Asunto(s)
Biología Evolutiva/tendencias , Técnicas de Cultivo de Embriones/tendencias , Desarrollo Embrionario , Animales , Blastómeros/citología , Biología Evolutiva/ética , Biología Evolutiva/métodos , Técnicas de Cultivo de Embriones/ética , Técnicas de Cultivo de Embriones/métodos , Investigaciones con Embriones/ética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Cuerpos Embrioides/citología , Desarrollo Embrionario/genética , Femenino , Fertilización In Vitro , Desarrollo Fetal , Feto/citología , Feto/embriología , Gastrulación , Edición Génica , Células Madre Embrionarias Humanas/citología , Humanos , Imagenología Tridimensional , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Organizadores Embrionarios/citología , Organizadores Embrionarios/embriología , Embarazo , Factores de TiempoRESUMEN
In amniotes, the development of the primitive streak and its accompanying 'organizer' define the first stages of gastrulation. Although these structures have been characterized in detail in model organisms, the human primitive streak and organizer remain a mystery. When stimulated with BMP4, micropatterned colonies of human embryonic stem cells self-organize to generate early embryonic germ layers 1 . Here we show that, in the same type of colonies, Wnt signalling is sufficient to induce a primitive streak, and stimulation with Wnt and Activin is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, markers of epithelial-to-mesenchymal transition and expression of the organizer-specific transcription factor GSC. Moreover, when grafted into chick embryos, human stem cell colonies treated with Wnt and Activin induce and contribute autonomously to a secondary axis while inducing a neural fate in the host. This fulfils the most stringent functional criteria for an organizer, and its discovery represents a milestone in human embryology.
Asunto(s)
Proteína Nodal/metabolismo , Organizadores Embrionarios/embriología , Organizadores Embrionarios/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Activinas/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Línea Celular , Embrión de Pollo , Transición Epitelial-Mesenquimal , Proteína Goosecoide/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ratones , Tejido Nervioso/citología , Tejido Nervioso/embriología , Tejido Nervioso/metabolismo , Organizadores Embrionarios/citología , Línea Primitiva/citología , Línea Primitiva/metabolismoRESUMEN
Organizers, which comprise groups of cells with the ability to instruct adjacent cells into specific states, represent a key principle in developmental biology. The concept was first introduced by Spemann and Mangold, who showed that there is a cellular population in the newt embryo that elicits the development of a secondary axis from adjacent cells. Similar experiments in chicken and rabbit embryos subsequently revealed groups of cells with similar instructive potential. In birds and mammals, organizer activity is often associated with a structure known as the node, which has thus been considered a functional homologue of Spemann's organizer. Here, we take an in-depth look at the structure and function of organizers across species and note that, whereas the amphibian organizer is a contingent collection of elements, each performing a specific function, the elements of organizers in other species are dispersed in time and space. This observation urges us to reconsider the universality and meaning of the organizer concept.
Asunto(s)
Organizadores Embrionarios/citología , Organizadores Embrionarios/fisiología , Anfibios/embriología , Animales , Aves/embriología , Tipificación del Cuerpo/fisiología , Embrión de Pollo , Embrión de Mamíferos , Embrión no Mamífero , Inducción Embrionaria/fisiología , Gástrula/citología , Humanos , Mamíferos/embriología , ConejosRESUMEN
During development in metazoan embryos, the fundamental embryonic axes are established by organizing centers that influence the fates of nearby cells. Among the spiralians, a large and diverse branch of protostome metazoans, studies have shown that an organizer sets up the dorsal-ventral axis, which arises from one of the four basic cell quadrants during development (the dorsal, D quadrant). Studies in a few species have also revealed variation in terms of how and when the D quadrant and the organizer are established. In some species the D quadrant is specified conditionally, via cell-cell interactions, while in others it is specified autonomously, via asymmetric cell divisions (such as those involving the formation of polar lobes). The third quartet macromere (3D) typically serves as the spiralian organizer; however, other cells born earlier or later in the D quadrant lineage can serve as the organizer, such as the 2d micromere in the annelid Capitella teleta or the 4d micromere in the mollusc Crepidula fornicata. Here we present work carried out in the snail C. fornicata to show that establishment of a single D quadrant appears to rely on a combination of both autonomous (via inheritance of the polar lobe) and conditional mechanisms (involving induction via the progeny of the first quartet micromeres). Through systematic ablation of cells, we show that D quadrant identity is established between 5th and 6th cleavage stages, as it is in other spiralians that use conditional specification. Subsequently, following the next cell cycle, organizer activity takes place soon after the birth of the 4d micromere. Therefore, unlike the case in other spiralians that use conditional specification, the specification of the D quadrant and the activity of the dorso-ventral organizer are temporally and spatially uncoupled. We also present data on organizer function in naturally-occurring and experimentally-induced twin embryos, which possess multiple D quadrants. We show that supernumerary D quadrants can arise in C. fornicata (either spontaneously or following polar lobe removal); when multiple D quadrants are present these do not exhibit effective organizer activity. We conclude that the polar lobe is not required for D quadrant specification, though it could play a role in effective organizer activity. We also tested whether the inheritance of the small polar lobe by the D quadrant is associated with the ability to laterally inhibit neighboring quadrants by direct contact in order to normally prevent supernumerary organizers from arising. Finally, we discuss the variation of spiralian organizers in a phylogenetic context.
Asunto(s)
Organismos Acuáticos/citología , Organismos Acuáticos/crecimiento & desarrollo , Gastrópodos/citología , Gastrópodos/embriología , Organizadores Embrionarios/citología , Organizadores Embrionarios/embriología , Animales , Fase de Segmentación del Huevo/citología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Factores de TiempoRESUMEN
The Kupffer's vesicle (KV) is the so-called left-right organizer in teleost fishes. KV is formed from dorsal forerunner cells (DFCs) and generates asymmetrical signals for breaking symmetry of embryos. It is unclear how DFCs or KV cells are prevented from intermingling with adjacent cells. In this study, we show that the Eph receptor gene ephb4b is highly expressed in DFCs whereas ephrin ligand genes, including efnb2b, are expressed in cells next to the DFC cluster during zebrafish gastrulation. ephb4b knockdown or mutation and efnb2b knockdown cause dispersal of DFCs, a smaller KV and randomization of laterality organs. DFCs often dynamically form lamellipodium-like, bleb-like and filopodium-like membrane protrusions at the interface, which attempt to invade but are bounced back by adjacent non-DFC cells during gastrulation. Upon inhibition of Eph/ephrin signaling, however, the repulsion between DFCs and non-DFC cells is weakened or lost, allowing DFCs to migrate away. Ephb4b/Efnb2b signaling by activating RhoA activity mediates contact and repulsion between DFCs and neighboring cells during gastrulation, preventing intermingling of different cell populations. Therefore, our data uncover an important role of Eph/ephrin signaling in maintaining DFC cluster boundary and KV boundary for normal left-right asymmetrical development.
Asunto(s)
Tipificación del Cuerpo , Embrión no Mamífero/citología , Efrinas/metabolismo , Morfogénesis , Organizadores Embrionarios/citología , Receptor EphB4/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Agregación Celular , Comunicación Celular , Movimiento Celular , Embrión no Mamífero/metabolismo , Lateralidad Funcional , Técnicas de Inactivación de Genes , Mesodermo/citología , Mutación/genética , Organizadores Embrionarios/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
An "organizer" is formally defined as a region, or group of cells in an embryo that can both induce (change the fate) and pattern (generate an organized set of structures) adjacent embryonic cells. To date, about four such regions have been demonstrated: the primary or Spemann organizer (Hensen's node in amniotes), the notochord, the zone of polarizing activity of the limb bud, and the mid-hindbrain boundary. Here we review the evidence for these and compare them with a few other regions which have been proposed to represent other organizers and we speculate on why so few such regions have been discovered.
Asunto(s)
Polaridad Celular , Embrión de Mamíferos/citología , Notocorda/citología , Organizadores Embrionarios/citología , Animales , HumanosRESUMEN
During animal gastrulation, the specification of the embryonic axes is accompanied by epithelio-mesenchymal transition (EMT), the first major change in cell shape after fertilization. EMT takes place in disparate topographical arrangements, such as the circular blastopore of amphibians, the straight primitive streak of birds and mammals or in intermediate gastrulation forms of other amniotes such as reptiles. Planar cell movements are prime candidates to arrange specific modes of gastrulation but there is no consensus view on their role in different vertebrate classes. Here, we test the impact of interfering with Rho kinase-mediated cell movements on gastrulation topography in blastocysts of the rabbit, which has a flat embryonic disc typical for most mammals. Time-lapse video microscopy, electron microscopy, gene expression and morphometric analyses of the effect of inhibiting ROCK activity showed - besides normal specification of the organizer region - a dose-dependent disruption of primitive streak formation; this disruption resulted in circular, arc-shaped or intermediate forms, reminiscent of those found in amphibians, fishes and reptiles. Our results reveal a crucial role of ROCK-controlled directional cell movements during rabbit primitive streak formation and highlight the possibility that temporal and spatial modulation of cell movements were instrumental for the evolution of gastrulation forms.
Asunto(s)
Movimiento Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Línea Primitiva/citología , Línea Primitiva/embriología , Quinasas Asociadas a rho/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Evolución Biológica , Tipificación del Cuerpo/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Movimiento Celular/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Gastrulación/efectos de los fármacos , Organizadores Embrionarios/citología , Organizadores Embrionarios/efectos de los fármacos , Línea Primitiva/efectos de los fármacos , Línea Primitiva/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Conejos , Tiazolidinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
The cortical hem, a source of Wingless-related (WNT) and bone morphogenetic protein (BMP) signaling in the dorsomedial telencephalon, is the embryonic organizer for the hippocampus. Whether the hem is a major regulator of cortical patterning outside the hippocampus has not been investigated. We examined regional organization across the entire cerebral cortex in mice genetically engineered to lack the hem. Indicating that the hem regulates dorsoventral patterning in the cortical hemisphere, the neocortex, particularly dorsomedial neocortex, was reduced in size in late-stage hem-ablated embryos, whereas cortex ventrolateral to the neocortex expanded dorsally. Unexpectedly, hem ablation also perturbed regional patterning along the rostrocaudal axis of neocortex. Rostral neocortical domains identified by characteristic gene expression were expanded, and caudal domains diminished. A similar shift occurs when fibroblast growth factor (FGF) 8 is increased at the rostral telencephalic organizer, yet the FGF8 source was unchanged in hem-ablated brains. Rather we found that hem WNT or BMP signals, or both, have opposite effects to those of FGF8 in regulating transcription factors that control the size and position of neocortical areas. When the hem is ablated a necessary balance is perturbed, and cerebral cortex is rostralized. Our findings reveal a much broader role for the hem in cortical development than previously recognized, and emphasize that two major signaling centers interact antagonistically to pattern cerebral cortex.
Asunto(s)
Tipificación del Cuerpo/genética , Neocórtex/embriología , Neocórtex/metabolismo , Organizadores Embrionarios/embriología , Organizadores Embrionarios/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Proliferación Celular , Femenino , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Hipocampo/citología , Hipocampo/metabolismo , Ratones , Neocórtex/citología , Tamaño de los Órganos , Organizadores Embrionarios/citología , Fenotipo , Transducción de Señal/genética , Células Madre/citología , Células Madre/metabolismo , Proteínas Wnt/metabolismoRESUMEN
BACKGROUND: The habenula and the thalamus are two critical nodes in the forebrain circuitry and they connect the midbrain and the cerebral cortex in vertebrates. The habenula is derived from the epithalamus and rests dorsally to the thalamus. Both epithalamus and thalamus arise from a single diencephalon segment called prosomere (p)2. Shh is expressed in the ventral midline of the neural tube and in the mid-diencephalic organizer (MDO) at the zona limitans intrathalamica between thalamus and prethalamus. Acting as a morphogen, Shh plays an important role in regulating cell proliferation and survival in the diencephalon and thalamic patterning. The molecular regulation of the MDO Shh expression and the potential role of Shh in development of the habenula remain largely unclear. RESULTS: We show that deleting paired-box and homeobox-containing gene Pax6 results in precocious and expanded expression of Shh in the prospective MDO in fish and mice, whereas gain-of-function of pax6 inhibits MDO shh expression in fish. Using gene expression and genetic fate mapping, we have characterized the expression of molecular markers that demarcate the progenitors and precursors of habenular neurons. We show that the thalamic domain is shifted dorsally and the epithalamus is missing in the alar plate of p2 in the Pax6 mutant mouse. Conversely, the epithalamus is expanded ventrally at the expense of the thalamus in mouse embryos with reduced Shh activity. Significantly, attenuating Shh signaling largely rescues the patterning of p2 and restores the epithalamus in Pax6 mouse mutants, suggesting that Shh acts downstream of Pax6 in controlling the formation of the habenula. Similar to that found in the mouse, we show that pax6 controls the formation of the epithalamus mostly via the regulation of MDO shh expression in zebrafish. CONCLUSIONS: Our findings demonstrate that Pax6 has an evolutionarily conserved function in establishing the temporospatial expression of Shh in the MDO in vertebrates. Furthermore, Shh mediates Pax6 function in regulating the partition of the p2 domain into the epithalamus and thalamus.
Asunto(s)
Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Habénula/embriología , Habénula/metabolismo , Proteínas Hedgehog/genética , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Vertebrados/embriología , Proteínas de Pez Cebra/genética , Animales , Biomarcadores/metabolismo , Tipificación del Cuerpo/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Habénula/citología , Proteínas Hedgehog/metabolismo , Ratones , Organizadores Embrionarios/citología , Organizadores Embrionarios/embriología , Factor de Transcripción PAX6 , Unión Proteica , Transducción de Señal/genética , Células Madre/citología , Células Madre/metabolismo , Tálamo/citología , Tálamo/embriología , Factores de Transcripción/metabolismo , Vertebrados/genética , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The vertebrate body plan follows stereotypical dorsal-ventral (D-V) tissue differentiation controlled by bone morphogenetic proteins (BMPs) and secreted BMP antagonists, such as Chordin. The three germ layers--ectoderm, mesoderm, and endoderm--are affected coordinately by the Chordin-BMP morphogen system. However, extracellular morphogen gradients of endogenous proteins have not been directly visualized in vertebrate embryos to date. In this study, we improved immunolocalization methods in Xenopus embryos and analyzed the distribution of endogenous Chordin using a specific antibody. Chordin protein secreted by the dorsal Spemann organizer was found to diffuse along a narrow region that separates the ectoderm from the anterior endoderm and mesoderm. This Fibronectin-rich extracellular matrix is called "Brachet's cleft" in the Xenopus gastrula and is present in all vertebrate embryos. Chordin protein formed a smooth gradient that encircled the embryo, reaching the ventral-most Brachet cleft. Depletion with morpholino oligos showed that this extracellular gradient was regulated by the Chordin protease Tolloid and its inhibitor Sizzled. The Chordin gradient, as well as the BMP signaling gradient, was self-regulating and, importantly, was able to rescale in dorsal half-embryos. Transplantation of Spemann organizer tissue showed that Chordin diffused over long distances along this signaling highway between the ectoderm and mesoderm. Chordin protein must reach very high concentrations in this narrow region. We suggest that as ectoderm and mesoderm undergo morphogenetic movements during gastrulation, cells in both germ layers read their positional information coordinately from a single morphogen gradient located in Brachet's cleft.
Asunto(s)
Ectodermo/embriología , Gástrula/embriología , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mesodermo/embriología , Transducción de Señal/fisiología , Animales , Ectodermo/citología , Gástrula/citología , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Mesodermo/citología , Morfolinos/farmacología , Organizadores Embrionarios/citología , Organizadores Embrionarios/embriología , Transducción de Señal/efectos de los fármacos , Metaloproteinasas Similares a Tolloid/genética , Metaloproteinasas Similares a Tolloid/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse ß-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of ß-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for ß-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of ß-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/ß-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/ß-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.
Asunto(s)
Endodermo/embriología , Endodermo/metabolismo , Proteínas HMGB/metabolismo , Organizadores Embrionarios/embriología , Organizadores Embrionarios/metabolismo , Factores de Transcripción SOXF/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Tipificación del Cuerpo , Diferenciación Celular , Linaje de la Célula , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Endodermo/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/deficiencia , Proteínas HMGB/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Organizadores Embrionarios/citología , Embarazo , Factores de Transcripción SOXF/deficiencia , Factores de Transcripción SOXF/genética , Tetraploidía , Factor de Transcripción 4 , beta Catenina/deficiencia , beta Catenina/genéticaRESUMEN
The organizer is one of the earliest structures to be established during vertebrate development and is crucial to subsequent patterning of the embryo. We have previously shown that the SoxB1 transcription factor, Sox3, plays a central role as a transcriptional repressor of zebrafish organizer gene expression. Recent data suggest that Fgf signaling has a positive influence on organizer formation, but its role remains to be fully elucidated. In order to better understand how Fgf signaling fits into the complex regulatory network that determines when and where the organizer forms, the relationship between the positive effects of Fgf signaling and the repressive effects of the SoxB1 factors must be resolved. This study demonstrates that both fgf3 and fgf8 are required for expression of the organizer genes, gsc and chd, and that SoxB1 factors (Sox3, and the zebrafish specific factors, Sox19a and Sox19b) can repress the expression of both fgf3 and fgf8. However, we also find that these SoxB1 factors inhibit the expression of gsc and chd independently of their repression of fgf expression. We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression. These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling. The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Organizadores Embrionarios/citología , Organizadores Embrionarios/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Animales , Secuencia Conservada , Evolución Molecular , Factor 3 de Crecimiento de Fibroblastos/genética , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Factor 8 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Proteína Goosecoide/metabolismo , Humanos , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Regiones Promotoras Genéticas/genéticaRESUMEN
Human embryonic stem cells differentiate into gastrula organizer cells that express typical markers and induce secondary axes when injected into frog embryos. Here, we report that these human organizer cells express DUXO (DUX of the Organizer), a novel member of the double-homeobox (DUX) family of transcription factors, a group of genes unique to placental mammals. Both of DUXO's homeodomains share high similarity with those of Siamois and Twin, the initial inducers of the amphibian gastrula organizer. DUXO overexpression in human embryoid bodies induces organizer related genes, whereas its knock down hampers formation of the organizer and its derivatives. Finally, we show that DUXO regulates GOOSECOID, the canonical organizer marker, in a direct manner, suggesting that DUXO is a major regulator of human organizer formation.
Asunto(s)
Células Madre Embrionarias/metabolismo , Gástrula/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Organizadores Embrionarios/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Células Madre Embrionarias/citología , Gástrula/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Datos de Secuencia Molecular , Organizadores Embrionarios/citología , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca(2+) channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.
Asunto(s)
Tipificación del Cuerpo , Embrión de Mamíferos/fisiología , Factores de Determinación Derecha-Izquierda/metabolismo , Organizadores Embrionarios/fisiología , Canales Catiónicos TRPP/metabolismo , Animales , Líquidos Corporales/fisiología , Calcio/metabolismo , Cilios/metabolismo , Cilios/fisiología , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/citología , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cinesinas/genética , Factores de Determinación Derecha-Izquierda/genética , Ratones , Ratones Mutantes , Mutación , Organizadores Embrionarios/citología , Transducción de Señal , Canales Catiónicos TRPP/genéticaRESUMEN
Under the European Commission's New Chemical Policy both currently used and new chemicals should be tested for their toxicities in several areas, one of which was reproductive/developmental toxicity. Thousands of chemicals will need testing which will require a large number of laboratory animals. In vitro systems (as pre-screens or as validated alternatives) appear to be useful tools to reduce the number of whole animals used or refine procedures and hence decrease the cost for the chemical industry. Validated in vitro systems exist for developmental toxicity/embryotoxicity testing. Indeed, three assays have recently been validated: the whole embryo culture (WEC), the rat limb bud micromass (MM), and the embryonic stem cell test (EST). In this article, the use of primary embryonic cell culture, and in particular micromass culture, including a relatively novel chick heart micromass (MM) culture system has been described and compared to the validated D3 mouse embryonic stem cell (ESC) test.
Asunto(s)
Pruebas de Toxicidad/métodos , Animales , Supervivencia Celular , Células Cultivadas , Embrión de Pollo , Biología Evolutiva , Drosophila/citología , Embrión de Mamíferos/citología , Embrión no Mamífero/citología , Células Madre Embrionarias/citología , Humanos , Hydra/crecimiento & desarrollo , Indicadores y Reactivos/metabolismo , Esbozos de los Miembros/citología , Mesencéfalo/citología , Ratones , Miocardio/citología , Organizadores Embrionarios/citología , Oxazinas/metabolismo , Oxidación-Reducción , Cultivo Primario de Células , Ratas , Teratógenos/toxicidad , Teratología , Xantenos/metabolismo , Xenopus laevisRESUMEN
Although vastly outnumbered, inhibitory interneurons critically pace and synchronize excitatory principal cell populations to coordinate cortical information processing. Precision in this control relies upon a remarkable diversity of interneurons primarily determined during embryogenesis by genetic restriction of neuronal potential at the progenitor stage. Like their neocortical counterparts, hippocampal interneurons arise from medial and caudal ganglionic eminence (MGE and CGE) precursors. However, while studies of the early specification of neocortical interneurons are rapidly advancing, similar lineage analyses of hippocampal interneurons have lagged. A "hippocampocentric" investigation is necessary as several hippocampal interneuron subtypes remain poorly represented in the neocortical literature. Thus, we investigated the spatiotemporal origins of hippocampal interneurons using transgenic mice that specifically report MGE- and CGE-derived interneurons either constitutively or inducibly. We found that hippocampal interneurons are produced in two neurogenic waves between E9-E12 and E12-E16 from MGE and CGE, respectively, and invade the hippocampus by E14. In the mature hippocampus, CGE-derived interneurons primarily localize to superficial layers in strata lacunosum moleculare and deep radiatum, while MGE-derived interneurons readily populate all layers with preference for strata pyramidale and oriens. Combined molecular, anatomical, and electrophysiological interrogation of MGE/CGE-derived interneurons revealed that MGE produces parvalbumin-, somatostatin-, and nitric oxide synthase-expressing interneurons including fast-spiking basket, bistratified, axo-axonic, oriens-lacunosum moleculare, neurogliaform, and ivy cells. In contrast, CGE-derived interneurons contain cholecystokinin, calretinin, vasoactive intestinal peptide, and reelin including non-fast-spiking basket, Schaffer collateral-associated, mossy fiber-associated, trilaminar, and additional neurogliaform cells. Our findings provide a basic blueprint of the developmental origins of hippocampal interneuron diversity.
Asunto(s)
Movimiento Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo , Interneuronas/clasificación , Interneuronas/metabolismo , Organizadores Embrionarios , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/fisiología , Análisis por Conglomerados , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Hipocampo/citología , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/embriología , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Organizadores Embrionarios/citología , Organizadores Embrionarios/embriología , Organizadores Embrionarios/crecimiento & desarrollo , Péptidos/genética , Péptidos/metabolismo , ARN Mensajero/metabolismo , Proteína Reelina , Factores de Tiempo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.
Asunto(s)
Células Madre Embrionarias/citología , Notocorda/citología , Organizadores Embrionarios/citología , Animales , Antígenos de Diferenciación/metabolismo , Benzamidas/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular , Embrión de Pollo , Dioxoles/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Proteínas Fluorescentes Verdes/biosíntesis , Factor Nuclear 3-beta del Hepatocito/farmacología , Factor Nuclear 3-beta del Hepatocito/fisiología , Proteínas de Homeodominio/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Ratones , Organizadores Embrionarios/metabolismo , Hormonas Peptídicas/farmacología , Hormonas Peptídicas/fisiología , Pirroles/farmacología , Receptores de Factores de Crecimiento/agonistas , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Transducción de Señal , Técnicas de Cultivo de Tejidos , Trasplante HeterólogoRESUMEN
Among spiral cleaving embryos (e.g. mollusks and annelids), it has long been known that one blastomere at the four-cell stage, the D cell, and its direct descendants play an important role in axial pattern formation. Various studies have suggested that the D quadrant acts as the organizer of the embryonic axes in annelids, although this has never been demonstrated directly. Here we show that D quadrant micromeres (2d and 4d) of the oligochaete annelid Tubifex tubifex are essential for embryonic axis formation. When 2d and 4d were ablated the embryo developed into a rounded cell mass covered with an epithelial cell sheet. To examine whether 2d and 4d are sufficient for axis formation they were transplanted to an ectopic position in an otherwise intact embryo. The reconstituted embryo formed a secondary embryonic axis with a duplicated head and/or tail. Cell lineage analyses showed that neuroectoderm and mesoderm along the secondary axis were derived from the transplanted D quadrant micromeres and not from the host embryo. However, endodermal tissue along the secondary axis originated from the host embryo. Interestingly, when either 2d or 4d was transplanted separately to host embryos, the reconstituted embryos failed to form a secondary axis, suggesting that both 2d and 4d are required for secondary axis formation. Thus, the Tubifex D quadrant micromeres have the ability to organize axis formation, but they lack the ability to induce neuroectodermal tissues, a characteristic common to chordate primary embryonic organizers.
Asunto(s)
Anélidos/embriología , Animales , Anélidos/citología , Blastómeros/trasplante , Colorantes Fluorescentes/administración & dosificación , Mesodermo/embriología , Microinyecciones , Placa Neural/embriología , Neurogénesis , Organizadores Embrionarios/citología , Especificidad de la EspecieRESUMEN
The role of microRNAs in embryonic cell fate specification is largely unknown. In vertebrates, the miR-430/427/302 family shows a unique expression signature and is exclusively expressed during early embryogenesis. Here, we comparatively address the embryonic function of miR-302 in human embryonic stem cells (hESCs) and its ortholog miR-427 in Xenopus laevis. Interestingly, we found that this miRNA family displays species-specific target selection among ligands of the Nodal pathway, with a striking conservation of the inhibitors, Lefties, but differential targeting of the activators, Nodals. The Nodal pathway plays a crucial role in germ layer specification. Accordingly, by gain and loss of function experiments in hESCs, we show that miR-302 promotes the mesendodermal lineage at the expense of neuroectoderm formation. Similarly, depletion of miR-427 in Xenopus embryos hinders the organizer formation and leads to severe dorsal mesodermal patterning defects. These findings suggest a crucial role for the miR-430/427/302 family in vertebrate embryogenesis by controlling germ layer specification.
Asunto(s)
Tipificación del Cuerpo , Linaje de la Célula , Endodermo/citología , Mesodermo/citología , MicroARNs/metabolismo , Xenopus laevis/embriología , Animales , Secuencia de Bases , Tipificación del Cuerpo/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Endodermo/efectos de los fármacos , Endodermo/embriología , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Factores de Determinación Derecha-Izquierda/metabolismo , Mesodermo/efectos de los fármacos , Mesodermo/embriología , Mesodermo/metabolismo , MicroARNs/genética , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Organizadores Embrionarios/citología , Organizadores Embrionarios/efectos de los fármacos , Organizadores Embrionarios/embriología , Especificidad de la Especie , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMEN
In vertebrates, the readily apparent left/right (L/R) anatomical asymmetries of the internal organs can be traced to molecular events initiated at or near the time of gastrulation. However, the earliest steps of this process do not seem to be universally conserved. In particular, how this axis is first defined in chicks has remained problematic. Here we show that asymmetric cell rearrangements take place within chick embryos, creating a leftward movement of cells around the node. It is the relative displacement of cells expressing sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8) that is responsible for establishing their asymmetric expression patterns. The creation of asymmetric expression domains as a passive effect of cell movements represents an alternative strategy for breaking L/R symmetry in gene activity.