RESUMEN
Adaxial cells, the progenitors of slow-twitch muscle fibres in zebrafish, exhibit a stereotypic migratory behaviour during somitogenesis. Although this process is known to be disrupted in various mutants, its precise nature has remained unclear. Here, using in vivo imaging and chimera analysis, we show that adaxial cell migration is a cell autonomous process, during which cells become polarised and extend filopodia at their leading edge. Loss of function of the Prdm1a transcription factor disrupts the polarisation and migration of adaxial cells, reflecting a role that is independent of its repression of sox6 expression. Expression of the M- and N-cadherins, previously implicated in driving adaxial cell migration, is largely unaffected by loss of Prdm1a function, suggesting that differential cadherin expression is not sufficient for adaxial cell migration.
Asunto(s)
Cadherinas/biosíntesis , Diferenciación Celular/genética , Proteínas de Unión al ADN/biosíntesis , Desarrollo Embrionario/genética , Proteínas Nucleares/biosíntesis , Proteínas de Pez Cebra/biosíntesis , Animales , Cadherinas/genética , Movimiento Celular/genética , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/crecimiento & desarrollo , Proteínas Nucleares/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genéticaRESUMEN
The availability of animal models of epileptic seizures provides opportunities to identify novel anticonvulsants for the treatment of people with epilepsy. We found that exposure of 2-day-old zebrafish embryos to the convulsant agent pentylenetetrazole (PTZ) rapidly induces the expression of synaptic-activity-regulated genes in the CNS, and elicited vigorous episodes of calcium (Ca(2+)) flux in muscle cells as well as intense locomotor activity. We then screened a library of â¼2000 known bioactive small molecules and identified 46 compounds that suppressed PTZ-inducedtranscription of the synaptic-activity-regulated gene fos in 2-day-old (2 dpf) zebrafish embryos. Further analysis of a subset of these compounds, which included compounds with known and newly identified anticonvulsant properties, revealed that they exhibited concentration-dependent inhibition of both locomotor activity and PTZ-induced fos transcription, confirming their anticonvulsant characteristics. We conclude that this in situ hybridisation assay for fos transcription in the zebrafish embryonic CNS is a robust, high-throughput in vivo indicator of the neural response to convulsant treatment and lends itself well to chemical screening applications. Moreover, our results demonstrate that suppression of PTZ-induced fos expression provides a sensitive means of identifying compounds with anticonvulsant activities.
Asunto(s)
Anticonvulsivantes/análisis , Anticonvulsivantes/uso terapéutico , Modelos Animales de Enfermedad , Epilepsia/tratamiento farmacológico , Pez Cebra/fisiología , Animales , Anticonvulsivantes/farmacología , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/embriología , Sistema Nervioso Central/patología , Evaluación Preclínica de Medicamentos , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/patología , Epilepsia/genética , Epilepsia/patología , Epilepsia/fisiopatología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hibridación in Situ , Larva/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Músculos/efectos de los fármacos , Músculos/embriología , Músculos/metabolismo , Músculos/patología , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Pentilenotetrazol , Picrotoxina/toxicidad , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
The Hedgehog (Hh) family of signalling molecules is essential for a wide range of developmental processes. Mammalian studies have implicated the Hedgehog pathway in the aetiology of anorectal malformations (ARMs), relatively common congenital anomalies caused by failures in the development of the cloaca. In this study we demonstrate that Hh signalling is absolutely required for the formation of the zebrafish cloaca and that the severity of the posterior gut abnormalities induced by a reduction in Hh activity is dependent on the levels of Hh signal transduction. The complete loss of all Hh activity results in the most severe defects and the critical period for Hh activity is between 34 and 74 hours post fertilisation. Using a range of mutant genotypes that cause notochord and floorplate abnormalities, we show that the source of the Hh signals required for posterior gut formation is the endoderm and not the notochord, as previously postulated in mammalian models of ARMs. We show that Adriamycin, a drug known to cause ARMs in rat, but not chick embryos, has no effect on the development of the zebrafish gastrointestinal tract. These studies establish the zebrafish as a model for ARMs, and for the elucidation of other pathways involved in hindgut developmental processes.
Asunto(s)
Cloaca/embriología , Cloaca/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis , Cloaca/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Mutación/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Alcaloides de Veratrum/farmacología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteína Gli2 con Dedos de ZincRESUMEN
Hedgehog (Hh) proteins are members of a family of secreted signaling factors that orchestrate the development of many organs and tissues including those of the gastrointestinal (GI) tract. The requirement for Hh activity is not limited to early development but underlies the homeostasis of a number of tissues, and abnormal activity of the Hh pathway is associated with several GI malignancies. Understanding the roles and mechanisms of action of Hh signaling both in development and postnatally should thus give novel insights into potential treatments for these diseases. Here we focus on the Hh signaling pathway and its role in GI tract development and maintenance and consider the diseases resulting from aberrant Hh activity.
Asunto(s)
Enfermedades Gastrointestinales/fisiopatología , Tracto Gastrointestinal/crecimiento & desarrollo , Proteínas Hedgehog/fisiología , Transducción de Señal/fisiología , Humanos , Páncreas/crecimiento & desarrollo , Páncreas/patología , Neoplasias Pancreáticas/patologíaRESUMEN
Myoblast fusion follows a defined sequence of events that is strikingly similar in vertebrates and invertebrates. Genetic analysis in Drosophila has identified many of the molecules that mediate the different steps in the fusion process; by contrast, the molecular basis of myoblast fusion during vertebrate embryogenesis remains poorly characterised. A key component of the intracellular fusion pathway in Drosophila is the protein encoded by the myoblast city (mbc) gene, a close homologue of the vertebrate protein dedicator of cytokinesis 1 (DOCK1, formerly DOCK180). Using morpholino antisense-oligonucleotide-mediated knockdown of gene activity in the zebrafish embryo, we show that the fusion of embryonic fast-twitch myoblasts requires the activities of Dock1 and the closely related Dock5 protein. In addition, we show that the adaptor proteins Crk and Crk-like (Crkl), with which Dock proteins are known to interact physically, are also required for myoblast fusion.