband and G.C. Tucker: Cell migration in the vertebrate embryo. Annu. Rev. Cell Biol. 1, 91-114 (1985).
375 Tomasek, J.J. and E.D. Hay: Analysis of the role of microfilaments and microtubules in the acquisition of bipolarity and the subsequent elongation of fibroblasts in hydrated collagen gels. J. Cell Biol. 99, 536-549 (1984). Tomasek, J.J., E.D. Hay and K. Fujiwara: Collagen modulates cell shape and cytoskeleton of embryonic corneal fibroblasts: distribution of actin, a-actinin and myosin. Dev. Biol. 92, 107-122 (1982). Trelstad, R.L., A. Hayashi, K. Hayashi and P.K. Donahoe: The epithelial-mesenchymal interface of the male rat Mullerian duct: loss of basement membrane integrity and ductal regression. Dev. Biol. 92, 27-40 (1982).
Wang, Y.-L.: Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J. Cell Biol. 101, 597-602 (1985). Zuk, A. and E.D. Hay: Changes in expression of the fll integrin family occur during transformation of lens epithelium to mesenchyme in 3D collagen gels. J. Cell Biol. 111, 2651 (1990). Zuk, A., K. Matlin and E.D. Hay: Type I collagen gel induces Madin-Darby canine kidney cells to become fusiform in shape and lose apical-basal polarity. J. Cell Biol. 108, 903-920 (1989).
