RESUMEN
The vertebrate nervous system arises through a series of inductive interactions, beginning with the induction of the neural plate and the rostrocaudal patterning of the neural tube. The process continues with dorsoventral patterning of the neural tube, during which floor plate cells and motor neurons are induced ventrally by interactions of the neural tube with the notochord, and dorsal cell types are induced via neural plate/ectodermal interactions. Later interactions result in the formation of interneurons as well as neuronal migrations. Recent progress, guided in part by knowledge of evolutionary conservation of transcription factors and signaling pathways, is beginning to reveal the cellular and molecular bases of each of these steps in neuronal patterning.
Asunto(s)
Sistema Nervioso/embriología , Vertebrados/embriología , Animales , Evolución Biológica , Tipificación del Cuerpo , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Sistema Nervioso/citología , Transducción de Señal , Vertebrados/genéticaRESUMEN
The study of cell lineages has been, and remains, of crucial importance in developmental biology. It requires the identification of a cell or group of cells and of all of their descendants during embryonic development. Here, we provide a brief survey of how different techniques for achieving this have evolved over the last 100 years.
Asunto(s)
Desarrollo Embrionario y Fetal , Morfogénesis , Sistema Nervioso/embriología , Animales , Biología Evolutiva/historia , Biología Evolutiva/tendencias , Historia del Siglo XX , Sistema Nervioso/citologíaRESUMEN
Previous analyses of labelled clones of cells within the developing nervous system of the mouse have indicated that descendants are initially dispersed rostrocaudally followed by more local proliferation, which is consistent with the progressing node's contributing descendants from a resident population of progenitor cells as it advances caudally. Here we electroporated an expression vector encoding green fluorescent protein into the chicken embryo near Hensen's node to test and confirm the pattern inferred in the mouse. This provides a model in which a proliferative stem zone is maintained in the node by a localized signal; those cells that are displaced out of the stem zone go on to contribute to the growing axis. To test whether fibroblast growth factor (FGF) signalling could be involved in the maintenance of the stem zone, we co-electroporated a dominant-negative FGF receptor with a lineage marker, and found that it markedly alters the elongation of the spinal cord primordium. The results indicate that FGF receptor signalling promotes the continuous development of the posterior nervous system by maintaining presumptive neural progenitors in the region near Hensen's node. This offers a potential explanation for the mixed findings on FGF in the growth and patterning of the embryonic axis.
Asunto(s)
Neuronas/fisiología , Organizadores Embrionarios/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Animales , Embrión de Pollo , Inducción Embrionaria , Transducción de Señal/fisiología , Médula Espinal/fisiologíaRESUMEN
Fluorescence lifetime imaging microscopy (FLIM) with phasor analysis provides easy visualization and analysis of fluorophores' lifetimes which is valuable for multiple applications including metabolic imaging, STED imaging, FRET imaging and functional imaging. However, FLIM imaging typically suffers from low photon budgets, leading to unfavorable signal to noise ratios which in many cases prevent extraction of information from the data. Traditionally, median filters are applied in phasor analysis to tackle this problem. This unfortunately degrades high spatial frequency FLIM information in the phasor analysis. These high spatial frequency components are typically edges of features and puncta, which applies to membranes, mitochondria, granules and small organelles in a biological sample. To tackle this problem, we propose a filtering strategy with complex wavelet filtering and Anscombe transform for FLIM phasor analysis. This filtering strategy preserves fine structures and reports accurate lifetimes in photon starved FLIM imaging. Moreover, this filter outperforms median filters and makes FLIM imaging with lower laser power and faster imaging possible.
RESUMEN
Magnetic resonance imaging (MRI) is a non-invasive imaging method that provides three-dimensional (3-D) images of the internal structure of opaque objects, such as humans and mice. In optimal situations, spatial resolution can approach the micron level. Arbitrarily oriented single-slice images can be obtained in seconds, with full 3-D volume images taking tens of minutes to collect. The exquisite sensitivity of MRI to the local physical and chemical environment provides a wide range of mechanisms giving rise to intrinsic contrast in the MR experiment, thus providing images with dramatic differences between different tissue types (e.g. white vs grey matter, myelinated vs unmyelinated fibres, and brain parenchyma vs ventricles). The recent advent of physiologically sensitive MRI contrast agents opens up a wealth of new avenues of study, even including the in vivo imaging of gene expression.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Animales , Humanos , Ratones , Vertebrados/crecimiento & desarrolloRESUMEN
Acetylcholine receptors become clustered at the neuromuscular junction during synaptogenesis, at least in part via lateral migration of diffusely expressed receptors. We have shown previously that electric fields initiate a specific receptor clustering event which is dependent on lateral migration in aneural muscle cell cultures (Stollberg, J., and S. E. Fraser. 1988. J. Cell Biol. 107:1397-1408). Subsequent work with this model system ruled out the possibility that the clustering event was triggered by increasing the receptor density beyond a critical threshold (Stollberg, J., and S. E. Fraser. 1990. J. Neurosci. 10:247-255). This leaves two possibilities: the clustering event could be triggered by the field-induced change in the density of some other molecule, or by a membrane voltage-sensitive mechanism (e.g., a voltage-gated calcium signal). Electromigration is a slow, linear process, while voltage-sensitive mechanisms respond in a rapid, nonlinear fashion. Because of this the two possibilities make different predictions about receptor clustering behavior in response to pulsed or alternating electric fields. In the present work we have studied subcellular calcium distributions, as well as receptor clustering, in response to such fields. Subcellular calcium distributions were quantified and found to be consistent with the predicted nonlinear response. Receptor clustering, however, behaves in accordance with the predictions of a linear response, consistent with the electromigration hypothesis. The experiments demonstrate that a local increase in calcium, or, more generally, a voltage-sensitive mechanism, is not sufficient and probably not necessary to trigger receptor clustering. Experiments with slowly alternating electric fields confirm the view that the clustering of acetylcholine receptors is initiated by a local change in the density of some non-receptor molecule.
Asunto(s)
Agregación de Receptores/fisiología , Receptores Colinérgicos/metabolismo , Animales , Calcio/fisiología , Simulación por Computador , Estimulación Eléctrica , Modelos Biológicos , Músculos/citología , Músculos/embriología , Músculos/metabolismo , Xenopus laevisRESUMEN
Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of approximately 2.3 X 10(-9) cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptor aggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusion-mediated aggregation.
Asunto(s)
Músculos/fisiología , Receptores de Concanavalina A/fisiología , Receptores Nicotínicos/fisiología , Citoesqueleto de Actina/fisiología , Animales , Bungarotoxinas/metabolismo , Calcio/fisiología , Células Cultivadas , Difusión , Electroforesis , Fluidez de la Membrana , Microtúbulos/fisiología , Tripsina/metabolismo , Grabación en Video , XenopusRESUMEN
A prospective lineage analysis was performed to determine the variety of cell types that could be formed by individual precursor cells of the developing frog retina. Fluorescent dextran was iontophoretically injected into single cells of the embryonic optic vesicle. After further development of the embryo, labeled descendants were observed in all three layers of the larval retina. Furthermore, different clones were composed of various combinations of all major cell types, including the glial Müller cells. Hence, single optic vesicle cells have the potential to form any type of retinal cell, suggesting that the interactions that specify the differentiation pathway of retinal cells must occur late in development.
Asunto(s)
Retina/citología , Células Madre/citología , Animales , Diferenciación Celular , Células Clonales/citología , Dextranos , Colorantes Fluorescentes , Iontoforesis , Lisina , Microinyecciones , Microscopía Fluorescente , Neuroglía/citología , Neuronas/citología , Retina/embriología , Rodaminas , Xenopus laevisRESUMEN
The organizer of the amphibian gastrula provides the neurectoderm with both neuralizing and posteriorizing (transforming) signals. In zebrafish, transplantations show that a spatially distinct transformer signal emanates from tissues other than the organizer. Cells of the germring (nonaxial mesendoderm) posteriorized forebrain progenitors when grafted nearby, resulting in an ectopic hindbrain-like structure; in contrast, cells of the organizer (axial mesendoderm) caused no posterior transformation. Local application of basic fibroblast growth factor, a candidate transformer in Xenopus, caused malformation but not hindbrain transformation in the forebrain. Thus, the zebrafish gastrula may integrate spatially distinct signals from the organizer and the germring to pattern the neural axis.
Asunto(s)
Tipificación del Cuerpo , Gástrula/fisiología , Mesencéfalo/embriología , Rombencéfalo/embriología , Pez Cebra/embriología , Animales , Trasplante de Células , Proteínas de Unión al ADN/biosíntesis , Proteína 2 de la Respuesta de Crecimiento Precoz , Ectodermo/citología , Ectodermo/fisiología , Ectodermo/trasplante , Endodermo/citología , Endodermo/fisiología , Endodermo/trasplante , Factor 2 de Crecimiento de Fibroblastos/farmacología , Mesodermo/citología , Mesodermo/fisiología , Mesodermo/trasplante , Morfogénesis , Proteínas Recombinantes/farmacología , Células Madre/fisiología , Factores de Transcripción/biosíntesisRESUMEN
Key events in vertebrate embryogenesis are difficult to observe in many species. High-resolution magnetic resonance imaging was used to follow cell movements and lineages in developing frog embryos. A single cell was injected at the 16-cell stage with a contrast agent, based on the gadolinium chelate gadolinium-diethylenetriamine pentaacetic acid-dextran. The labeled progeny cells could be followed uniquely in three-dimensional magnetic resonance images, acquired from the embryo over several days. The results show that external ectodermal and internal mesodermal tissues extend at different rates during amphibian gastrulation and neurulation.
Asunto(s)
Blastómeros/citología , Embrión no Mamífero/citología , Embriología/métodos , Gadolinio DTPA , Gástrula/citología , Imagen por Resonancia Magnética , Animales , Blastocisto/citología , Dextranos , Ectodermo/citología , Gadolinio , Mesodermo/citología , Microscopía , Morfogénesis , Ácido Pentético/análogos & derivados , Xenopus laevis/embriologíaRESUMEN
Neuromasts, the mechanoreceptors of the lateral line system of fishes and aquatic amphibians, have previously been thought to develop exclusively from embryonic epidermal placodes. Use of fate mapping techniques shows that neuromasts of the head and body of zebrafish, Siamese fighting fish, and Xenopus are also derived from neural crest. Neural crest migrates away from the neural tube in developing vertebrates to form much of the peripheral nervous system, pigment cells, and skeletal elements of the head. The data presented here demonstrate that neuromasts are derived from both neural crest and epidermal placodes.
Asunto(s)
Epidermis/embriología , Peces/embriología , Mecanorreceptores/embriología , Cresta Neural/embriología , Xenopus/embriología , Animales , Diferenciación Celular , Movimiento Celular , Sistema Nervioso Central/embriología , Embrión no Mamífero/citología , Células Epidérmicas , Colorantes Fluorescentes , Cresta Neural/citología , Sistema Nervioso Periférico/embriología , Pez Cebra/embriologíaRESUMEN
Removing the posterior (temporal) two-thirds of the Xenopus eye bud produces a remaining fragment, which becomes round and grows to a normal adult size eye. Electrophysiological and anatomical analyses showed that each of the two halves of this eye projected across the entire optic tectum in mirror image (double-nasal) fashion, and that fibers from each half-eye sorted out to form eye dominance stripes on the tectum. That both halves of the mirror-symmetric map were derived from only one animal, and from only one side of the head, rules out global markers such as right versus left and histocompatibility differences as causing the formation of these stripes.
Asunto(s)
Ojo/inervación , Visión Ocular/fisiología , Animales , Electrofisiología , Nervio Óptico/fisiología , Retina/fisiología , Colículos Superiores/fisiología , XenopusRESUMEN
The cells that make up the body column of hydra are extensively joined by gap junctions, capable of mediating the rapid exchange of small hydrophilic molecules between the cytoplasms of neighboring cells. Both the rate of transfer of small molecules through the gap junctions and the rate of return of gap junction coupling after grafting experiments are sufficiently rapid to mediate events in the patterning of hydra tissue. Antibodies to the major rat liver gap junction protein (27,000 daltons) recognize a gap junction antigen in hydra and are effective in eliminating junctional communication between hydra cells. The antibodies perturb the head inhibition gradient in grafting operations, suggesting that cell-cell communication via gap junctions is important in this defined tissue patterning process.
Asunto(s)
Comunicación Celular , Hydra/fisiología , Uniones Intercelulares/fisiología , Animales , Conexinas , Electrofisiología , Técnica del Anticuerpo Fluorescente , Técnicas Inmunológicas , Isoquinolinas , Proteínas de la Membrana/inmunología , MorfogénesisRESUMEN
Dynamic remodeling of retinal ganglion cell terminal arbors has been proposed to contribute to formation of the topographically ordered retinotectal projection. To test this directly, the growth of individual terminal arbors was observed in live X. laevis tadpoles using a confocal microscope to visualize their complex three-dimensional structure. During initial development, nasal and temporal retinal arbors covered overlapping tectal areas. Despite subsequent remodeling, the dimensions and positions of the temporal arbors remained relatively stable. In contrast, the nasal arbors grew caudally, as they extended caudal branches and retracted rostral branches. These results suggest that differences in the remodeling of the nasal and temporal arbors lead to the emergence of retino-topography along the rostrocaudal axis of the tectum. All the terminal arbors were dynamic, including those with stable dimensions, suggesting that continual remodeling of arbors may be a universal feature of neuronal projections.
Asunto(s)
Retina/citología , Células Ganglionares de la Retina/citología , Colículos Superiores/citología , Animales , Axones/ultraestructura , División Celular , Microscopía Fluorescente , Xenopus laevisRESUMEN
To explore the role of BDNF during Xenopus visual system development, the expression of BDNF and TrkB, as well as the effect of BDNF during retinal ganglion cell (RGC) development in culture, was examined. BDNF mRNA was found to be expressed in both the developing eye and tectum, peaking at stage 45/46. The expression of BDNF coincided with RGC expression of full-length trkB transcripts, suggesting a functional role for BDNF. In culture, BDNF significantly increased the number of RGCs. The ability of BDNF to rescue differentiated RGCs that had projected to the tectum, the time course of the effect, and the lack of mitogenic response indicate that this neurotrophin promotes survival. The expression of BDNF and TrkB and the responsiveness of RGCs to BDNF coincide with retinal axon terminal arborization and patterning. Our results indicate that BDNF is a relevant neurotrophin for Xenopus RGC development and suggest that it plays a role during visual system patterning.
Asunto(s)
Ojo/crecimiento & desarrollo , Proteínas del Tejido Nervioso/fisiología , Colículos Superiores/crecimiento & desarrollo , Xenopus/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factor Neurotrófico Derivado del Encéfalo , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Ojo/embriología , Expresión Génica , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Células Ganglionares de la Retina/citología , Colículos Superiores/embriología , Xenopus/embriologíaRESUMEN
Dynamic rearrangements of axon terminal arbors may be critical for establishing appropriate connections in the developing nervous system. Here, the changes in complex retinal axon arbors in the tecta of live Xenopus larvae were followed during the formation of the topographic retinotectal projection. Three-dimensional reconstructions of terminal arbors made with a confocal microscope at hourly intervals revealed rapid remodeling of arbor extensions. Shorter branches were extended and retracted very rapidly, suggesting that they probe the environment for the optimal sites to form stable branches. About 27% of longer branches were present throughout the entire observation period and may be sites of stabilized synaptic contacts. Treatment of the animals to block postsynaptic activity resulted in increased rates of arbor rearrangements, which may coincide with decreased synapse stability. These studies reveal the dynamic behavior of nerve arbors and provide estimates for the lifetimes of retinotectal branches.
Asunto(s)
Axones/fisiología , Retina/ultraestructura , Colículos Superiores/ultraestructura , Animales , Axones/ultraestructura , Electrofisiología , Larva/ultraestructura , N-Metilaspartato/farmacología , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/ultraestructura , Retina/efectos de los fármacos , Retina/crecimiento & desarrollo , Colículos Superiores/crecimiento & desarrollo , Xenopus laevis/crecimiento & desarrolloRESUMEN
In recent years, the classical approaches of experimental embryology have been used in combination with more modern techniques to investigate aspects of neurogenesis. This combination has advanced our knowledge of several areas of neuronal development, including the lineages of neuronal precursors, the segmentation of the nervous system, and the patterning of the neural tube.
Asunto(s)
Sistema Nervioso/embriología , Animales , Inducción Embrionaria , Desarrollo Embrionario y Fetal , Morfogénesis , Cresta Neural/citología , Notocorda/fisiología , Células Madre/citologíaRESUMEN
In the past few years, we have seen a surge of interest in the zebrafish as a model system for the study of embryonic induction and patterning. This review summarizes our current knowledge of the organization of zebrafish fate maps during early development. Recent advances have addressed the relationship between early cleavage planes and the future dorsal axis, the pattern of cell mixing during blastula and gastrula stages, and the morphogenesis of the trunk neural keel. In addition, refined fate maps have become available for the embryonic shield, the central nervous system, and the heart. In combination with recent advances in molecular and genetic manipulations, these fate maps set the stage for new, more incisive, experimental approaches.
Asunto(s)
Pez Cebra/embriología , Animales , Blastómeros/citología , Sistema Nervioso Central/embriología , Inducción Embrionaria , Gástrula/citología , Corazón/embriología , MorfogénesisRESUMEN
Dynamic processes of neural development, such as migrations of precursor cells, growth of axons and dendrites, and formation and modification of synapses, can be fully analyzed only with techniques that monitor changes over time. Although there has been long-standing motivation for following cellular and synaptic events in vivo (intravital microscopy), until recently few preparations have been studied, and then often only with great effort. Innovations in low-light and laser-scanning microscopies, coupled with developments of new dyes and of genetically encoded indicators, have increased both the breadth and depth of in situ imaging approaches. Here we present the motivations and challenges for dynamic imaging methods, offer some illustrative examples and point to future opportunities with emerging technologies.
Asunto(s)
Sistema Nervioso Central/embriología , Colorantes , Procesamiento de Imagen Asistido por Computador/métodos , Neuronas/citología , Animales , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Imagen Asistido por Computador/tendencias , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Microscopía Confocal/tendencias , Microscopía por Video/instrumentación , Microscopía por Video/métodos , Microscopía por Video/tendencias , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Neuronas/metabolismo , Técnicas de Cultivo de Órganos/instrumentación , Técnicas de Cultivo de Órganos/métodos , Técnicas de Cultivo de Órganos/tendenciasRESUMEN
The upper rhombic lip (URL), a germinal zone in the dorsoanterior hindbrain, has long been known to be a source for neurons of the vertebrate cerebellum. It was thought to give rise to dorsally migrating granule cell precursors (Figure 1e); however, recent fate mapping studies have questioned the exclusive contributions of the URL to granule cells. By taking advantage of the clarity of the zebrafish embryo during the stages of brain morphogenesis, we have followed the fate of neuronal precursor cells generated within the upper rhombic lip directly. Combining a novel GFP labeling strategy with in vivo time-lapse imaging, we find, contrary to the former view, that most URL-descendants migrate anterior toward the midhindbrain boundary (MHB) and then course ventrally along the MHB (Figure 1f). As the migrating neuronal precursors reach the MHB, they form ventrally extending projections, likely axons, and continue ventral migration to settle outside of the cerebellum, in the region of the ventral brainstem. Thus, we define a new pathway for URL-derived neuronal precursor cells consistent with the recent fate maps. In addition, our results strongly suggest that the MHB plays a crucial role, not only in induction and patterning of the cerebellar anlage, but also in organizing its later morphogenesis by influencing cell migration.