RESUMEN
Progress in understanding early human development has been impeded by the scarcity of reference datasets from natural embryos, particularly those with spatial information during crucial stages like gastrulation. We conducted high-resolution spatial transcriptomics profiling on 38,562 spots from 62 transverse sections of an intact Carnegie stage (CS) 8 human embryo. From this spatial transcriptomic dataset, we constructed a 3D model of the CS8 embryo, in which a range of cell subtypes are identified, based on gene expression patterns and positional register, along the anterior-posterior, medial-lateral, and dorsal-ventral axis in the embryo. We further characterized the lineage trajectories of embryonic and extra-embryonic tissues and associated regulons and the regionalization of signaling centers and signaling activities that underpin lineage progression and tissue patterning during gastrulation. Collectively, the findings of this study provide insights into gastrulation and post-gastrulation development of the human embryo.
Asunto(s)
Embrión de Mamíferos , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Imagenología Tridimensional , Humanos , Embrión de Mamíferos/metabolismo , Transcriptoma/genética , Gástrula/metabolismo , Gástrula/embriología , Transducción de Señal , Linaje de la Célula , Perfilación de la Expresión Génica , Tipificación del Cuerpo/genéticaRESUMEN
Chemical signalling is the primary means by which cells communicate in the embryo. The underlying principle refers to a group of ligand-producing cells and a group of cells that respond to this signal because they express the appropriate receptors1,2. In the zebrafish embryo, Wnt5b binds to the receptor Ror2 to trigger the Wnt-planar cell polarity (PCP) signalling pathway to regulate tissue polarity and cell migration3,4. However, it remains unclear how this lipophilic ligand is transported from the source cells through the aqueous extracellular space to the target tissue. In this study, we provide evidence that Wnt5b, together with Ror2, is loaded on long protrusions called cytonemes. Our data further suggest that the active Wnt5b-Ror2 complexes form in the producing cell and are handed over from these cytonemes to the receiving cell. Then, the receiving cell has the capacity to initiate Wnt-PCP signalling, irrespective of its functional Ror2 receptor status. On the tissue level, we further show that cytoneme-dependent spreading of active Wnt5b-Ror2 affects convergence and extension in the zebrafish gastrula. We suggest that cytoneme-mediated transfer of ligand-receptor complexes is a vital mechanism for paracrine signalling. This may prompt a reevaluation of the conventional concept of characterizing responsive and non-responsive tissues solely on the basis of the expression of receptors.
Asunto(s)
Seudópodos , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Proteínas Wnt , Pez Cebra , Animales , Gástrula/citología , Gástrula/embriología , Gástrula/metabolismo , Ligandos , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Pez Cebra/embriología , Pez Cebra/metabolismo , Polaridad Celular , Movimiento Celular , Seudópodos/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Comunicación ParacrinaRESUMEN
Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.
Asunto(s)
Desarrollo Embrionario , Estratos Germinativos , Células Madre Pluripotentes , Humanos , Diferenciación Celular , Implantación del Embrión , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Estratos Germinativos/citología , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Células Madre Pluripotentes/citología , Interleucina-6/metabolismo , Gástrula/citología , Gástrula/embriología , Amnios/citología , Amnios/embriología , Amnios/metabolismo , Ectodermo/citología , Ectodermo/embriología , Ectodermo/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismoRESUMEN
The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.
Asunto(s)
Animales Recién Nacidos , Embrión de Mamíferos , Desarrollo Embrionario , Gástrula , Análisis de la Célula Individual , Imagen de Lapso de Tiempo , Animales , Femenino , Ratones , Embarazo , Animales Recién Nacidos/embriología , Animales Recién Nacidos/genética , Diferenciación Celular/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Desarrollo Embrionario/genética , Gástrula/citología , Gástrula/embriología , Gastrulación/genética , Riñón/citología , Riñón/embriología , Mesodermo/citología , Mesodermo/enzimología , Neuronas/citología , Neuronas/metabolismo , Retina/citología , Retina/embriología , Somitos/citología , Somitos/embriología , Factores de Tiempo , Factores de Transcripción/genética , Transcripción Genética , Especificidad de Órganos/genéticaRESUMEN
During ontogeny, proliferating cells become restricted in their fate through the combined action of cell-type-specific transcription factors and ubiquitous epigenetic machinery, which recognizes universally available histone residues or nucleotides in a context-dependent manner1,2. The molecular functions of these regulators are generally well understood, but assigning direct developmental roles to them is hampered by complex mutant phenotypes that often emerge after gastrulation3,4. Single-cell RNA sequencing and analytical approaches have explored this highly conserved, dynamic period across numerous model organisms5-8, including mouse9-18. Here we advance these strategies using a combined zygotic perturbation and single-cell RNA-sequencing platform in which many mutant mouse embryos can be assayed simultaneously, recovering robust morphological and transcriptional information across a panel of ten essential regulators. Deeper analysis of central Polycomb repressive complex (PRC) 1 and 2 components indicates substantial cooperativity, but distinguishes a dominant role for PRC2 in restricting the germline. Moreover, PRC mutant phenotypes emerge after gross epigenetic and transcriptional changes within the initial conceptus prior to gastrulation. Our experimental framework may eventually lead to a fully quantitative view of how cellular diversity emerges using an identical genetic template and from a single totipotent cell.
Asunto(s)
Epigénesis Genética , Gástrula/embriología , Gástrula/metabolismo , Gastrulación/genética , Animales , Linaje de la Célula , Femenino , Gástrula/citología , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Mutación , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Análisis de la Célula Individual , Transcripción GenéticaRESUMEN
The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids-three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.
Asunto(s)
Tipificación del Cuerpo , Gástrula/citología , Células Madre Embrionarias Humanas/citología , Organoides/citología , Organoides/embriología , Somitos/citología , Somitos/embriología , Tipificación del Cuerpo/genética , Gástrula/embriología , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Técnicas In Vitro , Organoides/metabolismo , Transducción de Señal , Somitos/metabolismo , TranscriptomaRESUMEN
Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction1,2. However, recent work has revealed roles for basement membrane remodelling in global tissue morphogenesis3-5. Upon implantation, the epiblast and extraembryonic ectoderm of the mouse embryo become enveloped by a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis6,7. However, the mechanical role of the basement membrane in post-implantation embryogenesis remains unknown. Here we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating the expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis8,9 further regulates basement membrane remodelling by localizing Nodal signalling-and therefore the activity of matrix metalloproteinases and basement membrane perforations-to the posterior side of the embryo. Perforations on the posterior side are essential for primitive-streak extension during gastrulation by rendering the basement membrane of the prospective primitive streak more prone to breaching. Thus spatiotemporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.
Asunto(s)
Membrana Basal/embriología , Membrana Basal/metabolismo , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Animales , Membrana Basal/citología , Blastocisto/citología , Blastocisto/metabolismo , Embrión de Mamíferos/citología , Matriz Extracelular/metabolismo , Femenino , Gástrula/embriología , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ligandos de Señalización Nodal/metabolismo , Línea Primitiva/citología , Línea Primitiva/embriología , Línea Primitiva/metabolismoRESUMEN
Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.
Asunto(s)
Gástrula , Células Madre Embrionarias de Ratones/citología , Organoides/citología , Organoides/embriología , Análisis de la Célula Individual , Somitos/citología , Somitos/embriología , Transcriptoma , Animales , Colágeno , Combinación de Medicamentos , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Femenino , Gástrula/citología , Gástrula/embriología , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , Laminina , Masculino , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Organoides/metabolismo , Proteoglicanos , RNA-Seq , Somitos/metabolismo , Factores de TiempoRESUMEN
Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Gástrula/citología , Gástrula/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Análisis de la Célula Individual , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Cromatina/metabolismo , Desmetilación , Cuerpos Embrioides/citología , Endodermo/citología , Endodermo/embriología , Endodermo/metabolismo , Elementos de Facilitación Genéticos/genética , Epigenoma/genética , Eritropoyesis , Análisis Factorial , Gástrula/embriología , Gastrulación/fisiología , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN/análisis , Factores de Tiempo , Dedos de ZincRESUMEN
In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.
Asunto(s)
Tipificación del Cuerpo/fisiología , Organizadores Embrionarios/fisiología , Nicho de Células Madre/fisiología , Animales , Embrión de Pollo , Endodermo/embriología , Gástrula/embriología , Mesodermo/embriología , Sistema Nervioso , Notocorda/embriología , Organizadores Embrionarios/metabolismo , Nicho de Células Madre/genética , Células Madre/metabolismo , Células Madre/fisiologíaRESUMEN
The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes1, a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), when stimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids'2,3 suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending antero-posterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.
Asunto(s)
Tipificación del Cuerpo , Gástrula/citología , Gástrula/embriología , Células Madre Embrionarias de Ratones/citología , Organoides/citología , Organoides/embriología , Animales , Tipificación del Cuerpo/genética , Gástrula/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/genética , Técnicas In Vitro , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Organoides/metabolismo , Factores de TiempoRESUMEN
During Xenopus gastrulation, Wnt and FGF signaling pathways cooperate to induce posterior structures. Wnt target expression around the blastopore falls into two main categories: a horseshoe shape with a dorsal gap, as in Wnt8 expression; or a ring, as in FGF8 expression. Using ChIP-seq, we show, surprisingly, that the FGF signaling mediator Ets2 binds near all Wnt target genes. However, ß-catenin preferentially binds at the promoters of genes with horseshoe patterns, but further from the promoters of genes with ring patterns. Manipulation of FGF or Wnt signaling demonstrated that 'ring' genes are responsive to FGF signaling at the dorsal midline, whereas 'horseshoe' genes are predominantly regulated by Wnt signaling. We suggest that, in the absence of active ß-catenin at the dorsal midline, the DNA-binding protein TCF binds and actively represses gene activity only when close to the promoter. In contrast, genes without functional TCF sites at the promoter may be predominantly regulated by Ets at the dorsal midline and are expressed in a ring. These results suggest recruitment of only short-range repressors to potential Wnt targets in the Xenopus gastrula.
Asunto(s)
Gástrula/embriología , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteína Proto-Oncogénica c-ets-2/metabolismo , Factores de Transcripción TCF/metabolismo , Xenopus laevis/embriología , Animales , Sitios de Unión/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Unión Proteica/fisiología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismoRESUMEN
In Cnidaria, modes of gastrulation to produce the two body layers vary greatly between species. In the hydrozoan species Clytia hemisphaerica gastrulation involves unipolar ingression of presumptive endoderm cells from an oral domain of the blastula, followed by migration of these cells to fill the blastocoel with concomitant narrowing of the gastrula and elongation along the oral-aboral axis. We developed a 2D computational boundary model capable of simulating the morphogenetic changes during embryonic development from early blastula stage to the end of gastrulation. Cells are modeled as polygons with elastic membranes and cytoplasm, colliding and adhering to other cells, and capable of forming filopodia. With this model we could simulate compaction of the embryo preceding gastrulation, bottle cell formation, ingression, and intercalation between cells of the ingressing presumptive endoderm. We show that embryo elongation is dependent on the number of endodermal cells, low endodermal cell-cell adhesion, and planar cell polarity (PCP). When the strength of PCP is reduced in our model, resultant embryo morphologies closely resemble those reported previously following morpholino-mediated knockdown of the core PCP proteins Strabismus and Frizzled. Based on our results, we postulate that cellular processes of apical constriction, compaction, ingression, and then reduced cell-cell adhesion and mediolateral intercalation in the presumptive endoderm, are required and when combined, sufficient for Clytia gastrulation.
Asunto(s)
Cnidarios/embriología , Gástrula/embriología , Gastrulación/fisiología , Modelos Biológicos , Animales , Cnidarios/citología , Gástrula/citologíaRESUMEN
Transforming growth factor (TGF)ß/activin superfamily regulates diverse biological processes including germ layer specification and axis patterning in vertebrates. TGFß/activin leads to phosphorylation of Smad2 and Smad3, followed by regulation of their target genes. Activin treatment also induces the essential organizer gene chordin (chrd). The involvement of Smad2/3 in chrd expression has been unclear as to whether Smad2/3 involvement is direct or indirect and whether any cis-acting response elements for Smad2/3 are present in the proximal or distal regions of its promoter. In the present study, we isolated the -2250 bps portion of the chrd promoter, showing that it contained Smad2/3 direct binding sites at its distal portion, separate from the proximal locations of other organizer genes, goosecoid and cerberus. The pattern of transcription activation for the promoter (-2250 bps) was indistinguishable from that of the endogenous chrd in gastrula Xenopus embryos. Reporter gene assays and site-directed mutagenesis analysis of the chrd promoter mapped two active activin/Smad response elements (ARE1 and ARE2) for Smad2 and Smad3. For a differential chrd induction, Smad2 acted on both ARE1 and ARE2, but Smad3 was only active for ARE2. Collectively, the results demonstrate that the distal region of chrd promoter contains the direct binding cis-acting elements for Smad2 and Smad3, which differentially modulate chrd transcription in gastrula Xenopus embryos.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Gástrula/embriología , Gástrula/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Smad2/genética , Proteína smad3/genética , Activación Transcripcional , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Xenopus laevis is highly suitable as a toxicology animal model owing to its advantages in embryogenesis research. For toxicological studies, a large number of embryos must be handled simultaneously because they very rapidly develop into the target stages within a short period of time. To efficiently handle the embryos, a convenient embryo housing device is essential for fast and reliable assessment and statistical evaluation of malformation caused by toxicants. Here, we suggest 3D fabrication of single-egg trapping devices in which Xenopus eggs are fertilized in vitro, and the embryos are cultured. We used manual pipetting to insert the Xenopus eggs inside the trapping sites of the chip. By introducing a liquid circulating system, we connected a sperm-mixed solution with the chip to induce in vitro fertilization of the eggs. After the eggs were fertilized, we observed embryo development involving the formation of egg cleavage, blastula, gastrula, and tadpole. After the tadpoles grew inside the chip, we saved their lives by enabling their escape from the chip through reverse flow of the culture medium. The Xenopus chip can serve as an incubator to induce fertilization and monitor normal and abnormal development of the Xenopus from egg to tadpole.
Asunto(s)
Embrión no Mamífero/embriología , Fertilización In Vitro/métodos , Oocitos/citología , Xenopus laevis/embriología , Animales , Blástula/citología , Blástula/embriología , Blástula/fisiología , División Celular/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Femenino , Fertilización In Vitro/instrumentación , Gástrula/citología , Gástrula/embriología , Gástrula/fisiología , Larva/citología , Larva/crecimiento & desarrollo , Larva/fisiología , Locomoción/fisiología , Masculino , Oocitos/fisiología , Xenopus laevis/fisiologíaRESUMEN
Nanog has been implicated in establishment of pluripotency in mammals and in zygotic genome activation in zebrafish. In this study, we characterize the development of MZnanog (maternal and zygotic null) mutant zebrafish embryos. Without functional Nanog, epiboly is severely affected, embryo axes do not form and massive cell death starts at the end of gastrulation. We show that three independent defects in MZnanog mutants contribute to epiboly failure: yolk microtubule organization required for epiboly is abnormal, maternal mRNA fails to degrade owing to the absence of miR-430, and actin structure of the yolk syncytial layer does not form properly. We further demonstrate that the cell death in MZnanog embryos is cell-autonomous. Nanog is necessary for correct spatial expression of the ventral-specifying genes bmp2b, vox and vent, and the neural transcription factor her3 It is also required for the correctly timed activation of endoderm genes and for the degradation of maternal eomesa mRNA via miR-430. Our findings suggest that maternal Nanog coordinates several gene regulatory networks that shape the embryo during gastrulation.
Asunto(s)
Gástrula/embriología , Gastrulación/fisiología , Proteína Homeótica Nanog/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Supervivencia Celular/fisiología , Gástrula/citología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Proteína Homeótica Nanog/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saco Vitelino/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Apical constriction regulates epithelial morphogenesis during embryonic development, but how this process is controlled is not understood completely. Here, we identify a Rho guanine nucleotide exchange factor (GEF) gene plekhg5 as an essential regulator of apical constriction of bottle cells during Xenopus gastrulation. plekhg5 is expressed in the blastopore lip and its expression is sufficient to induce ectopic bottle cells in epithelia of different germ layers in a Rho-dependent manner. This activity is not shared by arhgef3, which encodes another organizer-specific RhoGEF. Plekhg5 protein is localized in the apical cell cortex via its pleckstrin homology domain, and the GEF activity enhances its apical recruitment. Plekhg5 induces apical actomyosin accumulation and cell elongation. Knockdown of plekhg5 inhibits activin-induced bottle cell formation and endogenous blastopore lip formation in gastrulating frog embryos. Apical accumulation of actomyosin, apical constriction and bottle cell formation fail to occur in these embryos. Taken together, our data indicate that transcriptional regulation of plekhg5 expression at the blastopore lip determines bottle cell morphology via local polarized activation of Rho by Plekhg5, which stimulates apical actomyosin activity to induce apical constriction.
Asunto(s)
Polaridad Celular , Gastrulación , Factores de Intercambio de Guanina Nucleótido/fisiología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Activinas/metabolismo , Actomiosina/metabolismo , Animales , Citoesqueleto/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Gástrula/embriología , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/genética , Movimiento , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
The larval pharynx of the cephalochordate Branchiostoma (amphioxus) is asymmetrical. The mouth is on the left, and endostyle and gill slits are on the right. At the neurula, Nodal and Hedgehog (Hh) expression becomes restricted to the left. To dissect their respective roles in gill slit formation, we inhibited each pathway separately for 20â min at intervals during the neurula stage, before gill slits penetrate, and monitored the effects on morphology and expression of pharyngeal markers. The results pinpoint the short interval spanning the gastrula/neurula transition as the critical period for specification and positioning of future gill slits. Thus, reduced Nodal signaling shifts the gill slits ventrally, skews the pharyngeal domains of Hh, Pax1/9, Pax2/5/8, Six1/2 and IrxC towards the left, and reduces Hh and Tbx1/10 expression in endoderm and mesoderm, respectively. Nodal auto-regulates. Decreased Hh signaling does not affect gill slit positions or Hh or Nodal expression, but it does reduce the domain of Gli, the Hh target, in the pharyngeal endoderm. Thus, during the neurula stage, Nodal and Hh cooperate in gill slit development - Hh mediates gill slit formation and Nodal establishes their left-right position.
Asunto(s)
Tipificación del Cuerpo , Branquias/metabolismo , Proteínas Hedgehog/metabolismo , Anfioxos/embriología , Anfioxos/metabolismo , Proteína Nodal/metabolismo , Animales , Benzodioxoles/farmacología , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Epistasis Genética/efectos de los fármacos , Gástrula/efectos de los fármacos , Gástrula/embriología , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Branquias/efectos de los fármacos , Proteínas Hedgehog/genética , Imidazoles/farmacología , Anfioxos/efectos de los fármacos , Anfioxos/genética , Larva/efectos de los fármacos , Larva/metabolismo , Mesodermo/efectos de los fármacos , Mesodermo/embriología , Mesodermo/metabolismo , Proteína Nodal/genética , Faringe/efectos de los fármacos , Faringe/embriología , Faringe/metabolismo , Piridinas/farmacología , Alcaloides de Veratrum/farmacologíaRESUMEN
Around the time of gastrulation in higher vertebrate embryos, inductive interactions direct cells to form central nervous system (neural plate) or sensory placodes. Grafts of different tissues into the periphery of a chicken embryo elicit different responses: Hensen's node induces a neural plate whereas the head mesoderm induces placodes. How different are these processes? Transcriptome analysis in time course reveals that both processes start by induction of a common set of genes, which later diverge. These genes are remarkably similar to those induced by an extraembryonic tissue, the hypoblast, and are normally expressed in the pregastrulation stage epiblast. Explants of this epiblast grown in the absence of further signals develop as neural plate border derivatives and eventually express lens markers. We designate this state as "preborder"; its transcriptome resembles embryonic stem cells. Finally, using sequential transplantation experiments, we show that the node, head mesoderm, and hypoblast are interchangeable to begin any of these inductions while the final outcome depends on the tissue emitting the later signals.
Asunto(s)
Sistema Nervioso Central/metabolismo , Inducción Embrionaria , Células Madre Embrionarias/metabolismo , Gástrula/metabolismo , Mesodermo/metabolismo , Placa Neural/metabolismo , Animales , Sistema Nervioso Central/embriología , Embrión de Pollo , Gástrula/embriología , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Mesodermo/embriología , Placa Neural/embriologíaRESUMEN
Ascidian species of the Phallusia and Ciona genera are distantly related, their last common ancestor dating several hundred million years ago. Although their genome sequences have extensively diverged since this radiation, Phallusia and Ciona species share almost identical early morphogenesis and stereotyped cell lineages. Here, we explored the evolution of transcriptional control between P. mammillata and C. robusta. We combined genome-wide mapping of open chromatin regions in both species with a comparative analysis of the regulatory sequences of a test set of 10 pairs of orthologous early regulatory genes with conserved expression patterns. We find that ascidian chromatin accessibility landscapes obey similar rules as in other metazoa. Open-chromatin regions are short, highly conserved within each genus and cluster around regulatory genes. The dynamics of chromatin accessibility and closest-gene expression are strongly correlated during early embryogenesis. Open-chromatin regions are highly enriched in cis-regulatory elements: 73% of 49 open chromatin regions around our test genes behaved as either distal enhancers or proximal enhancer/promoters following electroporation in Phallusia eggs. Analysis of this datasets suggests a pervasive use in ascidians of "shadow" enhancers with partially overlapping activities. Cross-species electroporations point to a deep conservation of both the trans-regulatory logic between these distantly-related ascidians and the cis-regulatory activities of individual enhancers. Finally, we found that the relative order and approximate distance to the transcription start site of open chromatin regions can be conserved between Ciona and Phallusia species despite extensive sequence divergence, a property that can be used to identify orthologous enhancers, whose regulatory activity can partially diverge.