RESUMEN
Convergent extension of the chordamesoderm is the best-examined gastrulation movement in Xenopus. Here we study general features of cell-cell contacts in this tissue by combining depletion of adhesion factors C-cadherin, Syndecan-4, fibronectin, and hyaluronic acid, the analysis of respective contact width spectra and contact angles, and La3+ staining of the pericellular matrix. We provide evidence that like in other gastrula tissues, cell-cell adhesion in the chordamesoderm is largely mediated by different types of pericellular matrix. Specific glycocalyx structures previously identified in Xenopus gastrula tissues are absent in chordamesoderm but other contact types like 10-20 nm wide La3+ stained structures are present instead. Knockdown of any of the adhesion factors reduces the abundance of cell contacts but not the average relative adhesiveness of the remaining ones: a decrease of adhesiveness at low contact widths is compensated by an increase of contact widths and an increase of adhesiveness proportional to width. From the adhesiveness-width relationship, we derive a model of chordamesoderm cell adhesion that involves the interdigitation of distinct pericellular matrix units. Quantitative description of pericellular matrix deployment suggests that reduced contact abundance upon adhesion factor depletion is correlated with excessive accumulation of matrix material in non-adhesive gaps and the loss of some contact types.
Asunto(s)
Gástrula , Notocorda , Animales , Gástrula/metabolismo , Xenopus laevis , Gastrulación , Adhesión Celular , Movimiento CelularRESUMEN
Morphogenetic movements during animal development involve repeated making and breaking of cell-cell contacts. Recent biophysical models of cell-cell adhesion integrate adhesion molecule interactions and cortical cytoskeletal tension modulation, describing equilibrium states for established contacts. We extend this emerging unified concept of adhesion to contact formation kinetics, showing that aggregating Xenopus embryonic cells rapidly achieve Ca2+-independent low-contact states. Subsequent transitions to cadherin-dependent high-contact states show rapid decreases in contact cortical F-actin levels but slow contact area growth. We developed a biophysical model that predicted contact growth quantitatively from known cellular and cytoskeletal parameters, revealing that elastic resistance to deformation and cytoskeletal network turnover are essential determinants of adhesion kinetics. Characteristic time scales of contact growth to low and high states differ by an order of magnitude, being at a few minutes and tens of minutes, respectively, thus providing insight into the timescales of cell-rearrangement-dependent tissue movements.
Asunto(s)
Cadherinas , Gástrula , Animales , Adhesión Celular , Xenopus laevis , Gástrula/metabolismo , Cadherinas/metabolismo , Moléculas de Adhesión CelularRESUMEN
Trogocytosis, in which cells nibble away parts of neighboring cells, is an intercellular cannibalism process conserved from protozoa to mammals. Its underlying molecular mechanisms are not well understood and are likely distinct from phagocytosis, a process that clears entire cells. Bi-directional contact repulsion induced by Eph/ephrin signaling involves transfer of membrane patches and full-length Eph/ephrin protein complexes between opposing cells, resembling trogocytosis. Here, we show that the phagocytic adaptor protein Gulp1 regulates EphB/ephrinB trogocytosis to achieve efficient cell rearrangements of cultured cells and during embryonic development. Gulp1 mediates trogocytosis bi-directionally by dynamic engagement with EphB/ephrinB protein clusters in cooperation with the Rac-specific guanine nucleotide exchange factor Tiam2. Ultimately, Gulp1's presence at the Eph/ephrin cluster is a prerequisite for recruiting the endocytic GTPase dynamin. These results suggest that EphB/ephrinB trogocytosis, unlike other trogocytosis events, uses a phagocytosis-like mechanism to achieve efficient membrane scission and engulfment.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Efrinas/metabolismo , Receptores de la Familia Eph/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Transducción de SeñalRESUMEN
Cleft-like boundaries represent a type of cell sorting boundary characterized by the presence of a physical gap between tissues. We studied the cleft-like ectoderm-mesoderm boundary in Xenopus laevis and zebrafish gastrulae. We identified the transcription factor Snail1 as being essential for tissue separation, showed that its expression in the mesoderm depends on noncanonical Wnt signaling, and demonstrated that it enables paraxial protocadherin (PAPC) to promote tissue separation through two novel functions. First, PAPC attenuates planar cell polarity signaling at the ectoderm-mesoderm boundary to lower cell adhesion and facilitate cleft formation. Second, PAPC controls formation of a distinct type of adhesive contact between mesoderm and ectoderm cells that shows properties of a cleft-like boundary at the single-cell level. It consists of short stretches of adherens junction-like contacts inserted between intermediate-sized contacts and large intercellular gaps. These roles of PAPC constitute a self/non-self-recognition mechanism that determines the site of boundary formation at the interface between PAPC-expressing and -nonexpressing cells.
Asunto(s)
Cadherinas/fisiología , Factores de Transcripción/fisiología , Proteínas de Xenopus/fisiología , Actinas/metabolismo , Animales , Tipificación del Cuerpo , Adhesión Celular , Polaridad Celular , Gástrula/embriología , Gástrula/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Protocadherinas , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis , Pez Cebra , Proteínas de Pez Cebra/fisiologíaRESUMEN
Morphogenetic processes often involve the rapid rearrangement of cells held together by mutual adhesion. The dynamic nature of this adhesion endows tissues with liquid-like properties, such that large-scale shape changes appear as tissue flows. Generally, the resistance to flow (tissue viscosity) is expected to depend on the cohesion of a tissue (how strongly its cells adhere to each other), but the exact relationship between these parameters is not known. Here, we analyse the link between cohesion and viscosity to uncover basic mechanical principles of cell rearrangement. We show that for vertebrate and invertebrate tissues, viscosity varies in proportion to cohesion over a 200-fold range of values. We demonstrate that this proportionality is predicted by a cell-based model of tissue viscosity. To do so, we analyse cell adhesion in Xenopus embryonic tissues and determine a number of parameters, including tissue surface tension (as a measure of cohesion), cell contact fluctuation and cortical tension. In the tissues studied, the ratio of surface tension to viscosity, which has the dimension of a velocity, is 1.8â µm/min. This characteristic velocity reflects the rate of cell-cell boundary contraction during rearrangement, and sets a limit to rearrangement rates. Moreover, we propose that, in these tissues, cell movement is maximally efficient. Our approach to cell rearrangement mechanics links adhesion to the resistance of a tissue to plastic deformation, identifies the characteristic velocity of the process, and provides a basis for the comparison of tissues with mechanical properties that may vary by orders of magnitude.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Embrión no Mamífero/citología , Modelos Biológicos , Morfogénesis/fisiología , Xenopus/embriología , Animales , Ficoll , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Tensión Superficial , ViscosidadRESUMEN
Xenopus provides a well-studied model of vertebrate gastrulation, but a central feature, the movement of the mesoderm to the interior of the embryo, has received little attention. Here, we analyze mesoderm involution at the Xenopus dorsal blastopore lip. We show that a phase of rapid involution - peak involution - is intimately linked to an early stage of convergent extension, which involves differential cell migration in the prechordal mesoderm and a new movement of the chordamesoderm, radial convergence. The latter process depends on Xenopus Brachyury, the expression of which at the time of peak involution is controlled by signaling through the ephrin receptor, EphA4, its ligand ephrinB2 and its downstream effector p21-activated kinase. Our findings support a conserved role for Brachyury in blastopore morphogenesis.
Asunto(s)
Proteínas Fetales/metabolismo , Gástrula/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Mesodermo/embriología , Receptor EphA4/metabolismo , Transducción de Señal/fisiología , Proteínas de Dominio T Box/metabolismo , Xenopus/embriología , Animales , Movimiento Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ , Microscopía Electrónica de Rastreo , Morfolinos/genética , Transducción de Señal/genéticaRESUMEN
ß-Catenin-independent Wnt signaling pathways have been implicated in the regulation of planar cell polarity (PCP) and convergent extension (CE) cell movements. Prickle, one of the core proteins of these pathways, is known to asymmetrically localize proximally at the adherens junction of Drosophila melanogaster wing cells and to locally accumulate within plasma membrane subdomains in cells undergoing CE movements during vertebrate development. Using mass spectrometry, we have identified the Ste20 kinase Mink1 as a Prickle-associated protein and found that they genetically interact during the establishment of PCP in the Drosophila eye and CE in Xenopus laevis embryos. We show that Mink1 phosphorylates Prickle on a conserved threonine residue and regulates its Rab5-dependent endosomal trafficking, a process required for the localized plasma membrane accumulation and function of Prickle. Mink1 also was found to be important for the clustering of Vangl within plasma membrane puncta. Our results provide a link between Mink and the Vangl-Prickle complex and highlight the importance of Prickle phosphorylation and endosomal trafficking for its function during Wnt-PCP signaling.
Asunto(s)
Proteínas con Dominio LIM/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endosomas/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/análisis , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/análisis , Transporte de Proteínas , Proteínas Supresoras de Tumor/análisis , Xenopus/embriología , Xenopus/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
BACKGROUND: The primordial organization of the metazoan body is achieved during gastrulation by the establishment of the germ layers. Adhesion differences between ectoderm, mesoderm, and endoderm cells could in principle be sufficient to maintain germ layer integrity and prevent intermixing. However, in organisms as diverse as fly, fish, or amphibian, the ectoderm-mesoderm boundary not only keeps these germ layers separated, but the ectoderm also serves as substratum for mesoderm migration, and the boundary must be compatible with repeated cell attachment and detachment. PRINCIPAL FINDINGS: We show that localized detachment resulting from contact-induced signals at the boundary is at the core of ectoderm-mesoderm segregation. Cells alternate between adhesion and detachment, and detachment requires ephrinB/EphB signaling. Multiple ephrinB ligands and EphB receptors are expressed on each side of the boundary, and tissue separation depends on forward signaling across the boundary in both directions, involving partially redundant ligands and receptors and activation of Rac and RhoA. CONCLUSION: This mechanism differs from a simple differential adhesion process of germ layer formation. Instead, it involves localized responses to signals exchanged at the tissue boundary and an attachment/detachment cycle which allows for cell migration across a cellular substratum.
Asunto(s)
Movimiento Celular/fisiología , Efrina-B1/metabolismo , Gástrula/fisiología , Estratos Germinativos/embriología , Receptor EphB1/metabolismo , Transducción de Señal/fisiología , Animales , Adhesión Celular/fisiología , Humanos , Ratones , Microscopía Confocal , Oligonucleótidos/genética , Plásmidos/genéticaRESUMEN
Epithelia are planar tissues that undergo major morphogenetic movements during development. These movements must work in the context of the mechanical properties of epithelia. Surprisingly little is known about these mechanical properties at the time and length scales of morphogenetic processes. We show that at a time scale of hours, Xenopus gastrula ectodermal epithelium mimics an elastic solid when stretched isometrically; strikingly, its area increases twofold in the embryo by such pseudoelastic expansion. At the same time, the basal side of the epithelium behaves like a liquid and exhibits tissue surface tension that minimizes its exposed area. We measure epithelial stiffness (~1 mN/m), surface tension (~0.6 mJ/m(2)), and epithelium-mesenchyme interfacial tensions and relate these to the folding of isolated epithelia and to the extent of epithelial spreading on various tissues. We propose that pseudoelasticity and tissue surface tension are main determinants of epithelial behavior at the scale of morphogenetic processes.
Asunto(s)
Xenopus/embriología , Animales , Movimiento Celular , Elasticidad , Células Epiteliales , GástrulaRESUMEN
The p21 activated kinases (Paks) are prominently involved in the regulation of cell motility. Using a kinase-dead mutant of xPak1, we show that during Xenopus gastrulation, the kinase activity of Pak1 is required upstream of Cdc42 for the establishment of cell polarity in the migrating mesendoderm. Overactivation of Pak1 function by the expression of constitutively active xPak1 compromises the maintenance of cell polarity, by indirectly inhibiting RhoA function. Inhibition of cell polarization does not affect the migration of single mesendoderm cells. However, Pak1 inhibition interferes with the guidance of mesendoderm migration by directional cues residing in the extracellular matrix of the blastocoel roof, and with mesendoderm translocation in the embryo.
Asunto(s)
Movimiento Celular/genética , Polaridad Celular/genética , Gástrula/embriología , Mesodermo/embriología , Xenopus laevis/embriología , Quinasas p21 Activadas/fisiología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Adhesión Celular/genética , Embrión no Mamífero , Gástrula/metabolismo , Mesodermo/metabolismo , Modelos Biológicos , Xenopus laevis/genética , Quinasas p21 Activadas/genética , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
Formation of tissue boundaries can be studied in a simple, inexpensive system, the Xenopus gastrula. Here, the internalized mesoderm and endoderm are separated from the ectodermal blastocoel roof by Brachet's cleft. Non-canonical Wnt signaling mediated by the Wnt receptor, Xfz-7, is essential for this tissue separation event. The function of Wnt pathway components and other factors in tissue separation at Brachet's cleft can be tested in a blastocoel roof assay. Small pieces of mesoderm or endoderm are placed on large blastocoel roof explants, and it is observed whether these test explants remain on the surface of their in vivo substratum, or sink into it.
Asunto(s)
Bioensayo/métodos , Gástrula , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Gástrula/anatomía & histología , Gástrula/fisiología , Morfogénesis , Receptores Acoplados a Proteínas G/genética , Proteínas de Xenopus/genética , Xenopus laevis/anatomía & histologíaRESUMEN
In the vegetal half of the Xenopus gastrula, cell populations differ with respect to migration on fibronectin substratum. We show that the paired-class homeodomain transcription factors Goosecoid (Gsc), Mix.1, and Siamois (Sia) are involved in the modulation of migration velocity and cell polarity. Mix.1 is expressed in the whole vegetal half and serves as a competence factor that is necessary, but not sufficient, for rapid cell migration and polarization. In the head mesoderm, Gsc and Sia are coexpressed with Mix.1, promoting rapid cell migration and polarization. Ectopic expression of Gsc and Sia in both vegetal and ventral regions often generates paradoxical effects; if a factor activates a certain motility trait in one region, it inhibits it in the other. Migration velocity and cell polarity are regulated independently. Fast and efficiently migrating multipolar cells and slow-moving polarized cells can be obtained by ectopic expression of these transcription factors in different combinations.