RESUMEN
The discovery of the amphibian gastrula organizer and its publication by Hans Spemann and Hilde Mangold in 1924 is a foundation of experimental embryology, and has shaped our understanding of embryonic induction and pattern formation in vertebrates until today. The original publication is a piece of scientific art, characterized by the meticulous hand drawings by Hilde Mangold, as well as the text that develops mechanistic concepts of modern embryology. While historic microphotographs of specimens got lost, the original microscope slides and Hilde Mangold's laboratory notebook have been secured in embryological collections until today. Here, we make the original data of the six embryonic specimens reported in 1924, as well as the laboratory notebook, available in an accessible digital format. Together, these data shed light on the scientific process that led to the discovery, and should help to make the experiments on the most important signalling center in early vertebrate development transparent for generations of embryologists to come.
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Gástrula , Animales , Microscopía/métodos , Organizadores Embrionarios , Historia del Siglo XX , Embriología/historia , Embriología/métodosRESUMEN
Morphogenetic movements during animal development involve repeated making and breaking of cell-cell contacts. Recent biophysical models of cell-cell adhesion integrate adhesion molecule interactions and cortical cytoskeletal tension modulation, describing equilibrium states for established contacts. We extend this emerging unified concept of adhesion to contact formation kinetics, showing that aggregating Xenopus embryonic cells rapidly achieve Ca2+-independent low-contact states. Subsequent transitions to cadherin-dependent high-contact states show rapid decreases in contact cortical F-actin levels but slow contact area growth. We developed a biophysical model that predicted contact growth quantitatively from known cellular and cytoskeletal parameters, revealing that elastic resistance to deformation and cytoskeletal network turnover are essential determinants of adhesion kinetics. Characteristic time scales of contact growth to low and high states differ by an order of magnitude, being at a few minutes and tens of minutes, respectively, thus providing insight into the timescales of cell-rearrangement-dependent tissue movements.
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Cadherinas , Gástrula , Animales , Adhesión Celular , Xenopus laevis , Gástrula/metabolismo , Cadherinas/metabolismo , Moléculas de Adhesión CelularRESUMEN
The establishment of anterior-posterior (AP) regional identity is an essential step in the appropriate development of the vertebrate central nervous system. An important aspect of AP neural axis formation is the inherent plasticity that allows developing cells to respond to and recover from the various perturbations that embryos continually face during the course of development. While the mechanisms governing the regionalization of the nervous system have been extensively studied, relatively less is known about the nature and limits of early neural plasticity of the anterior-posterior neural axis. This study aims to characterize the degree of neural axis plasticity in Xenopus laevis by investigating the response of embryos to a 180-degree rotation of their AP neural axis during gastrula stages by assessing the expression of regional marker genes using in situ hybridization. Our results reveal the presence of a narrow window of time between the mid- and late gastrula stage, during which embryos are able undergo significant recovery following a 180-degree rotation of their neural axis and eventually express appropriate regional marker genes including Otx, Engrailed, and Krox. By the late gastrula stage, embryos show misregulation of regional marker genes following neural axis rotation, suggesting that this profound axial plasticity is a transient phenomenon that is lost by late gastrula stages.
RESUMEN
In the primitive vertebrate gastrula, the boundary between ectoderm and mesoderm is formed by Brachet's cleft. Here we examine Brachet's cleft and its control by Eph/ephrin signaling in Xenopus at the ultrastructural level and by visualizing cortical F-actin. We infer cortical tension ratios at tissue surfaces and their interface in normal gastrulae and after depletion of receptors EphB4 and EphA4 and ligands ephrinB2 and ephrinB3. We find that cortical tension downregulation at cell contacts, a normal process in adhesion, is asymmetrically blocked in the ectoderm by Eph/ephrin signals from the mesoderm. This generates high interfacial tension that can prevent cell mixing across the boundary. Moreover, it determines an asymmetric boundary structure that is suited for the respective roles of ectoderm and mesoderm, as substratum and as migratory layers. The Eph and ephrin isoforms also control different cell-cell contact types in ectoderm and mesoderm. Respective changes of adhesion upon isoform depletion affect adhesion at the boundary to different degrees but usually do not prohibit cleft formation. In an extreme case, a new type of cleft-like boundary is even generated where cortical tension is symmetrically increased on both sides of the boundary.
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Efrinas , Gástrula , Animales , Ectodermo/metabolismo , Efrinas/metabolismo , Gástrula/metabolismo , Mesodermo/metabolismo , Xenopus laevis/metabolismoRESUMEN
The blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear ß-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.
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Blástula/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , Organizadores Embrionarios/embriología , Organizadores Embrionarios/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Biomarcadores , Blástula/citología , Desarrollo Embrionario/genética , Gástrula/embriología , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , Modelos Biológicos , Organogénesis , Xenopus laevisRESUMEN
The Brachyury gene encodes a transcription factor that is conserved across all animals. In non-chordate metazoans, brachyury is primarily expressed in ectoderm regions that are added to the endodermal gut during development, and often form a ring around the site of endoderm internalization in the gastrula, the blastopore. In chordates, this brachyury ring is conserved, but the gene has taken on a new role in the formation of the mesoderm. In this phylum, a novel type of mesoderm that develops into notochord and somites has been added to the ancestral lateral plate mesoderm. Brachyury contributes to a shift in cell fate from neural ectoderm to posterior notochord and somites during a major lineage segregation event that in Xenopus and in the zebrafish takes place in the early gastrula. In the absence of this brachyury function, impaired formation of posterior mesoderm indirectly affects the gastrulation movements of peak involution and convergent extension. These movements are confined to specific regions and stages, leaving open the question why brachyury expression in an extensive, coherent ring, before, during and after gastrulation, is conserved in the two species whose gastrulation modes differ considerably, and also in many other metazoan gastrulae of diverse structure.
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Ectodermo/crecimiento & desarrollo , Proteínas Fetales/genética , Gástrula/crecimiento & desarrollo , Morfogénesis/genética , Proteínas de Dominio T Box/genética , Animales , Endodermo/crecimiento & desarrollo , Proteínas Fetales/ultraestructura , Mesodermo/crecimiento & desarrollo , Notocorda/crecimiento & desarrollo , Proteínas de Dominio T Box/ultraestructura , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrollo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Mesoderm and endoderm internalization in the Xenopus embryo are based on a number of region-specific morphogenetic processes that co-act in the vegetal half of the gastrula. In the multilayered wall surrounding the blastocoel, the apical layer engages in bottle cell formation and associated invagination and involution movements, and in cell intercalation in the plane of the layer. Of these epithelial-type processes, only bottle cell formation has been analyzed mechanistically. In the deep layers of the blastocoel wall, cell-on-cell migration drives the internalization of mesoderm by various forms of involution and of the endodermal cell mass by vegetal rotation. In the mesoderm, cells migrate in a mesenchymal mode with the aid of locomotory protrusions, whereas cells of the vegetal cell mass resemble free bottle cells that engage in ingression-type amoeboid migration. Cells rearrange by differential migration leading to parallel or orthogonal forms of intercalation and respective types of convergent extension. The interaction of the various apical and deep layer processes gives rise to dorsal multilayer invagination, ventrolateral internal involution, peak involution and orthogonal convergent extension of the dorsal posterior mesoderm, vegetal rotation, and blastopore constriction. It is speculated how these multilayer gastrulation movements could be derived from mechanisms in invertebrate single-epithelium gastrulae.
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Embrión no Mamífero/fisiología , Endodermo/fisiología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/fisiología , Morfogénesis , Proteínas de Xenopus/metabolismo , Xenopus laevis/fisiología , Animales , Movimiento Celular , Embrión no Mamífero/citología , Endodermo/citología , Mesodermo/citología , Transducción de Señal , Proteínas de Xenopus/genética , Xenopus laevis/embriologíaRESUMEN
In Placentalia, the fetus depends upon an organized vascular connection with its mother for survival and development. Yet, this connection was, until recently, obscure. Here, we summarize how two unrelated tissues, the primitive streak, or body axis, and extraembryonic visceral endoderm collaborate to create and organize the fetal-placental arterial connection in the mouse gastrula. The primitive streak reaches into the extraembryonic space, where it marks the site of arterial union and creates a progenitor cell pool. Through contact with the streak, associated visceral endoderm undergoes an epithelial-to-mesenchymal transition, contributing extraembryonic mesoderm to the placental arterial vasculature, and to the allantois, or pre-umbilical tissue. In addition, visceral endoderm bifurcates into the allantois where, with the primitive streak, it organizes the nascent umbilical artery and promotes allantoic elongation to the chorion, the site of fetal-maternal exchange. Brachyury mediates streak extension and vascular patterning, while Hedgehog is involved in visceral endoderm's conversion to mesoderm. A unique CASPASE-3-positive cell separates streak- and non-streak-associated domains in visceral endoderm. Based on these new insights at the posterior embryonic-extraembryonic interface, we conclude by asking whether so-called primordial germ cells are truly antecedents to the germ line that segregate within the allantois, or whether they are placental progenitor cells. Incorporating these new working hypotheses into mutational analyses in which the placentae are affected will aid understanding a spectrum of disorders, including orphan diseases, which often include abnormalities of the umbilical cord, yolk sac, and hindgut, whose developmental relationship to each other has, until now, been poorly understood. This article is categorized under: Birth Defects > Associated with Preimplantation and Gastrulation Early Embryonic Development > Gastrulation and Neurulation.
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Arterias/embriología , Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Endodermo/embriología , Feto/embriología , Placenta/citología , Línea Primitiva/embriología , Animales , Femenino , Gástrula/citología , Gástrula/fisiología , Humanos , Ratones , EmbarazoRESUMEN
We characterized spontaneous Ca2+ signals in Oikopleura dioica embryos from pre-fertilization to gastrula stages following injection of GCaMP6 mRNA into unfertilized eggs. The unfertilized egg exhibited regular, transient elevations in intracellular Ca2+ concentration with an average duration of 4-6 s and an average frequency of about 1 every 2.5 min. Fertilization was accompanied by a longer Ca2+ transient that lasted several minutes. Thereafter, regular Ca2+ transients were reinstated that spread within seconds among blastomeres and gradually increased in duration (by about 50%) and decreased in frequency (by about 20%) by gastrulation. Peak amplitudes also exhibited a dynamic, with a transitory drop occurring at about the 4-cell stage and a subsequent rise. Each peak was preceded by about 15 s by a smaller and shorter Ca2+ increase (about 5% of the main peak amplitude, average duration 3 s), which we term the "minipeak". By gastrulation, Ca2+ transients exhibited a stereotyped initiation site on either side of the 32-64-cell embryo, likely in the nascent muscle precursor cells, and spread thereafter symmetrically in a stereotyped spatial pattern that engaged blastomeres giving rise to all the major tissue lineages. The rapid spread of the transients relative to the intertransient interval created a coordinated wave that, on a coarse time scale, could be considered an approximate synchronization. Treatment with the divalent cations Ni2+ or Cd2+ gradually diminished peak amplitudes, had only moderate effects on wave frequency, but markedly disrupted wave synchronization and normal development. The T-type Ca2+ channel blocker mibefradil similarly disrupted normal development, and eliminated the minipeaks, but did not affect wave synchronization. To assess the role of gap junctions in calcium wave spread and coordination, we first characterized the expression of two Oikopleura connexins, Od-CxA and Od-CxB, both of which are expressed during pre-gastrulation and gastrula stages, and then co-injected double-stranded inhibitory RNAs together with CGaMP6 to suppress connexin expression. Connexin mRNA knockdown led to a gradual increase in Ca2+ transient peak width, a decrease of interpeak interval and a marked disruption of wave synchronization. As seen with divalent cations and mibefradil, this desynchronization was accompanied by a disruption of normal development.
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Blastómeros/metabolismo , Señalización del Calcio/fisiología , Linaje de la Célula/fisiología , Uniones Comunicantes/metabolismo , Gastrulación/fisiología , Urocordados/embriología , Animales , Blastómeros/citología , Urocordados/citologíaRESUMEN
Reactions of embryonic tissues to a distributed and concentrated stretching are described and compared with the mechanics of the normal gastrulation movements. A role of mechanically stressed dynamic cell structures in the gastrulation, demarcation of notochord borders and in providing proportionality of the axial rudiments is demonstrated. A morphomechanical scheme of amphibian gastrulation is presented.
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Anfibios/embriología , Gástrula , Gastrulación , Notocorda/embriología , Estrés Mecánico , Anfibios/fisiología , Animales , Tipificación del Cuerpo , Movimiento Celular , Ectodermo/fisiología , Embriología/métodos , Resistencia a la Tracción , Xenopus laevisRESUMEN
Xenopus gastrulation movements are in large part based on the rearrangement of cells by differential cell-on-cell migration within multilayered tissues. Different patterns of migration-based cell intercalation drive endoderm and mesoderm internalization and their positioning along their prospective body axes. C-cadherin, fibronectin, integrins, and focal contact components are expressed in all gastrula cells and play putative roles in cell-on-cell migration, but their actual functions in this respect are not yet understood. The gastrula can be subdivided into two motility domains, and in the vegetal, migratory domain, two modes of cell migration are discerned. Vegetal endoderm cells show ingression-type migration, a variant of amoeboid migration characterized by the lack of locomotory protrusions and by macropinocytosis as a mechanism of trailing edge resorption. Mesendoderm and prechordal mesoderm cells use lamellipodia in a mesenchymal mode of migration. Gastrula cell motility can be dissected into traits, such as cell polarity, adhesion, mobility, or protrusive activity, which are controlled separately yet in complex, combinatorial ways. Cells can instantaneously switch between different combinations of traits, showing plasticity as they respond to substratum properties. This article is categorized under: Early Embryonic Development > Gastrulation and Neurulation.
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Tipificación del Cuerpo/genética , Ectodermo/citología , Endodermo/citología , Gástrula/citología , Mesodermo/citología , Xenopus laevis/embriología , Animales , Cadherinas/genética , Cadherinas/metabolismo , Movimiento Celular , Polaridad Celular , Ectodermo/crecimiento & desarrollo , Ectodermo/metabolismo , Embrión no Mamífero , Endodermo/crecimiento & desarrollo , Endodermo/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Gástrula/crecimiento & desarrollo , Gástrula/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Integrinas/genética , Integrinas/metabolismo , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Seudópodos/genética , Seudópodos/metabolismo , Seudópodos/ultraestructura , Transducción de Señal , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrollo , Xenopus laevis/metabolismoRESUMEN
The yolk syncytial layer (YSL) of Teleostei is a dynamic multifunctional temporary system. This paper describes the YSL structure of Misgurnus fossilis (Cobitidae) during its early developmental stages, studied using histological methods. YSL formation is prolonged. From the late blastula stage, the basal surface of the YSL is uneven and has protuberances, but becomes smoother during development. There are syncytial 'islands' with 1-2 yolk syncytial nuclei in the yolk mass. During epiboly, gastrulation and early segmentation, loach YSL is of different thickness in different regions along the dorso-ventral and antero-posterior axes of an embryo. The YSL is thickened in the dorsal region of gastrulae compared with the ventral region. Although the development of M. fossilis is similar to the development of zebrafish, there are important differences in YSL formation and organization that await further study and analysis. The study of YSL organization contributes to our knowledge of teleost developmental diversity and to the biology of temporary structures.
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Cipriniformes/embriología , Saco Vitelino/anatomía & histología , Saco Vitelino/citología , Animales , Blástula , Embrión no Mamífero , Femenino , Gástrula , MasculinoRESUMEN
Wnt/ß-catenin signaling is an important cell-to-cell signaling mechanism that controls gene expression during embryonic development and is critically implicated in human diseases. Developmental, cellular, and transcriptional responses to Wnt signaling are remarkably context-specific in different biological processes. While nuclear localization of ß-catenin is the key to activation of the Wnt/ß-catenin pathway and target gene expression, the molecular mechanisms of how the same Wnt/ß-catenin signaling pathway induces specific responses remain undetermined. Recent advances in high-throughput sequencing technologies and the availability of genome information for Xenopus tropicalis have enabled us to uncover a genome-wide view of Wnt/ß-catenin signaling in early vertebrate embryos, which challenges previous concepts about molecular mechanisms of Wnt target gene regulation. In this review, we summarize our experimental approaches, introduce the technologies we employed and focus on recent findings about Wnt target gene regulation from Xenopus research. We will also discuss potential functions of widespread ß-catenin binding in the genome that we discovered in this species.
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Desarrollo Embrionario/genética , Proteínas Wnt/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , Animales , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Genoma , Humanos , Unión Proteica , Transducción de Señal , Proteínas Wnt/metabolismo , Xenopus/genética , Xenopus/crecimiento & desarrollo , beta Catenina/metabolismoRESUMEN
Tissue or cell transplantation is an invaluable technique with a multitude of applications including studying the developmental potential of certain cell populations, dissecting cell-environment interactions, and identifying stem cells. One key technical requirement for performing transplantation assays is the capability of distinguishing the transplanted donor cells from the endogenous host cells and tracing the donor cells over time. The zebrafish has emerged as an excellent model organism for performing transplantation assays, thanks in part to the transparency of embryos and even adults when pigment mutants are employed. Using transgenic techniques and fast-evolving imaging technology, fluorescence-labeled donor cells can be readily identified and studied during development in vivo. In this chapter, we will discuss the rationale of different types of zebrafish transplantation in both embryos and adults and then focus on four detailed methods of transplantation: blastula/gastrula transplantation for mosaic analysis, hematopoietic stem cell transplantation, chemical screening using a transplantation model, and tumor transplantation.
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Diferenciación Celular/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Trasplante/métodos , Pez Cebra/genética , Animales , Blástula/crecimiento & desarrollo , Embrión no Mamífero , Gástrula/crecimiento & desarrollo , Pez Cebra/crecimiento & desarrolloRESUMEN
We present a plausible account of the origin of the archetypal vertebrate bauplan. We offer a theoretical reconstruction of the geometrically regular structure of the blastula resulting from the sequential subdivision of the egg, followed by mechanical deformations of the blastula in subsequent stages of gastrulation. We suggest that the formation of the vertebrate bauplan during development, as well as fixation of its variants over the course of evolution, have been constrained and guided by global mechanical biases. Arguably, the role of such biases in directing morphology-though all but neglected in previous accounts of both development and macroevolution-is critical to any substantive explanation for the origin of the archetypal vertebrate bauplan. We surmise that the blastula inherently preserves the underlying geometry of the cuboidal array of eight cells produced by the first three cleavages that ultimately define the medial-lateral, dorsal-ventral, and anterior-posterior axes of the future body plan. Through graphical depictions, we demonstrate the formation of principal structures of the vertebrate body via mechanical deformation of predictable geometrical patterns during gastrulation. The descriptive rigor of our model is supported through comparisons with previous characterizations of the embryonic and adult vertebrate bauplane. Though speculative, the model addresses the poignant absence in the literature of any plausible account of the origin of vertebrate morphology. A robust solution to the problem of morphogenesis-currently an elusive goal-will only emerge from consideration of both top-down (e.g., the mechanical constraints and geometric properties considered here) and bottom-up (e.g., molecular and mechano-chemical) influences.
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Blástula/embriología , Fenómenos Mecánicos , Vertebrados/embriología , Animales , Fenómenos Biomecánicos , Blastocisto , Desarrollo Embrionario , HumanosRESUMEN
Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi, Epipedobates machalilla, Gastrotheca riobambae, and Eleutherodactylus coqui, as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt (zvegt) mRNA was detected as a minor isoform besides the major maternal (mvegt) isoform of the X. laevis egg. In addition, α-amanitin-insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1, have been evolutionary conserved in frogs.
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Tipificación del Cuerpo , Endodermo/fisiología , Proteínas de Homeodominio/fisiología , ARN Mensajero/metabolismo , Proteínas de Dominio T Box/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Alfa-Amanitina/farmacología , Animales , Proteínas de Homeodominio/análisis , Proteínas de Dominio T Box/análisis , Proteínas de Dominio T Box/genética , Factores de Transcripción , Proteínas de Xenopus/análisis , Proteínas de Xenopus/genéticaRESUMEN
Key signalling pathways, such as canonical Wnt/ß-catenin signalling, operate repeatedly to regulate tissue- and stage-specific transcriptional responses during development. Although recruitment of nuclear ß-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling, mechanisms controlling stage- or tissue-specific transcriptional responses remain elusive. Here, a direct comparison of genome-wide occupancy of ß-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of ß-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling. We demonstrate that Wnt signalling regulates ß-catenin binding to Wnt target genes not only when they are transcriptionally regulated, but also in contexts in which their transcription remains unaffected. The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence ß-catenin recruitment. Our findings suggest a more general paradigm for Wnt-regulated transcriptional mechanisms, which is relevant for tissue-specific functions of Wnt/ß-catenin signalling in embryonic development but also for stem cell-mediated homeostasis and cancer. Chromatin association of ß-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes. Context-dependent mechanisms are crucial for transcriptional activation of Wnt/ß-catenin target genes subsequent to ß-catenin recruitment. Our conclusions therefore also imply that Wnt-regulated ß-catenin binding in one context can mark Wnt-regulated transcriptional target genes for different contexts.
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Regulación del Desarrollo de la Expresión Génica , Especificidad de Órganos/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/genética , beta Catenina/metabolismo , Animales , Secuencia de Bases , Proteínas Morfogenéticas Óseas/metabolismo , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Gástrula/metabolismo , Sitios Genéticos , Genoma , Modelos Biológicos , Unión Proteica/genética , Análisis de Secuencia de ARN , Transducción de Señal/genética , Transcripción Genética , Transcriptoma/genética , Xenopus/embriologíaRESUMEN
MicroRNAs (miRNAs) are known to play diverse roles in the regulation of vertebrate development. To investigate miRNA-target mRNA relationships in embryonic development, we have carried out small-RNA sequencing to identify miRNAs expressed in the early gastrula of Xenopus laevis. We identify a total of 180 miRNAs, and we have identified the locations of the miRNA precursor sequences in the X. laevis genome. Of these miRNAs, 141 represent miRs previously identified in Xenopus tropicalis. Alignment to human miRNAs led to the identification of 39 miRNAs that have not previously been described for Xenopus. We have also used a biochemical approach to isolate mRNAs that are associated with the RNA-Induced Silencing Complex (RISC) in early gastrulae and thus candidate targets of miRNA-dependent regulation. Interrogation of this RISC-associated mRNA pool by RT-PCR indicates that a number of genes essential for early patterning and specification may be under regulation by miRNAs. Smad1 transcripts are associated with the RISC; target prediction algorithms identify a single miRNA-binding site for miR-26, which is common to the 3'UTRs of Smad1a and Smad1b. Disruption of the interaction between miR-26 and the Smad1 3'UTR via a Target Protector Morpholino Oligonucleotide (TPMO) leads to a 2-fold increase in Smad1 protein accumulation, moderate increases in the expression of BMP4/Smad1 target genes, and a reduction in organizer gene expression, as well as a partially ventralized phenotype in approximately 25% of embryos. Overexpression of miR-26 resulted in moderately decreased expression of Smad1-dependent genes and an expansion of the region expressing the Organizer gene not1. Our findings indicate that interactions between miR-26 and the Smad1 3'UTR modulate Smad1 function in the establishment of axial patterning; they also establish a foundation for the functional analysis of miRNAs and their regulatory interactions during gastrulation.
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Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismo , Proteína Smad1/genética , Proteínas de Xenopus/genética , Xenopus/embriología , Xenopus/genética , Regiones no Traducidas 3'/genética , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Secuencia de Bases , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Gástrula/embriología , Inmunoprecipitación , MicroARNs/genética , Datos de Secuencia Molecular , Fenotipo , Unión Proteica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Proteína Smad1/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
At the gastrula phase of development, just after the onset of implantation, the embryo proper is characterized by extremely rapid cell proliferation. The importance of DNA repair is illustrated by embryonic lethality at this stage after ablation of the genes involved. Insight into mutation induction is called for by the fact that women often do not realize they are pregnant, shortly after implantation, a circumstance which may have important consequences when women are subjected to medical imaging using ionizing radiation. We screened gastrula embryos for DNA synthesis, nuclear morphology, growth, and chromosome aberrations (CA) shortly after irradiation with doses up to 2.5Gy. In order to obtain an insight into the importance of DNA repair for CA induction, we included mutants for the non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, as well as Parp1-/- and p53+/- embryos. With the pUR288 shuttle vector assay, we determined the radiation sensitivity for point mutations and small deletions detected in young adults. We found increased numbers of abnormal nuclei 5h after irradiation; an indication of disturbed development was also observed around this time. Chromosome aberrations 7h after irradiation arose in all genotypes and were mainly of the chromatid type, in agreement with a cell cycle dominated by S-phase. Increased frequencies of CA were found for NHEJ and HR mutants. Gastrula embryos are unusual in that they are low in exchange induction, even after compromised HR. Gastrula embryos were radiation sensitive in the pUR288 shuttle vector assay, giving the highest mutation induction ever reported for this genetic toxicology model. On theoretical grounds, a delayed radiation response must be involved. The compromised developmental profile after doses up to 2.5Gy likely is caused by both apoptosis and later cell death due to large deletions. Our data indicate a distinct radiation-sensitive profile of gastrula embryos, including some stage-specific aspects that are not as yet understood.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de la radiación , Análisis Mutacional de ADN , Gástrula/efectos de la radiación , Operón Lac , Recombinación Genética , Animales , Proliferación Celular , Aberraciones Cromosómicas , Reparación del ADN , Femenino , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Mutación , ProbabilidadRESUMEN
El pargo colorado (Lutjanus colorado) es una especie con un alto valor comercial en el mercado mexicano, con potencial para su cultivo. Hasta la fecha no existen estudios sobre su reproducción, cultivo larvario y engorda en cautiverio. El presente trabajo es el primer reporte sobre la descripción a detalle del desarrollo embrionario de la especie bajo condiciones de cultivo. Los huevos fertilizados viables del pargo colorado son pelágicos, esféricos, transparentes y con una sola gota de aceite. Midieron 0,77±0,09 mm de diámetro y la gota de aceite 0,14±0,01 mm. La primera división ocurrió a las 0,05 horas post fertilización (HPF). La eclosión se llevó a cabo a las 17,22 HPF bajo las condiciones del presente estudio. Las larvas recién eclosionadas midieron 1,8±0,1 mm de longitud total (LT). El desarrollo embrionario de esta especie fue similar a la descrita para especies de la misma familia. Los resultados del presente estudio aportan información básica para iniciar el desarrollo de la biotecnología para la producción de semilla de esta especie a escala comercial.
The Colorado snapper (Lutjanus colorado) is one of the most commercially important fish species in México and it is considered a suitable candidate for culture. Until now, no research has been carried out on its reproduction, larviculture and fattening in captivity. This study is the first description of embryonic development of this species under controlled conditions. Fertilized eggs of Colorado snapper are pelagic, spherical and transparent and contain one drop of oil. Eggs measured 0.77±0.09 mm and the drop of oil 0.14±0.01 mm. First cell division occurred at 0.05 h post-fertilization (HPF), hatching at 17.22 HPF under the above described conditions. Larvae total length (LT) was 1.8±0.1 mm. Embryonic development of this species was similar to other lutjanidae species. These results provide basic information for developing the necessary biotechnology for commercial seed production of the Colorado snapper.