RESUMEN

In Xenopus gastrula stage embryos, four isoforms of Tcf1 (B, C, D and E) are present with high amino acid sequence conservation compared to fish, mice and human. We studied possible functional differences between these Tcf1 isoforms during early Xenopus development. After overexpression of single Tcf1 isoforms, two distinct phenotypes were observed. Overexpression of the B or D isoforms of Tcf1, which both lack a C-clamp, enhances early canonical Wnt signaling and induces ectopic dorsal mesoderm at the expense of ventrolateral mesoderm prior to gastrulation, causing severe antero-dorzalization of embryos. Overexpression of the E-isoform, which contains a complete C-clamp, does not induce ectopic dorsal mesoderm, but rather leads to severe caudal truncation. Overexpression of the C-isoform, which contains a partial C-clamp, induces a similar phenotype. Mutation of a single amino acid in the C-clamp, known to produce a hypomorphic mutant in D. melanogaster, led to a gain of function in inducing ectopic organizer tissue, as observed after overexpression of the B or D isoforms of Tcf1. Depletion of the C-clamp exon from the zygotic mRNA pool, by injection of a morpholino oligo that targets the splice acceptor site of the exon containing the C-clamp, caused a severe shortening of the AP-axis. Furthermore, embryos showed poor development of the CNS, paraxial mesoderm and primary blood vessels. In situ hybridization analysis showed that Lef1 expression was downregulated at the mid-hindbrain boundary, in the otic vesicles and the branchial arches. The results indicate that in post-gastrula stage Xenopus embryos, the E-tail of Tcf1 is required for expression of Lef1 and for blood vessel formation.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Neovascularización Fisiológica/fisiología , Isoformas de Proteínas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Animales , Gastrulación/fisiología , Factor Nuclear 1-alfa del Hepatocito/genética , Mesodermo/metabolismo , Isoformas de Proteínas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xenopus/metabolismo , Proteínas de Xenopus/genética

RESUMEN

Given its fundamental role in development and cancer, the Wnt-ß-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of ß-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathway.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas de Unión al ADN/metabolismo , Membrana Nuclear/metabolismo , Proteínas Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Mutación , Membrana Nuclear/genética , Proteínas Oncogénicas/genética , Factor de Transcripción 4 , Factores de Transcripción/genética , Transcripción Genética , Ubiquitina-Proteína Ligasas , Xenopus laevis , beta Catenina/genética

RESUMEN

TCF1 belongs to the family of LEF1/TCF transcription factors that regulate gene expression downstream of Wnt/ß-catenin signaling, which is crucial for embryonic development and is involved in adult stem cell regulation and tumor growth. In early Xenopus embryos, tcf1 plays an important role in mesoderm induction and patterning. Foxd3 emerged as a potential tcf1 target gene in a microarray analysis of gastrula stage embryos. Because foxd3 and tcf1 are coexpressed during gastrulation, we investigated whether foxd3 is regulated by tcf1. By using morpholino-mediated knockdown, we show that during gastrulation foxd3 expression is dependent on tcf1. By chromatin immunoprecipitation, we also demonstrate direct interaction of ß-catenin/tcf complexes with the foxd3 gene locus. Hence, our results indicate that tcf1 acts as an essential activator of foxd3, which is critical for dorsal mesoderm formation in early embryos.

Asunto(s)

Factores de Transcripción Forkhead/metabolismo , Gastrulación , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Factores de Transcripción Forkhead/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Factor Nuclear 1-alfa del Hepatocito/biosíntesis , Mesodermo/embriología , Morfolinos , Transducción de Señal/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Proteínas de Xenopus/biosíntesis , Xenopus laevis/genética , Xenopus laevis/metabolismo , beta Catenina/metabolismo

RESUMEN

Tcf/Lef HMG box transcription factors are nuclear effectors of the canonical Wnt signaling pathway, which function in cell fate specification. Lef1 is required for the development of tissues and organs that depend on epithelial mesenchymal interactions. Here, we report the effects of lef1 loss of function on early development in X. tropicalis. Depletion of lef1 affects gene expression already during gastrulation and results in abnormal differentiation of cells derived from ectoderm and mesoderm. At tail bud stages, the epidermis was devoid of ciliated cells and derivatives of the neural crest, e.g. melanocytes and cephalic ganglia were absent. In the Central Nervous System, nerve fibers were absent or underdeveloped. The development of the paraxial mesoderm was affected; intersomitic boundaries were not distinct and development of the hypaxial musculature was impaired. The development of the pronephros and pronephric ducts was disturbed. Most striking was the absence of blood flow in lef1 depleted embryos. Analysis of blood vessel marker genes demonstrated that lef1 is required for the development of the major blood vessels and the heart.

Asunto(s)

Ectodermo/embriología , Ectodermo/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Factores de Transcripción TCF/metabolismo , Xenopus/embriología , Xenopus/metabolismo , Animales , Tipificación del Cuerpo , Diferenciación Celular , Vasos Coronarios , Ectodermo/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Exones/genética , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Mesodermo/citología , Miocardio/metabolismo , Especificidad de Órganos , Fenotipo , Factores de Transcripción TCF/genética , Xenopus/genética

RESUMEN

Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays > 60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.

Asunto(s)

Conexinas/química , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , Conexina 26 , Conexina 30 , Conexinas/genética , Conexinas/metabolismo , Uniones Comunicantes , Regulación del Desarrollo de la Expresión Génica , Humanos , Mesodermo/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Proteínas de Xenopus/metabolismo , Proteína beta1 de Unión Comunicante

RESUMEN

Wnt proteins function as morphogens that can form long-range concentration gradients to pattern developing tissues. Here, we show that the retromer, a multiprotein complex involved in intracellular protein trafficking, is required for long-range signaling of the Caenorhabditis elegans Wnt ortholog EGL-20. The retromer functions in EGL-20-producing cells to allow the formation of an EGL-20 gradient along the anteroposterior axis. This function is evolutionarily conserved, because Wnt target gene expression is also impaired in the absence of the retromer complex in vertebrates. These results demonstrate that the ability of Wnt to regulate long-range patterning events is dependent on a critical and conserved function of the retromer complex within Wnt-producing cells.

Asunto(s)

Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Glicoproteínas/fisiología , Complejos Multiproteicos/fisiología , Transducción de Señal , Proteínas Wnt/fisiología , Animales , Tipificación del Cuerpo , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/genética , Línea Celular , Expresión Génica , Glicoproteínas/análisis , Glicoproteínas/genética , Humanos , Mutación , Neuronas/citología , Neuronas/fisiología , Interferencia de ARN , Transgenes , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiología , Xenopus

RESUMEN

In frogs such as Rana and Xenopus, metamorphosis does not occur in the absence of a functional thyroid gland. Previous studies indicated that coordinated development in frogs requires tissue and stage-dependent type II and type III iodothyronine deiodinase expression patterns to obtain requisite levels of intracellular T(3) in tissues at the appropriate stages of metamorphosis. No type I iodothyronine deiodinase (D1), defined as T(4) or reverse T(3) (rT3) outer-ring deiodinase (ORD) activity with Michaelis constant (K(m)) values in the micromolar range and sensitivity to 6-propyl-2-thiouracil (6-PTU), could be detected in tadpoles so far. We obtained a X. laevis D1 cDNA clone from brain tissue. The complete sequence of this clone (1.1 kb, including poly A tail) encodes an ORF of 252 amino acid residues with high homology to other vertebrate D1 enzymes. The core catalytic center includes a UGA-encoded selenocysteine residue, and the 3' untranslated region (about 300 nt) contains a selenocysteine insertion sequence element. Transfection of cells with an expression vector containing the full-length cDNA resulted in generation of significant deiodinase activity in the homogenates. The enzyme displayed ORD activity with T(4) (K(m) 0.5 microm) and rT3 (K(m) 0.5 microm) and inner-ring deiodinase activity with T(4) (K(m) 0.4 microm). Recombinant Xenopus D1 was essentially insensitive to inhibition by 6-PTU (IC(50) > 1 mm) but was sensitive to gold thioglucose (IC(50) 0.1 mum) and iodoacetate (IC(50) 10 microm). Because the residue 2 positions downstream from the selenocysteine is Pro in Xenopus D1 but Ser in all cloned PTU-sensitive D1 enzymes, we prepared the Pro132Ser mutant of Xenopus D1. The mutant enzyme showed strongly increased ORD activity with T(4) and rT3 (K(m) about 4 microm) and was highly sensitive to 6-PTU (IC(50) 2 microm). Little native D1 activity could be detected in Xenopus liver, kidney, brain, and gut, but significant D1 mRNA expression was observed in juvenile brain and adult liver and kidney. These results indicate the existence of a 6-PTU-insensitive D1 enzyme in X. laevis tissues, but its role during tadpole metamorphosis remains to be defined.

Asunto(s)

Yoduro Peroxidasa/química , Yoduro Peroxidasa/genética , Mutación , Prolina/química , Serina/química , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dominio Catalítico , Cinética , Datos de Secuencia Molecular , Propiltiouracilo/farmacología , Ratas , Selenocisteína/química , Homología de Secuencia de Aminoácido , Xenopus laevis

RESUMEN

The Wnt-signaling cascade is required for several crucial steps during early embryogenesis, and its activity is modulated by various agonists and antagonists to provide spatiotemporal-specific signaling. Naked cuticle is a Wnt antagonist that itself is induced by Wnt signaling to keep Wnt signaling in check. Little is known about the regulation of this antagonist. We have recently shown that the protein phosphatase 2A regulatory subunit PR72 is required for the inhibitory effect of Naked cuticle on Wnt signaling. In the present study, we show that PR130, which has an N terminus that differs from that of PR72 but shares the same C terminus, also interacts with Naked cuticle but instead functions as an activator of the Wnt-signaling pathway, both in cell culture and during development. We find that PR130 modulates Wnt signal transduction by restricting the ability of Naked cuticle to function as a Wnt inhibitor. Our data establish PR130 as a modulator of the Wnt-signaling pathway and suggest a mechanism of Wnt signal regulation in which the inhibitory activity of Naked cuticle is determined by the relative level of expression of two transcripts of the same protein phosphatase 2A regulatory subunit.

Asunto(s)

Proteínas Portadoras/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Línea Celular , Humanos , Hibridación in Situ , Proteína Fosfatasa 2 , Xenopus/embriología

RESUMEN

Wnt signaling pathways have essential roles in developing embryos and adult tissue, and alterations in their function are implicated in many disease processes including cancers. The major nuclear transducers of Wnt signals are the Tcf/LEF family of transcription factors, which have binding sites for both the transcriptional co-repressor groucho, and the co-activator beta-catenin. The early Xenopus embryo expresses three maternally inherited Tcf/LEF mRNAs, and their relative roles in regulating the expression of Wnt target genes are not understood. We have addressed this by using antisense oligonucleotides to deplete maternal XTcf1 and XTcf4 mRNAs in oocytes. We find that XTcf1 represses expression of Wnt target genes ventrally and laterally, and activates their expression dorsally. Double depletions of XTcf1 and XTcf3 suggest that they act cooperatively to repress Wnt target genes ventrally. In contrast, XTcf4 has no repressive role but is required to activate expression of Xnr3 and chordin in organizer cells at the gastrula stage. This work provides evidence for distinct roles for XTcfs in regulating Wnt target gene expression.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor 1 de Transcripción de Linfocitos T/metabolismo , Factores de Transcripción TCF/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Embrión no Mamífero/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor 1 de Transcripción de Linfocitos T/genética , Factores de Transcripción TCF/genética , Proteína 1 Similar al Factor de Transcripción 7 , Proteína 2 Similar al Factor de Transcripción 7 , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/genética , Xenopus/embriología , Xenopus/metabolismo , Proteínas de Xenopus/genética

RESUMEN

Tcf/Lef transcription factors and beta-catenin mediate canonical Wnt signalling, which plays remarkably diverse roles in embryonic development, stem cell renewal and cancer progression. To investigate the molecular mechanisms allowing for these diverse yet specific functions, we studied the several distinct roles for Wnt/beta-catenin signalling in early Xenopus development: establishing the dorsal body axis; regulating mesoderm induction; and subsequent ventrolateral patterning. Our previous experiments and the expression patterns of Tcf/Lef factors during these embryonic stages led us to examine whether different Tcf/Lef factors mediate these distinct events downstream of canonical Wnt/beta-catenin signalling. By manipulating gene expression with morpholino-driven gene knockdown and capped RNA-mediated rescue, we show that genes encoding different Tcf/Lef transcription factors mediate distinct responses to Wnt signalling in early Xenopus development: Tcf1 and Tcf3 genes are non-redundantly required in mesoderm induction for mediating primarily transcriptional activation and repression, respectively; while ventrolateral patterning requires both Tcf1 and Lef1 genes to express sufficient levels of transcription-activating Tcf factors. Our investigation further identifies that motifs within their central domain, rather than their C-terminus, determine the particular molecular function of Tcf/Lef factors. These findings suggest that Tcf/Lef genes encode factors of different activities, which function together in antagonistic or synergistic ways to modulate the intensity and outcome of Wnt/beta-catenin signalling and to trigger tissue-specific responses.

Asunto(s)

Factor Nuclear 1-alfa del Hepatocito/fisiología , Factor de Unión 1 al Potenciador Linfoide/fisiología , Mesodermo/fisiología , Factores de Transcripción TCF/fisiología , Proteínas Wnt/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/fisiología , beta Catenina/fisiología , Secuencias de Aminoácidos , Animales , Tipificación del Cuerpo , Embrión no Mamífero/metabolismo , Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/genética , Factor de Unión 1 al Potenciador Linfoide/genética , Transducción de Señal , Factores de Transcripción TCF/genética , Proteína 1 Similar al Factor de Transcripción 7 , Proteínas de Xenopus/genética , Xenopus laevis/embriología

RESUMEN

BACKGROUND & AIMS: In the intestine, the canonical Wnt signaling cascade plays a crucial role in driving the proliferation of epithelial cells. Furthermore, aberrant activation of Wnt signaling is strongly associated with the development of colorectal cancer. Despite this evidence, little is known about the precise identity and localization of Wnts and their downstream effectors in the adult intestine. To address this issue, we examined the expression pattern of all Wnts, Frizzleds (Fzs), low-density lipoprotein receptor-related proteins, Wnt antagonists, and T-cell factors in the murine small intestine and colon and adenomas. METHODS: Embryonic, postnatal, and adult intestinal samples were subjected to in situ hybridization by using specific RNA probes for the various genes tested. RESULTS: Our analysis showed high expression of several signaling components (including Wnt-3, Wnt-6, Wnt-9b, Frizzled 4, Frizzled 6, Frizzled 7, low-density lipoprotein receptor-related protein 5, and secreted Frizzled-related protein 5) in crypt epithelial cells. We also detected Wnt-2b, Wnt-4, Wnt-5a, Wnt-5b, Frizzled 4, and Frizzled 6 in differentiated epithelial and mesenchymal cells of the small intestine and colon. Finally, several factors (Frizzled 4, T-cell factor 1, lymphoid enhancer factor, Dickkopf 2, Dickkopf 3, and Wnt-interacting factor) displayed differential expression in normal vs neoplastic tissue. CONCLUSIONS: Our study predicts a much broader role for Wnt signaling in gut development and homeostasis than was previously anticipated from available genetic studies and identifies novel factors likely involved in promoting canonical and noncanonical Wnt signals in the intestine.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Intestino Grueso/patología , Intestino Delgado/patología , Proteínas de Pez Cebra/genética , Adulto , Factores de Edad , Biopsia con Aguja , Diferenciación Celular , Femenino , Feto/patología , Humanos , Inmunohistoquímica , Hibridación in Situ , Recién Nacido , Péptidos y Proteínas de Señalización Intercelular/análisis , Intestino Grueso/embriología , Intestino Delgado/embriología , Masculino , Sensibilidad y Especificidad , Transducción de Señal , Técnicas de Cultivo de Tejidos , Proteínas Wnt

RESUMEN

The Fxr gene family is composed of three members, FMR1, FXR1 and FXR2. The FMR1 gene is involved in the fragile X syndrome, whereas for the other two members, no human disorder has been identified yet. An appropriate animal model to study in vivo gene function is essential to unravel the cellular function of the gene products FMRP, FXR1P and FXR2P, respectively. In Xenopus tropicalis both Fmr1 and Fxr1 were identified; however, unexpectedly Fxr2 was not. Here we describe the characterization of both Fmrp and Fxr1p in Xenopus tropicalis. Fmrp is expressed ubiquitously throughout the embryo during embryonic development, whereas Fxr1p shows a more tissue-specific expression particularly during late embryonic development. In adult frogs both proteins are highly expressed in most neurons of the central nervous system and in all spermatogenic cells in the testis. In addition, Fxr1p is also highly expressed in striated muscle tissue. Western blotting experiments revealed only one prominent isoform for both proteins using different tissue homogenates from adult frogs. Thus, for in vivo gene function studies, this relative simple animal model may serve as a highly advantageous and complementary model.

Asunto(s)

Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Discapacidad Intelectual/genética , Proteínas de Unión al ARN/genética , Proteínas de Xenopus/metabolismo , Xenopus/genética , Secuencia de Aminoácidos , Animales , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/química , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Modelos Animales , Datos de Secuencia Molecular , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia de Aminoácido , Proteínas de Xenopus/química , Proteínas de Xenopus/genética

RESUMEN

Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (approximately 62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals.

Asunto(s)

Conexinas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Somitos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Envejecimiento/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Biología Computacional , Conexinas/química , Conexinas/genética , Electrofisiología , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Filogenia , Alineación de Secuencia , Somitos/química , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrollo

RESUMEN

The Wnt signaling cascade is a central regulator of cell fate determination during embryonic development, whose deregulation contributes to oncogenesis. Naked cuticle is the first Wnt-induced antagonist found in this pathway, establishing a negative-feedback loop that limits the Wnt signal required for early segmentation. In addition, Naked cuticle is proposed to function as a switch, acting to restrict classical Wnt signaling and to activate a second Wnt signaling pathway that controls planar cell polarity during gastrulation movements in vertebrates. Little is known about the biochemical function of Naked cuticle or its regulation. Here we report that PR72, a Protein Phosphatase type 2A regulatory subunit of unknown function, interacts both physically and functionally with Naked cuticle. We show that PR72, like Naked cuticle, acts as a negative regulator of the classical Wnt signaling cascade, establishing PR72 as a novel regulator of the Wnt signaling pathway. Our data provide evidence that the inhibitory effect of Naked cuticle on Wnt signaling depends on the presence of PR72, both in mammalian cell culture and in Xenopus embryos. Moreover, PR72 is required during early embryonic development to regulate cell morphogenetic movements during body axis formation.

Asunto(s)

Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Transducción de Señal/fisiología , Proteínas de Xenopus/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio , Clonación Molecular , Proteínas del Citoesqueleto/metabolismo , Proteínas Dishevelled , Embrión no Mamífero/metabolismo , Ojo/embriología , Ojo/metabolismo , Gástrula/metabolismo , Humanos , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2 , Transactivadores/metabolismo , Proteínas Wnt , Xenopus , beta Catenina

RESUMEN

XTcf-3 functions as a transcriptional regulator in the canonical Wnt signaling cascade and can repress or activate downstream target genes. Expression of XTcf-3 is differentially regulated in time and place during development (Molenaar et al. [1998] Mech Dev. 75:151-154), but little is known about the mechanisms that control transcriptional activation and repression. A 15-kb genomic fragment of Tcf-3 sequences from Xenopus tropicalis was cloned, including the 5' untranslated region; exons 1, 2, and 3; and intron sequences. We used 5' deletion constructs for transgenesis and episomal luciferase assays in Xenopus to examine temporal and spatial regulation of the promoter during early development. A -3054/+34-bp Tcf-3 upstream region was identified that drives a green fluorescent protein (GFP) reporter transgene in a pattern similar to endogenous expression of XtTcf-3 from gastrula to tail bud stages. At stage 12, expression of the reporter is restricted to the middle and posterior neurectoderm. At stage 22, expression is strongest in the neural plate, the eye anlagen and branchial arches. At stage 35/36, expression is found in the head mesenchyme, the branchial arches, the heart, the mesencephalon, eyes, otic vesicles, notochord, somites and the lateral plate mesoderm. Part of the cis-acting elements driving this GFP reporter transgene expression map between -372 and -95 bp of the transcription start site. Furthermore, two TCF/LEF sites are necessary for full activity of the promoter during gastrula stages in episomal luciferase assays.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Regiones Promotoras Genéticas , Transactivadores , Factores de Transcripción/metabolismo , Xenopus/embriología , Regiones no Traducidas 5' , Animales , Animales Modificados Genéticamente , Mapeo Cromosómico , Clonación Molecular , Análisis Mutacional de ADN , Embrión no Mamífero , Desarrollo Embrionario , Exones , Gástrula , Eliminación de Gen , Genes Reporteros , Genoma , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Intrones , Luciferasas/metabolismo , Oocitos/metabolismo , Factores de Tiempo , Distribución Tisular/genética , Factores de Transcripción/genética , Transgenes , Xenopus/genética , Xenopus/metabolismo

RESUMEN

Wnt-1 belongs to the Wnt family of secreted glycoproteins inducing an intracellular signaling pathway involved in cell proliferation, differentiation, and pattern formation. The canonical branch is one of three known branches. This is also valid in vitro, and Wnts can be considered beneficial for culturing primary cells from organs, provided Wnts are available and applicable even with cells of different species. It was shown here that internally c-myc-tagged murine Wnt-1 produced in the heterologous host Escherichia coli was appropriate for inducing intracellular signaling of the canonical Wnt pathway in eukaryotic cells via stabilization of cytosolic beta-catenin. The pioneering injection of the protein into the blastocoels of Xenopus laevis embryos led to axis duplication and suppression of head formation. Applying the recombinant murine Wnt-1 to metanephric mesenchyme activated the tubulogenic program. The signal-inducing activity of the recombinant protein was also positively demonstrated in the TOP-flash reporter assay. Although Wnts were purified recently from the growth media of stably transfected eukaryotic cell lines, the production of active Wnt proteins in pro- or eukaryotic microorganisms reportedly has never been successful. Here soluble production in E. coli and translocation into the oxidizing environment of the periplasm were achieved. The protein was purified using the internal c-myc tag. The effect on the eukaryotic cells implies that activity was retained. Thus, this approach could make recombinant murine Wnt-1 available as a good starting point for other Wnts needed, for example, for maintaining and differentiating stem cells, organ restoration therapy, and tissue engineering.

Asunto(s)

Escherichia coli/metabolismo , Células Eucariotas/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Embrión no Mamífero , Escherichia coli/genética , Genes Reporteros , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Morfogénesis/fisiología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Wnt , Proteína Wnt1 , Proteínas de Xenopus , Xenopus laevis/fisiología

RESUMEN

We report the cloning and expression of Xenopus Tcf-1. The amino acid sequence of Tcf-1 of Xenopus laevis and Xenopus tropicalis is closely related to that of chicken, mouse and man. Thus, the family of Tcf/Lef proteins in the amphibian Xenopus comprises four members as in higher vertebrates. RT-PCR analysis revealed that Tcf-1 RNA encoding a beta-catenin binding isoform is maternally present as well as throughout early development. Different transcripts are expressed by alternative splicing. In cleavage and blastula stage embryos, Tcf-1 RNA is present at high levels in the animal hemisphere. During gastrulation Tcf-1 is differentially expressed with high levels in the animal cap and most of the marginal zone except for a narrow domain around the blastopore. At neurula stages expression is predominant in the neural plate. At tailbud stages expression is localized in specific areas of the brain, in the eyes, the otic vesicle, branchial arches and head mesenchyme, somites, tailbud, pronephros and pronephric duct.

Asunto(s)

Proteínas de Unión al ADN/genética , Embrión no Mamífero/metabolismo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Factor de Unión 1 al Potenciador Linfoide , Datos de Secuencia Molecular , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor 1 de Transcripción de Linfocitos T , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Xenopus , Proteínas de Xenopus , beta Catenina

RESUMEN

Wnt signaling functions repeatedly during embryonic development to induce different but specific responses. What molecular mechanisms ensure that Wnt signaling triggers the correct tissue-specific response in different tissues? Early Xenopus development is an ideal model for addressing this fundamental question, since there is a dramatic change in the response to Wnt signaling at the onset of zygotic gene transcription: Wnt signaling components encoded by maternal mRNA establish the dorsal embryonic axis; zygotically expressed Xwnt-8 causes almost the opposite, by promoting ventral and lateral and restricting dorsal mesodermal development. Although Wnt signaling can function through different signal transduction cascades, the same beta-catenin-dependent, canonical Wnt signal transduction pathway mediates Wnt signaling at both stages of Xenopus development. Here we show that, while the function of the transcription factor XTcf-3 is required for early Wnt signaling to establish the dorsal embryonic axis, closely related XLef-1 is required for Wnt signaling to pattern the mesoderm after the onset of zygotic transcription. Our results show for the first time that different transcription factors of the Lef/Tcf family function in different tissues to bring about tissue-specific responses downstream of canonical Wnt signaling.

Asunto(s)

Tipificación del Cuerpo/fisiología , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/metabolismo , Morfogénesis/fisiología , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas de Pez Cebra , Animales , Embrión no Mamífero/fisiología , Femenino , Impresión Genómica , Factor de Unión 1 al Potenciador Linfoide , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , ARN Mensajero/genética , Factores de Transcripción TCF , Factor de Transcripción 3 , Proteína 1 Similar al Factor de Transcripción 7 , Proteínas Wnt , Proteínas de Xenopus , Xenopus laevis , Cigoto/fisiología

RESUMEN

In the early Xenopus embryo, the dorsal axis is specified by a Wnt signal transduction pathway, involving the movement of beta-catenin into dorsal cell nuclei and its functional association with the LEF-type transcription factor XTcf3. The subsequent function of XTcf3 is uncertain. Overexpression data has suggested that it can be both an activator and repressor of downstream genes. XTcf3 mRNA is synthesized during oogenesis in Xenopus and is stored in the egg. To identify its role in dorsal axis specification, we depleted this maternal store in full-grown oocytes using antisense deoxyoligonucleotides, and fertilized them. The developmental effects of XTcf3 depletion, both on morphogenesis and the expression of marker genes, show that primarily, XTcf3 is an inhibitor, preventing both dorsal and ventral cells of the late blastula from expressing dorsal genes. We also show that simple relief from the repression is not the only factor required for dorsal gene expression. To demonstrate this, we fertilized eggs that had been depleted of both XTcf3 and the maternal transcription factor VegT. Dorsal genes normally repressed by XTcf3 are not activated in these embryos. These data show that normal dorsal gene expression in the embryo requires the transcriptional activator VegT, whilst XTcf3 prevents their inappropriate expression on the ventral side of the embryo.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/fisiología , Organizadores Embrionarios , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Proteínas de Xenopus , Animales , Proteína Axina , Blastocisto/fisiología , Tipificación del Cuerpo/fisiología , Proteínas del Citoesqueleto/metabolismo , Femenino , Proteína Goosecoide , Proteínas HMGB/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ligandos de Señalización Nodal , Oocitos/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/fisiología , Factores de Transcripción TCF , Transactivadores/metabolismo , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismo , Xenopus , beta Catenina

RESUMEN

Xenopus laevis type XVIII collagen occurs in three variants, 22 + 1285 amino acid residues (signal peptide + mature protein), 23 + 1581 residues and 23 + 1886 residues in length, differing in their N-terminal non-collagenous domains. The region showing highest homology to mammalian counterparts is the C-terminal endostatin domain. All three variants are expressed, at different levels, during early and late stages of development, as demonstrated by reverse transcription-polymerase chain reaction. Whole-mount in situ hybridization shows that the short variant is expressed at high levels in the developing eye, the central nervous system, the otic vesicle, the head mesenchyme, the branchial arches and the pronephros, and at the boundaries between somites. The middle variant is expressed in the head mesenchyme, the branchial arches, the peripheral nervous system, the pronephros and the pronephric duct, and at the somite boundaries. The longest variant is weakly expressed in the head mesenchyme and branchial arches.

Asunto(s)

Colágeno/genética , Regulación del Desarrollo de la Expresión Génica , Xenopus laevis/embriología , Empalme Alternativo , Animales , Clonación Molecular , Colágeno Tipo XVIII , ADN Complementario/metabolismo , Hibridación in Situ , Modelos Genéticos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa