Glutamate uptake by chick retina.
Ontology highlight
ABSTRACT:
SUBMITTER: Tunnicliff G
PROVIDER: S-EPMC1165738 | biostudies-other | 1975 Aug
REPOSITORIES: biostudies-other
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Project description:BackgroundLittle is known regarding the molecular pathways that underlie the process of retinal development. The purpose of this study was to identify proteins which may be involved in development of retina. We used a proteomics-based approach to identify proteins that are up- or down-regulated during the development of the embryo chick retina.ResultsTwo-dimensional gel electrophoresis was performed with the retina of embryo chicken, which was obtained from embryos of day 7 (ED7) and of day 11 (ED11). The protein spots showing significant differences were selected for identification by MALDI mass spectrometry. Thirteen proteins were differentially expressed; seven proteins were up-regulated in embryo retina of chicken at ED 11 and six proteins were down-regulated. Significant proteins were also evaluated in embryo day 15 (ED15). Some of identified proteins were known to regulate cell proliferation, cell death, transport, metabolism, organization and extracellular matrix, and others also included novel proteins.ConclusionWe identified thirteen proteins which differentially expressed in embryonal retina of chicken at day 7, as compared to the retina of embryo of day 11. They were various regulatory proteins for cellular signaling.
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Project description:To investigate the circadian regulation and acute illumination effects on the expression and secretion of retinoschisin from vertebrate retinas.Retinas were studied on the second day of constant darkness (DD) after several days of entrainment to 12-hour light/12-hour dark (LD) cycles in ovo or in vitro. Quantitative real-time PCR and Western immunoblotting were used to examine the mRNA and protein expressions of retinoschisin at different circadian time points. Pharmacologic treatments in whole retina and dissociated retinal cell cultures were used to investigate the cellular mechanisms underlying the circadian regulation of retinoschisin content and secretion. Different illumination conditions were given to examine changes in retinoschisin content in association with acute light/dark adaptation.The mRNA level, protein expression, and secretion of retinoschisin were under circadian control, all of which were higher at night and lower during the day. The Ras, MAP kinase Erk, CaMKII pathway served as part of the circadian output regulating the rhythmicity of retinoschisin. Blockage of L-type VGCCs dampened the retinoschisin rhythm, but inhibition of L-type VGCCs did not completely abolish the secretion of retinoschisin. The protein expression of retinoschisin also responded to acute illumination changes.The mRNA and protein expression, as well as retinoschisin secretion, are under circadian control. L-type VGCCs play a role in the circadian regulation of retinoschisin, but the molecular mechanism underlying retinoschisin secretion does not depend on L-type VGCCs. Protein expression of retinoschisin in response to acute illumination changes depends on previous light exposure experience.
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Project description:Cellular reprogramming resets the epigenetic landscape to drive shifts in transcriptional programmes and cell identity. The embryonic chick can regenerate a complete neural retina, after retinectomy, via retinal pigment epithelium (RPE) reprogramming in the presence of FGF2. In this study, we systematically analysed the reprogramming competent chick RPE prior to injury, and during different stages of reprogramming. In addition to changes in the expression of genes associated with epigenetic modifications during RPE reprogramming, we observed dynamic changes in histone marks associated with bivalent chromatin (H3K27me3/H3K4me3) and intermediates of the process of DNA demethylation including 5hmC and 5caC. Comprehensive analysis of the methylome by whole-genome bisulphite sequencing (WGBS) confirmed extensive rearrangements of DNA methylation patterns including differentially methylated regions (DMRs) found at promoters of genes associated with chromatin organization and fibroblast growth factor production. We also identified Tet methylcytosine dioxygenase 3 (TET3) as an important factor for DNA demethylation and retina regeneration, capable of reprogramming RPE in the absence of exogenous FGF2. In conclusion, we demonstrate that injury early in RPE reprogramming triggers genome-wide dynamic changes in chromatin, including bivalent chromatin and DNA methylation. In the presence of FGF2, these dynamic modifications are further sustained in the commitment to form a new retina. Our findings reveal active DNA demethylation as an important process that may be applied to remove the epigenetic barriers in order to regenerate retina in mammals.Abbreviationsbp: Base pair; DMR: Differentially methylated region; DMC: Differentially methylated cytosines; GFP: Green fluorescent protein; PCR: Polymerase chain reaction. TET: Ten-eleven translocation; RPE: retinal pigment epithelium.
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Project description:Gap junction (GJ) channels couple adjacent cells, allowing transfer of second messengers, ions, and molecules up to 1 kDa. These channels are composed by a multigene family of integral membrane proteins called connexins (Cx). In the retina, besides being essential circuit element in the visual processing, GJ channels also play important roles during its development. Herein, we analyzed Cx43, Cx45, Cx50, and Cx56 expression during chick retinal histogenesis. Cx exhibited distinct expression profiles during retinal development, except for Cx56, whose expression was not detected. Cx43 immunolabeling was observed at early development, in the transition of ventricular zone and pigmented epithelium. Later, Cx43 was seen in the outer plexiform and ganglion cell layers, and afterwards also in the inner plexiform layer. We observed remarkable changes in the phosphorylation status of this protein, which indicated modifications in functional properties of this Cx during retinal histogenesis. By contrast, Cx45 showed stable gene expression levels throughout development and ubiquitous immunoreactivity in progenitor cells. From later embryonic development, Cx45 was mainly observed in the inner retina, and it was expressed by glial cells and neurons. In turn, Cx50 was virtually absent in the chick retina at initial embryonic phases. Combination of PCR, immunohistochemistry and Western blot indicated that this Cx was present in differentiated cells, arising in parallel with the formation of the visual circuitry. Characterization of Cx expression in the developing chick retina indicated particular roles for these proteins and revealed similarities and differences when compared to other species.
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Project description:In the present study we explored the role of β-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear β-catenin in the CM and RPE after injury (retinectomy). β-Catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear β-catenin was maintained as long as FGF2 was present. However, nuclear β-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear β-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear β-catenin. Moreover, retinectomy followed by disruption of active β-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that β-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of β-catenin is a pre-requisite for chick retina regeneration.
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Project description:Glutamate in neurons is an important excitatory neurotransmitter, but it also is a key metabolite. We investigated how glutamate in a neural tissue is protected from catabolism. Flux analysis using (13)C-labeled fuels revealed that retinas use activities of the malate aspartate shuttle to protect >98% of their glutamate from oxidation in mitochondria. Isolation of glutamate from the oxidative pathway relies on cytosolic NADH/NAD(+), which is influenced by extracellular glucose, lactate, and pyruvate.
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Project description:To investigate whether and how the basic helix-loop-helix (bHLH) gene cNSCL2 is involved in retinal development.cNSCL2, the chick homologue of human NSCL2, was isolated and sequenced. In situ hybridization was used to examine its spatial and temporal expression pattern in the retina. Replication-competent retrovirus RCAS was used to drive cNSCL2 misexpression in the developing chick retina, and the effect of the misexpression was analyzed.Expression of cNSCL2 in the retina was restricted. Its mRNA was detected in amacrine and horizontal cells, but not in photoreceptor, bipolar, or ganglion cells. Retroviral-driven misexpression of cNSCL2 in the developing chick retina resulted in missing photoreceptor cells and gross deficits in the outer nuclear layer (ONL). These deficits were probably not because of decreased photoreceptor production, in that the ONL appeared normal in early developmental stages. TUNEL+ cells were detected in the ONL, indicating that photoreceptor cells underwent apoptosis in retinas misexpressing cNSCL2. Müller glial cells were far fewer in the experimental retina than in the control, indicating that cNSCL2 also caused Müller glia atrophy. The onset of Müller glia disappearance preceded that of photoreceptor degeneration.Expression of cNSCL2 in the chick retina was restricted to amacrine and horizontal cells. Misexpression of cNSCL2 caused severe retinal degeneration, and photoreceptor cells and Müller glia were particularly affected.
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Project description:The Reelin-Disabled 1 (Dab1)-signaling pathway plays a critical role in neuronal cell positioning in the brain. We have isolated two alternatively spliced variants of Dab1 from chick retina, an early form (chDab1-E) expressed in undifferentiated cells and a late form (chDab1-L) expressed in amacrine and ganglion cells. A key difference between the two forms is the exclusion in chDab1-E of two Src-related tyrosine kinase recognition sites implicated in Reelin-mediated Dab1 tyrosine phosphorylation. Retinal cultures transfected with a chDab1-L expression construct undergo a dramatic change in morphology, accompanied by the formation of numerous thin elongated processes, increased tyrosine phosphorylation, activation of Src family kinase(s) and increased levels of the axonal outgrowth protein growth-associated protein-43. In contrast, chDab1-E transfectants retain an undifferentiated morphology. Mutational analysis implicates a specific tyrosine (tyr-198) in the morphological and biochemical alterations associated with chDab1-L expression. We propose that alternative splicing of chDab1 represents an effective and flexible way of regulating the Reelin-Dab1-signaling pathway in a mixed cell population, by ensuring that secreted Reelin activates the signaling cascade only in target neuronal cells.
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Project description:BACKGROUND: The embryonic chick provides an excellent model system for studies of development. However, it has lacked an efficient loss-of-function method for studies of gene function. RESULTS: We show that avian retroviruses can deliver hairpins mediating RNA interference to the developing chick eye. These viruses 'knock down' specific genes in infected areas of the retina. The knock down persists as the retina matures and can be detected using in situ hybridization. Furthermore, the amount of retinal tissue affected can be controlled by manipulating the degree of infection. CONCLUSION: This technique provides a rapid and efficient loss-of-function option for studies in the developing chick retina.
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Project description:Retinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. We used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. We identified 136 cell types plus 14 positional or developmental intermediates distributed among the six classes conserved across vertebrates - photoreceptor, horizontal, bipolar, amacrine, retinal ganglion, and glial cells. To assess morphology of molecularly defined types, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Müller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral, and central-peripheral retinal axes. We also identified immature photoreceptor, horizontal cell, and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mouse, and primate retinal cell classes and types. Our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system.
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