oko meduzy mutations affect neuronal patterning in the zebrafish retina and reveal cell-cell interactions of the retinal neuroepithelial sheet.
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Mutations of the oko meduzy (ome) locus cause drastic neuronal patterning defect in the zebrafish retina. The precise, stratified appearance of the wild-type retina is absent in the mutants. Despite the lack of lamination, at least seven retinal cell types differentiate in oko meduzy. The ome phenotype is already expressed in the retinal neuroepithelium affecting morphology of the neuroepithelial cells. Our experiments indicate that previously unknown cell-cell interactions are involved in development of the retinal neuroepithelial sheet. In genetically mosaic animals, cell-cell interactions are sufficient to rescue the phenotype of oko meduzy retinal neuroepithelial cells. These cell-cell interactions may play a critical role in the patterning events that lead to differentiation of distinct neuronal laminae in the vertebrate retina. (+info)
The cytoskeletal network controls c-Jun expression and glucocorticoid receptor transcriptional activity in an antagonistic and cell-type-specific manner.
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The physical and functional link between adhesion molecules and the cytoskeletal network suggests that the cytoskeleton might mediate the transduction of cell-to-cell contact signals, which often regulate growth and differentiation in an antagonistic manner. Depolymerization of the cytoskeleton in confluent cell cultures is reportedly sufficient to initiate DNA synthesis. Here we show that depolymerization of the cytoskeleton is also sufficient to repress differentiation-specific gene expression. Glutamine synthetase is a glia-specific differentiation marker gene whose expression in the retinal tissue is regulated by glucocorticoids and is ultimately dependent on glia-neuron cell contacts. Depolymerization of the actin or microtubule network in cells of the intact retina mimics the effects of cell separation, repressing glutamine synthetase induction by a mechanism that involves induction of c-Jun and inhibition of glucocorticoid receptor transcriptional activity. Depolymerization of the cytoskeleton activates JNK and p38 mitogen-activated protein kinase and induces c-Jun expression by a signaling pathway that depends on tyrosine kinase activity. Induction of c-Jun expression is restricted to Muller glial cells, the only cells in the tissue that express glutamine synthetase and maintain the ability to proliferate upon cell separation. Our results suggest that the cytoskeletal network might play a part in the transduction of cell contact signals to the nucleus. (+info)
Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdgB) protein.
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The protein tyrosine kinase PYK2 has been implicated in signaling pathways activated by G-protein-coupled receptors, intracellular calcium, and stress signals. Here we describe the molecular cloning and characterization of a novel family of PYK2-binding proteins designated Nirs (PYK2 N-terminal domain-interacting receptors). The three Nir proteins (Nir1, Nir2, and Nir3) bind to the amino-terminal domain of PYK2 via a conserved sequence motif located in the carboxy terminus. The primary structures of Nirs reveal six putative transmembrane domains, a region homologous to phosphatidylinositol (PI) transfer protein, and an acidic domain. The Nir proteins are the human homologues of the Drosophila retinal degeneration B protein (rdgB), a protein implicated in the visual transduction pathway in flies. We demonstrate that Nirs are calcium-binding proteins that exhibit PI transfer activity in vivo. Activation of PYK2 by agents that elevate intracellular calcium or by phorbol ester induce tyrosine phosphorylation of Nirs. Moreover, PYK2 and Nirs exhibit similar expression patterns in several regions of the brain and retina. In addition, PYK2-Nir complexes are detected in lysates prepared from cultured cells or from brain tissues. Finally, the Nir1-encoding gene is located at human chromosome 17p13.1, in proximity to a locus responsible for several human retinal diseases. We propose that the Nir and rdgB proteins represent a new family of evolutionarily conserved PYK2-binding proteins that play a role in the control of calcium and phosphoinositide metabolism downstream of G-protein-coupled receptors. (+info)
Involvement of poly (ADP-ribose)-polymerase in the Pax-6 gene regulation in neuroretina.
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The quail Pax-6 gene is expressed from two promoters named P0 and P1. P0 promoter is under the control of a neuroretina-specific enhancer (EP). This enhancer activates the P0 promoter specifically in neuroretina cells and in a developmental stage-dependent manner. The EP enhancer binds efficiently, as revealed by southwestern experiments, to a 110 kDa protein present in neuroretina cells but not in Quail Embryos Cells and Retinal Pigmented Epithelium which do not express the P0-initiated mRNAs. To study the role of p110 in Pax-6 regulation, we have purified the p110 from neuroretina cells extracts. Based on the peptide sequence of the purified protein, we have identified the p110 as the poly(ADP-ribose) polymerase (PARP). Using bandshift experiments and footprinting studies, we present evidence that PARP is a component of protein complexes bound to the EP enhancer that increases the on rate of the protein complex formation to DNA. Using PARP inhibitors (3AB and 6.5 Hphe), we show that these products are able to inhibit EP enhancer activity in neuroretina cells. Finally, we demonstrate that these inhibitors are able to decrease the expression of the P0-initiated mRNA in the MC29-infected RPE cells which, in contrast to the RPE cells, accumulated the PARP in response to v-myc expression. Our results suggest that PARP is involved in the Pax-6 regulation. (+info)
Cloning of a bovine orphan transporter and its short splicing variant.
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We have isolated a cDNA (bv7-3) encoding a member of the Na+,Cl(-)-dependent transporter family and its short splicing variant (bv7-3s) by screening a bovine retina cDNA library. Sequence analysis revealed that bv7-3 encodes a protein of 729 amino acids and is a bovine homologue of the rat orphan transporter v7-3-2. bv7-3s contains 265 amino acids, sharing 252 N-terminal amino acids with bv7-3. Both mRNAs for bv7-3 and bv7-3s were detected in nervous system by Northern blot analysis. In immunofluorescence analysis in transfected HEK 293T cells, myc-tagged bv7-3 was mainly detected on the plasma membrane, whereas myc-tagged bv7-3s showed a pattern of intracellular membrane staining. (+info)
Cell junctions in the developing compound eye of the desert locust Schistocerca gregaria.
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Intercellular junctions in the developing retina of the locust Schistocerca gregaria have been examined by electron microscopy. Different types of junction appear in a well defined sequence during development. Five stages of ommatidial development are described. Close junctions and punctate junctions are present throughout development. Gap junctions appear transiently amongst the undifferentiated cells, before clearly defined preommatidia can be distinguished. The subsequent disappearance of gap junctions may be correlated with cell determination. Lanthanum studies confirm these findings. The later sequential appearance of adhesive junction types is described. These include septate desmosomes and two types of desmosomes. In the fully differentiated ommatidium only two types of junction remain, these are: desmosomes and rhabdomeric junctions. (+info)
The neuronal basis of a sensory analyser, the acridid movement detector system. I. Effects of simple incremental and decremental stimuli in light and dark adapted animals.
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1. The response of the movement detector (MD) system to proportionally constant incremental and decremental stimuli has been studied at various degrees of light and dark adaptation. Action potentials in the descending contralateral movement detector neurone were taken as the indicator of response. 2. Over a range of at least six log10 units of adapting luminance, the MD system behaves as an ON/OFF unit, giving responses to both incremental and decremental changes in the illumination of a 5 degrees target. 3. With increasing amplitudes of stimuli, both the ON and OFF responses saturate rapidly. Saturation is reached sooner at higher levels of light adaptation. At all levels of light adaptation, the OFF response is greater than the ON. The ratio for saturating stimuli is approximately constant at around 3:2. 4. At the brightest adapting luminances used (20 000 cd/m2) the ON response is reduced but not lost. At the lowest (0-004 cd/m2) the OFF response to a 5 degrees disc fails, but can be regained by increasing the test area to 10 degrees. 5. From what is known of the retina of locusts and other insects, it is thought that light and dark adaptation in the MD system can be adequately explained by events at the retinula cell. (+info)
Light-induced calcium influx into retinal axons is regulated by presynaptic nicotinic acetylcholine receptor activity in vivo.
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Visual activity is thought to be a critical factor in controlling the development of central retinal projections. Neuronal activity increases cytosolic calcium, which was hypothesized to regulate process outgrowth in neurons. We performed an in vivo imaging study in the retinotectal system of albino Xenopus laevis tadpoles with the fluorescent calcium indicator calcium green 1 dextran (CaGD) to test the role of calcium in regulating axon arbor development. We find that visual stimulus to the retina increased CaGD fluorescence intensity in retinal ganglion cell (RGC) axon arbors within the optic tectum and that branch additions to retinotectal axon arbors correlated with a local rise in calcium in the parent branch. We find three types of responses to visual stimulus, which roughly correlate with the ON, OFF, and SUSTAINED response types of RGC reported by physiological criteria. Imaging in bandscan mode indicated that patterns of calcium transients were nonuniform throughout the axons. We tested whether the increase in calcium in the retinotectal axons required synaptic activity in the retina; intraocular application of tetrodotoxin (10 microM) or nifedipine (1 and 10 microM) blocked the stimulus-induced increase in RGC axonal fluorescence. A second series of pharmacological investigations was designed to determine the mechanism of the calcium elevation in the axon terminals within the optic tectum. Injection of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) (20 mM) into the tectal ventricle reduced axonal calcium levels, supporting the idea that visual stimulation increases axonal calcium. Injection of BAPTA (20 mM) into the tectal ventricle to chelate extracellular calcium also attenuated the calcium response to visual stimulation, indicating that calcium enters the axon from the extracellular medium. Caffeine (10 mM) caused a large increase in axonal calcium, indicating that intracellular stores contribute to the calcium signal. Presynaptic nicotinic acetylcholine receptors (nAChRs) may play a role in axon arbor development and the formation of the topographic retinotectal projection. Injection of nicotine (10 microM) into the tectal ventricle significantly elevated RGC axonal calcium levels, whereas application of the nAChR antagonist alphaBTX (100 nM) reduced the stimulus-evoked rise in RGC calcium fluorescence. These data suggest that light stimulus to the retina increases calcium in the axon terminal arbors through a mechanism that includes influx through nAChRs and amplification by calcium-induced calcium release from intracellular calcium stores. Such a mechanism may contribute to developmental plasticity of the retinotectal system by influencing both axon arbor elaboration and the strength of synaptic transmission. (+info)