RESUMEN
Metazoan genomes are duplicated by the coordinated activation of clusters of replication origins at different times during S phase, but the underlying mechanisms of this temporal program remain unclear during early development. Rif1, a key replication timing factor, inhibits origin firing by recruiting protein phosphatase 1 (PP1) to chromatin counteracting S phase kinases. We have previously described that Rif1 depletion accelerates early Xenopus laevis embryonic cell cycles. Here, we find that in the absence of Rif1, patterns of replication foci change along with the acceleration of replication cluster activation. However, initiations increase only moderately inside active clusters. Our numerical simulations suggest that the absence of Rif1 compresses the temporal program towards more homogeneity and increases the availability of limiting initiation factors. We experimentally demonstrate that Rif1 depletion increases the chromatin-binding of the S phase kinase Cdc7/Drf1, the firing factors Treslin, MTBP, Cdc45, RecQL4, and the phosphorylation of both Treslin and MTBP. We show that Rif1 globally, but not locally, restrains the replication program in early embryos, possibly by inhibiting or excluding replication factors from chromatin.
Asunto(s)
Proteínas de Ciclo Celular , Origen de Réplica , Animales , Ciclo Celular , Proteínas de Ciclo Celular/genética , Cromatina/genética , Xenopus laevis/genéticaRESUMEN
One presenilin gene (PSEN) is expressed in the sea urchin embryo, in the vegetal pole of the gastrula and then mainly in cilia cells located around the digestive system of the pluteus, as we recently have reported. PSEN expression must be accurately regulated for correct execution of these two steps of development. While investigating PSEN expression changes in embryos after expansion of endoderm with LiCl or of ectoderm with Zn2+ by whole-mount in situ hybridization (WISH) and quantitative PCR (qPCR), we detected natural antisense transcription of PSEN. We then found that Endo16 and Wnt5, markers of endo-mesoderm, and of Hnf6 and Gsc, markers of ectoderm, are also sense and antisense transcribed. We discuss that general gene expression could depend on both sense and antisense transcription. This mechanism, together with the PSEN gene, should be included in gene regulatory networks (GRNs) that theorize diverse processes in this species. We suggest that it would also be relevant to investigate natural antisense transcription of PSEN in the field of Alzheimer's disease (AD) where the role of human PSEN1 and PSEN2 is well known.
Asunto(s)
Presenilinas , Erizos de Mar , Humanos , Animales , Presenilinas/genética , Hibridación in Situ , Expresión Génica , Erizos de Mar/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.
Asunto(s)
Origen de Réplica , Proteínas de Unión a Telómeros , Animales , Replicación del ADN , Momento de Replicación del ADN , Fase S/genética , Proteínas de Unión a Telómeros/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Retinitis pigmentosa is an inherited retinal dystrophy that ultimately leads to blindness due to the progressive degeneration of rod photoreceptors and the subsequent non-cell autonomous death of cones. Rhodopsin is the most frequently mutated gene in this disease. We here developed rhodopsin gene editing-based models of retinitis pigmentosa in two Xenopus species, Xenopus laevis and Xenopus tropicalis, by using CRISPR/Cas9 technology. In both of them, loss of rhodopsin function results in massive rod cell degeneration characterized by progressive shortening of outer segments and occasional cell death. This is followed by cone morphology deterioration. Despite these apparently similar degenerative environments, we found that Müller glial cells behave differently in Xenopus laevis and Xenopus tropicalis. While a significant proportion of Müller cells re-enter into the cell cycle in Xenopus laevis, their proliferation remains extremely limited in Xenopus tropicalis. This work thus reveals divergent responses to retinal injury in closely related species. These models should help in the future to deepen our understanding of the mechanisms that have shaped regeneration during evolution, with tremendous differences across vertebrates.
Asunto(s)
Retinitis Pigmentosa , Rodopsina , Animales , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Células Ependimogliales/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinitis Pigmentosa/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Presenilins (PSENs) are widely expressed across eukaryotes. Two PSENs are expressed in humans, where they play a crucial role in Alzheimer's disease (AD). Each PSEN can be part of the γ-secretase complex, which has multiple substrates, including Notch and amyloid-ß precursor protein (AßPP) - the source of amyloid-ß (Aß) peptides that compose the senile plaques during AD. PSENs also interact with various proteins independently of their γ-secretase activity. They can then be involved in numerous cellular functions, which makes their role in a given cell and/or organism complex to decipher. We have established the Paracentrotus lividus sea urchin embryo as a new model to study the role of PSEN. In the sea urchin embryo, the PSEN gene is present in unduplicated form and encodes a protein highly similar to human PSENs. Our results suggest that PSEN expression must be precisely tuned to control the course of the first mitotic cycles and the associated intracellular Ca2+ transients, the execution of gastrulation and, probably in association with ciliated cells, the establishment of the pluteus. We suggest that it would be relevant to study the role of PSEN within the gene regulatory network deciphered in the sea urchin.
Asunto(s)
Presenilinas , Erizos de Mar/crecimiento & desarrollo , Animales , Presenilina-1 , Presenilinas/genética , Erizos de Mar/genéticaRESUMEN
Xenopus is an attractive model system for regeneration studies, as it exhibits an extraordinary regenerative capacity compared to mammals. It is commonly used to study body part regeneration following amputation, for instance of the limb, the tail, or the retina. Models with more subtle injuries are also needed for human degenerative disease modeling, allowing for the study of stem cell recruitment for the regeneration of a given cellular subtype. We present here a model to ablate photoreceptor cells in the Xenopus retina. This method is based on the nitroreductase/metronidazole (NTR/MTZ) system, a combination of chemical and genetic tools, allowing for the conditional ablation of targeted cells. This type of approach establishes Xenopus as a powerful model to study cellular regeneration and stem cell regulation.
Asunto(s)
Metronidazol/farmacología , Nitrorreductasas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Xenopus/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Fluorescentes Verdes/metabolismo , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/metabolismo , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Rodopsina/genética , TransgenesRESUMEN
BACKGROUND: Amacrine interneurons that modulate synaptic plasticity between bipolar and ganglion cells constitute the most diverse cell type in the retina. Most are inhibitory neurons using either GABA or glycine as neurotransmitters. Although several transcription factors involved in amacrine cell fate determination have been identified, mechanisms underlying amacrine cell subtype specification remain to be further understood. The Prdm13 histone methyltransferase encoding gene is a target of the transcription factor Ptf1a, an essential regulator of inhibitory neuron cell fate in the retina. Here, we have deepened our knowledge on its interaction with Ptf1a and investigated its role in amacrine cell subtype determination in the developing Xenopus retina. METHODS: We performed prdm13 gain and loss of function in Xenopus and assessed the impact on retinal cell fate determination using RT-qPCR, in situ hybridization and immunohistochemistry. RESULTS: We found that prdm13 in the amphibian Xenopus is expressed in few retinal progenitors and in about 40% of mature amacrine cells, predominantly in glycinergic ones. Clonal analysis in the retina reveals that prdm13 overexpression favours amacrine cell fate determination, with a bias towards glycinergic cells. Conversely, knockdown of prdm13 specifically inhibits glycinergic amacrine cell genesis. We also showed that, as in the neural tube, prdm13 is subjected to a negative autoregulation in the retina. Our data suggest that this is likely due to its ability to repress the expression of its inducer, ptf1a. CONCLUSIONS: Our results demonstrate that Prdm13, downstream of Ptf1a, acts as an important regulator of glycinergic amacrine subtype specification in the Xenopus retina. We also reveal that Prdm13 regulates ptf1a expression through a negative feedback loop.
Asunto(s)
Células Amacrinas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Neurogénesis/fisiología , Retina/embriología , Proteínas de Xenopus/metabolismo , Células Amacrinas/citología , Animales , Retroalimentación Fisiológica/fisiología , Regulación del Desarrollo de la Expresión Génica , Glicina/metabolismo , Retina/metabolismo , Xenopus laevisRESUMEN
Mitotic arrest deficient 2 (Mad2) belongs to the spindle assembly checkpoint (SAC), a mechanism that blocks progression of the cell cycle until microtubule attachment to kinetochores is complete. It has been found to be involved in the resistance of cancer cells to "anti-mitotic" drugs such as paclitaxel. Mad2 controls meiotic progression, but its role during sea urchin development had never been investigated. Furthermore, the existence of a SAC in this species had never been proved. The present data show that a Mad2 protein, highly homologous to that of humans, is expressed in this species. Mad2 expression increases during development, becoming confined to the endomesoderm at gastrula stages. The level of Mad2 expression is enhanced in embryos that do not gastrulate after treatment with anti-mitotic drugs, lithium or inhibition of the ERK pathway. Mis-aligned and lagging chromosomes were induced after injection of an anti-Mad2 antibody or a Mad2 morpholino. Our results point to the role of a non-canonical SAC involving Mad2 in the control of mitotic divisions of the sea urchin embryo.
Asunto(s)
Proteínas Mad2/metabolismo , Mitosis/fisiología , Erizos de Mar/crecimiento & desarrollo , Huso Acromático/fisiología , Animales , Cinetocoros , Proteínas Mad2/genética , Erizos de Mar/metabolismoRESUMEN
Thyroid hormones (TH) have been mainly associated with post-embryonic development and adult homeostasis but few studies report direct experimental evidence for TH function at very early phases of embryogenesis. We assessed the outcome of altered TH signaling on early embryogenesis using the amphibian Xenopus as a model system. Precocious exposure to the TH antagonist NH-3 or impaired thyroid receptor beta function led to severe malformations related to neurocristopathies. These include pathologies with a broad spectrum of organ dysplasias arising from defects in embryonic neural crest cell (NCC) development. We identified a specific temporal window of sensitivity that encompasses the emergence of NCCs. Although the initial steps in NCC ontogenesis appeared unaffected, their migration properties were severely compromised both in vivo and in vitro. Our data describe a role for TH signaling in NCCs migration ability and suggest severe consequences of altered TH signaling during early phases of embryonic development.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Cresta Neural/citología , Cresta Neural/metabolismo , Transducción de Señal/efectos de los fármacos , Receptores beta de Hormona Tiroidea/metabolismo , Hormonas Tiroideas/metabolismo , Animales , Derivados del Benceno/farmacología , Biomarcadores/metabolismo , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Cresta Neural/efectos de los fármacos , Fenotipo , Xenopus laevis/embriologíaRESUMEN
The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability.
Asunto(s)
División Celular , Momento de Replicación del ADN , Inestabilidad Genómica , Retina/citología , Células Madre/fisiología , Transactivadores/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Xenopus , Proteínas Señalizadoras YAPRESUMEN
Live imaging studies of the processes of demyelination and remyelination have so far been technically limited in mammals. We have thus generated a Xenopus laevis transgenic line allowing live imaging and conditional ablation of myelinating oligodendrocytes throughout the CNS. In these transgenic pMBP-eGFP-NTR tadpoles the myelin basic protein (MBP) regulatory sequences, specific to mature oligodendrocytes, are used to drive expression of an eGFP (enhanced green fluorescent protein) reporter fused to the Escherichia coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous prodrug metronidazole (MTZ) to a cytotoxin. Using two-photon imaging in vivo, we show that pMBP-eGFP-NTR tadpoles display a graded oligodendrocyte ablation in response to MTZ, which depends on the exposure time to MTZ. MTZ-induced cell death was restricted to oligodendrocytes, without detectable axonal damage. After cessation of MTZ treatment, remyelination proceeded spontaneously, but was strongly accelerated by retinoic acid. Altogether, these features establish the Xenopus pMBP-eGFP-NTR line as a novel in vivo model for the study of demyelination/remyelination processes and for large-scale screens of therapeutic agents promoting myelin repair.
Asunto(s)
Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/fisiopatología , Modelos Animales de Enfermedad , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Regeneración Nerviosa/fisiología , Xenopus laevis/anatomía & histología , Xenopus laevis/fisiología , Animales , HumanosRESUMEN
Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/ß-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.
Asunto(s)
Proliferación Celular , Proteínas Hedgehog/fisiología , Retina/embriología , Retina/crecimiento & desarrollo , Vía de Señalización Wnt/fisiología , Animales , Animales Modificados Genéticamente , Proliferación Celular/efectos de los fármacos , Antagonismo de Drogas , Embrión no Mamífero , Inhibidores Enzimáticos/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Indoles/farmacología , Modelos Biológicos , Organogénesis/efectos de los fármacos , Organogénesis/genética , Organogénesis/fisiología , Oximas/farmacología , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Retina/efectos de los fármacos , Retina/metabolismo , Teratógenos/farmacología , Alcaloides de Veratrum/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Xenopus laevis/embriologíaRESUMEN
In all species, fertilization triggers in the egg a rapid and transient increase of intracellular free calcium (Cai), but how this signal is generated following sperm and egg interaction has not been clearly characterised yet. In sea urchin, a signalling pathway involving tyrosine kinase and PLCγ has been proposed to be at the origin of the fertilization Cai signal. We report here that injection of src homology-2 (SH2) domains of the sea urchin PLCγ inhibits in a competitive manner the endogenous PLCγ, alters both the amplitude and duration of the fertilization Cai wave, but does not abrogate it. Our results suggest that PLCγ acts in conjunction with a cADPr pathway and G-proteins of the Gαq type to trigger the fertilization Cai wave, and reinforce a crucial role for PLCγ at mitosis and cytokinesis.
Asunto(s)
ADP-Ribosa Cíclica/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Óvulo/metabolismo , Fosfolipasa C gamma/metabolismo , Erizos de Mar/fisiología , Animales , Secuencia de Bases , Señalización del Calcio , Células Cultivadas , Femenino , Fertilización , Masculino , Datos de Secuencia Molecular , Fosfolipasa C gamma/genética , Interacciones Espermatozoide-Óvulo , Transgenes/genética , Dominios Homologos src/genéticaRESUMEN
Mycobacterium liflandii has been responsible for an emerging infection reported in the international trade of Western clawed frogs (Silurana tropicalis). This study shows that this mycolactone-producing Mycobacterium (MPM) has expanded its distribution range to France. The results of this study suggest that the use of in vitro fertilization to maintain genetic lines could be a temporary solution for valuable S. tropicalis propagation.
Asunto(s)
Anuros , Infecciones por Mycobacterium/veterinaria , Mycobacterium/clasificación , Animales , Animales de Laboratorio , Anuros/genética , Hepatopatías/microbiología , Hepatopatías/veterinaria , Reproducción/fisiología , Enfermedades Cutáneas Bacterianas/microbiología , Enfermedades Cutáneas Bacterianas/veterinaria , Enfermedades del Bazo/microbiología , Enfermedades del Bazo/veterinariaRESUMEN
Neural stem cell research suffers from a lack of molecular markers to specifically assess stem or progenitor cell properties. The organization of the Xenopus ciliary marginal zone (CMZ) in the retina allows the spatial distinction of these two cell types: stem cells are confined to the most peripheral region, while progenitors are more central. Despite this clear advantage, very few genes specifically expressed in retinal stem cells have been discovered so far in this model. To gain insight into the molecular signature of these cells, we performed a large-scale expression screen in the Xenopus CMZ, establishing it as a model system for stem cell gene profiling. Eighteen genes expressed specifically in the CMZ stem cell compartment were retrieved and are discussed here. These encode various types of proteins, including factors associated with proliferation, mitotic spindle organization, DNA/RNA processing, and cell adhesion. In addition, the publication of this work in a special issue on Xenopus prompted us to give a more general illustration of the value of large-scale screens in this model species. Thus, beyond neural stem cell specific genes, we give a broader highlight of our screen outcome, describing in particular other retinal cell markers that we found. Finally, we present how these can all be easily retrieved through a novel module we developed in the web-based annotation tool XenMARK, and illustrate the potential of this powerful searchable database in the context of the retina.
Asunto(s)
Biomarcadores/análisis , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Células-Madre Neurales/citología , Retina/citología , Animales , Secuencia de Bases , Biomarcadores/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Células-Madre Neurales/metabolismo , Reacción en Cadena de la Polimerasa , Retina/metabolismo , XenopusRESUMEN
Cell lines are useful tools to facilitate in vitro studies of many biological and molecular processes. We describe a new permanent fibroblast-type cell line obtained from disaggregated Xenopus tropicalis limb bud. The cell line population doubling time was ~24 h. Its karyotype was genetically stable with a chromosome number of 2n = 21 and a chromosome 10 trisomy. These cells could be readily transfected and expressed transgenes faithfully. We obtained stable transformants using transposon-based gene transfer technology. These cells responded to thyroid hormone and thus can provide a complementary research tool to study thyroid hormone signaling events. In conclusion, this cell line baptized "Speedy" should prove useful to couple in vitro and in vivo biological studies in the X. tropicalis frog model.
Asunto(s)
Línea Celular , Xenopus/genética , Animales , Elementos Transponibles de ADN , Orden Génico , Técnicas de Transferencia de Gen , Genes Reporteros , Vectores Genéticos/genética , Cariotipo , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacos , Hormonas Tiroideas/farmacología , Transfección , Transgenes , Xenopus/metabolismoRESUMEN
Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by motor neuron loss and skeletal muscle atrophy. The loss of function of the smn1 gene, the main supplier of survival motor neuron protein (SMN) protein in human, leads to reduced levels of SMN and eventually to SMA. Here, we ask if the amphibian Xenopus tropicalis can be a good model system to study SMA. Inhibition of the production of SMN using antisense morpholinos leads to caudal muscular atrophy in tadpoles. Of note, early developmental patterning of muscles and motor neurons is unaffected in this system as well as acetylcholine receptors clustering. Muscular atrophy seems to rather result from aberrant pathfinding and growth arrest and/or shortening of motor axons. This event occurs in the absence of neuronal cell bodies apoptosis, a process comparable to that of amyotrophic lateral sclerosis. Xenopus tropicalis is revealed as a complementary animal model for the study of SMA.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Atrofia Muscular Espinal/genética , Proteína 1 para la Supervivencia de la Neurona Motora/biosíntesis , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Modelos Animales de Enfermedad , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Receptores Colinérgicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , XenopusRESUMEN
The precise localization of gene expression within the developing embryo, and how it changes over time, is one of the most important sources of information for elucidating gene function. As a searchable resource, this information has up until now been largely inaccessible to the Xenopus community. Here, we present a new database of Xenopus gene expression patterns, queryable by specific location or region in the embryo. Pattern matching can be driven either from an existing in situ image, or from a user-defined pattern based on development stage schematic diagrams. The data are derived from the work of a group of 21 Xenopus researchers over a period of 4 days. We used a novel, rapid manual annotation tool, XenMARK, which exploits the ability of the human brain to make the necessary distortions in transferring data from the in situ images to the standard schematic geometry. Developmental Dynamics 238:1379-1388, 2009. (c) 2009 Wiley-Liss, Inc.
Asunto(s)
Bases de Datos Genéticas , Regulación del Desarrollo de la Expresión Génica , Expresión Génica , Xenopus laevis/embriología , Xenopus laevis/genética , Animales , Humanos , Programas Informáticos , Xenopus laevis/anatomía & histologíaRESUMEN
Stable integration of foreign DNA into the frog genome has been the purpose of several studies aimed at generating transgenic animals or producing mutations of endogenous genes. Inserting DNA into a host genome can be achieved in a number of ways. In Xenopus, different strategies have been developed which exhibit specific molecular and technical features. Although several of these technologies were also applied in various model organizms, the attributes of each method have rarely been experimentally compared. Investigators are thus confronted with a difficult choice to discriminate which method would be best suited for their applications. To gain better understanding, a transgenesis workshop was organized by the X-omics consortium. Three procedures were assessed side-by-side, and the results obtained are used to illustrate this review. In addition, a number of reagents and tools have been set up for the purpose of gene expression and functional gene analyses. This not only improves the status of Xenopus as a powerful model for developmental studies, but also renders it suitable for sophisticated genetic approaches. Twenty years after the first reported transgenic Xenopus, we review the state of the art of transgenic research, focusing on the new perspectives in performing genetic studies in this species.
Asunto(s)
Técnicas de Transferencia de Gen , Técnicas Genéticas , Xenopus/genética , Animales , Animales Modificados Genéticamente , Vectores GenéticosRESUMEN
In vertebrates, the actin-binding proteins tropomyosins are encoded by four distinct genes that are expressed in a complex pattern during development and muscle differentiation. In this study, we have characterized the transcriptional machinery of the alpha-tropomyosin (alpha-Tm) gene in muscle cells. Promoter analysis revealed that a 284-bp proximal promoter region of the Xenopus laevis alpha-Tm gene is sufficient for maximal activity in the three muscle cell types. The transcriptional activity of this promoter in the three muscle cell types depends on both distinct and common cis-regulatory sequences. We have identified a 30-bp conserved sequence unique to all vertebrate alpha-Tm genes that contains an MCAT site that is critical for expression of the gene in all muscle cell types. This site can bind transcription enhancer factor-1 (TEF-1) present in muscle cells both in vitro and in vivo. In serum-deprived differentiated smooth muscle cells, TEF-1 was redistributed to the nucleus, and this correlated with increased activity of the alpha-Tm promoter. Overexpression of TEF-1 mRNA in Xenopus embryonic cells led to activation of both the endogenous alpha-Tm gene and the exogenous 284-bp promoter. Finally, we show that, in transgenic embryos and juveniles, an intact MCAT sequence is required for correct temporal and spatial expression of the 284-bp gene promoter. This study represents the first analysis of the transcriptional regulation of the alpha-Tm gene in vivo and highlights a common TEF-1-dependent regulatory mechanism necessary for expression of the gene in the three muscle lineages.