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E D Hay

Publications and source records attributed to E D Hay.

At least 19 recordsLinked to original sources
PubMed1996

Effects of electroporation on the tubulin cytoskeleton and directed migration of corneal fibroblasts cultured within collagen matrices.

Electroporation provides a useful method for loading fibroblasts with fluorescent probes for the cytoskeleton, but the possible deleterious effects of this loading technique on cell motility are unknown. We have used conventional and confocal microscopy of living cells and immunohistochemistry to examine the migration and cytoskeleton of chick embryo corneal fibroblasts electroporated while cultured within collagen gels. Fibroblasts cultured in collagen (1 mg/ml) are successfully electroloaded (0.5-1.0 kVcm-1/960 microF in DMEM/F12/20 mM Hepes, pH 7.2) with dextran (4-150 kDa) and immunoglobulin, but subsequently display uncoordinated pseudopodia and hence are unable to migrate effectively in any one direction. The lack of directed movement is due to depolymerization of microtubules and/or a perinuclear collapse of vimentin filaments, seemingly caused by millimolar levels of Ca2+ ions derived from culture medium following electroporation. Fibroblasts loaded in a buffer which resembles intracellular fluid (< or = 10 microM Ca2+) maintain their cytoskeleton and continue to migrate, when returned to culture medium within 10 min. Using this novel approach, we have loaded fibroblasts migrating through extracellular matrix (ECM) with rhodamine phalloidin and monitored the behavior of the labeled actin cortex by confocal microscopy. During migration phalloidin-actin accumulates near the base of pseudopodia and at the rear of the cell where it is subsequently left behind. We conclude that electroporation is a valuable technique for loading fibroblasts to study migration within ECM, provided that the conditions used support stability of the tubulin cytoskeleton.

Actins↗
PubMed1995

Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced.

In this review, we define the two major tissue types, epithelium and mesenchyme, and we describe the transformations (transdifferentiations) of epithelium to mesenchyme (EMT) and mesenchyme to epithelium (MET) that occur during embryonic development. The differentiation of the metanephric blastema provides a striking example of MET. Differentiation of metanephric epithelium is promoted by matrix molecules and receptors (nidogen, laminins, alpha 6 integrins), hepatic growth factor/scatter factor, and products of the genes wnt-1, wnt-4, and Pax-2. Transformation of MDCK epithelium to mesenchyme-like cells is promoted in vitro by antibodies to E-cadherin, products of v-src, v-ras, and v-mos, and by manipulation of the epithelium on collagen gels. Suspension in collagen gel, transforming growth factors, and c-fos have also been shown to promote EMT in epithelia. We present studies from our laboratory showing that alpha 5 beta 1 integrin has a role in the EMT of lens epithelium that is brought about by suspension in collagen gel. Our laboratory has also shown that transfection with the E-cadherin gene induces embryonic corneal fibroblasts to undergo MET and that this MET is enhanced by interaction of the differentiating epithelium with living fibroblasts. This review calls attention to the roles that EMT and MET might have in kidney pathologies and urges further study of the involvement of these phenomena in renal development, renal injury, and renal malignancy.

Animals↗
PubMed1995

An overview of epithelio-mesenchymal transformation.

Epithelium is the tissue phenotype of early embryos and primitive adults of the chordate phylum. A second tissue type, however, is produced by epithelial-mesenchymal transformation (EMT) in higher chordates, such as vertebrata. Mesenchymal cells have the ability, which true epithelia do not, to invade and migrate through the extracellular matrix (ECM) to create dramatic cell transpositions. The first-formed or primary mesenchymal cells in amniote vertebrates migrate from the primitive streak to differentiate into the mesodermal and endodermal epithelia. Definitive mesenchyme with connective tissue and muscle potentials arises from the epithelial mesoderm at about the same time as the neural crest mesenchyme forms from the ectoderm. Later on in embryogenesis. EMT is used to remodel unwanted epithelia, such as that of the palate medial edges. We discuss the mechanisms by which epithelial cells transform into mesenchyme and vice versa. On the one hand, cells activate putative mesenchymal master genes, turn off epithelial genes, and acquire motility machinery that allows them to interact in 3 dimensions (3D) with ECM via actin cortex while sliding their endoplasm into their new front ends. On the other hand, primary mesenchymal cells can reactivate epithelial regulatory genes, such as E-cadherin, turn off the motility machinery for invading ECM, and reexpress apical-basal polarity. We review the genes, such as FSP1, src, ras, and fos, that are activated in cells transforming to mesenchyme and the genes their neighbors activate to induce EMT, such as those for TGF beta, NT-3, and sonic hedgehog. Suspension in 3D collagen gels can induce adult epithelium to undergo EMT; alpha 5 beta 1 integrin is activated on surfaces in contact with collagen, including apical surfaces that do not normally express integrins. In vivo, it is possible that pathological manipulations of a cell's environment likewise induce EMT. Of the examples we give, the creation of invasive metastatic carcinoma cells by EMT is the most fearful. Interestingly, transfection of either metastatic cells or normal embryonic fibroblasts with the E-cadherin gene converts them to the epithelial phenotype. It may be possible in the future to manipulate the tissue phenotype of diseased cells to the advantage of the animal.

Animals↗
PubMed1994

Expression of beta 1 integrins changes during transformation of avian lens epithelium to mesenchyme in collagen gels.

Remarkably, a number of definitive epithelia, such as that of the anterior lens, give rise when suspended within 3D gels of type I collagen, to elongate, bipolar shaped cells that exhibit the ultrastructure, polarity, and migratory ability of mesenchymal cells. They begin producing type I collagen and stop producing crystallins, type IV collagen, and laminin. Here, we investigated changes in beta 1 integrins and their extracellular matrix (ECM) ligands during this transdifferentiation. The former free surface of the lens epithelium that is now in contact with collagen begins within a day to stain intensely for beta 1 and it is this surface rather than the surface facing the basement membrane that gives rise to mesenchymal cells. Immunoprecipitation experiments reveal a large increase in the beta 1 integrin subunit on mesenchymal cells as compared to the epithelium of origin. The alpha 5 integrin subunit, which is barely detectable in the lens, increases in the mesenchymal cells and alpha 3 continues to be expressed at about the same level as in the epithelium. alpha 6, the epithelial integrin subunit, and laminin, its ECM ligand, are not detected immunohistochemically or biochemically in the mesenchyme. Rather, the mesenchymal cells secrete abundant fibronectin, the major ECM ligand for alpha 5 beta 1. RGD peptides do not inhibit the transformation but antibodies to beta 1 do perturb the emigration of mesenchymal cells from the lens apical surface. We conclude that the beta 1 integrins newly expressed on the apical epithelial surface interact with the surrounding 3D collagen gel to help bring about this unusual epithelial-mesenchymal transition.

Amino Acid Sequence↗
PubMed1994

Cytoplasmic loading of dyes, protein and plasmid DNA using an impact-mediated procedure.

We describe a method and apparatus designed to rapidly and reproducibly produce transient, survivable plasma membrane disruptions--"wounds"--in order to gain access to the cytoplasm of eukaryotic cells growing in culture. Compressed gas is used to propel glass beads, dispersed as a uniform aerosol, at adherent cells growing on a culture substratum. The impact of beads with the cells creates plasma membrane wounds. Macromolecules, such as dyes, proteins and plasmid DNAs, diffuse from the extracellular environment directly into the cytoplasmic compartment of the cell through these wounds. Resealing of the plasma membrane, necessary for cell survival, traps macromolecules within the cytoplasm of the cell.

3T3 Cells↗
PubMed1993

Origin of extraembryonic mesoderm in experimental animals: relevance to chorionic mosaicism in humans.

Confined chorionic mosaicism, a discordance in the karyotype between the fetus and placenta, occurs in 1% of chorionic villus sampling (CVS) cases. While the cytogenetic discrepancies occurring between different fetal tissues may pose clinical dilemmas, they can also be viewed as a natural experiment to determine early cell lineage relationships in the human. We reviewed extensive data in experimental animals to define the origin of the human extraembryonic mesoderm. The extraembryonic mesoderm in humans is an important component of the CVS culture preparation. Previously, the extraembryonic mesoderm was thought to originate in the cytotrophoblast or primitive streak. More recent evidence supports its origin from the yolk sac, which does not always correlate with the fetal karyotype. We formulated a model of early human cell lineage and employed it to clarify clinical cases of chorionic mosaicism in two large published studies.

Animals↗
PubMed1993

Tissue-specific expression of type XII collagen during mouse embryonic development.

Polyclonal antibodies were raised in rabbits against a fusion peptide representing a portion of the amino-terminal non-triple-helical domain of mouse type XII collagen. The antibodies reacted with bands of 220 and 350 kDa on Western blots of mouse tissue extracts. Immunohistochemical analyses of mouse embryos demonstrated that type XII collagen is expressed mainly in dense connective tissues of tendons, ligaments, dermis, cornea, blood vessel walls, meninges, and developing membranous bones. Comparison of skin extracts and medium of cultured mouse skin fibroblasts by Western blotting showed that while tissue contain short 220 kDa type XII collagen polypeptides as well as the long form, cultured cells produce mainly the long form with 350 kDa polypeptides.

Animals↗
PubMed1993

Extracellular matrix alters epithelial differentiation.

Extracellular matrix (ECM) induces and maintains the differentiation of epithelial cells, not by totally altering their state of differentiation, but by activating overt differentiation. Recent studies of cultured mammary cells provide an elegant molecular analysis of this kind of progressive cell differentiation. Other studies show that ECM can not only activate and enhance a differentiated state, but can even alter it in bringing about transformation of epithelium to mesenchyme.

Animals↗
PubMed1993

Retinoic acid inhibits formation of mesenchyme from lens epithelium in collagen gels.

PURPOSE: To examine the possibility that retinoic acid (RA), a stabilizer of the epithelial phenotype, would inhibit formation of mesenchymal cells from avian lens epithelium in three-dimensional collagen. METHODS: Lens epithelia from 11-day-old chick embryos were cultured for 6 days in collagen gels in the presence of RA. The number of mesenchymal cells emigrating into the gels was quantitatively compared with control cultures to which RA was not added. RESULTS: It was found that few fibroblast-like cells form at the highest dose used (10(-5) M RA) and outgrowth approaches control levels at lower doses of RA. The mesenchymal cells that form after RA treatment are not ultrastructurally different from those of controls. Many have well-developed rough endoplasmic reticulum and undoubtedly produce the collagen fibrils that accumulate around the cells. Others, although spindle-shaped, still exhibit lenslike cytoplasm. New basement membrane is deposited on the former free surface of RA-treated lens epithelium, but is not present at the former free surface of control epithelium. CONCLUSIONS: It is possible that RA inhibition of lens transformation to fibroblast-like cells is at least partly due to the ability of RA to stimulate production of basement membrane components by epithelia. More studies of RA action on epithelial-mesenchymal transformation in collagen gels may reveal additional mechanisms. It is also suggested that mesenchymal genes similar to those activated in lens epithelium by suspension in collagen may turn on in pathologic transformations (ie, in anterior capsular cataract, fibroblast-like cells arise from lens epithelium.

Animals↗
PubMed1992

Type X collagen is transcriptionally activated and specifically localized during sternal cartilage maturation.

Type X collagen is an extracellular matrix protein which is synthesized by chondrocytes when they undergo hypertrophy. We present evidence here that the expression of type X collagen in the developing chick sternum is controlled primarily by transcriptional mechanisms. Using chondrocyte nuclei isolated from 15-, 16-, 17- and 18-day chick embryonic sterna, nuclear run-off assays demonstrate that type X collagen gene transcription begins at day 16 in chondrocytes isolated from the cephalic portion. This occurs two days prior to mineralization of this tissue as observed by alizarin red staining. The rate of type X transcription increases dramatically through days 17 and 18. Western blot analyses of extracts of freshly isolated sternal chondrocytes from the same stages show that intracellular levels of the type X protein follow the same time course. Immunostaining with a monoclonal antibody specific for type X collagen demonstrates that the initial appearances of hypertrophic cells and pericellular type X collagen occur at embryonic day 16 in the cephalic portion of sterna. Observation of immunostained cephalic sternal sections from day 18 embryos by confocal microscopy reveals that type X collagen is localized in a capsule-like configuration around each hypertrophic chondrocyte.

Animals↗
PubMed1992

Epithelial-mesenchymal transformation during palatal fusion: carboxyfluorescein traces cells at light and electron microscopic levels.

During the fusion of rodent embryo palatal shelves, the cells of the outer epithelial layer slough off, allowing the cells of the medial edge basal layer to form a midline seam that undergoes epithelial-mesenchymal transformation, as judged by electron microscopy and immunohistochemistry. In this study, we analyze the fate of the transformed cells using a lipid soluble dye to label the medial edge epithelium in situ. Prefusion E14 mouse palates were exposed in vitro or in vivo to a fluoresceinated lipid soluble marker, carboxydichlorofluorescein diacetate succinimidyl ester (CCFSE), which localizes in epithelia as a lipid insoluble compound that does not pass into the connective tissue compartment. The midline seam that formed after 24 hours contained labelled epithelial cells that were replaced by individually labelled mesenchymal cells where the seam transformed. By light microscopy, the labelled cells were seen to contain intensely fluorescent bodies that do not react for acid phosphatase. We were able for the first time to identify these structures by electron microscopy as CCFSE isolation bodies. The cells with isolation bodies are clearly healthy and able to participate in subsequent development of the palate. At 4 days after labelling, individual CCFSE containing cells present in the palate mesenchyme occupy both midline and lateral areas and can clearly be classified as fibroblasts by electron microscopy. CCFSE is a far more useful marker than another lipid soluble marker, DiI, for following cells, because the cells can be fixed and identified both at the light and electron microscope levels. Interestingly, if labelled palatal shelves are not allowed to fuse in vitro, the basal epithelial cells do not form mesenchyme after sloughing, indicating that formation of the epithelial midline seam is necessary to trigger its epithelial-mesenchymal transformation.

Animals↗
PubMed1991

Collagen I, laminin, and tenascin: ultrastructure and correlation with avian neural crest formation.

We have investigated the distribution of type I collagen, tenascin, and laminin in younger chick embryos than have previously been studied in detail. The initial appearance of type I collagen, but not tenascin and laminin, is exactly correlated with the beginning of neural crest migration, suggesting a role for collagen I in the migration. Light microscopy of whole mounts of 2-day-old chick embryos reveals that type I collagen is expressed in a rostral to caudal gradient; it localizes to the notochord sheath before accumulating around the neural tube and somites. Collagen I and tenascin also associate with central somite cells. Surprisingly, no extracellular matrix can be detected among the early sclerotomal cells, which suggests that little or no cell migration is involved in this epithelial-mesenchymal transformation. Electron microscopy using peroxidase antiperoxidase reveals that tenascin is present in nonstriated, 10 nm wide fibrils and in interstitial bodies, both of which have previously been reported to contain fibronectin. However, collagen I only occurs in the 10 nm fibrils and larger striated fibrils. This is the first ultrastructural study to assign tenascin to fibrils and interstitial bodies and to describe its appearance and disappearance from embryonic basement membranes. The discussion emphasizes the possible importance of type I collagen in neural crest cell migration and compares the ultrastructural associations of the ECM molecules present at this early embryonic stage.

Animals↗
PubMed1990

Role of cell-matrix contacts in cell migration and epithelial-mesenchymal transformation.

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 fibroblasts 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.

Actin Cytoskeleton↗
PubMed1989

Extracellular matrix, cell skeletons, and embryonic development.

During embryonic development, the extracellular matrix (ECM) promotes the production of differentiated products by epithelial cells and the migration of mesenchymal cells, and probably also plays a role in epithelial-mesenchymal transformation. Here we examine the role of the cell skeleton (actin, microtubules, intermediate filaments) in mediating matrix effects on mesenchymal cell morphology, migration, and formation. The interaction of both epithelial cells and mesenchymal cells with ECM seems to involve the actin cortex, which is best developed in the base of the epithelial cell, where it attaches to underlying matrix via membrane-intercalated receptors. To interact with the matrix, the fibroblast has appropriate ECM receptors and an actin cortex around the whole cell. The actin cortex is absolutely required for assumption of bipolar shape, elongation, and movement through the matrix. Since the cortex seems to be anchored to the matrix, it is unlikely that it moves during cell migration. A new hypothesis states that the microtubule- and intermediate filament-rich endoplasm, containing the nucleus, moves past the actin cortex-receptor-matrix complex into the newly synthesized front end of the mesenchymal cell to effect forward movement. When epithelial cells transform into mesenchyme in the embryo, or when they are induced to do this in vitro, they switch from the keratin intermediate filament profile to one rich in vimentin, and the effect of cell matrix interaction on cell shape is profoundly altered. Vimentin-actin interactions with ECM may be a major factor in the ability of a cell to become mesenchymal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Cell migration into neural tube lumen provides evidence for the "fixed cortex" theory of cell motility.

We present a model of cell motility based on emigration of neural crest cells into the neural tube lumen under in vitro conditions (10% fetal calf serum or YIGSR) that inhibit their normal emigration from the base of the neuroepithelium into surrounding extracellular matrix (ECM). Ultrastructural observations reveal that cells lining the lumen are joined by zonulae adherentes (ZA), which are points of strong intercellular attachment, and thereby serve as markers for fixed regions of plasmalemma and cortical actin. Three major observations of the relationship of cells to the ZA support the "fixed cortex" model of mesenchymal cell migration. First, cells extend apical cell processes past the ZA into the lumen. To do this, they must make new apical plasmalemma and actin cortex that the endoplasm slides into. Second, elongated cells are observed in the lumen that are still attached via ZA to the neuroepithelium. This indicates that all of the endoplasm finally slides past the ZA. Third, numerous cytoplasmic pieces, often attached to each other and to the neuroepithelium via ZA, are found at the site where cells appear to have detached from the epithelium after entering the lumen. Since the ZA is fixed in location, the endoplasm must have slid past it into newly manufactured anterior cortex and plasmalemma, with the trailing end of the cell finally snapping off. The "fixed cortex" theory of cell migration agrees with existing data in that it predicts the polarized insertion of new plasmalemma and actin at the leading end of the cell, but it differs significantly from existing theories of mesenchymal cell migration in that it states that the cell surface remains firmly attached to the substratum while the myosin-rich endoplasm slides past it.

Animals↗
PubMed1989

Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse.

The disappearance of palatal medial edge epithelium (MEE) after fusion of secondary palatal shelves is often cited as a classical example of embryonic remodeling by programmed cell death. We reinvestigated this phenomenon in 16-day rat embryos, using light and electron microscopy. We confirm reports that the periderm of the two-layered MEE begins to slough after shelves assume horizontal positions. In vitro, peridermal cells are not able to slough and are trapped during the adhesion process. In vivo, however, surface cells shed before the shelves in the anterior palate adhere, allowing junctions to form between opposing basal epithelial cells. Midline seams so formed consist of two layers of basal cells, all of which appear healthy. Even though its cells are dividing, growth of the seam fails to keep pace with palatal growth and it thins to one layer of cells, and then breaks up into small islands. The basal lamina disappears and elongating MEE cells extend filopodia into adjacent connective tissue. Electron micrographs reveal transitional steps in loss of epithelial characteristics and gain of fibroblast-like features by transforming MEE cells. One such feature, observed with the aid of immunofluorescence, is the turn of the mesenchymal cytoskeletal protein, vimentin. No cell death or macrophages are observed after adhesion and thinning over most of the palate. These data indicate that MEE is an ectoderm that retains the ability to transform into mesenchymal cells. Epithelial-mesenchymal transformation may be expressed in other embryonic remodelings (R.L. Trelstad, A. Hayashi, K. Hayashi, and P.K. Donahue, 1982, Dev. Biol. 92, 27), resulting in heretofore unsuspected conservation of embryonic cell populations.

Animals↗
PubMed1989

Type I collagen gel induces Madin-Darby canine kidney cells to become fusiform in shape and lose apical-basal polarity.

In the embryo, epithelia give rise to mesenchyme at specific times and places. Recently, it has been reported (Greenburg, G., and E. D. Hay. 1986. Dev. Biol. 115:363-379; Greenberg, G., and E. D. Hay. 1988. Development (Camb.). 102:605-622) that definitive epithelia can give rise to fibroblast-like cells when suspended within type I collagen gels. We wanted to know whether Madin-Darby canine kidney (MDCK) cells, an epithelial line, can form mesenchyme under similar conditions. Small explants of MDCK cells on basement membrane were suspended within or placed on top of extracellular matrix gels. MDCK cells on basement membrane gel are tall, columnar in shape, and ultrastructurally resemble epithelia transporting fluid and ions. MDCK explants cultured on type I collagen gel give rise to isolated fusiform-shaped cells that migrate over the gel surface. The fusiform cells extend pseudopodia and filopodia, lose cell membrane specializations, and develop an actin cortex around the entire cell. Unlike true mesenchymal cells, which express vimentin and type I collagen, fusiform cells produce both keratin and vimentin, continue to express laminin, and do not turn on type I collagen. Fusiform cells are not apically-basally polarized, but show mesenchymal cell polarity. Influenza hemagglutinin and virus budding localize to the front end or entire cell surface. Na,K-ATPase occurs intracellularly and also symmetrically distributes on the cell surface. Fodrin becomes diffusely distributed along the plasma membrane, ZO-1 cannot be detected, and desmoplakins distribute randomly in the cytoplasm. The loss of epithelial polarity and acquisition of mesenchymal cell polarity and shape by fusiform MDCK cells on type I collagen gel was previously unsuspected. The phenomenon may offer new opportunities for studying cytoplasmic and nuclear mechanisms regulating cell shape and polarity.

Animals↗