Embryonic Chick Neural Retina Research Articles

Growth hormone and retinal ganglion cell function: QNR/D cells as an experimental model

Retinal ganglion cells (RGCs) have been shown to be sites of growth hormone (GH) production and GH action in the embryonic (embryo day 7, ED7) chick neural retina. Primary RGC cell cultures were previously used to determine autocrine or paracrine actions of GH in the retina, but the antibody used in their immunopanning (anti-Thy-1) is no longer available. We have therefore characterized an immortalized neural retina (QNR/D) cell line derived from ED7 embryonic quail as a replacement experimental model. These cells express the GH gene and have GH receptor (GHR)-immunoreactivity. They are also immunoreactive for RGC markers (islet-1, calretinin, RA4) and neural fibers (neurofilament, GAP 43, vimentin) and they express the genes for Thy-1, neurotrophin 3 (NTF3), neuritin 1 (NRN1) and brn3 (POU4F). These cells are also electrically active and therefore resemble the RGCs in the neural retina. They are also similarly responsive to exogenous GH, which induces overexpression of the neurotrophin 3 and insulin-like growth factor (IGF) 1 genes and stimulates cell survival, as in the chick embryo neural retina. QNR/D cells are therefore a useful experimental model to assess the actions of GH in retinal function.

Cellular interfacial and surface tensions determined from aggregate compression tests using a finite element model

Although previous studies suggested that the interfacial tension gamma(cc) acting along cell-cell boundaries and the effective viscosity mu of the cell cytoplasm could be measured by compressing a spherical aggregate of cells between parallel plates, the mechanical understanding necessary to extract this information from these tests-tests that have provided the surface tension sigma(cm) acting along cell-medium interfaces-has been lacking. These tensions can produce net forces at the subcellular level and give rise to cell motions and tissue reorganization, the rates of which are regulated by mu. Here, a three-dimensional (3D) cell-based finite element model provides insight into the mechanics of the compression test, where these same forces are at work, and leads to quantitative relationships from which the effective viscosity mu of the cell cytoplasm, the tension gamma(cc) that acts along internal cell-cell interfaces and the surface tension sigma(cp) along the cell-platen boundaries can be determined from force-time curves and aggregate profiles. Tests on 5-day embryonic chick mesencephalon, neural retina, liver, and heart aggregates show that all of these properties vary significantly with cell type, except gamma(cc), which is remarkably constant. These properties are crucial for understanding cell rearrangement and tissue self-organization in contexts that include embryogenesis, cancer metastases, and tissue engineering.

Formation of retinal pigment epithelium in vitro by transdifferentiation of neural retina cells.

Chick embryonic neural retina (NR) dedifferentiates in culture and can transdifferentiate spontaneously into retinal pigment epithelium (RPE). Both, primary RPE and transdifferentiated RPE (RPEt), are characterized by pigmentation, expression of RPE-specific protein, eRPEAG and lack of expression of the neural cell adhesion molecule, NCAM. In contrast, NR cells are unpigmented and express NCAM but not eRPE(AG). Functionally, both primary RPE and the RPEt cells display a pH(i) response to bFGF, which is different from that of the NR. We used these characteristics to distinguish cell types in primary cultures of chick NR and follow the changes in phenotype that occur during transdifferentiation. We show that the RPEt forms as small "islands" in the packed regions of the primary, "mother" NR cell sheets, in a stochastic process. Because of a small number of cells involved in the initiation of the transdifferentiation we refer to it as a "leader effect" to contrast it with the "community effect" which requires many competent cells to be present in a group to be able to respond to an inductive signal. The RPEt then expands centrifugally and underneath the surrounding NR sheet. To determine if the RPEt maintains its identity in isolation while displaying the RPE-typical phenotypic plasticity, we explanted the islands of RPEt and treated half of them with bFGF. The untreated RPEt maintained its closely packed, polygonal pigmented phenotype but the bFGF-treated RPEt transdifferentiated into a non-pigmented, NR-like phenotype, indicating that RPEt encompasses the full differentiation repertoire of native RPE.

PTP1B regulates neurite extension mediated by cell-cell and cell-matrix adhesion molecules.

N-cadherin and beta1-integrin adhesion and signaling play important roles in growth cone adhesion and guidance. Each of these adhesion receptor systems is composed of multiprotein complexes, and both adhesion and downstream signaling events are regulated through the interaction of protein tyrosine kinases and phosphatases with many of the proteins that make up these complex systems. Work from our laboratory reported that the nonreceptor protein tyrosine phosphatase PTP1B is localized to adherens junctions and focal adhesion complexes and regulates both N-cadherin- and beta1-integrin-mediated adhesion. PTP1B appears to modulate integrin-mediated adhesion through regulation of src activation and cadherin-mediated adhesion through dephosphorylation of beta-catenin. We have continued these studies and report that PTP1B is localized to the tips of growing neurites and that introduction of a noncatalytic mutant of PTP1B into PC12 cells results in inhibition of N-cadherin- and beta1-integrin-mediated neurite outgrowth but is without effect on neurite outgrowth on poly-L-lysine. Moreover, suppressing the level of PTP1B in primary embryonic chick neural retina cells using antisense oligonucleotides also inhibits N-cadherin- and beta1-integrin-mediated neurite outgrowth. Neither of these techniques reduces the levels of expression of either adhesion receptor. We conclude that PTP1B is a regulatory component of the molecular complex required for both N-cadherin and beta1-integrin-mediated axon growth.

Cl(-)-dependent excitotoxicity is associated with 3H2O influx in chick embryonic retina.

The aim of this study was to show that Cl(-)-dependent excitotoxicity, with its characteristic cell swelling, involves actual water influx into the intracellular compartment. Taking advantage of the Ca2+ omission paradigm of Cl(-)-dependent excitotoxicity, in the chick embryonic neural retina ex vivo, which is associated with toxicity levels (lactate dehydrogenase (LDH) release) considerably higher than those seen after simple exposure of the retinas to glutamate agonists, we have demonstrated that an intracellular water intake of 4.2 microl into retinal cells is associated with 13.3% total retinal LDH release. The fact that mannitol blocks both water inflow and LDH release appears to link both events from a pathogenic point of view.

Ca2+-dependent kainate excitotoxicity in the chick embryonic neural retina ex vivo.

The chick embryonic neural retina ex vivo has been singled out as a unique example of Cl(-)-dependent/Ca2+-independent excitotoxicity. However, after continuous incubation with 100 microM kainate, we have demonstrated the susceptibility of the chick retina to Ca2+-mediated damage, which becomes apparent after 12 h of exposure to the agonist in the absence of Cl-. Of the 20.8% lactate dehydrogenase released after 24 h incubation with kainate, some 11% is Cl(-)-dependent and the rest (9.8%) is presumably Ca2+-dependent. Upon omission of both Cl- and Ca2+, a 5% residual toxicity can still be detected after 24 h. This can be overcome by inclusion of EGTA in the incubation medium to neutralize Ca2+ released during incubation. A Ca2+-dependent toxicity mechanism is then operative in the embryonic chick retina ex vivo.

Characterization of a chicken retinoid X receptor-gamma gene promoter and identification of sequences that direct expression in retinal cells.

Development of the cellular complexity of the vertebrate neural retina relies on an intricate interplay between extracellular signals and intracellular factors. In particular, transcription factors play a key role in determining the competence of cells to respond to extracellular signals. We have previously shown that, in the developing chick neural retina, expression of the retinoid X receptor-gamma (RXR-gamma2) nuclear receptor gene is restricted to photoreceptors. To characterize the mechanisms that regulate expression of this gene in the neural retina, we isolated a chicken RXR-gamma genomic clone containing the RXR-gamma2 promoter and mapped the transcription initiation site by means of ribonuclease protection. We analysed promoter activity by transient transfection of luciferase reporter gene constructs into cultured cells isolated from embryonic-chick neural retina or facial mesenchyme, which does not normally express detectable RXR-gamma2 transcripts. The DNA fragment lying between nucleotides -657 and +37 with respect to the transcription initiation site had basal promoter activity in both cell types. The fragment lying between nucleotides -1198 and -991 directed 10-20-fold higher levels of luciferase activity in neural retina cells, but only basal levels in facial mesenchyme cells. This 208 bp fragment also enhanced the activity of the simian-virus-40 promoter, when placed upstream in either orientation. Electrophoretic-mobility-shift assays using this 208 bp fragment demonstrated the formation of four neural retina-specific protein-DNA complexes. These results indicate that regulation of RXR-gamma2 transcription in the developing chick neural retina involves the binding of one or more neural retina-specific protein factors to an enhancer element located approx. 1 kbp upstream of the transcription initiation site.

Characterization of a chicken retinoid X receptor-γ gene promoter and identification of sequences that direct expression in retinal cells

Development of the cellular complexity of the vertebrate neural retina relies on an intricate interplay between extracellular signals and intracellular factors. In particular, transcription factors play a key role in determining the competence of cells to respond to extracellular signals. We have previously shown that, in the developing chick neural retina, expression of the retinoid X receptor-γ (RXR-γ2) nuclear receptor gene is restricted to photoreceptors. To characterize the mechanisms that regulate expression of this gene in the neural retina, we isolated a chicken RXR-γ genomic clone containing the RXR-γ2 promoter and mapped the transcription initiation site by means of ribonuclease protection. We analysed promoter activity by transient transfection of luciferase reporter gene constructs into cultured cells isolated from embryonic-chick neural retina or facial mesenchyme, which does not normally express detectable RXR-γ2 transcripts. The DNA fragment lying between nucleotides -657 and +37 with respect to the transcription initiation site had basal promoter activity in both cell types. The fragment lying between nucleotides -1198 and -991 directed 10-20-fold higher levels of luciferase activity in neural retina cells, but only basal levels in facial mesenchyme cells. This 208 bp fragment also enhanced the activity of the simian-virus-40 promoter, when placed upstream in either orientation. Electrophoretic-mobility-shift assays using this 208 bp fragment demonstrated the formation of four neural retina-specific protein-DNA complexes. These results indicate that regulation of RXR-γ2 transcription in the developing chick neural retina involves the binding of one or more neural retina-specific protein factors to an enhancer element located approx. 1 kbp upstream of the transcription initiation site.

Inhibition of β1-integrin expression reduces clone size during early retinogenesis

We have used an antisense retrovirus strategy to test the role of β1-integrin in the early period of clone development in the embryonic chick neural retina. Analyses of clone size and dispersion demonstrated a marked effect of reducing the levels of expressed β1-integrin on this critical phase of retina cell and tissue development.

Suppression of fibroblast growth factor 2 expression by antisense oligonucleotides inhibits embryonic chick neural retina cell differentiation and survival in vivo.

During retinal differentiation, fibroblast growth factor 2 (FGF2) expression increases in retinal neurons following the sequential appearance of the neuronal layers. The function of the developmental increase of endogenous FGF2 in the developing chick retina was investigated by using an antisense strategy, using both optic vesicle cultures and in ovo-intravitreal microinjections. The former model allowed us to study the consequences of FGF2 down-regulation on early ganglion cell differentiation, whereas, in the latter model, subsequent development stages and terminal maturation of the retina were studied. FGF2 inhibition resulted in reduced ganglion cell differentiation, as visualized by the expression of the ganglion cell-specific RA4 and Islet-1 markers in optic vesicle cultures. Eyes intravitreally injected with the FGF2-specific antisense oligonucleotide exhibited profound retinal differentiation defects: thinning of the ganglion and outer nuclear (photoreceptors) cell layers and increased cell death in ganglion cell and inner nuclear layers. These results indicate that the loss of endogenous FGF2 cannot be compensated for in the retina and suggest that, although many other sources of FGF exist in the eye, the main role of the increase in endogenous FGF2 observed during retinal development is to intrinsically stimulate neuron differentiation and to protect neurons against cell death.

The Neurotoxic Effects of Ochratoxin-A Are Reduced by Protein Binding but Are Not Affected byl-Phenylalanine

Recentin vivoinvestigations indicate that the mycotoxin ochratoxin A (OTA) is a neurotoxicant during prenatal stages. In line within vivodata, in our embryonic chick brain and neural retina cell cultures the markers for neuritic outgrowth and differentiation (NF68 and 160 kDa, MAP2 and MAP5) were especially negatively affected.In vivoOTA is nearly completely bound to serum constituents. In our culture system binding of OTA to BSA evoked a significant shift of the concentration–effect relationships in meningeal and brain cell cultures. As a result of the albumin binding the OTA IC5 and IC50 values of all parameters increased by nearly the same value (about 15-fold in brain and 32-fold in meningeal cell cultures). One of the mechanisms responsible for OTA toxicity is thought to be the competitive inhibition versus Phe of Phe-dependent enzymes. Therefore, in addition, we investigated the effects ofl-phenylalanine (Phe) and its influence on OTA toxicity in brain and neural retina cell cultures. Phe itself was found to differently affect brain and neural retina cell cultures. However, in both cultures OTA toxicity is not diminished by Phe. Therefore, our data indicate that at least in our cultures competition with Phe-dependent processes does not play a role in OTA toxicity.

Enhancement of apoptosis in developing chick neural retina cells by basic fibroblast growth factor.

To evaluate the role of various growth factors in naturally occurring cell death during development of the neural retina, we examined the effects of such factors on the nuclear morphology and the size of DNA in cultured chick embryonic neural retina cells. Basic fibroblast growth factor (bFGF) increased internucleosomal cleavage of DNA and nuclear fragmentation in a time- and dose-dependent manner. The effect was inhibited by anti-bFGF antibody, suramin, and cycloheximide. Epidermal growth factor, platelet-derived growth factor, nerve growth factor, tumor necrosis factor-alpha, and dexamethasone had no effect. These results provide evidence that bFGF may eventually act as a lethal factor inducing apoptotic cell death during the development of the neural retina in chick embryo.

Reduction of ochratoxin A toxicity by heat-induced epimerization. In vitro effects of ochratoxins on embryonic chick meningeal and other cell cultures

The widespread contamination of food by mycotoxins may present a serious hazard to human and animal health. The present study was designed to determine the toxic potential of three structurally related ochratoxins: ochratoxin A (OTA), ochratoxin B (OTB) and the heat-induced 3 S-epimer of OTA (3S-OTA) recently discovered in roasted coffee and human serum. The toxicity was determined using serum-free cell cultures of embryonic chick meningeal fibroblasts, taking the effects on mitochondrial and lysosomal activity and culture protein content as an index for toxicity. OTA, OTB and 3S-OTA were toxic. However, the concentration necessary to induce comparable effects were nearly 19- and 10-fold higher for OTB and 3S-OTA, respectively, than those for OTA. In a next step the sensitivity of serum-free cell cultures of embryonic chick neural retina and brain were compared in relation to meningeal cell cultures. In the present study, no indications for differences in sensitivity could be detected. Furthermore, our study suggest that the OTA-induced toxic effects are not due to the inhibition by OTA of phenylalanine-tRNA synthetase.

Neurotrophic effects of transferrin on embryonic chick brain and neural retina cell cultures

The viability and differentiation promoting effects of various transferrins [iron-saturated (holo) and iron-depleted (apo) human and chick ovo (conalbumin)-transferrins, and bovine apo-transferrin] were studied, using serum-free, flat-sedimented cell cultures of embryonic chick brain and neural retina. The effects of transferrin (Tf) on the cell cultures depended on the type of Tf used and the parameter measured.Significant differences between brain and neural retina cultures in the effects of apo-ovoTf and iron [supplemented as ammonium-iron (III) citrate] were detected. Maximal levels of mitochondrial activity were observed in the presence of 2mg/l apo-ovoTf in neural retina cell cultures. In brain cell cultures, 40mg ovoTf/l were needed to achieve maximal levels. In brain, but not in neural, retina cell cultures ovoTf and optimal concentrations of Fe3+ exhibited similar effects on biochemical parameters of cell function and differentiation. Although, in the absence of ovoTf, neuronal outgrowth on areas not covered by glial cells was inhibited in both cell cultures, the differences were more prominent in neural retina cell cultures. Our data strongly suggest that Tf plays a key role in processes not connected directly with its iron transport capability.

Hormonal and non-hormonal regulation of glutamine synthetase in the developing neural retina

Two isoforms of the glucocorticoid receptor, with apparent molecular mass of 90 and 95 kDa, are expressed in embryonic chicken neural retina. The 95-kDa receptor represents a hyperphosphorylated form of the 90-kDa receptor. Activation of the glucocorticoid receptor by cortisol results in a dose-dependent increase in receptor phosphorylation, translocation of receptor molecules into the nucleus and a decline in the total amount of the receptor. Activation of the glucocorticoid receptor can also be observed in the developing retinal tissue in ovo. At late embryonic ages, when the systemic level of glucocorticoids increases, a substantial quantity of receptor molecules becomes translocated into the nucleus, the relative level of the 95-kDa isoform increases, and the total amount of receptor declines. Activation of the receptor molecules in ovo correlates directly with an increase in transcription of the glucocorticoid-inducible gene, glutamine synthetase. The close correlation between the increase in systemic glucocorticoids, activation of glucocorticoid receptor molecules and induction of glutamine synthetase gene transcription suggests that glucocorticoids are directly involved in the developmental control of glutamine synthetase expression. Long-term organ culturing of embryonic retinal tissue in the absence of hormone results in an increase in glutamine synthetase expression. This increase, which is only 5 to 10% of that observed in ovo, is not mediated by activated receptor molecules and represents a mechanism for non-hormonal regulation of glutamine synthetase.

Insulin-like growth factor-I stimulates neurogenesis in chick retina by regulating expression of the alpha 6 integrin subunit.

Insulin-like growth factor I (IGF-I) strongly stimulates the generation of differentiated neurons in cultures of neuroepithelial cells of the embryonic chick neural retina in the presence of a laminin-1 tissue culture substrate. Treatment of cultured neuroepithelial cells with IGF-I rapidly up-regulated the mRNA coding for the alpha 6 integrin subunit whereas specific reduction of alpha 6 subunit levels by treatment with an alpha 6 integrin antisense oligonucleotide resulted in reduced neuronal differentiation in vitro. Although IGF-I immunoreactivity is seen throughout the neural retina, expression of IGF-I mRNA is confined to the pigment epithelium during the period of neurogenesis in vivo. Neutralization of the endogenous IGF-I with a blocking antibody down-regulated levels of alpha 6 integrin mRNA and reduced the production of differentiated retinal neurons in vivo. These data indicate a role for IGF-I in the generation of retinal neurons mediated by the interaction of laminin with its alpha 6 integrin subunit-containing receptor.

Distribution of heparan sulfate proteoglycans in embryonic chicken neural retina and isolated inner limiting membrane.

Quantitative distribution of proteoglycans was studied in retinal neural epithelium and its basement membrane (inner limiting membrane). Heparan sulfate proteoglycans (HSPGs) were primarily associated with both inner and outer plexiform (synaptic) layers, and inner limiting membrane (ILM), as determined by autoradiographs of lyase-digested cryosections. Based on distribution of 35S-sulfate-labeled proteoglycans, the isolated ILM contained on average approximately three fourths of its proteoglycans as HSPGs and one fourth as chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs), whereas the remaining retina contained approximately equal amounts of the two proteoglycans (PGs). Immunohistochemical staining indicates that the core proteins of the HSPGs in the ILM are distinct from those of the plexiform layers. The photoreceptor layer, which other studies have shown to contain much of the extracellular CS/DSPGs, was not examined. Enrichment of distinct HSPGs in the ILM and plexiform layers support the conclusion that the HSPGs may be intimately involved in the different developmental events characterizing the two regions: development and extension of ganglion cell axons in the former, synaptogenesis and neuronal function in the latter.

Changes in Crystallin Expression During Transdifferentiation and Subsequent Ageing of Embryonic Chick Neural Retina In Vitro: Comparison with Lens Epithelium

We have shown that cultured day-old chick lens epithelial cells undergo changes in crystallin expression during lens fibre (lentoid body) differentiation and ageing in serial subculture which are similar to those found in the adult lens in vivo. Here we have maintained neural retina cells, which transdifferentiate in vitro, for 250 days in serial subculture, in order to determine whether the tissue of origin affects the sequence of changes in crystallin expression and capacity for lentoid body formation both during fibre formation in primary culture and ageing in vitro. Alpha and β-crystallins were detectable before δ-crystallin in primary cultures of both day-old chick lens epithelium and neural retina from 8-day embryos, but while β 2 (26 kDa) was detected in pre-lentoid lens epithelial cultures it was not detected until after lentoids formed in neural retina cultures. The relative proportions of the α- and β-crystallin polypeptides were similar in lentoid-rich lens epithelium and neural retina cultures, and both cultures underwent similar changes in serial subculture: a loss of lentoid forming capacity, an early preferential loss of δ-crystallin expression followed by a failure to accumulate first α- and then β-crystallins. The order in which the β-crystallin polypeptides were lost differed between the cultures. There is evidence for rapid turnover of α- and β-crystallins while actin is the major component expressed in both types of aged cultures. Thus lens and retinal cells show some initial differences in the sequence of crystallin expression in primary cultures and in the eventual characteristics of aged cultures, but during the period beginning with lentoid formation and ending with the onset of senescence, lens cells from either source follow a broadly similar programme of ageing changes which are similar to those which occur during lens ageing in vivo.

1102 Muscarinic acetylcholine responses in early embryonic chick neural retina

1102 Muscarinic acetylcholine responses in early embryonic chick neural retina

Distribution of the novel developmentally-regulated protein EAP-300 in the embryonic chick nervous system

In a previous study we described a 300 kDa, developmentally regulated protein identified in embryonic chick neural retina with a monoclonal antibody. Because this protein has been shown to be undetectable in the adult nervous system, and the monoclonal antibody is species-specific, the protein has been named embryonal avian polypeptide of 300 kDa (EAP-300). In the present study we have analyzed the histological expression of EAP-300 during chick embryogenesis. In the developing nervous system, EAP-300 expression was detected as early as Stage 5 (19 h), and was subsequently down-regulated to undetectable levels in the adult. Of particular interest was the association of EAP-300 with putative barriers of neuronal growth, such as the telencephalon/diencephalon glial knot, the dorsal midline in the mesencephalon and the midline in myelencephalon, and the spinal cord roof plate. EAP-300 was also shown to be expressed by Bergmann glia during the period of granule cell migration in the cerebellum. The expression of EAP-300 by radial astrocytes was confirmed in culture by immunofluorescent co-labeling with a MAb to EAP-300 and the R5 MAb, which is a radial astrocyte-specific marker. It has also been shown that EAP-300, when immunopurified from embryonic brain under non-dissociating conditions, co-purifies with a neural keratan sulfate proteoglycan that is also associated with CNS barrier structures during brain development. The restricted expression of EAP-300 in nervous tissue, particularly in CNS barrier structures, suggests that EAP-300 may play an important, but transient, role in the development of the chick nervous system.