RESUMEN
Fibronectin (FN) is reported to be important for early morphogenetic movements in a variety of vertebrate embryos, but the cellular basis for this requirement is unclear. We have used confocal and digital time-lapse microscopy to analyze cell behaviors in Xenopus gastrulae injected with monoclonal antibodies directed against the central cell-binding domain of fibronectin. Among the defects observed is a disruption of fibronectin matrix assembly, resulting in a failure of radial intercalation movements, which are required for blastocoel roof thinning and epiboly. We identified two phases of FN-dependent cellular rearrangements in the blastocoel roof. The first involves maintenance of early roof thinning in the animal cap, and the second is required for the initiation of radial intercalation movements in the marginal zone. A novel explant system was used to establish that radial intercalation in the blastocoel roof requires integrin-dependent contact of deep cells with fibronectin. Deep cell adhesion to fibronectin is sufficient to initiate intercalation behavior in cell layers some distance from the substrate. Expression of a dominant-negative beta1 integrin construct in embryos results in localized depletion of the fibronectin matrix and thickening of the blastocoel roof. Lack of fibronectin fibrils in vivo is correlated with blastocoel roof thickening and a loss of deep cell polarity. The integrin-dependent binding of deep cells to fibronectin is sufficient to drive membrane localization of Dishevelled-GFP, suggesting that a convergence of integrin and Wnt signaling pathways acts to regulate radial intercalation in Xenopus embryos.
Asunto(s)
Movimiento Celular , Polaridad Celular , Embrión no Mamífero/citología , Fibronectinas/metabolismo , Gástrula/citología , Integrinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Dishevelled , Integrina beta1/metabolismo , Modelos Biológicos , Fosfoproteínas/aislamiento & purificación , Xenopus laevisRESUMEN
BACKGROUND: Cranial neural-crest (CNC) cells originate from the lateral edge of the anterior neuroepithelium and migrate to form parts of the peripheral nervous system, muscles, cartilage, and bones of the face. Neural crest-cell migration involves the loss of adhesion from the surrounding neuroepithelium and a corresponding increase in cell adhesion to the extracellular matrix (ECM) present in migratory pathways. While proteolytic activity is likely to contribute to the regulation of neural crest-cell adhesion and migration, the role of a neural crest-specific protease in these processes has yet to be demonstrated. We previously showed that CNC cells express ADAM 13, a cell surface metalloprotease/disintegrin. Proteins of this family are known to act in cell-cell adhesion and as sheddases. ADAMs have also been proposed to degrade the ECM, but this has not yet been shown in a physiological context. RESULTS: Using a tissue transplantation technique, we show that Xenopus CNC cells overexpressing wild-type ADAM 13 migrate along the same hyoid, branchial, and mandibular pathways used by normal CNC cells. In contrast, CNC cell grafts that express protease-defective ADAM 13 fail to migrate along the hyoid and branchial pathways. In addition, ectopic expression of wild-type ADAM 13 results in a gain-of-function phenotype in embryos, namely the abnormal positioning of trunk neural-crest cells. We further show that explanted embryonic tissues expressing wild-type, but not protease-defective, ADAM 13 display decreased cell-matrix adhesion. Purified ADAM 13 can cleave fibronectin, and tissue culture cells that express wild-type, but not protease-defective, ADAM 13 can remodel a fibronectin substrate. CONCLUSIONS: Our findings support the hypothesis that the protease activity of ADAM 13 plays a critical role in neural crest-cell migration along defined pathways. We propose that the ADAM 13-dependent modification of ECM and/or other guidance molecules is a key step in the directed migration of the CNC.
Asunto(s)
Movimiento Celular/fisiología , Proteínas de la Membrana/metabolismo , Cresta Neural/citología , Proteínas de Xenopus , Xenopus laevis/embriología , Proteínas ADAM , Animales , Células Cultivadas , Sistema Nervioso Central/embriología , Fibronectinas/metabolismo , Colorantes Fluorescentes/metabolismo , Immunoblotting , Hibridación in Situ , Proteínas de la Membrana/genética , Microinyecciones , Modelos Moleculares , Morfogénesis , Cresta Neural/fisiología , Fenotipo , Trasplante de Tejidos , Xenopus laevis/genética , Xenopus laevis/fisiologíaRESUMEN
Cellular interactions with laminin are important for numerous morphogenetic events. In Xenopus, the first of these is neurulation. The integrin alpha6 subunit mediates an attachment of the cells of the neural plate to the underlying basal lamina. A disruption of this interaction results in embryos that fail to neurulate (T. E. Lallier et al., 1996, Development 122, 2539-2554). Here we provide evidence supporting the specificity of this phenomenon and characterize developmental events as either disrupted or unaffected by a perturbation of alpha6 integrin expression. First, reduction of alpha6 integrin expression does not halt mitotic division throughout the embryo, indicating that the neural defects observed are not simply a global perturbation of all developmental processes. Second, a gene associated with dorsal mesoderm formation, brachyury, is expressed normally in alpha6 integrin-perturbed embryos. Third, the expression of BMP4, noggin, chordin, and follistatin, all of which are critical for neural induction, are at near normal levels. In addition, several genes expressed shortly after neural induction (N-CAM, nrp1, and Xanf1) are not perturbed in nonneurulating embryos. Interestingly, expression of one neural-specific gene (synaptobrevin), which is normally detectable late in neurulation, is abolished in these alpha6 integrin-perturbed embryos. Furthermore, the spatial expression of several transcripts is expanded in alpha6 integrin-perturbed embryos (orthodenticle and engrailed). Taken together, these data indicate that while alpha6 integrin-mediated interactions with laminin are required for neurulation, they are not required for the initial processes of neural induction. However, these cell-extracellular matrix interactions appear to be important in later inductive events and rostrocaudal patterning of the neural tube.
Asunto(s)
Antígenos CD/fisiología , Sistema Nervioso/embriología , Xenopus/embriología , Animales , Adhesión Celular , Movimiento Celular , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/fisiología , Inducción Embrionaria , Integrina alfa6 , Oligonucleótidos Antisentido , Xenopus/fisiologíaRESUMEN
Active zones are the sites along nerve terminals where synaptic vesicles dock and undergo calcium-dependent exocytosis during synaptic transmission. Here we show, by immunofluorescent staining with antibodies generated against Xenopus laevis integrins, that alpha3beta1 integrin is concentrated at the active zones of Xenopus motor nerve terminals. Because integrins can link extracellular matrix molecules to cytoskeletal elements and participate in the formation of signaling complexes, the localization of integrin at active zones suggests that it may play a role in the adhesion of the nerve terminals to the synaptic basal lamina, in the formation and maintenance of active zones, and in some of the events associated with calcium-dependent exocytosis of neurotransmitter. Our findings also indicate that the integrin composition of the terminal Schwann cells differs from that of the motor nerve terminals, and this may account at least in part for differences in their adhesiveness to the synaptic basal lamina.
Asunto(s)
Integrinas/análisis , Neuronas Motoras/citología , Terminaciones Nerviosas/ultraestructura , Unión Neuromuscular/citología , Animales , Anticuerpos Monoclonales , Adhesión Celular , Colagenasas , Integrina alfa3beta1 , Neuronas Motoras/ultraestructura , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/inervación , Unión Neuromuscular/ultraestructura , Células de Schwann/citología , Células de Schwann/ultraestructura , Sinapsis/ultraestructura , Xenopus laevisRESUMEN
The thrombospondins (TSPs) are a family of extracellular matrix (ECM) glycoproteins that modulate many cell behaviors including adhesion, migration, and proliferation. Here we report the molecular cloning of the Xenopus homologs of TSP-1 and TSP-3, and the developmental patterns of expression of Xenopus TSP-1, TSP-3, and TSP-4 mRNAs. Xenopus TSP-1 and TSP-3 protein sequences each share approximately 80% amino acid identity with their mammalian counterparts. TSP-1 mRNAs are detectable at low levels in fertilized eggs indicating that this TSP is a maternally deposited transcript. Zygotic expression of TSP-1, TSP-3, and TSP-4 begins at the end of gastrulation and transcripts encoding each protein accumulate through the tadpole stages of development. Whole mount in situ hybridizations reveal that each TSP mRNA is localized in the embryo with distinct, developmentally regulated patterns of expression. TSP-1 mRNAs are detected in a wide range of tissues including the floor plate of the neural tube, epidermis, somites, notochord and, most notably, alternating rhombomeres. Transcripts encoding TSP-3 are expressed in the notochord, floor plate, sensorial layer of the epidermis and sensory epithelia. TSP-4 mRNAs are restricted to somitic mesoderm and skeletal muscle. These data suggest that the TSPs represent a functionally diverse family of ECM proteins with tissue-specific functions during embryogenesis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Músculos/metabolismo , Sistema Nervioso/metabolismo , Notocorda/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Embrión no Mamífero/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Músculos/embriología , Sistema Nervioso/embriología , Factores de Tiempo , Distribución Tisular , Xenopus laevisRESUMEN
Integrin receptors containing an alpha 4 subunit mediate cell-cell adhesion by binding to VCAM and MadCAM-1 in addition to supporting cell-extracellular matrix (ECM) adhesion by binding to the alternatively spliced V-region of fibronectin (FN). Studies in chick and mouse embryos have implicated these integrins in neural crest migration, myotube formation, heart development, and placentation. Because integrin-FN adhesive interactions have been shown to play essential roles in mammalian development, studies were initiated of integrin alpha 4 in amphibian embryos, which are better suited to experimental analyses of the earliest stages of embryogenesis. Here, the cDNA cloning and pattern of expression of the Xenopus laevis homolog of integrin alpha 4 is reported. Xenopus alpha 4 is 55% identical at the amino-acid level to both its human and mouse counterparts, including conservation of an alpha 4-specific protease cleavage site, 11 potential N-linked glycosylation sites, and 24 cysteine residues. In situ hybridization analysis reveals that transcripts encoding alpha 4 are expressed in epidermis and the branchial arches. Although alpha 4 transcripts can be detected as early as gastrulation, the protein is observed only after tailbud stages of development and is spatially restricted to the epidermis and gills of tadpole stage embryos. From these data it is concluded that Xenopus integrin alpha 4 has structural features in common with other vertebrate alpha 4 homologs, but is detected in a more restricted tissue distribution during development than alpha 4 in other species.
Asunto(s)
Antígenos CD/genética , Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica , Transcripción Genética , Secuencia de Aminoácidos , Animales , Antígenos CD/biosíntesis , Antígenos CD/química , Clonación de Organismos , Epidermis/embriología , Epidermis/fisiología , Biblioteca de Genes , Humanos , Integrina alfa4 , Integrinas/genética , Mesodermo/fisiología , Ratones , Datos de Secuencia Molecular , Morfogénesis , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
Integrins containing the alpha2 and alpha3 subunits associate with the beta1 subunit to form distinct receptors with partially overlapping adhesive specificities. We report the cloning and sequence of cDNAs that encode the Xenopus orthologues of integrins alpha2 and alpha3 and the expression of these subunits during embryogenesis. Integrin alpha2 and alpha3 mRNAs are first expressed in the dorsal mesoderm and developing notochord at gastrulation. We also show that alpha3 mRNAs are expressed in the entire marginal zone of gastrulae dorsalized with LiCl but that this localization is lost in embryos ventralized by ultraviolet light. Immunoblots reveal that the alpha3 protein is expressed throughout early development, however, the alpha2 protein is not detected until late tailbud stages. Injection of full-length alpha3 transcripts into the animal poles of fertilized eggs results in embryonic defects in paraxial mesoderm attributed to the failure of somites to form segments. Injection of the alpha3 transcripts into the vegetal pole and overexpression of a 5'-truncated alpha3 control construct have no apparent affect on development or somite formation. These data suggest that normal position-specific expression of integrins is important in maintaining the proper organization of tissues during early amphibian morphogenesis.
Asunto(s)
Antígenos CD/genética , Integrinas/genética , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Antígenos CD/inmunología , Northern Blotting , Tipificación del Cuerpo/genética , Clonación Molecular , ADN Complementario , Regulación del Desarrollo de la Expresión Génica , Humanos , Integrina alfa2 , Integrina alfa3 , Integrinas/inmunología , Datos de Secuencia Molecular , Notocorda/metabolismo , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Xenopus laevis/embriologíaRESUMEN
Embryonic development involves a series of cell adhesive interactions that provide mechanical and instructive information required for morphogenesis. The ADAMs family of membrane-anchored proteins, containing a disintegrin and metalloprotease domain, is well suited for participating in such developmental events. They encode not only a potential adhesive function, through an integrin-binding disintegrin domain, but also a potential antiadhesive function, through a zinc-dependent metalloprotease domain. In order to investigate the role of ADAMs in early development we cloned a cDNA encoding a novel member of the ADAM family from a Xenopus laevis neurula stage library. We call this cDNA, and the 915-amino-acid protein it encodes, ADAM 13, X-ADAM 13 RNA is expressed during embryogenesis from the midblastula stage through tadpole stage 45. X-ADAM 13 is localized to somitic mesoderm and cranial neural crest cells during gastrulation, neurulation, and in tail bud stages. Sequence analyses of the X-ADAM 13 metalloprotease and disintegrin domains indicate that the protein is likely to be involved in both proteolytic and cell-adhesive functions. The X-ADAM 13 sequence is most closely related to that of mouse meltrin alpha, which is implicated in myoblast fusion. Our data suggest that X-ADAM 13 may be involved in neural crest cell adhesion and migration as well as myoblast differentiation.
Asunto(s)
Proteínas de la Membrana/genética , Cresta Neural/metabolismo , Somitos/metabolismo , Proteínas de Xenopus , Células 3T3 , Proteínas ADAM , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario , Desintegrinas/genética , Desintegrinas/fisiología , Embrión no Mamífero/metabolismo , Humanos , Mamíferos , Proteínas de la Membrana/fisiología , Metaloendopeptidasas/genética , Metaloendopeptidasas/fisiología , Ratones , Datos de Secuencia Molecular , Cresta Neural/citología , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Xenopus laevis/embriologíaRESUMEN
Integrins mediate cell-ECM interactions essential for morphogenesis, however, the extent to which integrin adhesive activities are regulated in the embryo has not been addressed. We report that integrin-dependent cell adhesion to the Arg-Gly-Asp (RGD) containing central cell-binding domain of fibronectin is required for gastrulation in Xenopus. Although all cells of the early embryo retain the ability to attach to this region, only involuting cells arising from the dorsal and ventral lips of the blastopore are able to spread and migrate on fibronectin in vitro. This change in adhesive behavior is mimicked by treating animal cap cells with activin-A. Activin-induced changes in adhesion are independent of new transcription, translation, or changes in receptor expression at the cell surface. We demonstrate that ectopic expression of integrin alpha4beta1 in animal cap cells results in attachment to the non RGD-containing V-region of fibronectin. Further, these cells acquire the ability to spread on the V-region following activin induction. Thus, alpha4beta1 adhesion to the V-region, like endogenous integrin binding to the central cell-binding domain, is responsive to activin signalling. These data indicate that cell adhesion to the central cell-binding domain is regulated in both space and time, and is under the control of inductive signals that initiate gastrulation movements. We suggest that position-specific inductive interactions are likely to represent a novel and general mechanism by which integrin adhesion is modulated throughout development.
Asunto(s)
Adhesión Celular , Inducción Embrionaria , Fibronectinas/metabolismo , Gástrula/fisiología , Integrinas/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Activinas , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Fibronectinas/química , Inhibinas/farmacología , Integrina alfa4beta1 , Mesodermo/metabolismo , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Pruebas de Precipitina , Transducción de Señal , Xenopus laevis/embriologíaRESUMEN
The integrin alpha 6 subunit pairs with both the beta 1 and beta 4 subunits to form a subfamily of laminin receptors. Here we report the cDNA cloning and primary sequence for the Xenopus homologue of the mammalian integrin alpha 6 subunit. We present data demonstrating the spatial and temporal expression of alpha 6 mRNA and protein during early development. Initially, alpha 6 transcripts are expressed in the dorsal ectoderm and future neural plate at the end of gastrulation. Later in development, alpha 6 mRNAs are expressed in a variety of neural derivatives, including the developing sensory placodes (otic and olfactory) and commissural neurons within the neural tube. Integrin alpha 6 is also expressed in the elongating pronephric duct as well as a subset of the rhombencephalic neural crest, which will form the Schwann cells lining several cranial nerves (VII, VIII and X). In vivo expression of an alpha 6 antisense transcript in the animal hemisphere leads to a reduction in alpha 6 protein expression, a loss of adhesion to laminin, and severe defects in normal development. In 35% of cases, reduced levels of alpha 6 expression result in embryos that complete gastrulation normally but arrest at neurulation prior to the formation of the neural plate. In an additional 22% of cases, embryos develop with severe axial defects, including complete loss of head or tail structures. In contrast, overexpression of the alpha 6 subunit by injection of full-length mRNA has no apparent effect on embryonic development. Co-injection of antisense and sense plasmid constructs results in a partial rescue of the antisense-generated phenotypes. These data indicate that the integrin alpha 6 subunit is critical for the early development of the nervous system in amphibians.
Asunto(s)
Antígenos CD/fisiología , Sistema Nervioso/embriología , Células 3T3 , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Complementario , Ectodermo , Femenino , Expresión Génica , Humanos , Hibridación in Situ , Integrina alfa6 , Laminina/metabolismo , Ratones , Datos de Secuencia Molecular , Sistema Nervioso/metabolismo , ARN Mensajero , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transfección , Xenopus laevisRESUMEN
During Xenopus laevis gastrulation, the basic body plan of the embryo is generated by movement of the marginal zone cells of the blastula into the blastocoel cavity. This morphogenetic process involves cell adhesion to the extracellular matrix protein fibronectin (FN). Regions of FN required for the attachment and migration of involuting marginal zone (IMZ) cells were analyzed in vitro using FN fusion protein substrates. IMZ cell attachment to FN is mediated by the Arg-Gly-Asp (RGD) sequence located in the type III-10 repeat and by the Pro-Pro-Arg-Arg-Ala-Arg (PPRRAR) sequence in the type III-13 repeat of the Hep II domain. IMZ cells spread and migrate persistently on fusion proteins containing both the RGD and synergy site sequence Pro-Pro-Ser-Arg-Asn (PPSRN) located in the type III-9 repeat. Cell recognition of the synergy site is positionally regulated in the early embryo. During gastrulation, IMZ cells will spread and migrate on FN whereas presumptive pre-involuting mesoderm, vegetal pole endoderm, and animal cap ectoderm will not. However, animal cap ectoderm cells acquire the ability to spread and migrate on the RGD/synergy region when treated with the mesoderm inducing factor activin-A. These data suggest that mesoderm induction activates the position-specific recognition of the synergy site of FN in vivo. Moreover, we demonstrate the functional importance of this site using a monoclonal antibody that blocks synergy region-dependent cell spreading and migration on FN. Normal IMZ movement is perturbed when this antibody is injected into the blastocoel cavity indicating that IMZ cell interaction with the synergy region is required for normal gastrulation.
Asunto(s)
Adhesión Celular , Fibronectinas/fisiología , Gástrula/fisiología , Xenopus laevis/embriología , Activinas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Movimiento Celular , Cartilla de ADN/química , Inducción Embrionaria , Regulación del Desarrollo de la Expresión Génica , Heparina/fisiología , Inhibinas/fisiología , Datos de Secuencia Molecular , Morfogénesis , Oligopéptidos , ARN Mensajero/genética , Relación Estructura-ActividadRESUMEN
In early amphibian development, interactions between fibronectin and both ectoderm and mesoderm cells are critical in the progression of gastrulation movements. In the Pleurodeles waltl embryo, it has been established that ectoderm cells of the animal hemisphere organize a fibrillar-extracellular matrix containing fibronectin. Mesoderm cells migrate along the blastocoel roof using these fibronectin fibrils as substratum. Fibronectin is an adhesive glycoprotein which possesses multiple cell-binding domains. From previous studies, it is clear that amphibian ectoderm and mesoderm cells interact with fibronectin in an RGD-dependent manner, whereas the contributions of RGD-independent domains in the adhesive behaviors of gastrula cells has not been defined. To study this question, we have used bacterially expressed Pleurodeles waltl fibronectin-fusion proteins. The approach consisted of in vitro adhesion assays with either isolated cells or tissue fragments of embryos dissected at the onset of gastrulation. Tissues were obtained from regions of the embryo which represent presumptive ectoderm cells or from the dorsal-marginal zone which contains cells of the presumptive cephalic, chordal and somitic mesoderm. The results show that both the RGD-dependent and the Hep II domains of fibronectin mediate attachment and spreading of isolated cells. Both regions cooperate to control the proper expansion of a sheet of dorsal mesoderm cells. The Hep II domain promotes the migration of cells ahead of the mesoderm-cell sheet.
Asunto(s)
Adhesión Celular/fisiología , Fibronectinas/metabolismo , Oligopéptidos/metabolismo , Pleurodeles/embriología , Animales , Sitios de Unión , Movimiento Celular , Ectodermo/citología , Embrión no Mamífero/citología , Fibronectinas/química , Fibronectinas/genética , Gástrula/citología , Mesodermo/citología , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Mesodermal cell migration during amphibian gastrulation is dependent on cellular interactions with fibronectin. One mechanism whereby cells bind fibronectin is through alpha v-containing integrin heterodimers. In order to investigate the role of alpha v in amphibian gastrulation, we have cloned the Pleurodeles homologue of the integrin alpha v subunit using homology PCR. The deduced amino acid sequence is 73 and 74% identical with the human and chick homologues, respectively. The 4.8-kb mRNA is expressed during oogenesis and persists throughout development. Messenger RNA and protein are widely expressed in oocytes and embryos while cell surface expression is spatially regulated. The protein first appears on the plasma membrane of fully grown oocytes. Fertilization results in the progressive loss of alpha v membrane localization. Before and during gastrulation, the integrin alpha v subunit is expressed on the surface of mesodermal cells. These data show that alpha v expression is developmentally regulated by a post-translational mechanism which correlates with the onset of mesodermal cell migration at gastrulation.
Asunto(s)
Integrinas/genética , Pleurodeles/crecimiento & desarrollo , Pleurodeles/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clonación Molecular , Cartilla de ADN/genética , Femenino , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Integrina alfaV , Integrinas/metabolismo , Mesodermo/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Oogénesis/genética , Pleurodeles/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Integrin-mediated cellular adhesion to components of the extracellular matrix (ECM) is important in a number of morphogenetic events that occur during vertebrate embryogenesis. Recent studies suggest that the focal adhesion kinase pp125FAK is involved in the regulation of integrin-dependent signaling processes triggered by cell adhesion to the ECM. We report the cDNA cloning and sequence analysis of the Xenopus homolog of pp125FAK. We also describe temporal and spatial patterns of FAK expression during early development. Xenopus FAK shares greater than 90% identity with its avian and mammalian homologs. FAK mRNA and protein are present in the fertilized egg and in cleavage stage embryos. During gastrulation, FAK protein expression increases significantly and is detected in mesoderm, marginal zone ectoderm, and cells of the blastocoel roof. Later in development, FAK is prominently expressed at intersomitic junctions, in the brain, and in several cranial nerves. Phosphotyrosyl-FAK is first detected during gastrulation, suggesting that the phosphorylation of FAK on tyrosine is developmentally regulated. These data indicate that FAK is likely to participate in a variety of integrin-ECM-dependent signaling events during morphogenesis.
Asunto(s)
Moléculas de Adhesión Celular/genética , Proteínas Tirosina Quinasas/genética , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/metabolismo , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mapeo Restrictivo , Xenopus laevis/embriologíaRESUMEN
The full length sequence of the Xenopus integrin alpha 5 subunit is reported. Analysis of cloned cDNA fragments reveals that alternative polyadenylation of alpha 5 mRNA occurs in the embryo. Furthermore, a variant form of the alpha 5 mRNA is expressed which encodes an integrin alpha 5 subunit with a truncated cytoplasmic domain. Integrin alpha 5 mRNA and protein are expressed in oocytes, eggs and throughout development. Spatial expression of alpha 5 mRNAs is first detected by whole mount in situ hybridization in presumptive neural crest cells and in the somitic mesoderm from the midgastrula stage onwards. In contrast, the alpha 5 protein is present on newly formed plasma membranes beginning at first cleavage. During neurulation, the integrin alpha 5 subunit disappears from the outer layer of the ectoderm, the notochord and the neural tube and accumulates in the sensorial layer of the ectoderm, the somites and the neural crest cells. These results provide evidence for the position specific regulation of alpha subunit expression in early vertebrate embryos.
Asunto(s)
Antígenos CD/genética , Fase de Segmentación del Huevo , Regulación del Desarrollo de la Expresión Génica/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Variación Genética , Integrina alfa5 , Datos de Secuencia Molecular , ARN Mensajero/genética , Xenopus laevisRESUMEN
The extracellular matrix supports the adhesion and migration of cells during morphogenesis and influences cell differentiation. Cell interactions with the extracellular matrix are mediated in large part by members of the integrin family of cell-surface receptors. Recent progress in this area has resulted in the identification of multiple integrins, many of which are expressed in position-specific patterns during vertebrate development. The contributions of these receptors to specific developmental events are now being investigated in a variety of systems using a combination of genetic, molecular and immunological approaches.
Asunto(s)
Adhesión Celular/fisiología , Matriz Extracelular/fisiología , Vertebrados/crecimiento & desarrollo , Anfibios , Animales , Anticuerpos , Adhesión Celular/genética , Marcación de Gen , Humanos , Integrinas/antagonistas & inhibidores , Integrinas/genética , Integrinas/fisiología , Ratones , ARN sin Sentido/genética , Vertebrados/genética , Vertebrados/fisiologíaRESUMEN
During embryogenesis cells modulate their adhesion to other cells and the surrounding extracellular matrix, in part, through the combination of integrins they express. In order to identify integrins that may mediate morphogenetic cell movements in the early Xenopus embryo, we have used polymerase chain reaction methods to isolate cDNAs encoding Xenopus integrin beta subunits. Based on deduced amino acid sequence, they are identified as homologs of human integrins beta 1, beta 2, beta 3 and beta 6. We also report the cloning and sequencing of cDNAs covering the complete coding region of Xenopus beta 3. Embryonic patterns of expression for these integrin beta subunit mRNAs have been examined both by RNase protection analysis and by whole mount in situ hybridization. In the early embryo the beta 1 subunit is encoded by a maternally transcribed mRNA expressed in all cells, but is most abundant in ectoderm and mesoderm. In contrast, Xenopus beta 3 mRNA is detected in the epidermis, bottle cells of the neural groove, and a subset of cells arising from the ventral blood islands. The beta 2 and beta 6 mRNAs are expressed at high levels in late tailbud stages, although very low levels of beta 6 are also detected in eggs and early embryos. These data provide evidence that multiple integrins are expressed at the earliest stages of vertebrate development coincident with the onset of morphogenesis.
Asunto(s)
Cadenas beta de Integrinas , Integrinas/genética , Xenopus laevis/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , Técnicas de Cultivo , ADN , Embrión no Mamífero/metabolismo , Humanos , Integrina beta1 , Integrina beta3 , Integrinas/biosíntesis , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Xenopus laevis/metabolismoRESUMEN
Partial cDNA clones encoding approximately the carboxy terminal half of Pleurodeles fibronectin (FN) were isolated. They account for 4.7 Kbp of the 3' region of the FN mRNA. The cDNA nucleotide sequence comprises all three alternatively spliced segments designated EIIIA, EIIIB and V-segment, respectively. All three segments are included in FN mRNA synthesized during early embryogenesis whereas, from the tailbud stage onward the V-region was partially excluded. The isolation of Pleurodeles cDNA clones including the three different spliced EIIIA, EIIIB and V segment raises new possibilities for the study of the precise role of specific regions of FN in early amphibian development.
Asunto(s)
ADN Complementario/genética , Fibronectinas/genética , Pleurodeles/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Desarrollo Embrionario y Fetal/genética , Expresión Génica , Variación Genética , Datos de Secuencia Molecular , Pleurodeles/embriología , Mapeo RestrictivoRESUMEN
Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, alpha 3, alpha 4 and alpha 6 transcripts begins during gastrulation. Integrin alpha 5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin beta 1 protein synthesis remains constant following induction of stage 8 animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990) Development 108, 289-298.). Here we report that integrin alpha 3, alpha 4 and alpha 6 mRNA levels increase following induction with 10 U/ml activin-A whereas alpha 5, beta 1 and beta 3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that alpha 3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, alpha 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, alpha 3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, alpha 3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.
Asunto(s)
Anfibios/embriología , Embrión no Mamífero/fisiología , Gástrula/fisiología , Expresión Génica/genética , Integrinas/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Animales , Hibridación in Situ , Datos de Secuencia Molecular , Sistema Nervioso/embriología , Reacción en Cadena de la Polimerasa , Xenopus laevisRESUMEN
A new member of the thrombospondin gene family, designated thrombospondin-4, has been identified in the Xenopus laevis genome. The predicted amino acid sequence indicates that the protein is similar to the other members of this gene family in the structure of the type 3 repeats and the COOH-terminal domain. Thrombospondin-4 contains four type 2 repeats and lacks the type 1 repeats that are found in thrombospondin-1 and 2. The amino-terminal domain of thrombospondin-4 has no significant homology with the other members of the thrombospondin gene family or with other proteins in the database. RNAse protection analysis establishes that the initial expression of Xenopus thrombospondin-4 is observed during neurulation. Levels of mRNA expression increase twofold during tailbud stages but decrease by the feeding tadpole stage. The size of the thrombospondin-4 message is 3.3 Kb and 3.4 Kb in the frog and human, respectively. Northern blot analysis of human tissues reveals high levels of thrombospondin-4 expression in heart and skeletal muscle, low levels in brain, lung and pancreas and undetectable levels in the placenta, liver and kidney. These data establish the existence of a new member of the thrombospondin gene family that may participate in the genesis and function of cardiac and skeletal muscle.