Epithelial-Mesenchymal Transitions in Development and Disease

The epithelial to mesenchymal transition (EMT) plays crucial roles in the formation of the body plan and in the differentiation of multiple tissues and organs. EMT also contributes to tissue repair, but it can adversely cause organ fibrosis and promote carcinoma progression through a variety of mechanisms. EMT endows cells with migratory and invasive properties, induces stem cell properties, prevents apoptosis and senescence, and contributes to immunosuppression. Thus, the mesenchymal state is associated with the capacity of cells to migrate to distant organs and maintain stemness, allowing their subsequent differentiation into multiple cell types during development and the initiation of metastasis. The epithelial to mesenchymal transition (EMT) plays crucial roles in the formation of the body plan and in the differentiation of multiple tissues and organs. EMT also contributes to tissue repair, but it can adversely cause organ fibrosis and promote carcinoma progression through a variety of mechanisms. EMT endows cells with migratory and invasive properties, induces stem cell properties, prevents apoptosis and senescence, and contributes to immunosuppression. Thus, the mesenchymal state is associated with the capacity of cells to migrate to distant organs and maintain stemness, allowing their subsequent differentiation into multiple cell types during development and the initiation of metastasis. Most adult tissues and organs arise from a series of conversions of epithelial cells to mesenchymal cells, through the epithelial to mesenchymal transition (EMT) and the reverse process (mesenchymal to epithelial transition [MET]). Epithelial cells establish close contacts with their neighbors and an apicobasal axis of polarity through the sequential arrangement of adherens junctions, desmosomes, and tight junctions. The epithelial cell layer maintains global communication through gap junctional complexes, and it remains separated from adjacent tissues by a basal lamina. Epithelia have the capacity to function as barriers or in absorption. Conversely, mesenchymal or stromal cells are loosely organized in a three-dimensional extracellular matrix and comprise connective tissues adjacent to epithelia. The conversion of epithelial cells to mesenchymal cells is fundamental for embryonic development and involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties. This review presents the events in development that involve EMT and discusses its relevance in tissue homeostasis, tissue repair, fibrosis, and carcinoma progression. We also examine the impact of EMT on drug resistance and explore recent findings that reinforce the concept of EMT as a major driver of morphogenesis and tumor progression. The transition of epithelial to mesenchymal cells is not irreversible, as several rounds of EMT and MET are necessary for the final differentiation of specialized cell types and the acquisition of the complex three-dimensional structure of internal organs. Accordingly, these sequential rounds are referred to as primary, secondary, and tertiary EMT (Figure 1). Examples of primary EMT include those evident during mammalian implantation, during gastrulation in various metazoans, and in the neural crest of all vertebrates. Although the morphogenetic movements associated with gastrulation vary among metazoans, it is the universal process by which the body plan is established. The necessary changes in cell shape are followed by internalization of the mesendoderm, convergence to the midline, and extension along the anteroposterior axis. A crucial structure in all organisms is the region where cells involute or ingress (the ventral furrow in Drosophila, the blastopore in Xenopus, and the primitive streak in the chick and mouse). In vertebrates, this region contains an organizing center known as the Spemann organizer in Xenopus, the shield in fish, and the node in birds and mammals. To understand how gastrulation proceeds, it is necessary to consider the successive inductive processes that occur before or at the time when the organizer forms and to identify the molecular elements involved. Interestingly, some of the most important elements are conserved throughout evolution. The sea urchin is amenable to detailed fate mapping and molecular embryology approaches, leading to the generation of an extensive gene regulatory network at gastrulation (Oliveri et al., 2008Oliveri P. Tu Q. Davidson E.H. Global regulatory logic for specification of an embryonic cell lineage.Proc. Natl. Acad. Sci. USA. 2008; 105: 5955-5962Crossref PubMed Scopus (169) Google Scholar) (Figure 2A). Key in this network are the transcription factors Snail and Twist, which are evolutionary conserved repressors of E-cadherin and inducers of EMT (Peinado et al., 2007Peinado H. Olmeda D. Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?.Nat. Rev. Cancer. 2007; 7: 415-428Crossref PubMed Scopus (1420) Google Scholar). In the sea urchin, Snail inhibits E-cadherin transcription and promotes cadherin endocytosis as well as the delamination of primary mesenchyme cells (PMCs) by EMT (Wu et al., 2007Wu S.Y. Ferkowicz M. McClay D.R. Ingression of primary mesenchyme cells of the sea urchin embryo: a precisely timed epithelial mesenchymal transition. Birth Defects Res.C Embryo Today. 2007; 81: 241-252Crossref PubMed Scopus (15) Google Scholar), whereas its inhibition blocks PMC ingression. In turn, inhibition of Twist function delays the ingression of PMCs (Wu et al., 2008Wu S.Y. Yang Y.P. McClay D.R. Twist is an essential regulator of the skeletogenic gene regulatory network in the sea urchin embryo.Dev. Biol. 2008; 319: 406-415Crossref PubMed Scopus (14) Google Scholar). (A) The gene regulatory network governing EMT during gastrulation in the sea urchin embryo. A specification step involving Wnt8 signaling leads to HesC repression, switching on the EMT regulatory program, and inducing the ingression of the primary mesenchymal cells (PMCs). Alx1, aristaless-like 1. (B) Mesoderm invagination in Drosophila. Twist and Snail pathways cooperate to modulate cell adhesion and cytoskeletal changes to undergo gastrulation movements and mesoderm spreading. The arrows indicate the flow of the pathway, not direct transcriptional regulation. Abl, Abelson kinase; Htl, Heartless (Drosophila FGF receptor); Dof, downstream of FGFR; Fog/Cta, folded in gastrulation/concertina. (C) Genetic pathways controlling gastrulation in amniotes. Convergence of signaling pathways at the posterior part of the embryo leads to primitive streak formation and initiation of the EMT as well as the mesodermal fate program. Snail genes are key regulators of the EMT program during gastrulation in amniotes as they control cell-cell adhesion, cell shape, and motility. Additional mechanisms such as endocytosis, lysosomal targeting, and degradation of the E-cadherin protein together with the control of basement membrane integrity explain the rapid and drastic changes occurring in ingressing cells during gastrulation. The induction of endodermal and mesodermal fates is mainly governed by the FGF and Nodal pathways through specific regulators and the contribution of some of the genes involved in the EMT program. EPB4L5, FERM and actin-binding domains-containing band 4.1 superfamily member; FLRT3, Fibronectin-leucine-rich-transmembrane protein-3; Net-1, neuroepithelial transforming factor 1; MMP, metalloproteinases; p38IK, p38 interacting kinase. As in sea urchin, Twist and Snail are crucial factors in fly gastrulation (Figure 2B). Apical constriction is necessary for ventral furrow formation and cell invagination, and this process requires Twist and its target, T48. These proteins are recruited to the adherens junctions, producing rapid changes in cell shape in conjunction with RhoGEF2, a Rho GTP-exchange factor and cytoskeletal regulator that concentrates at the site of apical constriction (Kolsch et al., 2007Kolsch V. Seher T. Fernandez-Ballester G.J. Serrano L. Leptin M. Control of Drosophila gastrulation by apical localization of adherens junctions and RhoGEF2.Science. 2007; 315: 384-386Crossref PubMed Scopus (148) Google Scholar). Snail is also required for ventral furrow formation, the cells of which express string, a cdc25 homolog essential for entry into mitosis. Snail-dependent string inhibition generates the mitotic block necessary for gastrulation to occur (Grosshans and Wieschaus, 2000Grosshans J. Wieschaus E. A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila.Cell. 2000; 101: 523-531Abstract Full Text Full Text PDF PubMed Google Scholar). Simultaneously, Snail represses E-cadherin transcription (Oda et al., 1998Oda H. Tsukita S. Takeichi M. Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation.Dev. Biol. 1998; 203: 435-450Crossref PubMed Scopus (160) Google Scholar) and generates the pulses of myosin contraction required for apical constriction while Twist maintains the constricted state between pulses (Martin et al., 2009Martin A.C. Kaschube M. Wieschaus E.F. Pulsed actin-myosin network contractions drive apical constriction.Nature. 2009; 457: 495-499Crossref PubMed Scopus (345) Google Scholar). In vertebrates, T48 is not conserved, and Twist is not crucial for gastrulation, suggesting that Snail may fulfill all of these functions. In Xenopus, the Spemann organizer is induced by the Nieuwkoop center, a group of dorsal blastula cells characterized by the nuclear accumulation of β-catenin. Wnt signaling initiates the process, and Goosecoid is induced in the Spemann organizer by its target, Siamois, and by several transforming growth factor β (TGFβ) superfamily members, including Nodal (Gilbert, 2006Gilbert S.F. Developmental Biology.Eighth Edition. Sinauer Associates, Inc., Sunderland, MA2006Google Scholar). In amniotes, activation of Wnt signaling confers competence to the posterior part of the embryo in the formation of the primitive streak (Figure 2C). Subsequently, members of the TGFβ superfamily, including Nodal and Vg1, induce gastrulation. Nodal signaling, together with fibroblast growth factor (FGF), controls the specification of the mesendoderm in all vertebrates (Figure 2C). Thus, in preparation for EMT, numerous signaling pathways help establish an organizing center that in turn controls morphogenetic movements and specification (Heisenberg and Solnica-Krezel, 2008Heisenberg C.P. Solnica-Krezel L. Back and forth between cell fate specification and movement during vertebrate gastrulation.Curr. Opin. Genet. Dev. 2008; 18: 311-316Crossref PubMed Scopus (45) Google Scholar). There are two main Snail genes in vertebrates, Snail1 and Snail2 (called SNAI1 and SNAI2 in humans). They are induced by TGFβ superfamily members, and FGF signaling is necessary to maintain their expression and for gastrulation to proceed (Barrallo-Gimeno and Nieto, 2005Barrallo-Gimeno A. Nieto M.A. The Snail genes as inducers of cell movement and survival: implications in development and cancer.Development. 2005; 132: 3151-3161Crossref PubMed Scopus (687) Google Scholar, Ciruna and Rossant, 2001Ciruna B. Rossant J. FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak.Dev. Cell. 2001; 1: 37-49Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Snail-deficient embryos fail to gastrulate, and “mesodermal” cells are unable to downregulate E-cadherin accumulate at the streak (Carver et al., 2001Carver E.A. Jiang R. Lan Y. Oram K.F. Gridley T. The mouse snail gene encodes a key regulator of the epithelial-mesenchymal transition.Mol. Cell. Biol. 2001; 21: 8184-8188Crossref PubMed Scopus (367) Google Scholar, Nieto et al., 1994Nieto M.A. Sargent M.G. Wilkinson D.G. Cooke J. Control of cell behavior during vertebrate development by Slug, a zinc finger gene.Science. 1994; 264: 835-839Crossref PubMed Google Scholar). Snail proteins are not essential for mesodermal fate specification as a “mesodermal” population expressing the appropriate markers still forms in Snail mutant mice, although cells fail to migrate because it cannot undergo EMT (Carver et al., 2001Carver E.A. Jiang R. Lan Y. Oram K.F. Gridley T. The mouse snail gene encodes a key regulator of the epithelial-mesenchymal transition.Mol. Cell. Biol. 2001; 21: 8184-8188Crossref PubMed Scopus (367) Google Scholar). Furthermore, diploblasts (animals derived from only two germ layers) that do not form mesoderm also express snail in the regions of involution or ingression during endoderm formation. Hence, Snail activity is not associated with the mesodermal lineage but rather with changes in cell shape, cell adhesion, and cell movements, consistent with the notion that cell fate specification and morphogenetic movements are independent processes even though they occur simultaneously. Given that gastrulation is a very rapid process, the regulation of E-cadherin transcription alone is insufficient. E-cadherin is also controlled at the protein level by the P38 interacting protein (IP), p38-MAP kinase, and the FERM protein (EPB4.1L5) (Figure 2C) (Zohn et al., 2006Zohn I.E. Li Y. Skolnik E.Y. Anderson K.V. Han J. Niswander L. p38 and a p38-interacting protein are critical for downregulation of E-cadherin during mouse gastrulation.Cell. 2006; 125: 957-969Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, Hirano et al., 2008Hirano M. Hashimoto S. Yonemura S. Sabe H. Aizawa S. EPB41L5 functions to post-transcriptionally regulate cadherin and integrin during epithelial-mesenchymal transition.J. Cell Biol. 2008; 182: 1217-1230Crossref PubMed Scopus (40) Google Scholar, Lee et al., 2007Lee J.D. Silva-Gagliardi N.F. Tepass U. McGlade C.J. Anderson K.V. The FERM protein Epb4.1l5 is required for organization of the neural plate and for the epithelial-mesenchymal transition at the primitive streak of the mouse embryo.Development. 2007; 134: 2007-2016Crossref PubMed Scopus (41) Google Scholar). Other transcription factors such as Eomesodermin (Eomes) and Mesp1 and 2 are important for EMT during mouse gastrulation (Figure 2C). Eomes is a T-box transcription factor expressed in the posterior epiblast prior to streak formation, in the streak and in nascent mesendoderm at gastrulation. In turn, the basic helix-loop-helix transcription factors Mesp1 and 2 are also expressed in the posterior epiblast of the mouse embryo. Mesodermal delamination from the streak is blocked in mice lacking Eomes in the epiblast and in double Mesp1/Mesp2 mutants (Kitajima et al., 2000Kitajima S. Takagi A. Inoue T. Saga Y. MesP1 and MesP2 are essential for the development of cardiac mesoderm.Development. 2000; 127: 3215-3226PubMed Google Scholar, Arnold et al., 2008Arnold S.J. Hofmann U.K. Bikoff E.K. Robertson E.J. Pivotal roles for eomesodermin during axis formation, epithelium-to-mesenchyme transition and endoderm specification in the mouse.Development. 2008; 135: 501-511Crossref PubMed Scopus (96) Google Scholar). This is consistent with the ability of Mesp proteins to induce Snail and EMT in differentiated embryonic stem cells (Lindsley et al., 2008Lindsley R.C. Gill J.G. Murphy T.L. Langer E.M. Cai M. Mashayekhi M. Wang W. Niwa N. Nerbonne J.M. Kyba M. et al.Mesp1 coordinately regulates cardiovascular fate restriction and epithelial-mesenchymal transition in differentiating ESCs.Cell Stem Cell. 2008; 3: 55-68Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Cells need to break the basal membrane to successfully delaminate from the primitive streak. A pathway mediated by the RhoGEF protein Net1 induces RhoA downregulation in the primitive streak and disrupts the interaction between epiblast cells and the underlying the basal membrane (Figure 2C) (Nakaya et al., 2008Nakaya Y. Sukowati E.W. Wu Y. Sheng G. RhoA and microtubule dynamics control cell-basement membrane interaction in EMT during gastrulation.Nat. Cell Biol. 2008; 10: 765-775Crossref PubMed Scopus (141) Google Scholar). Snail factors contribute to basal membrane degradation by activating metalloproteases (Jorda et al., 2005Jorda M. Olmeda D. Vinyals A. Valero E. Cubillo E. Llorens A. Cano A. Fabra A. Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor.J. Cell Sci. 2005; 118: 3371-3385Crossref PubMed Scopus (133) Google Scholar) and by repressing some components such as Laminin5 and its receptors (Haraguchi et al., 2008Haraguchi M. Okubo T. Miyashita Y. Miyamoto Y. Hayashi M. Crotti T.N. McHugh K.P. Ozawa M. Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane paroteins.J. Biol. Chem. 2008; 283: 23514-23523Crossref PubMed Scopus (41) Google Scholar). Importantly, the integrity of the basal membrane must be maintained in areas outside of the primitive streak and the transmembrane protein FLRT3 seems to offer protection against its disruption in addition to regulating cell fate (Figure 2C) (Egea et al., 2008Egea J. Erlacher C. Montanez E. Burtscher I. Yamagishi S. Hess M. Hampel F. Sanchez R. Rodriguez-Manzaneque M.T. Bösl M.R. et al.Genetic ablation of FLRT3 reveals a novel morphogenetic function for the anterior visceral endoderm in suppressing mesoderm differentiation.Genes Dev. 2008; 22: 3349-3362Crossref PubMed Scopus (28) Google Scholar). Unlike in the sea urchin or in Drosophila, the gene regulatory networks operating at gastrulation in vertebrates are far from complete. Future studies of EMT would benefit from the application of genome-wide approaches and in vivo cell imaging. Indeed, attempts to define the transcriptome in the gastrulating mouse embryo have already provided interesting information (Mitiku and Baker, 2007Mitiku N. Baker J.C. Genomic analysis of gastrulation and organogenesis in the mouse.Dev. Cell. 2007; 13: 897-907Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). After gastrulation in vertebrates, the epidermal and neural territories are progressively defined along the rostrocaudal axis, and the neural crest forms at the boundary between these two territories. Neural crest cells undergo EMT within the dorsal neural epithelium, and individual cells migrate before giving rise to different derivatives, including craniofacial structures, most of the peripheral nervous system, some endocrine cells, and melanocytes. Our understanding of how the neural crest territory is specified has increased substantially over the last 10 years, although the gene regulatory network operating during neural crest development is less complete than at gastrulation (Sauka-Spengler and Bronner-Fraser, 2008Sauka-Spengler T. Bronner-Fraser M. A gene regulatory network orchestrates neural crest formation.Nat. Rev. Mol. Cell Biol. 2008; 9: 557-568Crossref PubMed Scopus (308) Google Scholar). A complex set of inductive events initiated before and during gastrulation define a distinct territory at the junction between neural and nonneural ectoderm. The neural crest territory is delimited by FGF, Wnt, Notch, and retinoic acid signaling pathways in cooperation with opposing gradients of bone morphogenetic protein 4 (BMP4) and its antagonists Noggin, Follistatin, and Chordin. Although canonical Wnt signaling is important for both the induction and stabilization of neural crest precursors and for their delamination, noncanonical Wnt signaling is important for neural crest migration (De Calisto et al., 2005De Calisto J. Araya C. Marchant L. Riaz C.F. Mayor R. Essential role of non-canonical Wnt signalling in neural crest migration.Development. 2005; 132: 2587-2597Crossref PubMed Scopus (177) Google Scholar, Carmona-Fontaine et al., 2008Carmona-Fontaine C. Matthews H.K. Kuriyama S. Moreno M. Dunn G.A. Parsons M. Stern C.D. Mayor R. Contact inhibition of locomotion in vivo controls neural crest directional migration.Nature. 2008; 456: 957-961Crossref PubMed Scopus (240) Google Scholar). Thus, most of the signaling pathways involved in defining the neural crest territory are common to those used during gastrulation. The presumptive neural crest is modified by the neural plate border specifiers Msx1, Msx2, Dlx3, Dlx5, Pax3, Pax7, and Zic1, which induce genes that trigger different programs in the neural crest such as Snail and Sox factors, FoxD3, AP2, c-Myc, and Id. Although organized in a hierarchy, these factors can also regulate expression of each other (Sauka-Spengler and Bronner-Fraser, 2008Sauka-Spengler T. Bronner-Fraser M. A gene regulatory network orchestrates neural crest formation.Nat. Rev. Mol. Cell Biol. 2008; 9: 557-568Crossref PubMed Scopus (308) Google Scholar). Cadherin-mediated adhesion also plays a major role in neural crest cell EMT, where delamination from the neural fold involves the downregulation of N-cadherin and Cadherin 6 as well as the de novo expression of type II cadherins, such as Cadherin 7 and 11 (Nakagawa and Takeichi, 1995Nakagawa S. Takeichi M. Neural crest cell-cell adhesion controlled by sequential and subpopulation-specific expression of novel cadherins.Development. 1995; 121: 1321-1332Crossref PubMed Google Scholar, Vallin et al., 1998Vallin J. Girault J.M. Thiery J.P. Broders F. Xenopus cadherin-11 is expressed in different populations of migrating neural crest cells.Mech. Dev. 1998; 75: 171-174Crossref PubMed Scopus (48) Google Scholar). The less adhesive type II cadherins allow crest cells to migrate away from the neural tube (Chu et al., 2006Chu Y.S. Eder O. Thomas W.A. Simcha I. Pincet F. Ben-Ze'ev A. Perez E. Thiery J.P. Dufour S. Prototypical type I E-cadherin and type II cadherin-7 mediate very distinct adhesiveness through their extracellular domains.J. Biol. Chem. 2006; 281: 2901-2910Crossref PubMed Scopus (70) Google Scholar). In the chick embryo, the onset of delamination involves N-cadherin protein cleavage by the ADAM 10 protease. The membrane bound fragment generated is then further digested by γ-secretase. It then translocates to the nucleus together with β-catenin where it activates cyclin D1 and promotes exit from the G1 phase, a prerequisite for the emigration of these cells (Shoval et al., 2007Shoval I. Ludwig A. Kalcheim C. Antagonistic roles of full-length N-cadherin and its soluble BMP cleavage product in neural crest delamination.Development. 2007; 134: 491-501Crossref PubMed Scopus (116) Google Scholar). Interestingly, Snail factors prevent entry into the S phase of the cycle by repressing cyclin D transcription (Vega et al., 2004Vega S. Morales A.V. Ocana O.H. Valdes F. Fabregat I. Nieto M.A. Snail blocks the cell cycle and confers resistance to cell death.Genes Dev. 2004; 18: 1131-1143Crossref PubMed Scopus (390) Google Scholar), probably synchronizing the premigratory crest population so that all the cells can simultaneously enter into the S phase upon N-cadherin cleavage and cyclin D activation. At least in the chick embryo, Cadherin 6B (Cad6B) is transiently expressed at the premigratory phase, and its downregulation leads to premature neural crest cell migration, whereas its overexpression induces accumulation of crest cells at the dorsal border of neural tube (Coles et al., 2007Coles E.G. Taneyhill L.A. Bronner-Fraser M. A critical role for Cadherin6B in regulating avian neural crest emigration.Dev. Biol. 2007; 312: 533-544Crossref PubMed Scopus (50) Google Scholar). The precise timing of Cad6B downregulation is directly controlled by Snail2 (Taneyhill et al., 2007Taneyhill L.A. Coles E.G. Bronner-Fraser M. Snail2 directly represses cadherin6B during epithelial-to-mesenchymal transitions of the neural crest.Development. 2007; 134: 1481-1490Crossref PubMed Scopus (97) Google Scholar). In addition to cadherins, small GTPases also play an important role in neural crest EMT as they do during gastrulation. RhoV downregulation affects Sox9, Snail2, and Twist expression in Xenopus embryos (Guemar et al., 2007Guemar L. de Santa Barbara P. Vignal E. Maurel B. Fort P. Faure S. The small GTPase RhoV is an essential regulator of neural crest induction in Xenopus.Dev. Biol. 2007; 310: 113-128Crossref PubMed Scopus (27) Google Scholar). Rac1 can induce Snail2 expression in the neural crest in Xenopus, and its dominant negative or activated forms markedly affect crest cell delamination. RhoA has the opposite effect, as its activated form abrogates crest cell delamination (Broders-Bondon et al., 2007Broders-Bondon F. Chesneau A. Romero-Oliva F. Mazabraud A. Mayor R. Thiery J.P. Regulation of XSnail2 expression by Rho GTPases.Dev. Dyn. 2007; 236: 2555-2566Crossref PubMed Scopus (18) Google Scholar) and RhoB lies downstream of Snail2 and Sox5 in the chick neural crest (del Barrio and Nieto, 2002del Barrio M.G. Nieto M.A. Overexpression of Snail family members highlights their ability to promote chick neural crest formation.Development. 2002; 129: 1583-1593PubMed Google Scholar, Perez-Alcala et al., 2004Perez-Alcala S. Nieto M.A. Barbas J.A. LSox5 regulates RhoB expression in the neural tube and promotes generation of the neural crest.Development. 2004; 131: 4455-4465Crossref PubMed Scopus (63) Google Scholar). RADIL, a downstream effector of Rap GTPase that links the plasma membrane with the actin cytoskeleton, controls neural crest cell adhesion and migration in zebrafish (Smolen et al., 2007Smolen G.A. Schott B.J. Stewart R.A. Diederichs S. Muir B. Provencher H.L. Look A.T. Sgroi D.C. Peterson R.T. Haber D.A. A Rap GTPase interactor, RADIL, mediates migration of neural crest precursors.Genes Dev. 2007; 21: 2131-2136Crossref PubMed Scopus (17) Google Scholar), whereas RhoA and Rac1 control both FoxD3 and Snail1 expression. Thus, the small Rho-GTPases play a major role in establishing a transcription factor autoregulatory network at the time of neural crest specification and EMT. As in gastrulation, in vivo cell imaging analysis of chick neural crest has provided interesting information about the individual cell movements and the contacts that crest cells maintain or create while migrating (Teddy and Kulesa, 2004Teddy J.M. Kulesa P.M. In vivo evidence for short- and long-range cell communication in cranial neural crest cells.Development. 2004; 131: 6141-6151Crossref PubMed Scopus (111) Google Scholar). Elegant analyses at the single-cell resolution in Xenopus embryos have shown that contact inhibition of locomotion mediated by Wnt noncanonical signaling is a crucial mechanism for the directional movements of neural crest cells toward their target tissue (Carmona-Fontaine et al., 2008Carmona-Fontaine C. Matthews H.K. Kuriyama S. Moreno M. Dunn G.A. Parsons M. Stern C.D. Mayor R. Contact inhibition of locomotion in vivo controls neural crest directional migration.Nature. 2008; 456: 957-961Crossref PubMed Scopus (240) Google Scholar). In summary, analogous signaling pathways operate during EMT in gastrulation and neural crest formation. It is worth noting here that while defects in individual genes lead to very strong EMT phenotypes at gastrulation, there is a high degree of cooperation and plasticity during neural crest development. The existence of regulatory loops among the different EMT inducers in the neural tube explains why the absence of one player may be compensated for by the others. For instance, although Snail is crucial for gastrulation in all metazoans analyzed (Carver et al., 2001Carver E.A. Jiang R. Lan Y. Oram K.F. Gridley T. The mouse snail gene encodes a key regulator of the epithelial-mesenchymal transition.Mol. Cell. Biol. 2001; 21: 8184-8188Crossref PubMed Scopus (367) Google Scholar, Nieto et al., 1994Nieto M.A. Sargent M.G. Wilkinson D.G. Cooke J. Control of cell behavior during vertebrate development by Slug, a zinc finger gene.Science. 1994; 264: 835-839Crossref PubMed Google Scholar, Wu et al., 2007Wu S.Y. Ferkowicz M. McClay D.R. Ingression of primary mesenchyme cells of the sea urchin embryo: a precisely timed epithelial mesenchymal transition. Birth Defects Res.C Embryo Today. 2007; 81: 241-252Crossref PubMed Scopus (15) Google Scholar), mice mutant for Snail1 and Snail2 still generate neural crest even though they develop multiple craniofacial defects (Murray and Gridley, 2006Murray S.A. Gridley T. Snail family genes are required for left-right asymmetry determination, but not neural crest formation, in mice.Proc. Natl. Acad. Sci. USA. 2006; 103: 10300-10304Crossref PubMed Scopus (65) Google Scholar). This plasticity and cooperation endows the system with robustness, perhaps reflecting the importance of the neural crest as an evolutionary novelty fundamental for the development of the vertebrate head. The primary EMTs are followed by differentiation events that generate different cell types. Indeed, the migratory neural crest cells follow stereotyped pathways and then differentiate into neurons, cartilage, or bone cells, and mesodermal cells subdivide into axial, paraxial, intermediate, and lateral mesoderm after gastrulation. These populations condense into transient epithelial structures through a MET process, thereby forming the notochord, the somites, the precursors of the urogenital system and the somatopleure and splanchnopleure, respectively (Figure 1B). Except for the notochord and in response to signals from their microenvironment, these secondary epithelia undergo a secondary EMT to generate mesenchymal cells with a more restricted differentiation potential. The repression of Snail factors controls the timing of presomitic axial mesoderm MET, which leads to somite epithelialization (Dale et al., 2006Dale J.K. Malapert P. Chal J. Vilhais-Neto G. Maroto M. Johnson T. Jayasinghe S. Trainor P. Herrmann B. Pourquie O. Oscillations of the snail genes in the presomitic mesoderm coordinate segmental patterning and morphogen