Epithelium-mesenchyme Transition Research Articles

CDC42-mediated Wnt signaling facilitates odontogenic differentiation of DPCs during tooth root elongation

BackgroundCDC42 is a member of Rho GTPase family, acting as a molecular switch to regulate cytoskeleton organization and junction maturation of epithelium in organ development. Tooth root pattern is a highly complicated and dynamic process that dependens on interaction of epithelium and mesenchyme. However, there is a lack of understanding of the role of CDC42 during tooth root elongation.MethodsThe dynamic expression of CDC42 was traced during tooth development through immunofluorescence staining. Then we constructed a model of lentivirus or inhibitor mediated Cdc42 knockdown in Herwig’s epithelial root sheath (HERS) cells and dental papilla cells (DPCs), respectively. Long-term influence of CDC42 abnormality was assessed via renal capsule transplantation and in situ injection of alveolar socket.ResultsCDC42 displayed a dynamic spatiotemporal pattern, with abundant expression in HERS cells and apical DPCs in developing root. Lentivirus-mediated Cdc42 knockdown in HERS cells didn’t disrupt cell junctions as well as epithelium-mesenchyme transition. However, inhibition of CDC42 in DPCs undermined cell proliferation, migration and odontogenic differentiation. Wnt/β-catenin signaling as the downstream target of CDC42 modulated DPCs’ odontogenic differentiation. The transplantation and in situ injection experiments verified that loss of CDC42 impeded root extension via inhibiting the proliferation and differentiation of DPCs.ConclusionsWe innovatively revealed that CDC42 was responsible for guiding root elongation in a mesenchyme-specific manner. Furthermore, CDC42-mediated canonical Wnt signaling regulated odontogenic differentiation of DPCs during root formation.

Transición epitelio – mesenquima y cáncer.

Cancer cell migration and invasion are critical components of metastatic disease, the leading cause of death in cancer patients. The epithe-lium-mesenchyme-transition (EMT) and mesenchyme-epithelium-transition (MET) are pathways involved in cancer metastasis. This process involves the degradation of cell-cell and cell-extracellular matrix junctions and the subse-quent loss of regulation of binding proteins such as E-cadherin. Cells undergo a reorganization of the cytoskeleton. These alterations are associated with a change in cell shape from epithelial to mesenchymal morphology. Understand-ing EMT and MET’s molecular and cellular basis provides fundamental insights into cancer etiology and may lead to new therapeutic strategies. In this review, we discuss some of the regulatory mechanisms and pathological role of epitheli-al-mesenchymal plasticity, focusing on the knowledge about the complexity and dynamics of this phenomenon in cancer

ERK Signaling Pathway Is Constitutively Active in NT2D1 Non-Seminoma Cells and Its Inhibition Impairs Basal and HGF-Activated Cell Proliferation.

c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype», while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium-mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

Prolactin increases cell migration of MCF-7 cells without inducing an epithelium-mesenchyme transition

Prolactin increases cell migration of MCF-7 cells without inducing an epithelium-mesenchyme transition

Implication of Ataxia-Telangiectasia-mutated kinase in epithelium-mesenchyme transition.

Impairment of genome instability drives the development of cancer by disrupting anti-cancer barriers. Upon genotoxic insults, DNA damage responsive factors, notably ATM kinase, is crucial to protect genomic integrity while promoting cell death. Meanwhile, cytotoxic therapy-inducing DNA lesions is double-edged sword by causing cancer metastasis based on animal models and clinical observations. The underlying mechanisms for the procancer effect of cytotoxic therapies are poorly understood. Here, we report that cancer cells subjected to cytotoxic treatments elicit dramatic alteration of gene expression controlling the potential of epithelium-mesenchyme transition (EMT). Resultantly, EMT-dependent cell mobility is potently induced upon DNA damage. This stimulation of EMT is mainly Ataxia-Telangiectasia-mutated (ATM)-dependent, as the chemical inhibitor specifically inhibiting ATM kinase activity can suppress the EMT gene expression and thus cell mobility. At last, we show that cancer cells with ATM activation display increased metastatic potential in ovarian cancer tissues. Taken together, we reveal a novel role of ATM in promoting metastatic potential of cancer cells by favoring EMT gene expression.

MicroRNA-203 Modulates the Radiation Sensitivity of Human Malignant Glioma Cells

We investigated whether miR-203 could modulate the radiation sensitivity of glioblastoma (GBM) cells and which target gene(s) could be involved. Three human malignant glioma (MG) cell lines and normal human astrocytes were transfected with control microRNA, pre-miR-203, or antisense miR-203. Real-time PCR (RT-PCR), clonogenic assays, immunofluorescence, and invasion/migration assays were performed. To predict the target(s), bioinformatics analyses using microRNA target databases were performed. Overexpression of miR-203 increased the radiation sensitivity of all 3 human MG cell lines and prolonged radiation-induced γ-H2AX foci formation. Bioinformatics analyses suggested that miR-203 could be involved in post-transcriptional control of DNA repair, PI3K/AKT, SRC, and JAK/STAT3 and the vascular signaling pathway. Western blot analysis validated the fact that miR-203 downregulated ATM, RAD51, SRC, PLD2, PI3K-AKT, JAK-STAT3, VEGF, HIF-1α, and MMP2. Overexpression of miR-203 inhibited invasion and migration potentials, downregulated SLUG and Vimentin, and upregulated Claudin-1 and ZO1. These data demonstrate that miR-203 potentially controls DNA damage repair via the PI3K/AKT and JAK/STAT3 pathways and may collectively contribute to the modulation of radiation sensitivity in MG cells by inhibiting DNA damage repair, prosurvival signaling, and epithelium-mesenchyme transition. Taken together, these findings demonstrate that miR-203 could be a target for overcoming the radiation resistance of GBM.

Development, Triploblastism, Physics of Wetting and the Cambrian Explosion

The Cambrian explosion is characterized by the sudden outburst of organized animal plans, which occurred circa 530 M years ago. Around that time, many forms of animal life appeared, including several which have since disappeared. There is no general consensus about "why" this happened, and why it had any form of suddenness. However, all organized animal plans share a common feature: they are triploblastic, i.e., composed of 3 layers of tissue, endoderm, ectoderm and mesoderm. I show here that, within simple hypotheses, the formation of the mesoderm has intrinsically a physical exponential dynamics, leading rapidly to triploblastism, and eventually, to animal formation. A novel physico-mathematical framework including epithelium-mesenchyme transition, visco-elastic constitutive equations, and conservation laws, is presented which allows one to describe gastrulation as a self-wetting phenomenon of a soft solid onto itself. This phenomenon couples differentiation and migration during gastrulation, and leads in a closed form to an exponential scaling law for the formation of the mesoderm. Therefore, the Cambrian explosion might have started, actually, by a true viscoelastic "explosion": the exponential run-away of mesenchymal cells.

Abstract 1487: Evaluation of the relationship between e-cadherin expression and proliferative activity at the invasive front of esophageal squamous cell carcinoma.

Abstract Introduction Esophageal squamous cell carcinoma (ESCC) has a poor prognosis mainly because it is usually in an advanced stage at the time of diagnosis. The epithelium-mesenchyme transition (EMT), when epithelial cells loss their original characteristics and acquire mesenchymal phenotype, is indicated first by the loss of E-cadherin expression. The EMT is considered the main event in tumor progression and metastasis. In vitro studies have demonstrated that e-cadherin downregulation can lead to cell proliferation. This finding has not been evaluated in human tumors. This study aimed to elucidate whether tumor cells that have undergone EMT at the invasive front of the ESCC are in proliferative state. Material and Methods Samples of 58 ESCC cases were double stained by immunohistochemistry (IHQ) for E-cadherin (BD Bioscience) and Ki67 (ROCHE) antibodies using the Automated System Ventana BenchMark XT (ROCHE). IHC analysis was performed using Aperio ScanScope XT (APERIO) with the Algorithm ColorDeconvolution. In each case E-cadherin and Ki67 expression were evaluated in the tumor core and at the invasive front, separately. Positivity was divided into strong, moderate and weak positive according to intensity staining. For each case a score was given based on the formula with the percentages of each positivity group [Score = 1x (% Weak) + 2x (% Moderate) + 3x (% Strong)]. Results and Discussion Analysis of the IHQ double-stain for E-cadherin showed that 51 (87.9%) cases had a high expression at the tumor core when compared with invasive front and in 7 (12.1%) cases were found high expression at the invasion front (p<0,001). The median values for E-cadherin double-stain scores were 184.2 and 163.0 at invasive front and tumor core, respectively (p<0.001). Analyses of the Ki67 double-stain showed that 19 (32.7%) cases showed a high expression at tumor core and 39 (67.3%) cases showed a high expression at the invasive front (p<0,001). Proliferation rates, represented by Ki67 expression, showed median values of 8.1 and 6.8 at the invasive front and tumor core, respectively (p=0.052). Spearman correlation between the IHQ double and single-stain was moderately positive (p <0.001). Conclusion Tumor cells at the invasive front showed an increased proliferation rate as they lose the E-cadherin expression and at the tumor core showed an higher expression of E-cadherin and a lower expression of Ki67. However, some cases showed a different behavior with a higher expression of E-cadherin at the invasive front and a elevated rate proliferation at tumor core. A more complex mechanism in the dynamics of E-cadherin and Ki67 expressions could be acting during EMT. Further studies are necessary to the better understanding of the complexity of this mechanism. Citation Format: Juliano Jampietro, Yukie Sato-Kuwabara, José Humberto H.T.G. Fregnani, Felipe Coimbra, Claudia Malheiros Coutinho Camillo, Fernando Augusto Soares. Evaluation of the relationship between e-cadherin expression and proliferative activity at the invasive front of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2013-1487

A tale of two collagen receptors, integrin β1 and discoidin domain receptor 1, in epithelial cell differentiation

As increase in collagen deposition is no longer taken as simply a consequence but, rather, an inducer of disease progression; therefore, the understanding of collagen signal transduction is fundamentally important. Cells contain at least two types of collagen receptors: integrins and discoidin domain receptors (DDRs). The integrin heterodimers α(1)β(1), α(2)β(1), α(10)β(1), and α(11)β(1) are recognized as the non-tyrosine kinase collagen receptors. DDR1 and 2, the tyrosine kinase receptors of collagen, are specifically expressed in epithelium and mesenchyme, respectively. While integrin β(1) and DDR1 are both required for cell adhesion on collagen, their roles in epithelial cell differentiation during development and disease progression seem to counteract each other, with integrin β(1) favoring epithelium mesenchyme transition (EMT) and DDR1 inducing epithelial cell differentiation. The in vitro evidence shows that the integrin β(1) and DDR1 exert opposing actions in regulation of membrane stability of E-cadherin, which itself is a critical regulator of epithelial cell differentiation. Here, we review the functional roles of integrin β(1) and DDR1 in regulation of epithelial cell differentiation during development and disease progression, and explore the underlining mechanisms regarding to the regulation of membrane stability of E-cadherin.

IL-18 neutralization ameliorates obstruction-induced epithelial–mesenchymal transition and renal fibrosis

Ureteral obstruction results in renal fibrosis in part due to inflammatory injury. The role of interleukin-18 (IL-18), an important mediator of inflammation, in the genesis of renal fibrosis was studied using transgenic mice overexpressing human IL-18-binding protein. In addition, HK-2 cells were analyzed following direct exposure to IL-18 compared to control media. Two weeks after ureteral obstruction, the kidneys of wild-type mice had a significant increase in IL-18 production, collagen deposition, alpha-smooth muscle actin and RhoA expression, fibroblast and macrophage accumulation, chemokine expression, and transforming growth factor-beta1 (TGF-beta1) and tumor necrosis factor-alpha (TNF-alpha) production, whereas E-cadherin expression was simultaneously decreased. The transgenic mice with neutralized IL-18 activity exhibited significant reductions in these indicators of obstruction-induced renal fibrosis and epithelial- mesenchymal transition, without demonstrating alterations in TGF-beta1 or TNF-alpha activity. Similarly, the HK-2 cells exhibited increased alpha-smooth muscle actin expression and collagen production, and decreased E-cadherin expression in response to IL-18 stimulation without alterations in TNF-alpha or TGF-beta1 activity. Our study demonstrates that IL-18 is a significant mediator of obstruction-induced renal fibrosis and epithelial- mesenchymal transition independent of downstream TGF-beta1 or TNF-alpha production.

Fibroblast Growth Factor 2 Reactivates G1 Checkpoint in SK-N-MC Cells via Regulation of p21, Inhibitor of Differentiation Genes (Id1-3), and Epithelium-Mesenchyme Transition-Like Events

We have recently demonstrated that fibroblast growth factor (FGF)-2 promotes neuroblastoma cell differentiation and overrides their mitogenic response to IGF-I. However, the mechanisms involved are unknown. SK-N-MC cells were cultured with FGF-2 (50 ng/ml) and/or IGF-I (100 ng/ml) up to 48 h. Fluorescence-activated cell sorting analysis indicated that FGF-2 promotes G1/G0 cell cycle phase arrest. Gene expression by RT2-PCR and cellular localization showed up-regulation of p21. We then investigated whether FGF-2-induced differentiation of SK-N-MC cells (by GAP43 and NeuroD-6 expression) involves epithelium-mesenchyme transition interconversion. Real-time PCR (RT2-PCR) showed modulation of genes involved in maintenance of the epithelial phenotype and cell-matrix interactions (E-cadherin, Snail-1, MMPs). Zymography confirmed FGF-2 up-regulated MMP2 and induced MMP9, known to contribute to neuronal differentiation and neurite extension. Id1-3 expression was determined by RT2-PCR. FGF-2 induced Id2, while down-regulating Id1 and Id3. FGF-2 induced nuclear accumulation of ID2 protein, while ID1 and ID3 remained cytoplasmic. RNA interference demonstrated that Id3 regulates differentiation and cell cycle (increased Neuro-D6 and p21 mRNA), while d Id2 modulates epithelium-mesenchyme transition-like events (increased E-cadherin mRNA). In conclusion, we have shown for the first time that FGF-2 induces differentiation of neuroblastoma cells via activation of a complex gene expression program enabling modulation of cell cycle, transcription factors, and suppression of the cancer phenotype. The use of RNA interference indicated that Id-3 is a key regulator of these events, thus pointing to a novel therapeutic target for this devastating childhood cancer.

Ajuba LIM Proteins Are Snail/Slug Corepressors Required for Neural Crest Development in Xenopus

Snail family transcriptional repressors regulate epithelial mesenchymal transitions during physiological and pathological processes. A conserved SNAG repression domain present in all vertebrate Snail proteins is necessary for repressor complex assembly. Here, we identify the Ajuba family of LIM proteins as functional corepressors of the Snail family via an interaction with the SNAG domain. Ajuba LIM proteins interact with Snail in the nucleus on endogenous E-cadherin promoters and contribute to Snail-dependent repression of E-cadherin. Using Xenopus neural crest as a model of in vivo Snail- or Slug-induced EMT, we demonstrate that Ajuba LIM proteins contribute to neural crest development as Snail/Slug corepressors and are required for in vivo Snail/Slug function. Because Ajuba LIM proteins are also components of adherens junctions and contribute to their assembly or stability, their functional interaction with Snail proteins in the nucleus suggests that Ajuba LIM proteins are important regulators of epithelia dynamics communicating surface events with nuclear responses.

Snail Family Transcription Factors Are Implicated in Thyroid Carcinogenesis

E-Cadherin (CDH1) expression is reduced in thyroid carcinomas by primarily unknown mechanisms. In several tissues, SNAIL (SNAI1) and SLUG (SNAI2) induce epithelial-mesenchymal transition by altering target gene transcription, including CDH1 repression, but these transcription factors have not been studied in thyroid carcinoma. Recently, our group has provided direct evidence that ectopic SNAI1 expression induces epithelial and mesenchymal mouse tumors. SNAI1, SNAI2, and CDH1 expression were analyzed in thyroid-derived cell lines and samples of human follicular and papillary thyroid carcinoma by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. The effect of SNAI1 expression on CDH1 transcription was analyzed by reverse transcriptase-polymerase chain reaction and Western blotting in ori-3 cells. Thyroid carcinoma development was analyzed in CombitTA-Snail mice, in which SNAI1 levels are up-regulated. SNAI1 and SNAI2 were not expressed in cells derived from normal thyroid tissue, or in normal human thyroid samples, but were highly expressed in cell lines derived from thyroid carcinomas, in human thyroid carcinoma samples, and their metastases. SNAI1 expression in ori-3 cells repressed CDH1 transcription. Combi-TA mice developed papillary thyroid carcinomas, the incidence of which was increased by concomitant radiotherapy. In conclusion, SNAI1 and SNAI2 are ectopically expressed in thyroid carcinomas, and aberrant expression in mice is associated with papillary carcinoma development.

Activation of NF-κB by Akt upregulates Snail expression and induces epithelium mesenchyme transition

Carcinoma progression is associated with the loss of epithelial features, and the acquisition of mesenchymal characteristics and invasive properties by tumour cells. The loss of cell-cell contacts may be the first step of the epithelium mesenchyme transition (EMT) and involves the functional inactivation of the cell-cell adhesion molecule E-cadherin. Repression of E-cadherin expression by the transcription factor Snail is a central event during the loss of epithelial phenotype. Akt kinase activation is frequent in human carcinomas, and Akt regulates various cellular mechanisms including EMT. Here, we show that Snail activation and consequent repression of E-cadherin may depend on AKT-mediated nuclear factor-kappaB (NF-kappaB) activation, and that NF-kappaB induces Snail expression. Expression of the NF-kappaB subunit p65 is sufficient for EMT induction, validating this signalling module during EMT. NF-kappaB pathway activation is associated with tumour progression and metastasis of several human tumour types; E-cadherin acts as a metastasis suppressor protein. Thus, this signalling and transcriptional network linking AKT, NF-kappaB, Snail and E-cadherin during EMT is a potential target for antimetastatic therapeutics.

Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail.

Snail is a transcription repressor that plays a central role in the epithelium-mesenchyme transition (EMT), by which epithelial cells lose their polarity. Claudins and occludin are integral membrane proteins localized at tight junctions, which are responsible for establishing and maintaining epithelial cell polarity. We examined the relationship between Snail and the promoter activity of claudins and occludin. When Snail was overexpressed in cultured mouse epithelial cells, EMT was induced with concomitant repression of the expression of claudins and occludin not only at the protein but also at the mRNA level. We then isolated the promoters of genes encoding claudins and occludin, in which multiple E-boxes were identified. Transfection experiments with various promoter constructs as well as electrophoretic mobility assays revealed that Snail binds directly to the E-boxes of the promoters of claudin/occludin genes, resulting in complete repression of their promoter activity. Because the gene encoding E-cadherin was also reported to be repressed by Snail, we concluded that EMT was associated with the simultaneous repression of the genes encoding E-cadherin and claudins/occludin (i.e. the expression of adherens and tight junction adhesion molecules, respectively).

Roles of lumican and keratocan on corneal transparency.

Lumican and keratocan are members of the small leucine-rich proteoglycan (SLRP) family, and are the major keratan sulfate (KS) proteoglycans in corneal stroma. Both lumican and keratocan are essential for normal cornea morphogenesis during embryonic development and maintenance of corneal topography in adults. This is attributed to their bi-functional characteristic (protein moiety binding collagen fibrils to regulate collagen fibril diameters, and highly charged glycosaminoglycan (GAG) chains extending out to regulate interfibrillar spacings) that contributes to their regulatory role in extracellular matrix assembly. The absence of lumican leads to formation of cloudy corneas in homozygous knockout mice due to altered collagenous matrix characterized by larger fibril diameters and disorganized fibril spacing. In contrast, keratocan knockout mice exhibit thin but clear cornea with insignificant alteration of stromal collaegenous matrix. Mutations of keratocan cause cornea plana in human, which is often associated with glaucoma. These observations suggest that lumican and keratocan have different roles in regulating formation of stromal extracellular matrix. Experimental evidence indicates that lumican may have additional biological functions, such as modulation of cell migration and epithelium-mesenchyme transition in wound healing and tumorgenesis, besides regulating collagen fibrillogenesis.

In situ hybridisation of chick embryos with p53-specific probe and their immunostaining with anti-p53 antibodies.

Tumor-suppressor protein p53 is an important regulator of cell cycle and apoptosis. On the level of embryo extracts it has been shown earlier that both p53 protein and mRNA are expressed in developing chicken. Here we describe the expression patterns of p53 mRNA and protein in developing chicken embryos (stages 2-12) using in situ hybridisation and immunostaining with p53-specific monoclonal antibody Mab421. p53 mRNA is equally localised all over the embryo in the stages observed. According to electron microscopy data a subfraction of p53 mRNA is bound to dissolving yolk granules expressing acid phosphatase activity characteristic for lysosomes. Protein p53 is synthesised starting from the medium primitive streak stage (stage 3) and reaches its maximum level at the full primitive streak stage. During these stages protein p53 is distributed evenly across the embryos. After gastrulation p53 protein remains visible at higher levels only in certain anlages and areas. In developing nervous system the expression is observable in neuroectoderm, during the closure of the neural tube and in mesenchyme in the area of migrating neural crest cells. In cardiogenesis protein p53 is expressed during formation of tubular heart in the epimyocardium, endocardium and cardiac jelly. p53 protein localises in the neurocoele (obviously connected with cellular debris) and cardiac jelly. Our data support the role of p53 in early development, especially during embryo gastrulation, the development of central nervous system, neural crest and heart. In some cases increased p53 amounts colocalise with the areas of intensive epithelium-mesenchyme transition.

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.