RESUMEN

Organoids are carrying the promise of modeling complex disease phenotypes and serving as a powerful basis for unbiased drug screens, potentially offering a more efficient drug-discovery route. However, unsolved technical bottlenecks of reproducibility and scalability have prevented the use of current organoids for high-throughput screening. Here, we present a method that overcomes these limitations by using deep-learning-driven analysis for phenotypic drug screens based on highly standardized micropattern-based neural organoids. This allows us to distinguish between disease and wild-type phenotypes in complex tissues with extremely high accuracy as well as quantify two predictors of drug success: efficacy and adverse effects. We applied our approach to Huntington's disease (HD) and discovered that bromodomain inhibitors revert complex phenotypes induced by the HD mutation. This work demonstrates the power of combining machine learning with phenotypic drug screening and its successful application to reveal a potentially new druggable target for HD.

Asunto(s)

Aprendizaje Profundo , Enfermedad de Huntington , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Ensayos Analíticos de Alto Rendimiento , Evaluación Preclínica de Medicamentos , Reproducibilidad de los Resultados , Organoides

RESUMEN

The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP's potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and nonneuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington's Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP's transcriptional activity can partially rescue the HD phenotype.

Asunto(s)

Ectodermo , Enfermedad de Huntington , Proteínas Señalizadoras YAP , Proteínas de Ciclo Celular/metabolismo , Ectodermo/metabolismo , Humanos , Neurogénesis , Neurulación , Transducción de Señal/genética , Proteínas Señalizadoras YAP/genética

RESUMEN

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the huntingtin gene (HTT). Although HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced activin signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFß signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of activin response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the 2D gastruloid, leading to ectopic subcellular localization of TGFß receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first 2 weeks of human development, as modeled by our 2D gastruloids.

Asunto(s)

Linaje de la Célula , Polaridad Celular , Estratos Germinativos/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Proteína Huntingtina/metabolismo , Activinas/metabolismo , Animales , Línea Celular , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Estratos Germinativos/citología , Estratos Germinativos/embriología , Células Madre Embrionarias Humanas/citología , Humanos , Proteína Huntingtina/genética , Ratones , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Expansión de Repetición de Trinucleótido

RESUMEN

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington's disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening.

Asunto(s)

Ectodermo/fisiología , Células Madre Embrionarias/fisiología , Enfermedad de Huntington , Neurulación/fisiología , Telencéfalo/crecimiento & desarrollo , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Línea Celular , Humanos , Neurogénesis , Telencéfalo/fisiología

RESUMEN

Apolipoprotein CI (ApoCI) belongs to the Apolipoprotein superfamily, members of which are involved in lipid transport, uptake and homeostasis. Excessive ApoCI has been implicated in atherosclerosis and Alzheimer's disease in humans. In this study we report the isolation of Xenopus laevis apoCI and describe the expression pattern of this gene during early development, using reverse transcription polymerase chain reaction and whole mount in situ hybridization. Xenopus apoCI is enriched in the dorsal ectoderm during gastrulation, and is subsequently expressed in sensory placodes, neural tube and cranial neural crest. These data suggest as yet uncharacterized roles for ApoCI during early vertebrate embryogenesis.

Asunto(s)

Apolipoproteína C-I/genética , Animales , Clonación Molecular , Gastrulación , Hibridación in Situ , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Xenopus laevis/embriología

RESUMEN

Huntington's Disease (HD) is a neurodegenerative disorder that results from the abnormal expansion of poly-glutamine (polyQ) repeats in the Huntingtin (HTT) gene. Although HTT has been linked to a variety of cellular events, it is still not clear what the physiological functions of the protein are. Because of its critical role during mouse embryonic mouse development, we investigated the functions of Htt during early Xenopus embryogenesis. We find that reduction of Htt levels affects cilia polarity and function and causes whole body paralysis. Moreover, Htt loss of function leads to abnormal development of trigeminal and motor neurons. Interestingly, these phenotypes are partially rescued by either wild-type or expanded HTT. These results show that the Htt activity is required for normal embryonic development, and highlight the usefulness of the Xenopus system for investigating proteins involved in human diseases.

Asunto(s)

Cilios/fisiología , Proteínas del Tejido Nervioso/fisiología , Neurogénesis/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Polaridad Celular/genética , Polaridad Celular/fisiología , Cilios/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteína Huntingtina , Ratones , Modelos Animales , Modelos Neurológicos , Datos de Secuencia Molecular , Morfolinos/genética , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/fisiología

RESUMEN

The TGFß signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFß ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFß1 signaling. Coco synergizes with TGFß1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFß1 and enhances TGFß1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFß signaling.

Asunto(s)

Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animales , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Luciferasas , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Huntington's disease (HD) is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT). HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel HTT mRNA splice isoforms that are expressed in normal and HTT-expanded human embryonic stem cell (hESC) lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease.

Asunto(s)

Exones , Enfermedad de Huntington , Proteínas del Tejido Nervioso , Animales , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad de la Especie

RESUMEN

The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of ß-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to ß-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of ß-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of ß-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to ß-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the ß-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of ß-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.

Asunto(s)

Factor de Transcripción Activador 2/genética , Carcinogénesis/genética , Factor Nuclear 1-alfa del Hepatocito/genética , Neoplasias/genética , beta Catenina/genética , Factor de Transcripción Activador 2/metabolismo , Animales , Línea Celular Tumoral , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Humanos , Neoplasias/patología , Regiones Promotoras Genéticas , Transducción de Señal , Activación Transcripcional/genética , Vía de Señalización Wnt/genética , Xenopus laevis

RESUMEN

The Exon Junction Complex (EJC) plays a critical role in multiple posttranscriptional events, including RNA subcellular localization, nonsense-mediated decay (NMD), and translation. We previously reported that knockdown of the EJC core component Eukaryotic initiation factor 4a3 (Eif4a3) results in full-body paralysis of embryos of the frog, Xenopus laevis. Here, we explore the cellular and molecular mechanisms underlying this phenotype. We find that cultured muscle cells derived from Eif4a3 morphants do not contract, and fail to undergo calcium-dependent calcium release in response to electrical stimulation or treatment with caffeine. We show that ryr (ryanodine receptor) transcripts are incorrectly spliced in Eif4a3 morphants, and demonstrate that inhibition of Xenopus Ryr function similarly results in embryonic paralysis. These results suggest that the EJC mediates muscle cell function via regulation of pre-mRNA splicing during early vertebrate embryogenesis.

Asunto(s)

Factor 4A Eucariótico de Iniciación/genética , Precursores del ARN/metabolismo , Empalme del ARN , Canal Liberador de Calcio Receptor de Rianodina/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animales , Factor 4A Eucariótico de Iniciación/metabolismo , Exones , Degradación de ARNm Mediada por Codón sin Sentido/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo

RESUMEN

BACKGROUND: Xenopus embryonic skin is composed of the superficial layer with defined apicobasal polarity and the inner layer lacking the apical domain. Multiciliated cells (MCCs) originate in the inner layer of the epidermal ectoderm and subsequently migrate to the surface. How MCCs acquire the apicobasal polarity and intercalate into the superficial layer during neurulation is largely unknown. As Rab11-dependent vesicle trafficking has been implicated in ciliary membrane assembly and in apical domain formation in epithelial cells, we assessed the involvement of Rab11 in MCC development. RESULTS: Here we report that Rab11 is specifically enriched and becomes apically polarized in skin MCCs. Interference with Rab11 function by overexpression of a dominant negative mutant or injection of a specific morpholino oligonucleotide inhibited MCC intercalation into the superficial layer. Dominant negative Rab11-expressing MCC precursors revealed intrinsic apicobasal polarity, characterized by the apical domain, which is not normally observed in inner layer cells. Despite the presence of the apical domain, the cells with inhibited Rab11 function were randomly oriented relative to the plane of the tissue, thereby demonstrating a defect in planar polarity. CONCLUSIONS: These results establish a requirement for Rab11 in MCC development and support a two-step model, in which the initial polarization of MCC precursors is critical for their integration into the superficial cell layer.

Asunto(s)

Movimiento Celular/genética , Polaridad Celular/genética , Cilios/genética , Epidermis/embriología , Xenopus laevis/embriología , Proteínas de Unión al GTP rab/fisiología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Cilios/metabolismo , Cilios/fisiología , Embrión no Mamífero , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Células Epidérmicas , Epidermis/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Modelos Biológicos , Xenopus laevis/genética , Xenopus laevis/metabolismo , Xenopus laevis/fisiología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo

RESUMEN

Specification of the dorsoventral (DV) axis is critical for the subsequent differentiation of regional fate in the primary germ layers of the vertebrate embryo. We have identified a novel factor that is essential for dorsal development in embryos of the frog Xenopus laevis. Misexpression of Xenopus mab21-like 3 (Xmab21l3) dorsalizes gastrula-stage mesoderm and neurula-stage ectoderm, while morpholino-mediated knockdown of Xmab21l3 inhibits dorsal differentiation of these embryonic germ layers. Xmab21l3 is a member of a chordate-specific subclass of a recently characterized gene family, all members of which contain a conserved, but as yet ill-defined, Mab21 domain. Our studies suggest that Xmab21l3 functions to repress ventralizing activity in the early vertebrate embryo, via regulation of BMP/Smad and Ras/ERK signaling.

Asunto(s)

Tipificación del Cuerpo/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genética , Animales , Proteína Morfogenética Ósea 2/farmacología , Ectodermo/efectos de los fármacos , Ectodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Filogenia , Regiones Promotoras Genéticas/genética , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Proteínas de Xenopus/genética

RESUMEN

The establishment and maintenance of cellular identity are ultimately dependent upon the accurate regulation of gene expression, the process by which genetic information is used to synthesize functional gene products. The post-transcriptional, pre-translational regulation of RNA constitutes RNA processing, which plays a prominent role in the modulation of gene expression in differentiated animal cells. The multi-protein Exon Junction Complex (EJC) serves as a critical signaling hub within the network that underlies many RNA processing events. Here, we identify a requirement for the EJC during early vertebrate embryogenesis. Knockdown of the EJC component Eukaryotic initiation factor 4a3 (Eif4a3) in embryos of the frog Xenopus laevis results in full-body paralysis, with defects in sensory neuron, pigment cell, and cardiac development; similar phenotypes are seen following knockdown of other "core" EJC protein constituents. Our studies point to an essential role for the EJC in the development of neural plate border derivatives.

Asunto(s)

Desarrollo Embrionario/fisiología , Factor 4A Eucariótico de Iniciación/fisiología , Vertebrados/embriología , Animales , Desarrollo Embrionario/genética , Factor 4A Eucariótico de Iniciación/genética , Exones/genética , Inmunohistoquímica , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Procesamiento Postranscripcional del ARN/genética , Vertebrados/genética , Vertebrados/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genética , Xenopus laevis/metabolismo

RESUMEN

In the frog, Xenopus laevis, fibroblast growth factor (FGF) signaling is required for both mesoderm formation and the morphogenetic movements that drive the elongation of the notochord, a dorsal mesodermal derivative; the coordination of these distinct roles is mediated by the Xenopus Ctr1 (Xctr1) protein: maternal Xctr1 is required for mesodermal differentiation, while the subsequent loss of Xctr1 promotes morphogenesis. The signaling cascade activated by FGF in the presence of Ctr1 has been well characterized; however, the Xctr1-independent, FGF-responsive network remains poorly defined. We have identified Xenopus Marginal Coil (Xmc) as a gene whose expression is highly enriched following Xctr1 knockdown. Zygotic initiation of Xmc expression in vivo coincides with a decrease in maternal Xctr1 transcripts; moreover, Xmc loss-of-function inhibits Xctr1 knockdown-mediated elongation of FGF-treated animal cap explants, implicating Xmc as a key effector of Xctr1-independent gastrular morphogenesis.

Asunto(s)

Proteínas de Transporte de Catión/fisiología , Proteínas de la Membrana/fisiología , Morfogénesis/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Xenopus laevis/genética , Animales , Proteínas de Transporte de Catión/genética , Transportador de Cobre 1 , Proteínas de la Membrana/genética , Morfogénesis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Xenopus/genética

RESUMEN

Embryogenesis involves two distinct processes. On the one hand, cells must specialize, acquiring fates appropriate to their positions (differentiation); on the other hand, they must physically construct the embryo through coordinated mechanical activity (morphogenesis). In early vertebrate development, fibroblast growth factor (FGF) regulates multiple embryonic events, including germ layer differentiation and morphogenesis; the cellular components that direct FGF signaling to evoke these different responses remain largely unknown. We show here that the copper transporter 1 (Ctr1) protein is a critical router of FGF signals during early embryogenesis. Ctr1 both promotes the differentiation and inhibits the morphogenesis of mesoderm and neurectoderm in embryos of the frog Xenopus laevis, thereby coordinating normal development. Signal sorting by Ctr1 involves the activation of the Ras-MAP kinase cascade and appears to be independent of its role in copper transport. Mouse embryonic stem (ES) cells deficient for Ctr1 (Ctr1(-/-)) retain characteristics of pluripotency under conditions that favor differentiation in wild-type ES cells, indicating a conserved role for Ctr1 during amphibian and mammalian cell fate determination. Our studies support a model in which vertebrate Ctr1 functions as a key regulator of the differentiation capacity of both stem and progenitor cell populations.

Asunto(s)

Proteínas de Transporte de Catión/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Madre Embrionarias/citología , Morfogénesis , Xenopus laevis/embriología , Animales , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Transportador de Cobre 1 , Embrión no Mamífero/citología , Regulación del Desarrollo de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Xenopus , Xenopus laevis/genética

RESUMEN

The molecular basis of vertebrate germ layer formation has been the focus of intense scrutiny for decades, and the inductive interactions underlying this process are well defined. Only recently, however, have studies demonstrated that the regulated inhibition of ectopic germ layer formation is also crucial for patterning the early vertebrate embryo. We report here the characterization of Xema (Xenopus Ectodermally-expressed Mesendoderm Antagonist), a novel member of the Foxi-subclass of winged-helix transcription factors that is involved in the suppression of ectopic germ layer formation in the frog, Xenopus laevis. Xema transcripts are restricted to the animal pole ectoderm during early Xenopus development. Ectopic expression of Xema RNA inhibits mesoderm induction, both by growth factors and in the marginal zone, in vivo. Conversely, introduction of antisense morpholino oligonucleotides directed against the Xema transcript stimulates the expression of a broad range of mesodermal and endodermal marker genes in the animal pole. Our studies demonstrate that Xema is both necessary and sufficient for the inhibition of ectopic mesendoderm in the cells of the presumptive ectoderm, and support a model in which Fox proteins function in part to restrict inappropriate germ layer development throughout the vertebrate embryo.

Asunto(s)

Ectodermo/metabolismo , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Mesodermo/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Ectodermo/citología , Ectodermo/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Factores de Transcripción Forkhead , Gástrula/citología , Gástrula/efectos de los fármacos , Mesodermo/citología , Mesodermo/efectos de los fármacos , Datos de Secuencia Molecular , Alineación de Secuencia , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/química , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/deficiencia , Proteínas de Xenopus/genética , Xenopus laevis/genética

RESUMEN

The winged-helix transcription factor HNF3beta/FoxA2 is expressed in embryonic organizing centers of the gastrulating mouse, frog, fish, and chick. In the mouse, HNF3beta is required for the formation of the mammalian node and notochord, and can induce ectopic floor plate formation when misexpressed in the developing neural tube; HNF3beta expression in the extraembryonic endoderm is also necessary for the proper morphogenesis of the mammalian primitive streak. In the frog Xenopus laevis, several lines of evidence suggest that the related winged-helix factor Pintallavis functions as the ortholog of mammalian HNF3beta in both axial mesoderm and neurectoderm; the role of Xenopus HNF3beta itself, however, has not been clearly defined, and is the subject of this study. HNF3beta is widely expressed in the vegetal pole but, as previously suggested, is excluded from the gastrula-stage mesoderm. We find that expression of an HNF3beta-Engrailed repressor fusion protein induces ectopic axes and inhibits head formation in Xenopus embryos, while ectopic HNF3beta inhibits mesoderm and anterior endoderm formation in explant assays and in vivo. Our studies suggest that HNF3beta target genes function to limit the extent of mesoderm formation in the Xenopus gastrula, and point to related roles for Xenopus HNF3beta and the extraembryonic component of mammalian HNF3beta during vertebrate gastrulation.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Gástrula/fisiología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción , Xenopus laevis/embriología , Animales , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Silenciador del Gen , Factor Nuclear 3-beta del Hepatocito , Inmunohistoquímica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Xenopus laevis/fisiología

RESUMEN

Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. We investigate the nature of Fgf response element (FRE) in a posterior neural gene, Xcad3 (Xenopus caudal homologue) that plays a crucial role of posterior neural development. We provide evidence that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s).

Asunto(s)

Proteínas Fetales/metabolismo , Factores de Crecimiento de Fibroblastos/fisiología , Elementos de Respuesta/fisiología , Transducción de Señal/fisiología , Proteínas de Xenopus , Animales , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Proteínas HMGB/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Factores de Transcripción SOXB1 , Médula Espinal/embriología , Médula Espinal/metabolismo , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/metabolismo , Xenopus

RESUMEN

Members of the Src family of non-receptor tyrosine kinases play a critical role in mesoderm formation in the frog, Xenopus laevis, acting as required mediators downstream of the fibroblast growth factor receptor. At least four members of this gene family, Src, Fyn, Yes, and Laloo, are expressed during early embryonic development. Ectopic expression of Laloo and Fyn, but not Src, induce mesoderm in ectodermal explants, indicating that these factors are non-redundant during early vertebrate development. Here we investigate the basis for the differential activity of the Src and Laloo kinases during mesoderm formation. We demonstrate that although both Src and Laloo physically interact with the substrate protein SNT-1/FRS2alpha only Laloo phosphorylates SNT-1, an event previously shown to be required for the activity of the latter and for mesoderm induction in vivo. We show that Src is enzymatically capable of stimulating mesoderm formation, as an activated Src construct both phosphorylates SNT-1 and induces mesoderm in explant cultures. However, a chimeric Laloo construct containing a Src C-terminal tail is inactive, suggesting that the early embryo contains a specific Laloo-activating, or Src-inactivating, factor. Finally, through further chimeric analysis, we provide evidence to suggest that differences in Laloo and Src activity are also mediated by the SH2, SH3, and kinase domains of these molecules.

Asunto(s)

Mesodermo/metabolismo , Proteínas de Xenopus , Familia-src Quinasas/metabolismo , Animales , Western Blotting , Fosforilación , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-fyn , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Xenopus laevis , Dominios Homologos src , Familia-src Quinasas/fisiología