Elizabeth D. Hay

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 cells 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.

The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it.

This review centers on the role of the mesenchymal cell in development. The creation of this cell is a remarkable process, one where a tightly knit, impervious epithelium suddenly extends filopodia from its basal surface and gives rise to migrating cells. The ensuing process of epithelial–mesenchymal transformation (EMT) creates the mechanism that makes it possible for the mesenchymal cell to become mobile, so as to leave the epithelium and move through the extracellular matrix. EMT is now recognized as a very important mechanism for the remodeling of embryonic tissues, with the power to turn an epithelial somite into sclerotome mesenchyme, and the neural crest into mesenchyme that migrates to many targets. Thus, the time has come for serious study of the underlying mechanisms and the signaling pathways that are used to form the mesenchymal cell in the embryo. In this review, I discuss EMT centers in the embryo that are ready for such serious study and review our current understanding of the mechanisms used for EMT in vitro, as well as those that have been implicated in EMT in vivo. The purpose of this review is not to describe every study published in this rapidly expanding field but rather to stimulate the interest of the reader in the study of the role of the mesenchymal cell in the embryo, where it plays profound roles in development. In the adult, mesenchymal cells may give rise to metastatic tumor cells and other pathological conditions that we will touch on at the end of the review. Developmental Dynamics 233:706–720, 2005.

Cell contact during early morphogenesis in the chick embryo.

Abstract The primitive epithelial layers of the chick blastoderm, the epiblast and hypoblast, are underlain by discontinuous basement laminae. Primary mesenchymal cells, which comprise the mesoblast that migrates laterally from the primitive streak, make close junctions (intercellular space, 25–100 A) and tight junctions (no apparent extracellular space) with the basal surfaces of the epiblast and hypoblast. Close and tight junctions are also prominent between mesenchymal cells, especially along their trailing edges. Filopodia are most numerous on the leading edges of the migrating cells as would be expected if the close and tight junctions played a role in contact inhibition, perhaps by mediating electrical coupling between the cells. The tight junctions within mesoblast, epiblast, and hypoblast are at first focal in nature (maculae occludentes), but subsequently those between cells in the same tissue become more extensive while those between cells in unlike tissues are disrupted. When the primary mesenchyme organizes itself into an epithelium (somite, lateral, and intermediate mesoderm), the trailing ends of the cells, which contain the Golgi organelles, are directed toward the middle of the mass where zonulae occludentes appear next to the newly created free surface. Subsequently, secondary mesenchyme derives from the mesodermal epithelium. The migrating cells are connected by broad tight junctions (fasciae occludentes) which seem to persist as maculae occludentes after the presumptive connective tissue cells differentiate. Within the epithelia, maculae adhaerentes (desmosomes) are particularly well developed at later stages and probably contribute to tissue-specific intercellular adhesion.

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.

AN AUTORADIOGRAPHIC AND ELECTRON MICROSCOPIC STUDY OF COLLAGEN SYNTHESIS IN DIFFERENTIATING CARTILAGE.

SummaryThe synthesis of the proline-rich collagen component of cartilage matrix has been studied by autoradiography using both the light and electron microscope. Amblystoma maculatum larvae had their forelimbs amputated, were allowed to regenerate for 12–15 days, and then injected intraperitoneally with tritiated proline. The animals were fixed at various times (1 min. to 28 days) after the injection and sections of the developing limbs were coated for autoradiography by dipping in Ilford L 4 or Gevaert 3.07 emulsion. The sequential labeling of the organelles of the cartilage cell which occurred is illustrated in light and electron micrographs. Radioactive products first appeared in the ergastoplasm and were associated with the cisternae of the endoplasmic reticulum. Twenty to thirty minutes after the injection, labeled material began to appear in the Golgi zone. There, the newly synthesized protein accumulated within large vacuoles. The fibrillar material within the vacuoles may represent collagen and the more amorphous material, mucoprotein. The vacuoles subsequently (∼2 hrs. later) discharge their labeled contents into the extracellular space. The secreted protein is probably soluble collagen (tropocollagen) for it diffuses readily through the matrix to polymerize into striated collagen fibrils some distance from the cell. These findings contradict some widely held opinions that the fibrillar component of the matrix arises by excortication and appositional growth of fibrils originating from the ectoplasm of chondrocytes. It seems reasonable to conclude that the secretory pathway by which extracellular proteins are produced in cartilage is analogous to that suggested for epithelial gland cells.

Development of the vertebrate cornea.

Publisher Summary The development of the avian cornea involves a remarkable continuum of highly coordinated events. The corneal epithelium is the first of the component tissues to differentiate. This ectodermal derivative, with the help of the lens, secretes the highly ordered primary stroma composed of orthogonally arranged collagen fibrils embedded in a chondroitin sulfate (CS)-rich matrix. The fibroblasts of the avian cornea may produce the first fibronectin to appear within the stroma, although fibronectin is present earlier on the posterior stromal surface and lens. Several striking events occur together during the period of stromal condensation. An enzyme—hyaluronidase—appears, which may remove hyaluronic acid (HA), contributing to the dehydration that begins next to the endothelium and leads to the compaction of the posterior stroma. The dehydration of the stroma and acquisition of transparency are triggered by thyroxine. Thyroxine accelerates interdigitation of endothelial cells and conceivably tells this tissue to begin pumping water out of the cornea. The mitogenic effect of thyroxine on mammalian corneal epithelium in situ is mimicked by fibroblast growth factor (FGF) and epidermal growth factor (EFG), both of which stimulate stratification.

DIRECT EVIDENCE FOR A ROLE OF β-CATENIN/LEF-1 SIGNALING PATHWAY IN INDUCTION OF EMT

Epithelial‐mesenchymal transformation (EMT) is an important process in development that is characterized by loss of E‐cadherin, β‐catenin relocalization, and acquisition of elongated cell shape and ability to invade ECM. β‐catenin has been shown to activate LEF‐1 transcription during EMT induced in vitro by c‐Fos. Here, we ask whether or not LEF‐1 directly introduced into epithelial cells in an adenovirus construct can induce EMT. In normal epithelial cell lines, such as HCE and MDCK cells, that contain functional APC, nuclear β‐catenin induced by exogenous LEF‐1 is rapidly exported and EMT is not induced. Leptomycin‐B blocks β‐catenin nuclear export, but no EMT occurs due to toxicity. Addition of Wnt‐1 to normal epithelial cell lines stabilizes cytoplasmic β‐catenin that LEF‐1 then transports to nuclei, causing a small amount of EMT. Our experiments demonstrated, however, that overexpressed LEF‐1 upregulates nuclear β‐catenin and promotes dramatic EMT in DLD‐1 epithelial tumors that retain nuclear β‐catenin. This EMT is reversible if the LEF‐1 virus is removed. Thus, our results demonstrate that LEF‐1 can induce EMT directly when its transcription activity is activated by stable nuclear β‐catenin. Normal adult epithelial cells appear to use APC to keep β‐catenin out of the nucleus, thereby avoiding pathologies such as metastases due to LEF/β‐catenin‐induced EMT.

Snail and Slug Promote Epithelial-Mesenchymal Transition through β-Catenin–T-Cell Factor-4-dependent Expression of Transforming Growth Factor-β3

Members of the Snail family of transcription factors have been shown to induce epithelial-mesenchymal transition (EMT), a fundamental mechanism of embryogenesis and progressive disease. Here, we show that Snail and Slug promote formation of beta-catenin-T-cell factor (TCF)-4 transcription complexes that bind to the promoter of the TGF-beta3 gene to increase its transcription. Subsequent transforming growth factor (TGF)-beta3 signaling increases LEF-1 gene expression causing formation of beta-catenin-lymphoid enhancer factor (LEF)-1 complexes that initiate EMT. TGF-beta1 or TGF-beta2 stimulates this signaling mechanism by up-regulating synthesis of Snail and Slug. TGF-beta1- and TGF-beta2-induced EMT were found to be TGF-beta3 dependent, establishing essential roles for multiple TGF-beta isoforms. Finally, we determined that beta-catenin-LEF-1 complexes can promote EMT without upstream signaling pathways. These findings provide evidence for a unified signaling mechanism driven by convergence of multiple TGF-beta and TCF signaling molecules that confers loss of cell-cell adhesion and acquisition of the mesenchymal phenotype.

Origin of the blastema in regenerating limbs of the newt Triturus viridescens: An autoradiographic study using tritiated thymidine to follow cell proliferation and migration☆

Abstract The present study has utilized autoradiography to detect incorporation of tritiated thymidine by the cells of the regenerating Triturus viridescens limb during blastema formation and to follow the subsequent migration of cells labeled by the isotope. New information was obtained on sites of DNA synthesis during blastema formation and the role of the internal tissues and epithelium was re-evaluated by tracing, for the first time, the actual fate of labeled cells during normal regeneration. Three series of experiments were performed. In the first, regenerating limbs 1–28 days post amputation were fixed the same day that the animals were injected with tritiated thymidine. This experiment revealed that DNA synthesis begins 4–5 days after amputation in the dedifferentiating muscle, endomysium, epimysium, periosteum, nerve sheaths, and loose connective tissue of the stump for a distance ∼ 1 mm proximal to the amputation surface. The number of cells in the inner tissues synthesizing DNA increases rapidly 10–20 days post amputation. The epidermis which migrates over the wound surface ceases to synthesize DNA within about 2 days. Epithelium proximal to the amputation surface, however, reaches a high level of DNA synthesis 8 days after amputation, and its cells migrate distally to increase the size of the apical cap 10–15 days post amputation. During blastema formation, no more than 2% of the cells in the apical cap incorporate thymidine, these rather feebly. After the blastema is established and the regenerate has begun to elongate, the apical cap thins out and its cells begin to synthesize DNA again. In the second experimental series, limbs were treated with tritiated thymidine during regeneration (5, 10, and 15 days post amputation) and representative limbs were fixed at daily intervals after treatment. The dedifferentiating inner cells incorporated a large amount of thymidine on the day of injection, whereas the apical epithelium did not. Almost all the blastema cells that appeared subsequently were labeled, and it can be concluded that they were derived from the dedifferentiating internal tissues. In the third experimental series, animals were injected with tritiated thymidine before their limbs were amputated. Epidermis is the only limb tissue that labels under these circumstances. Blood cells are labeled in their sites of origin, the liver and spleen. After amputation the labeled epidermis migrates over the wound surface and forms an apical cap which remains well labeled throughout blastema formation. The isotope becomes diluted in that area of proliferating proximal epithelium shown in Series I to be actively synthesizing new DNA. Blood cells labeled at the time of injection extravasate into the wounded limb tissues, but disappear from the limb 12–15 days post amputation The blastema cells which form in these limbs that contained labeled blood and apical epithelium are not labeled. In the discussion, the concept that the morphological and physiological changes which take place in dedifferentiating tissues make possible a phase of active cellular proliferation, is emphasized. The apical epidermal cap does not exhibit the DNA synthesis or any of the other essential features of dedifferentiating cells and, when this epidermis is labeled by appropriate treatment, no transformation of epithelial cells into blastema cells can be demonstrated. Indeed, the cells of the apical cap give evidence of being more highly differentiated than epidermis elsewhere.

Transforming Growth Factor-β Signaling during Epithelial-Mesenchymal Transformation: Implications for Embryogenesis and Tumor Metastasis

The molecular mechanisms of epithelial-mesenchymal transformation (EMT) have long been studied to gain a greater understanding of this distinct change in cellular morphology. Early studies of the developing embryo have designated the involvement of Wnt signaling in EMT, through an activated complex of the lymphoid-enhancing factor-1 (LEF-1) transcription factor and the cell adhesion molecule β-catenin. However, more recent studies have implicated a significant role of the transforming growth factor-β (TGF-β) in causing EMT in both development and pathology. The ability of TGF-β isoforms to signal through a variety of molecules such as Smads, phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) creates an incredible complexity as to their role in this transition. Here we assess the biochemical signaling pathways of TGF-β and their potential cross-interaction with traditional Wnt signaling molecules to bring about EMT during embryogenesis and tumor metastasis.