RESUMEN
The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knockdown (KD) or knockout (KO), we combine the inactivation of the Arp2/3 inhibitory protein arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit CYFIP2, all of which enhance the polymerization of cortical branched actin. Inactivation of the three negative regulators of cortical branched actin increases migration persistence of human breast MCF10A cells and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO cells, but not in triple KD cells, the 'super-migrator' phenotype was associated with a heterogenous downregulation of vimentin (VIM) expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells largely restored normal persistence of single cell migration, suggesting that vimentin downregulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.
Asunto(s)
Actinas , Pez Cebra , Animales , Humanos , Actinas/metabolismo , Vimentina/genética , Vimentina/metabolismo , Pez Cebra/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Movimiento Celular/fisiología , Proteínas Portadoras/metabolismoRESUMEN
Simultaneous imaging of multiple labels in tissues is key to studying complex biological processes. Although strategies for color multiphoton excitation have been established, chromatic aberration remains a major problem when multiple excitation wavelengths are used in a scanning microscope. Chromatic aberration introduces a spatial shift between the foci of beams of different wavelengths that varies across the field of view, severely degrading the performance of color imaging. In this work, we propose an adaptive correction strategy that solves this problem in two-beam microscopy techniques. Axial chromatic aberration is corrected by a refractive phase mask that introduces pure defocus into one beam, while lateral chromatic aberration is corrected by a piezoelectric mirror that dynamically compensates for lateral shifts during scanning. We show that this light-efficient approach allows seamless chromatic correction over the entire field of view of different multiphoton objectives without compromising spatial and temporal resolution and that the effective area for beam-mixing processes can be increased by more than 1 order of magnitude. We illustrate this approach with simultaneous three-color, two-photon imaging of developing zebrafish embryos and fixed Brainbow mouse brain slices over large areas. These results establish a robust and efficient method for chromatically corrected multiphoton imaging.
RESUMEN
Cilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing, and development. Reduced actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR (Bin/amphiphysin/Rvs) domain that we named MiniBAR (also known as KIAA0355/GARRE1), which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia, resulting from abnormal Rac-GTP/Rho-GTP levels and increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies, including left-right asymmetry defects. Thus, MiniBAR is a dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis.
Asunto(s)
Proteínas del Citoesqueleto , Pez Cebra , Animales , Pez Cebra/metabolismo , Proteínas del Citoesqueleto/metabolismo , Transducción de Señal , Proteínas Portadoras/metabolismo , Cilios/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Mapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain and tissue physiology. Current microscopy methods are limited either in sensitivity or in spatio-temporal resolution. In this work, we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. First, we propose a novel experimental scheme for color TSFG microscopy, which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band of hemoglobin. We show that there is a strong three-photon (3P) resonance related to the Soret band of hemoglobin in THG and TSFG signals from zebrafish and human RBCs, and that this resonance is sensitive to RBC oxygenation state. We demonstrate that our color TSFG implementation enables specific detection of flowing RBCs in zebrafish embryos and is sensitive to RBC oxygenation dynamics with single-cell resolution and microsecond pixel times. Moreover, it can be implemented on a 3P microscope and provides label-free RBC-specific contrast at depths exceeding 600 µm in live adult zebrafish brain. Our results establish a new multiphoton contrast extending the palette of deep-tissue microscopy.
RESUMEN
Morphogenesis, wound healing, and some cancer metastases depend upon the migration of cell collectives that need to be guided to their destination as well as coordinated with other cell movements. During zebrafish gastrulation, the extension of the embryonic axis is led by the mesendodermal polster that migrates toward the animal pole, followed by the axial mesoderm that undergoes convergence and extension. Here, we investigate how polster cells are guided toward the animal pole. Using a combination of precise laser ablations, advanced transplants, and functional as well as in silico approaches, we establish that each polster cell is oriented by its immediate follower cells. Each cell perceives the migration of followers, through E-cadherin/α-catenin mechanotransduction, and aligns with them. Therefore, directional information propagates from cell to cell over the whole tissue. Such guidance of migrating cells by followers ensures long-range coordination of movements and developmental robustness.
Asunto(s)
Mecanotransducción Celular , Pez Cebra , Animales , Movimiento Celular/fisiología , Mesodermo , alfa CateninaRESUMEN
To investigate the role of mechanical constraints in morphogenesis and development, we have developed a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. The local shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the zebrafish embryo during gastrulation, our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.
Asunto(s)
Embrión no Mamífero/citología , Imagenología Tridimensional/métodos , Resistencia al Corte , Animales , Agregación Celular , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Embrión no Mamífero/metabolismo , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Pez CebraRESUMEN
Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns.
Asunto(s)
Gástrula , Pez Cebra , Animales , Ectodermo , Morfogénesis , FotonesRESUMEN
Being optically clear, the zebrafish embryo is a nice model system to analyze cell migration in vivo. This chapter describes a combination of injection and cell transplant procedures that allows creation of mosaic embryos, containing a few cells labeled differently from their neighbors. Rapid 5D confocal imaging of these embryos permits to simultaneously track and quantify the movement of large cell groups, as well as analyze the cellular or subcellular dynamics of transplanted cells during their migration. In addition, expression of a candidate gene can be modified in transplanted cells. Comparing behavior of these cells to control or neighboring cells allows determination of the role of the candidate gene in cell migration. We describe the procedure, focusing on one specific cell population during gastrulation, but it can easily be adapted to other cell populations and other migration events during early embryogenesis.
Asunto(s)
Movimiento Celular/fisiología , Embrión no Mamífero/citología , Microscopía Confocal/métodos , Animales , Pez CebraRESUMEN
Germ-layer formation during gastrulation is both a fundamental step of development and a paradigm for tissue formation and remodeling. However, the cellular and molecular basis of germ-layer segregation is poorly understood, mostly because of the lack of direct in vivo observations. We used mosaic zebrafish embryos to investigate the formation of the endoderm. High-resolution live imaging and functional analyses revealed that endodermal cells reach their characteristic innermost position through an active, oriented, and actin-based migration dependent on Rac1, which contrasts with the previously proposed differential adhesion cell sorting. Rather than being attracted to their destination, the yolk syncytial layer, cells appear to migrate away from their neighbors. This migration depends on N-cadherin that, when imposed in ectodermal cells, is sufficient to trigger their internalization without affecting their fate. Overall, these results lead to a model of germ-layer formation in which, upon N-cadherin expression, endodermal cells actively migrate away from their epiblastic neighbors to reach their internal position, revealing cell-contact avoidance as an unexplored mechanism driving germ-layer formation.
Asunto(s)
Cadherinas/metabolismo , Movimiento Celular , Endodermo/citología , Pez Cebra/embriología , Proteína de Unión al GTP rac1/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Citoesqueleto/fisiologíaRESUMEN
Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.
Asunto(s)
Movimiento Celular/fisiología , Trasplante de Células , Pez Cebra/embriología , Animales , Gastrulación , Estudios Prospectivos , Imagen de Lapso de TiempoRESUMEN
TORC2 is a serine-threonine kinase complex conserved through evolution that recently emerged as a new regulator of actin dynamics and cell migration. However, knockout in mice of its core components Sin1 and Rictor is embryonic lethal, which has limited in vivo analyses. Here, we analysed TORC2 function during early zebrafish development, using a morpholino-mediated loss of function of sin1. Sin1 appears required during gastrulation for migration of the prechordal plate, the anterior most mesoderm. In absence of Sin1, cells migrate both slower and less persistently, which can be correlated to a reduction in actin-rich protrusions and a randomisation of the remaining protrusions. These results demonstrate that, as established in vitro, the TORC2 component Sin1 controls actin dynamics and cell migration in vivo. We furthermore establish that Sin1 is required for protrusion formation downstream of PI3K, and is acting upstream of the GTPase Rac1, since expression of an activated form of Rac1 is sufficient to rescue sin1 loss of function.
Asunto(s)
Gastrulación , Mesodermo/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Actinas/metabolismo , Animales , Animales Modificados Genéticamente/crecimiento & desarrollo , Animales Modificados Genéticamente/metabolismo , Movimiento Celular , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/antagonistas & inhibidores , Oligonucleótidos Antisentido/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Imagen de Lapso de Tiempo , Pez Cebra/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Movimiento Celular/genética , Seudópodos/genética , Seudópodos/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Dictyostelium/genética , Dictyostelium/metabolismo , Embrión no Mamífero , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Ratones , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pez Cebra/genéticaRESUMEN
Collective cell migration is key to morphogenesis, wound healing, or cancer cell migration. However, its cellular bases are just starting to be unraveled. During vertebrate gastrulation, axial mesendoderm migrates in a group, the prechordal plate, from the embryonic organizer to the animal pole. How this collective migration is achieved remains unclear. Previous work has suggested that cells migrate as individuals, with collective movement resulting from the addition of similar individual cell behavior. Through extensive analyses of cell trajectories, morphologies, and polarization in zebrafish embryos, we reveal that all prechordal plate cells show the same behavior and rely on the same signaling pathway to migrate, as expected if they do so individually. However, by using cell transplants, we demonstrate that prechordal plate migration is a true collective process, as isolated cells do not migrate toward the animal pole. They are still polarized and motile but lose directionality. Directionality is restored upon contact with the endogenous prechordal plate. This contact dependent orientation relies on E-cadherin, Wnt-PCP signaling, and Rac1. Importantly, groups of cells also need contact with the endogenous plate to orient correctly, showing an instructive role of the plate in establishing directionality. Overall, our results lead to an original model of collective migration in which directional information is contained within the moving group rather than provided by extrinsic cues, and constantly maintained in cells by contacts with their neighbors. This self-organizing model could account for collective invasion of new territories, as observed in cancer strands, without requirement for any attractant in the colonized tissue.
Asunto(s)
Movimiento Celular/fisiología , Endodermo/fisiología , Mesodermo/fisiología , Morfogénesis/fisiología , Transducción de Señal/fisiología , Animales , Cadherinas/metabolismo , Polaridad Celular/fisiología , Endodermo/citología , Hibridación in Situ , Mesodermo/citología , Imagen de Lapso de Tiempo , Vía de Señalización Wnt/fisiología , Pez CebraRESUMEN
The Frizzled receptor and Dishevelled effector regulate mitotic spindle orientation in both vertebrates and invertebrates, but how Dishevelled orients the mitotic spindle is unknown. Using the Drosophila S2 cell "induced polarity" system, we find that Dishevelled cortical polarity is sufficient to orient the spindle and that Dishevelled's DEP domain mediates this function. This domain binds a C-terminal domain of Mud (the Drosophila NuMA ortholog), and Mud is required for Dishevelled-mediated spindle orientation. In Drosophila, Frizzled-Dishevelled planar cell polarity (PCP) orients the sensory organ precursor (pI) spindle along the anterior-posterior axis. We show that Dishevelled and Mud colocalize at the posterior cortex of pI, Mud localization at the posterior cortex requires Dsh, and Mud loss-of-function randomizes spindle orientation. During zebrafish gastrulation, the Wnt11-Frizzled-Dishevelled PCP pathway orients spindles along the animal-vegetal axis, and reducing NuMA levels disrupts spindle orientation. Overall, we describe a Frizzled-Dishevelled-NuMA pathway that orients division from Drosophila to vertebrates.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , División Celular/fisiología , Polaridad Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Receptores Frizzled/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Fosfoproteínas/metabolismo , Pez Cebra , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Línea Celular , Linaje de la Célula , Proteínas Dishevelled , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Dineínas/metabolismo , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/fisiología , Receptores Frizzled/genética , Gastrulación , Inhibidores de Disociación de Guanina Nucleótido/genética , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas Asociadas a Matriz Nuclear/genética , Fosfoproteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Huso Acromático/metabolismo , Pez Cebra/anatomía & histología , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
During gastrulation, dramatic movements rearrange cells into three germ layers expanded over the entire embryo [1-3]. In fish, both endoderm and mesoderm are specified as a belt at the embryo margin. Mesodermal layer expansion is achieved through the combination of two directed migrations. The outer ring of precursors moves toward the vegetal pole and continuously seeds mesodermal cells inside the embryo, which then reverse their movement in the direction of the animal pole [3-6]. Unlike mesoderm, endodermal cells internalize at once and must therefore adopt a different strategy to expand over the embryo [7, 8]. With live imaging of YFP-expressing zebrafish endodermal cells, we demonstrate that in contrast to mesoderm, internalized endodermal cells display a nonoriented/noncoordinated movement fit by a random walk that rapidly disperses them over the yolk surface. Transplantation experiments reveal that this behaviour is largely cell autonomous, induced by TGF-beta/Nodal, and dependent on the downstream effector Casanova. At midgastrulation, endodermal cells switch to a convergence movement. We demonstrate that this switch is triggered by environmental cues. These results uncover random walk as a novel Nodal-induced gastrulation movement and as an efficient strategy to transform a localized cell group into a layer expanded over the embryo.
Asunto(s)
Movimiento Celular/fisiología , Endodermo/citología , Gastrulación/fisiología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente/embriología , Animales Modificados Genéticamente/fisiología , Inducción Embrionaria/fisiología , Endodermo/fisiología , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteína Nodal , Factores de Transcripción SOX , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/metabolismoRESUMEN
Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of Galphai depends on Partner of Inscuteable (Pins). We establish here that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Galphai, regulates cortical localization of the subunits Galphai and Gbeta13F. Ric-8, Galphai and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis.
Asunto(s)
División Celular/fisiología , Drosophila/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Neuronas Aferentes/fisiología , Animales , Animales Modificados Genéticamente , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Receptores Frizzled/fisiología , Subunidades beta de la Proteína de Unión al GTP/fisiología , Proteínas de Unión al GTP/genética , Huso Acromático/metabolismo , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Cell migration plays an essential role in many morphogenetic processes, and its deregulation has many dramatic consequences. Yet how migration is controlled during normal development is still a largely unresolved question. We examined this process in the case of the posterior lateral line (PLL), a mechanosensory system present in fish and amphibians. In zebrafish, the embryonic PLL comprises seven to eight sense organs (neuromasts) aligned from head to tail along the flank of the animal and is formed by a primordium that originates from a cephalic placode. This primordium migrates along a stereotyped pathway toward the tip of the tail and deposits in its wake discrete groups of cells, each of which will become a neuromast. We show that a trail of SDF1-like chemokine is present along the pathway of the primordium and that a CXCR4-like chemokine receptor is expressed by the migrating cells. The inactivation of either the ligand or its receptor blocks migration, whereas in mutants in which the normal SDF1 trail is absent, the primordium path is redirected to the next, more ventral sdf1 expression domain. In all cases, the sensory axons remain associated to the primordium, indicating that the extension of the neurites to form the PLL nerve depends on the movement of the primordium. We conclude that both the formation and the innervation of this system depend on the SDF1-CXCR4 system, which has also been implicated in several migration events in humans, including metastasis formation and lymphocyte homing.
Asunto(s)
Quimiocinas CXC/fisiología , Neuronas Aferentes/citología , Receptores CXCR4/fisiología , Órganos de los Sentidos/embriología , Pez Cebra/genética , Animales , Axones/ultraestructura , Linaje de la Célula , Movimiento Celular , Quimiocina CXCL12 , Quimiocinas CXC/análisis , Quimiocinas CXC/genética , Inducción Embrionaria , Neuritas/ultraestructura , Receptores CXCR4/análisis , Receptores CXCR4/genética , Órganos de los Sentidos/inervación , Pez Cebra/embriologíaRESUMEN
The vertebrate head skeleton is derived in part from neural crest cells, which physically interact with head ectoderm, mesoderm and endoderm to shape the pharyngeal arches. The cellular and molecular nature of these interactions is poorly understood, and we explore here the function of endoderm in this process. By genetic ablation and reintroduction of endoderm in zebrafish, we show that it is required for the development of chondrogenic neural crest cells, including their identity, survival and differentiation into arch cartilages. Using a genetic interference approach, we further identify Fgf3 as a critical component of endodermal function that allows the development of posterior arch cartilages. Together, our results reveal for the first time that the endoderm provides differential cues along the anteroposterior axis to control ventral head skeleton development and demonstrate that this function is mediated in part by Fgf3.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Cráneo/embriología , Proteínas de Pez Cebra , Animales , Región Branquial/embriología , Cartílago Articular/embriología , Diferenciación Celular , Endodermo , Factor 3 de Crecimiento de Fibroblastos , Cabeza/embriología , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Morfogénesis , Cresta Neural/citología , Factores de Transcripción SOX , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
Endoderm originates from a large endomesodermal field requiring Nodal signalling. The mechanisms that ensure segregation of endoderm from mesoderm are not fully understood. We first show that the timing and dose of Nodal activation are crucial for endoderm formation and the endoderm versus mesoderm fate choice, because sustained Nodal signalling is required to ensure endoderm formation but transient signalling is sufficient for mesoderm formation. In zebrafish, downstream of Nodal signals, three genes encoding transcription factors (faust, bonnie and clyde and the recently identified gene casanova) are required for endoderm formation and differentiation. However their positions within the pathway are not completely established. In the present work, we show that casanova is the earliest specification marker for endodermal cells and that its expression requires bonnie and clyde. Furthermore, we have analysed the molecular activities of casanova on endoderm formation and found that it can induce endodermal markers and repress mesodermal markers during gastrulation, as well as change the fate of marginal blastomeres to endoderm. Overexpression of casanova also restores endoderm markers in the absence of Nodal signalling. In addition, casanova efficiently restores later endodermal differentiation in these mutants, but this process requires, in addition, a partial activation of Nodal signalling.