Epithelio-mesenchymal Transformation Research Articles

Abstract 1045: CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development

Abstract Among women, breast cancer continues to be the most common cancer, with metastasis being the leading cause of mortality in cancer patients around the world (DIZON et al. 2016). Epithelial to mesenchymal transition (EMT) - the process by which epithelial cells shift to the mesenchymal state, has been implicated in many aspects of breast cancer tumorigenesis, metastasis and drug resistance ). The accumulation of a large body of data on the association of EMT with cancer over the years has not resulted in EMT being an active target for therapeutic development. This is due in part to the lack of appropriate in vitro models. Here we have exploited some of the basic biology of EMT, to create an advanced in vitro metastatic breast cancer reporter cell line model for use in both basic research and discovery of new EMT inhibitors. During EMT, E-cadherin protein expression is down regulated in cancer cells in association with the loss of cell-to-cell adhesion, apico-basal polarity and a change to a spindle-shaped morphology. By installing an emerald green fluorescent protein (EmGFP) tag on the C-terminus of the e-cadherin (ECAD) gene in the epithelial BT-474 breast cancer cell line via CRISPR/Cas9 genome editing, end-point or real-time EMT status of cells can be tracked under defined conditions. The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the BT-474 ECAD EmGFP cell line, shows that it responds to EMT induction. The subsequent EMT status of cells can be monitored in real time by observing and measuring ECAD EmGFP expression, in addition, the resulting mesenchymal cells have increased invasive capacities. Furthermore, this EMT reporter cell line shows sensitivity to the MEK1/2 inhibitor U0126 - thereby providing the basis for the use of this cell line in high-throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic breast cancer. The BT-474 ECAD EmGFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis. References DIZON, D. S., L. KRILOV, E. COHEN, T. GANGADHAR, P. A. GANZ et al., 2016 Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer From the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011. HAY, E. D., 1995 An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 154: 8-20. Citation Format: Metewo S. Enuameh, Weiguo Shu, Robert Newman. CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1045.

Protein analysis of the plaques in congenital cataracts

PurposeTo identify the protein composition of posterior capsular plaques (PCP) in congenital idiopathic unilateral cataract with anterior vitreolenticular interface dysgenesis (AVLID).MethodsSamples of PCP's were collected during cataract surgery in children with congenital unilateral cataract presenting with a PCP and with vitreolenticular adherences observed during posterior capsulorhexis procedure. Proteome analysis was performed on the collected PCPs.ResultsPCP collection and proteome analysis was feasible from four children. A large portion of the proteins found in the PCPs were similar to the proteins known to be present in lens epithelial cells and fibers. Proteins like vimentin, fibronectin, actin, collagen type I, collagen type VI, lumican and keratin were also found which typically are present in mesenchymal tissue but not in lens tissue or capsule.ConclusionsCases of unilateral idiopathic congenital cataract with AVLID typically present with a PCP. The protein composition in these PCPs, measured by means of proteome analysis, includes atypical proteins for lens and lens capsule and may be suggestive for an epitheliomesenchymal transformation (EMT).

03-P097 Is the process of epithelio-mesenchymal transformation involved in the dental lamina regression?

03-P097 Is the process of epithelio-mesenchymal transformation involved in the dental lamina regression?

Reorganization of cytoskeleton as a basis of morphogenesis

A brief review of the cytoskeleton dynamic structure in cells of two types: fibroblasts and epitheliocytes. Differences have been described between the functions of y-actin filaments and beta-actomyosin bundles. Tubulogenesis and angiogenesis have been considered as consequences of partial epitheliomesenchymal transformation and neoplastic transformation as a consequence of gamma-actomyosin bundles disturbance.

Cytomegalovirus-induced embryopathology: mouse submandibular salivary gland epithelial-mesenchymal ontogeny as a model

BackgroundHuman studies suggest, and mouse models clearly demonstrate, that cytomegalovirus (CMV) is dysmorphic to early organ and tissue development. CMV has a particular tropism for embryonic salivary gland and other head mesenchyme. CMV has evolved to co-opt cell signaling networks so to optimize replication and survival, to the detriment of infected tissues. It has been postulated that mesenchymal infection is the critical step in disrupting organogenesis. If so, organogenesis dependent on epithelial-mesenchymal interactions would be particularly vulnerable. In this study, we chose to model the vulnerability by investigating the cell and molecular pathogenesis of CMV infected mouse embryonic submandibular salivary glands (SMGs).ResultsWe infected E15 SMG explants with mouse CMV (mCMV). Active infection for up to 12 days in vitro results in a remarkable cell and molecular pathology characterized by atypical ductal epithelial hyperplasia, apparent epitheliomesenchymal transformation, oncocytic-like stromal metaplasia, β-catenin nuclear localization, and upregulation of Nfkb2, Relb, Il6, Stat3, and Cox2. Rescue with an antiviral nucleoside analogue indicates that mCMV replication is necessary to initiate and maintain SMG dysmorphogenesis.ConclusionmCMV infection of embryonic mouse explants results in dysplasia, metaplasia, and, possibly, anaplasia. The molecular pathogenesis appears to center around the activation of canonical and, perhaps more importantly, noncanonical NFκB. Further, COX-2 and IL-6 are important downstream effectors of embryopathology. At the cellular level, there appears to be a consequential interplay between the transformed SMG cells and the surrounding extracellular matrix, resulting in the nuclear translocation of β-catenin. From these studies, a tentative framework has emerged within which additional studies may be planned and performed.

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo.

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.

Molecular mechanisms of hormone-mediated Müllerian duct regression: involvement of beta-catenin.

Regression of the Müllerian duct in the male embryo is one unequivocal effect of anti-Müllerian hormone, a glycoprotein secreted by the Sertoli cells of the testis. This hormone induces ductal epithelial regression through a paracrine mechanism originating in periductal mesenchyme. To probe the mechanisms of action of anti-Müllerian hormone, we have studied the sequence of cellular and molecular events involved in duct regression. Studies were performed in male rat embryos and in transgenic mice overexpressing or lacking anti-Müllerian hormone, both in vivo and in vitro. Anti-Müllerian hormone causes regression of the cranial part of the Müllerian duct whereas it continues to grow caudally. Our work shows that this pattern of regression is correlated with a cranial to caudal gradient of anti-Müllerian hormone receptor protein, followed by a wave of apoptosis spreading along the Müllerian duct as its progresses caudally. Apoptosis is also induced by AMH in female Müllerian duct in vitro. Furthermore, apoptotic indexes are increased in Müllerian epithelium of transgenic mice of both sexes overexpressing the human anti-Müllerian hormone gene, exhibiting a positive correlation with serum hormone concentration. Inversely, apoptosis is reduced in male anti-Müllerian hormone-deficient mice. We also show that apoptosis is a decisive but not sufficient process, and that epitheliomesenchymal transformation is an important event of Müllerian regression. The most striking result of this study is that anti-Müllerian hormone action in peri-Müllerian mesenchyme leads in vivo and in vitro to an accumulation of cytoplasmic beta-catenin. The co-localization of beta-catenin with lymphoid enhancer factor 1 in the nucleus of peri-Müllerian mesenchymal cells, demonstrated in primary culture, suggests that overexpressed beta-catenin in association with lymphoid enhancer factor 1 may alter transcription of target genes and may lead to changes in mesenchymal gene expression and cell fate during Müllerian duct regression. To our knowledge, this is the first report that beta-catenin, known for its role in Wnt signaling, may mediate anti-Müllerian hormone action.

Epithelio-mesenchymal transformation in the embryonic face: implications for craniofacial malformations

A high percentage of craniofacial malformations is ascribed to abnormalities in cell populations derived from the neural crest and ganglionic placodes. Both cell populations originate from the ectoderm of the head-neck area by means of the mechanism of epithelio-mesenchymal transformation (EMT). Neural crest cells are multipotent and form the majority of the mesenchymal compartment of the head-neck area. EMT of the neural crest will stop shortly after closure of the neural tube. The current opinion about ganglionic placodal cells is that they are derived from the surface ectodermal placodes after closure of the cranial neural tube, and transform into neurons of the sensory ganglia only. However, additional EMT sites of the cranial neural and surface ectoderm have also been found to produce multipotent cells. Because of the fact that most craniofacial malformations develop after closure of the cranial neural tube, in this study we focused on the EMT of the surface ectoderm of the head-neck area during the stages of outgrowth of facial swellings and branchial arches. The surface ectoderm was labeled by injecting various tracker dyes into the amniotic cavity of chick embryos after closure of the anterior neuropore, to exclude labeling of the neural ectoderm and the neural crest. After various incubation periods, ranging from 0–24 h, labeled cells were found in the mesenchymal compartment scattered over the embryonic face and neck, originating from non-placodal as well as placodal surface ectoderm. Thus, besides the neural ectoderm and neural crest, it could be shown that the entire surface ectoderm of the face and neck contributes cells to the underlying mesenchymal tissue, and that this phenomenon occurs in a higher frequency at sites of outgrowing swellings. As a consequence, the pathogenesis of congenital craniofacial malformations should be reconsidered.

Induction of epithelio-mesenchymal transformation of quail embryonic neural cells by inhibition of atypical protein kinase-C.

Epithelio-mesenchymal transition, which involves the re-organisation of cell-cell adhesion molecules and the actin cytoskeleton, can be induced in embryonic neural epithelium in vitro by protein kinase-C inhibitors. A non-inhibitory analogue, BIM V, and potent inhibitors of other kinases are not active. This suggests a central role for C-kinases, although the powerful specific C-kinase inhibitors BIM I and Ro 31-8220 show lower than expected activity. Co-inhibition by several kinases is unlikely to account for this, since no potentiation occurs when these are combined with potent inhibitors of other kinases. BIM I and Ro 31-8220 strongly inhibit only conventional calcium-regulated C-kinases; this and the lack of effect of TMB-8, which inhibits calcium release, suggests that novel and/or atypical isoforms are involved. Various potentiators and activators of conventional and novel C-kinases have no obvious effect alone and fail to reduce the effect of staurosporine, suggesting that atypical C-kinases are critical. The presence of C-kinase isoforms in the E2 embryonic neural tissues has been probed on Western blots, revealing immunoreactivity for the atypical isoforms iota (or lambda) and zeta and the alpha, gamma, epsilon and mu isoforms. Immunofluorecent localisation on sections of embryos has shown the widespread distribution of conventional and novel isoforms but only the atypical isoforms lambda and zeta are enriched at the apical margins of the neural and other epithelia; they overlap with the cell-cell adhesion molecule N-cadherin and with F-actin. Thus, epithelio-mesenchymal transition in the embryonic neural epithelium in vitro is induced by inhibiting protein kinase activity, probably via an atypical protein kinase-C; atypical protein kinase-C isoforms are present in the tissue at the appropriate developmental stage and subcellular site in cells capable of epithelio-mesenchymal transition.

Scatter Factor/Hepatocyte Growth Factor (SF/HGF) Induces Emigration of Myogenic Cells at Interlimb Levelin Vivo

The initiating event in the migration of myogenic cells to the limb buds is an epitheliomesenchymal transformation of cells located at the lateral edge of the dermomyotome. Recently, a targeted mutation ofc-metin mice demonstrated an essential role of this tyrosine kinase receptor and its ligand, scatter factor/hepatocyte growth factor (SF/HGF), in the migration of myogenic cells to the limb buds. Here, we show that ectopic application of exogenous SF/HGF induces emigration ofPax-3-positive myogenic cells into the lateral plate mesoderm. During this process, the lateral portions of the dermomyotomes deepithelialize and the basement membrane disintegrates. Detaching myogenic cells do not lose N-cadherin from their surfaces. We conclude that an HGF/SF- andc-met-mediated signal detaches myogenic precursor cells from the somites and thus plays a necessary role in the initiation of myoblast migration.

Control of Epitheliomesenchymal Transformation: II. Cross-Modulation of Cell Adhesion and Cytoskeletal Systems in Embryonic Neural Cells

Protein kinase (PK) inhibitors like staurosporine induce precocious epitheliomesenchymal transformation (EMT) of quail embryo neuralanlagencultured on the extracellular matrix (ECM) molecule fibronectin (Newgreen and Minichiello,Dev. Biol.170, 91–101; 1995). We show here that this also occurs on laminin, vitronectin, and collagen type I and type IV, but not on polylysine or BSA. In the absence of cell–ECM adhesion, staurosporine produced a rapid increase in, rather than a loss of, cohesion in isolated neuralanlagenand increased the rate of reaggregation of dissociated neural cells. In the absence of cell–cell adhesions (i.e., with dissociated cells), almost all neural cells rapidly adhered to fibronectinin vitroand staurosporine made only a marginal difference to this. Almost no neural cells spread on fibronectin in control conditions but staurosporine stimulated spreading by almost all cells. When cell–cell adhesions were present, neuralanlageexplants were more difficult to dislodge by controlled shear from fibronectin substrates in the presence of staurosporine than under control conditions, but in both experiments and controls dislodgment was due to the cell bodies breaking proximal to the cell–fibronectin adhesion sites. EMT in neural cells in culture, both spontaneous and induced by staurosporine, involved rapid reduction in circumferential F-actin fibers and an increase in pancytoplasmic G-actin, as shown by fluorescent phalloidin and DNase I labeling. Loss of immunoreactivity for N-cadherin at cell–cell junctions also occurred in both spontaneous and induced EMT. However, in spontaneous EMT the cadherin changes preceded the actin changes, whereas in induced EMT, the reverse occurred. The results suggest that the molecules involved in EMT which are affected by PK inhibitors are cytoskeletal and link elements. These results also suggest that balanced cross-modulation among cell–cell adhesion molecules, cell–ECM adhesion molecules, and cytoskeletal molecules can trigger and orchestrate EMT, without direct genetic supervision.

Control of Epitheliomesenchymal Transformation. I. Events in the Onset of Neural Crest Cell Migration Are Separable and Inducible by Protein Kinase Inhibitors

Neural tubes isolated from quail embryos prior to epitheliomesenchymal transformation (EMT) and neural crest (NC) cell migration, when explanted onto fibronectin surfaces, replicated properties of normal NC morphogenesis such as (i) cell outgrowth, (ii) loss of A-CAM (N-cadherin) junctions and adoption of mesenchymal form, and (iii) development of HNK-1 immunoreactivity. The timetable of property (i) was essentially normal but the outgrowing cells were initially mainly epithelial, unlike NC outgrowth in vivo and in cultures of older neural tubes. Mesenchymal properties (ii) and (iii) were progressively and variably retarded relative to the in vivo timetable. Achievement of these properties by EMT was principally related to proximity to the neural tube and to preexisting mesenchymal cells, rather than being related temporally to the outgrowth timetable. This EMT, combined with a higher mitotic rate in the mesenchyme cells, resulted in the outgrowth passing from mainly epithelial at 16 hr to mainly mesenchymal at 48 hr in vitro. Immediate precocious EMT and outgrowth of A-CAM negative mesenchymal cells from pre-EMT neural tubes was stimulated by the protein kinase inhibitors staurosporine and bisindolymaleimide in a cycloheximide-independent manner. EMT could be induced not only on the dorsal (i.e., NC) side, but also on the ventral side of the neural tube, but the ventral cells were less sensitive than the dorsal cells, and with developmental age became still less sensitive while the dorsal cells became more sensitive. The results suggest that the complex events of EMT in the NC system are not obligatorily coregulated, can be triggered by epigenetic events involving differential protein phosphorylation, and may be controlled via intraneural signaling.

Epithelio-mesenchymal transformation during formation of the mesoderm in the mammalian embryo.

The earliest example of epithelio-mesenchymal transformation during embryonic development is the generation of the third germ layer, the mesoderm, from the epiblast (or primitive ectoderm), which marks the beginning of gastrulation. Although it has been regarded as most likely that the principles of this transformation in invertebrates and lower vertebrates also apply to amniotes, morphological and molecular details of mesoderm formation in birds and, in particular, in mammals, which may support this assumption, have only recently been clarified. This chapter thus brings together the light- and electron-microscopical morphology of epithelio-mesenchymal transformation during initial mesoderm formation in the mammalian embryo. Cellular differentiation during this process with regard to the cytoskeleton, cell adhesion molecules and the extracellular matrix are also covered as are cell kinetic studies and the candidate growth factors and genes most likely to be involved in the regulation of mesoderm formation in mammals. Finally, a model is presented which summarizes these morphological and molecular changes and which links the promoting and inhibiting influences of regulatory factors to some of the changes observed during epithelio-mesenchymal transformation.

Morphology of incipient mesoderm formation in the rabbit embryo: a light- and retrospective electron-microscopic study.

Mesoderm formation is a hallmark of vertebrate gastrulation and, at the same time, one of the prime examples for epithelio-mesenchymal transformation. Recent advances in experimental embryology and molecular biology have clarified the role of growth factors and genes in this process; however, its microscopic anatomy in higher vertebrates is still far from clear. Therefore, the present study describes the morphology of mesoderm formation in the rabbit embryo, a species which may be representative for both the avian and the mammalian embryo in this respect. Serial semithin sections were correlated with topographical landmarks in surface views of embryonic discs at 6.4, 6.5, and 6.6 days post conceptionem, and selected semithin sections were reembedded for ultrastructural analysis. Mesoderm cells are shown to be generated by ingression of bottle-shaped epiblast cells in the area of the posterior node and the primitive streak. Here, basal endocytotic pits and absence or discontinuity of the basal lamina are taken as suggestive evidence for specific removal of extracellular matrix material. Within the bottle-shaped cells most organelles are concentrated in a narrow apical neck which will subsequently constitute the 'trailing end' of the ingressing mesoderm cells. These features support the assumption that most principles of epithelio-mesenchymal transformation seen during primary mesenchyme formation in the sea urchin also apply to mesoderm formation in vertebrates. However, transient tripartite zonula adherens-type junctions are formed apically between ingressing mesoderm cells and the neighboring epiblast cells. They are interpreted here as being responsible for maintaining supracellular integrity of the embryonic disc during the shedding of mesoderm cells in the amniote embryo.

Palate development.

In all vertebrates, the secondary palate arises as bilateral outgrowths from the maxillary processes. In birds and most reptiles, these palatal shelves grow initially horizontally, but do not fuse with each other resulting in physiological cleft palate. In crocodilians, shelf fusion occurs resulting in an intact secondary palate. Mammalian palatal shelves initially grow vertically down the side of the tongue, but elevate at a precise time to a horizontal position above the dorsum of the tongue and fuse with each other to form an intact palate. Palatal shelf-elevation is the result of an intrinsic shelf elevating force, chiefly generated by the progressive accumulation and hydration of hyaluronic acid. In all vertebrates the nasal epithelium differentiates into pseudostratified ciliated columnar cells and the oral epithelia differentiates into stratified squamous cells, but the medial edge epithelial (MEE) phenotype differs in different groups. In mammals, the MEE of opposing shelves adhere to each other to form an epithelial seam which then disrupts by cell death and cell migration into the mesenchyme accompanied by an epitheliomesenchymal transformation. In birds, the MEE keratinize resulting in cleft palate whereas, in alligators, the MEE migrate onto the nasal aspect of the palate. In all vertebrates, this regional, temporal and species-specific epithelial differentiation is specified by the underlying mesenchyme. Signalling of this interaction is complex but involves both extracellular matrix and soluble factors e.g. minor collagen types, tenascin, EGF, TGF alpha, TGF beta, PDGF, FGF. These soluble growth factors have a biphasic effect: directly on the epithelia and on the mesenchyme where they stimulate or inhibit cell division and synthesis of specific extracellular matrix molecules. The extracellular matrix molecules (and bound growth factors) synthesized by the mesenchymal cells may then directly affect the epithelium. These signals cause differential gene expression via second messenger systems e.g. cAMP, cGMP, Ca2+, pH, pI etc. Molecular markers for nasal, medial and oral epithelial cell differentiation include the types of cytokeratin intermediate filaments and specific cell surface molecules recognized by monoclonal antibodies: the genes for such molecules are probably expressed in response to mesenchymal signals. Using such an approach, it is possible to go from a morphological description of palate development to a cellular analysis of the mechanisms involved and then to identification of candidate genes that may be important for screening and diagnosis of cleft palate.

The relationship between emerging neural crest cells and basement membranes in the trunk of the mouse embryo: a TEM and immunocytochemical study

The earliest stage of neural crest cell (NCC) migration is characterized by an epitheliomesenchymal transformation, as the cells leave the neural tube. There is evidence that in a number of cell systems this transformation is accompanied by alteration or depletion of associated basement membranes. This study examines the ultrastructural relationship between mouse NCCs and adjacent basement membranes during the earliest stages of migration from the neural tube. Basement membranes were identified by transmission electron microscopy (TEM) and immunofluorescence using antibodies to type-IV collagen. The ultrastructural features of NCCs and their relationship with surrounding tissues were also examined using TEM. In the dorsal region of the neural tube, from which NCCs originate, the basement membrane was depleted or absent, and with the immunofluorescence technique it was shown that this pattern was reflected in a deficit of type-IV collagen. TEM observations indicated that ultrastructurally NCCs differ from their neuroepithelial neighbours only in overall cell shape and their relationship to other cells and the extracellular matrix.

Control of the onset of migration of neural crest cells in avian embryos. Role of Ca++-dependent cell adhesions.

To investigate the control of the timing in the epithelio-mesenchymal transformation of the neural crest into a migrating population, neural anlagen (neural tube plus crest) were isolated from 2-day quail embryos by proteases in the presence of CA++ and explanted onto substrates favourable for neural crest cell migration. Explants isolated before normal migration had commenced required 3-8 h in vitro before neural crest cells started migration, but explants obtained at migratory stages showed an immediate onset of migration. The schedule was similar to that expected in vivo. When pre-migratory neural anlagen were isolated by protease in Ca++-and Mg++-free (CMF) medium, or when the protease was followed by a brief (5 min) exposure to CMF medium, neural crest cell migration commenced without delay, and the cohesion of the anlagen was impaired. Ca++-free medium duplicated the effects of CMF, but neither Mg++-free medium nor CMF treatment before treatment with protease stimulated migration and reduced cohesion. Precocious neural crest cell migration and reduced cohesion also followed when neural anlagen of pre-migratory stages were cultured with membrane Ca++-channel antagonists D600 and Nifedipine, without any exernal Ca++-depletion. The decrease of cohesion of these tissues is consistent with results in other systems where protease/Ca++-depletion inactivates Ca++-dependent cell-cell adhesive mechanisms. Therefore, we suggest that Ca++-dependent cell-cell adhesions play a part in preventing neural crest cells from migrating precociously and that the timed inactivation of this adhesion system normally helps trigger the onset of migration. The results with blockers of Ca++-channels suggest that Ca++ levels may be involved in regulating this system.