RESUMEN
We report the cloning and expression patterns of three novel zebrafish Roundabout homologs. The Roundabout (robo) gene encodes a transmembrane receptor that is essential for axon guidance in Drosophila and Robo family members have been implicated in cell migration. Analysis of extracellular domains and conserved cytoplasmic motifs shows that zebrafish Robo1 and Robo2 are orthologs of mammalian Robo1 and Robo2, respectively, while zebrafish Robo3 is likely to be an ortholog of mouse Rig-1. The three zebrafish robos are expressed in distinct but overlapping patterns during embryogenesis. They are highly expressed in the developing nervous system, including the olfactory system, visual system, hindbrain, cranial ganglia, spinal cord, and posterior lateral line primordium. They are also expressed in several nonneuronal tissues, including somites and fin buds. The timing and patterns of expression suggest roles for zebrafish robos in axon guidance and cell migration. Wiley-Liss, Inc.
Asunto(s)
Axones/metabolismo , Axones/fisiología , Regulación del Desarrollo de la Expresión Génica , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Movimiento Celular , Clonación Molecular , Citoplasma/metabolismo , ADN Complementario/metabolismo , Drosophila , Ojo/embriología , Ganglios/embriología , Hibridación in Situ , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transducción de Señal , Médula Espinal/embriología , Factores de Tiempo , Distribución Tisular , Pez Cebra , Proteínas RoundaboutRESUMEN
As growing retinotectal axons navigate from the eye to the tectum, they sense guidance molecules distributed along the optic pathway. Mutations in the zebrafish astray gene severely disrupt retinal axon guidance, causing anterior-posterior pathfinding defects, excessive midline crossing, and defasciculation of the retinal projection. Eye transplantation experiments show that astray function is required in the eye. We identify astray as zebrafish robo2, a member of the Roundabout family of axon guidance receptors. Retinal ganglion cells express robo2 as they extend axons. Thus, robo2 is required for multiple axon guidance decisions during establishment of the vertebrate visual projection.
Asunto(s)
Axones/fisiología , Proteínas del Tejido Nervioso/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/fisiología , Células Ganglionares de la Retina/fisiología , Colículos Superiores/embriología , Alelos , Animales , Tipificación del Cuerpo , Mapeo Cromosómico , Cruzamientos Genéticos , Ojo/embriología , Ojo/trasplante , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes , Hibridación in Situ , Masculino , Mutación , Proteínas del Tejido Nervioso/fisiología , Fenotipo , Retina/embriología , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Colículos Superiores/citología , Vías Visuales/embriología , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez CebraRESUMEN
It has been hypothesized that taste buds are induced by contact with developing cranial nerve fibers late in embryonic development, since descriptive studies indicate that during embryonic development taste cell differentiation occurs concomitantly with or slightly following the advent of innervation. However, experimental evidence delineating the role of innervation in taste bud development is sparse and equivocal. Using two complementary experimental approaches, we demonstrate that taste cells differentiate fully in the complete absence of innervation. When the presumptive oropharyngeal region was taken from a donor axolotl embryo, prior to its innervation and development of taste buds, and grafted ectopically on to the trunk of a host embryo, the graft developed well-differentiated taste buds. Although grafts were invaded by branches of local spinal nerves, these neurites were rarely found near ectopic taste cells. When the oropharyngeal region was raised in culture, numerous taste buds were generated in the complete absence of neural elements. Taste buds in grafts and in explants were identical to those found in situ both in terms of their morphology and their expression of calretinin and serotonin immunoreactivity. Our findings indicate that innervation is not necessary for complete differentiation of taste receptor cells. We propose that taste buds are either induced in response to signals from other tissues, such as the neural crest, or arise independently through intrinsic patterning of the local epithelium.
Asunto(s)
Orofaringe/inervación , Papilas Gustativas/embriología , Ambystoma , Animales , Calbindina 2 , Diferenciación Celular , Técnicas de Cultivo , Embrión no Mamífero , Proteínas del Tejido Nervioso/análisis , Proteína G de Unión al Calcio S100/análisis , Serotonina/análisis , Papilas Gustativas/citología , Papilas Gustativas/trasplante , Trasplante Heterotópico , Tubulina (Proteína)/análisisRESUMEN
Navigating growth cones need signal transduction machinery to amplify and transmit the effects of extracellular signals throughout the growth cone. In culture, many drugs that affect second messengers are known to modulate neurite extension (with different effects on different neurons), and gradients of calcium influx and cyclic nucleotide analogs can cause growth cones to turn. However, it is not clear which of these responses are physiologically relevant, as axons grow through much more complex environments in vivo. The "exposed brain" preparation in Xenopus embryos provides an experimentally tractable system in which it is possible to study growth, pathfinding, and target recognition of retinal growth cones in vivo, while pharmacologically manipulating their signal transduction systems. These growth cones can also be easily studied in explant culture. We describe preliminary results of parallel in vivo and in vitro experiments using an array of drugs that perturb transduction molecules. Surprisingly, calcium ionophores and cyclic nucleotide analogs have no significant effect on retinal axon growth or pathfinding. Several agents including herbimycin A, ML-7, mastoparan, and RHC80267 inhibit retinal axon growth, both in vivo and in vitro, suggesting that tyrosine kinases, myosin, heterotrimeric G-proteins, and diacylglycerol lipase are important for retinal growth cones navigating in the optic pathway.
Asunto(s)
Axones/fisiología , Transducción de Señal , Vertebrados/fisiología , Animales , Vías Nerviosas/embriología , Retina/embriología , Sistemas de Mensajero Secundario , Colículos Superiores/embriología , Xenopus/embriologíaRESUMEN
The retinotectal map in Xenopus forms very early: retinal axons are topographically ordered along the dorsoventral axis of the tectum by stage 39, as they first arrive. To test whether topographic cues are present even earlier, we forced retinal axons to innervate the tectum prematurely by transplanting stage 28 eye primordia into stage 20 hosts, then assayed dorsoventral topography using focal injections of lipophilic dye into dorsal and ventral retina at donor stages 39-40. Unoperated and isochronic control projections showed normal dorsoventral ordering both in the optic tract and in the tectum. In contrast, projections from heterochronically transplanted eyes were ordered in the tract, but spread out upon entering the tectum and did not show significant dorsoventral ordering. Individual axons entering the tectum precociously often made abnormally abrupt and topographically incorrect turns. Thus, the topographical cues normally expressed in the tectum at stage 39 appear to be absent a few hours earlier. However, this lack of cues is only temporary, since heterochronic transplants allowed to survive to donor stages 45-46 showed normal topography. The absence of tectal topography at a stage when retinal axons can navigate to the young tectum strongly suggests that the molecules that provide tectal topographical signals are distinct from those used for pathfinding in the diencephalon and target recognition at the tectum.
Asunto(s)
Retina/embriología , Colículos Superiores/embriología , Animales , Axones , Movimiento Celular/fisiología , Procesamiento de Imagen Asistido por Computador , Microinyecciones , Microscopía Fluorescente , Retina/citología , Retina/trasplante , Colículos Superiores/citología , XenopusAsunto(s)
Axones , Retina/embriología , Colículos Superiores/embriología , Xenopus laevis/embriología , Animales , Moléculas de Adhesión Celular/fisiología , Embrión no Mamífero , Inducción Embrionaria , Proteínas de la Matriz Extracelular/fisiología , Proteínas del Ojo/fisiología , Morfogénesis , Proteínas del Tejido Nervioso/fisiología , Vías Visuales/embriología , Xenopus laevis/metabolismoRESUMEN
We have developed an exposed brain preparation for observing growth cone pathfinding behavior while performing in vivo pharmacological manipulations, and we used it to test whether Xenopus retinal growth cones need filopodia to navigate. Time-lapse video observation showed that cytochalasin B acted quickly and reversibly when applied; cytochalasin B-treated growth cones lacked filopodia, but had active lamellipodia and continued to advance slowly. Whereas normal retinotectal axons visualized with horseradish peroxidase turn caudally in the mid-diencephalon to reach the tectum, cytochalasin B-treated axons grew past the normal turning point and, instead, continued straight within the diencephalon. In dose-response experiments, pathfinding became abnormal in the same concentration range in which filopodia disappeared. These results suggest that filopodia are necessary for retinal growth cones to respond to guidance signals in the diencephalon.
Asunto(s)
Encéfalo/embriología , Embrión no Mamífero/fisiología , Neuronas/fisiología , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Citocalasina B/farmacología , Citocalasinas/farmacología , Relación Dosis-Respuesta a Droga , Vías Nerviosas/efectos de los fármacos , Neuronas/efectos de los fármacos , Factores de Tiempo , Xenopus laevisRESUMEN
Given the appropriate multicell electrophysiological techniques, small networks of cultured neurons (microcultures) are well suited to long-term studies of synaptic plasticity. To this end, we have developed an apparatus for optical recording from cultured vertebrate neurons using voltage-sensitive fluorescent dyes (Chien, C.-B., and J. Pine. 1991. J. Neurosci. Methods. 38:93-105). We evaluate here the usefulness of this technique for recording action potentials and synaptic potentials in microcultures of neurons from the rat superior cervical ganglion (SCG). After extensive dye screening and optimization of conditions, we chose the styryl dye RH423, which gave fast linear fluorescence changes of approximately 1%/100 mV for typical recordings. The root mean square noise of the apparatus (limited by shot noise) was typically 0.03%, equivalent to 3 mV of membrane potential. Illumination for at least 100 flashes of 100 ms each caused no noticeable photodynamic damage. Our results show that voltage-sensitive dyes can be used to record from microcultures of vertebrate neurons with high sensitivity. Dye signals were detected from both cell bodies and neurites. Signals from presumptive dendrites showed hyperpolarizations and action potentials simultaneous with those in the cell body, while those from presumptive axons showed delayed propagating action potentials. Subthreshold synaptic potentials in the cell body were occasionally detectable optically; however, they were usually masked by signals from axons passing through the same pixel. This is due to the complex anatomy of SCG microcultures, which have many crisscrossing neurites that often pass over cell bodies. Given a simpler microculture system with fewer neurites, it should be possible to use dye recording to routinely measure subthreshold synaptic strengths.
Asunto(s)
Potenciales de Acción , Colorantes Fluorescentes , Ganglios Simpáticos/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Animales Recién Nacidos , Células Cultivadas , Dendritas/fisiología , Hexametonio , Compuestos de Hexametonio/farmacología , Neuronas/efectos de los fármacos , Ratas , Ratas Endogámicas , Espectrometría de Fluorescencia/métodos , Sinapsis/efectos de los fármacosRESUMEN
Voltage-sensitive dyes offer the promise of noninvasive multicell recording of electrical activity, and should therefore be useful for studying the synaptic interactions of small networks of cultured neurons. We have designed and built a system for recording from microcultures of 1-15 neurons from the rat superior cervical ganglion (SCG), using voltage-sensitive fluorescent dyes of the styryl class. The apparatus comprises a standard inverted epifluorescence microscope; a mercury arc lamp with an optical feedback regulator; a 256-pixel fiber-optic camera with individual photodiode detectors and very low-noise amplifiers; and a personal computer-based data acquisition system. Its dark noise and illumination fluctuations are low enough that at typical fluorescence levels for these cells, it is limited by shot noise (the inherent physical limit of detection). Recording from SCG neurons, the signal-to-noise ratio is high enough to see large subthreshold synaptic potentials without signal averaging. This apparatus should be useful for studying long-term synaptic plasticity in cultures of vertebrate neurons, and several of its features should apply to optical recording from other preparations.