Donald F. Newgreen

Carcinoma Invasion and Metastasis: A Role for Epithelial-Mesenchymal Transition?

Carcinogenesis involves the accretion of unprogrammed genetic and epigenetic changes, which lead to dysregulation of the normal control of cell number. But a key clinical turning point in carcinoma progression is the establishment by emigrant cells of secondary growth sites (i.e., metastasis). The metastatic “cascade” comprises numerous steps, including escape from the primary tumor site, penetration of local stroma, entry of local vascular or lymphatic vessels (intravasation), aggregation with platelets, interaction with and adhesion to distant endothelia, extravasation, recolonization, and expansion ( 1), all the time avoiding effective immune clearance and being able to survive in these multiple contexts...

The fallacy of epithelial mesenchymal transition in neoplasia

Epithelial mesenchymal transition has been postulated as a versatile mechanism which facilitates cellular repositioning and redeployment during embryonic development, tissue reconstruction after injury, carcinogenesis, and tumor metastasis. The hypothesis originates from parallels drawn between the morphology and behavior of locomotory and sedentary cells in vitro and in various normal and pathologic processes in vivo. This review analyzes data from several studies on embryonic development, wound healing, and the pathology of human tumors, including work from our own laboratory, to assess the validity of the proposal. It is concluded that there is no convincing evidence for conversion of epithelial cells into mesenchymal cell lineages in vivo and that the biological repertoire of normal and malignant cells is sufficient to account for the events and processes observed, without needing to invoke radical changes in cell identity.

Fibronectin in early avian embryos: Synthesis and distribution along the migration pathways of neural crest cells

SummaryImmunoperoxidase labelling for fibronectin (FN) in chick embryos showed FN-positive basement membranes surrounding the neural crest cell population prior to crest-cell migration. At cranial levels, crest cells migrated laterally into a large cell-free space. Initially they moved as a tongue of cells contacting the FN-positive basement membrane of the ectoderm, but later the crest cell population expanded into space further from the ectoderm, until eventually the entire cranial cell-free space was occupied by mesenchyme cells. This was accompanied by the appearance of FN among the crest cells. At trunk levels, crest cells entered a relatively small space already containing FN-positive extracellular material. At later stages the migration of trunk crest cells broadly matched the distribution of FN. In vitro, chick and quail embryo ectoderm, endoderm, somites, notochord and neural tube synthesized and organized fibrous FN-matrices, as shown by immunofluorescence. Ectoderm and endoderm deposited this matrix only on the substrate face. The FN content of endoderm and neural tube matrices was transient, the immunofluorescence intensity declining after 1–2 days in culture. Some crest cells of cranial and sacral axial levels synthesized FN. Our data suggests that these were the earliest crest cells to migrate from these levels. This ability may be the first expression of mesenchymal differentiation in these crest cells, and in vivo enable them to occupy a large space. Almost all crest cells from cervico-lumbar axial levels were unable to synthesize FN. In vivo, this inability may magnify the response of these crest cells to FN provided by the neighbouring embryonic tissues.

Vimentin and Epithelial-Mesenchymal Transition in Human Breast Cancer – Observations in vitro and in vivo

Breast cancer is a highly prevalent disease among women worldwide. While the expression of certain proteins within these tumours is used for prognosis and selection of therapies, there is a continuing need for additional markers to be identified. A considerable amount of current literature, based predominantly on cell culture systems, suggests that a major mechanism responsible for the progression of breast cancer is due to tumour cells losing their epithelial features and gaining mesenchymal properties. These events are proposed to be very similar to the epithelial-mesenchymal transition (EMT) process that has been well characterised in embryonic development. For the developmental and putative cancer EMT, the cell intermediate filament status changes from a keratin-rich network which connects to adherens junctions and hemidesmosomes, to a vimentin-rich network connecting to focal adhesions. This review summarises observations of vimentin expression in breast cancer model systems, and discusses the potential role of EMT in human breast cancer progression, and the prognostic usefulness of vimentin expression.

Enteric nervous system: development and developmental disturbances--part 2.

This review, which is presented in two parts, summarizes and synthesizes current views on the genetic, molecular, and cell biological underpinnings of the early embryonic phases of enteric nervous system (ENS) formation and its defects. Accurate descriptions of the phenotype of ENS dysplasias, and knowledge of genes which, when mutated, give rise to the disorders (see Part 1 in the previous issue of this journal), are not sufficient to give a real understanding of how these abnormalities arise. The often indirect link between genotype and phenotype must be sought in the early embryonic development of the ENS. Therefore, in this, the second part, we provide a description of the development of the ENS, concentrating mainly on the origin of the ENS precursor cells and on the cell migration by which they become distributed throughout the gastrointestinal tract. This section also includes experimental evidence on the controls of ENS formation derived from classic embryological, cell culture, and molecular genetic approaches. In addition, for reasons of completeness, we also briefly describe the origins of the interstitial cells of Cajal, a cell population closely related anatomically and functionally to the ENS. Finally, a brief sketch is presented of current notions on the developmental processes between the genes and the morphogenesis of the ENS, and of the means by which the known genetic abnormalities might result in the ENS phenotype observed in Hirschsprungs disease.

Epithelium-Mesenchyme Transition during Neural Crest Development

The neural crest is the organ system whose presence defines vertebrates. The onset of migration of neural crest cells is an archetypal epithelium to mesenchyme transition (EMT), and this event identifies the cell lineage. Little is known yet of the establishment of the neural crest, although the zinc finger gene Slug seems to be involved in specifying EMT competence. The details, especially the temporal order of events in neural crest EMT, vary between different species and between different axial levels, but several important features have emerged from observations in situ and experiments in vitro and in vivo. EMT seems to be strongly associated with decrease in cell-cell adhesion, and particularly with loss of N-cadherin on the surface of neural crest cells at the time of onset of migration. The related adhesion molecule T-cadherin is also present, but correlated changes have not yet been described, while the unrelated adhesion molecule N-CAM also declines on neural crest cells, but with a time course unrelated to EMT. The extracellular matrix is also important: EMT-related changes in matrix receptor (i.e. integrin) activity are recorded in avian crest cells, while the nature of the matrix itself changes in urodele amphibians. Changes in cell shape and in cell motility also occur at the time of EMT, consistent with changes in the cytoskeleton. These concerted changes can be triggered by TGF-beta family growth factors, of which dorsalin-I appears particularly important. These may act through pathways involving controlled alterations in phosphorylation to effect the complex of responses that make up EMT. Although much remains to be understood, the spatiotemporal definability of this system makes it a very useful model for studying EMTs in general.

Ultrastructural and tissue-culture studies on the role of fibronectin, collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo

SummaryThe initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15–40 nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin (⪖ 3 nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker (⪖ 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen.Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates. However, partially desulphated chondroitin sulphate (5mg/ml) strongly retarded the migration of NC cells.The in vivo and in vitro studies suggest that fibronectin may dictate the pathways of NC cell migration by acting as a highly preferred physical substrate. However, the utilization of these pathways may be reduced by the presence of proteoglycans bearing undersulphated chondroitin sulphate.

Ryk-deficient mice exhibit craniofacial defects associated with perturbed Eph receptor crosstalk

Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-α/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-β2, Tgf-β3 and activin-βA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor ‘related to tyrosine kinases’ (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction–associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.

Enteric neural crest-derived cells: origin, identification, migration, and differentiation.

Neurons and glial cells forming the enteric nervous system (ENS) arise from neural crest cells that migrate away from two different rostrocaudal levels of the neural axis, the vagal and sacral regions. The vagal region is defined as the post-otic hindbrain level with somites 1–7 (Le Douarin, 1982), and the sacral region is caudal to somite 28 in chick embryos and caudal to somite 24 in embryonic mice.

Neurovascular congruence results from a shared patterning mechanism that utilizes Semaphorin3A and Neuropilin-1.

Peripheral nerves and blood vessels have similar patterns in quail forelimb development. Usually, nerves extend adjacent to existing blood vessels, but in a few cases, vessels follow nerves. Nerves have been proposed to follow vascular smooth muscle, endothelium, or their basal laminae. Focusing on the major axial blood vessels and nerves, we found that when nerves grow into forelimbs at E3.5-E5, vascular smooth muscle was not detectable by smooth muscle actin immunoreactivity. Additionally, transmission electron microscopy at E5.5 confirmed that early blood vessels lacked smooth muscle and showed that the endothelial cell layer lacks a basal lamina, and we did not observe physical contact between peripheral nerves and these endothelial cells. To test more generally whether lack of nerves affected blood vessel patterns, forelimb-level neural tube ablations were performed at E2 to produce aneural limbs; these had completely normal vascular patterns up to at least E10. To test more generally whether vascular perturbation affected nerve patterns, VEGF(165), VEGF(121), Ang-1, and soluble Flt-1/Fc proteins singly and in combination were focally introduced via beads implanted into E4.5 forelimbs. These produced significant alterations to the vascular patterns, which included the formation of neo-vessels and the creation of ectopic avascular spaces at E6, but in both under- and overvascularized forelimbs, the peripheral nerve pattern was normal. The spatial distribution of semaphorin3A protein immunoreactivity was consistent with a negative regulation of neural and/or vascular patterning. Semaphorin3A bead implantations into E4.5 forelimbs caused failure of nerves and blood vessels to form and to deviate away from the bead. Conversely, semaphorin3A antibody bead implantation was associated with a local increase in capillary formation. Furthermore, neural tube electroporation at E2 with a construct for the soluble form of neuropilin-1 caused vascular malformations and hemorrhage as well as altered nerve trajectories and peripheral nerve defasciculation at E5-E6. These results suggest that neurovascular congruency does not arise from interdependence between peripheral nerves and blood vessels, but supports the hypothesis that it arises by a shared patterning mechanism that utilizes semaphorin3A.