Chick Embryo Retina Research Articles

Activin Family Members in the Developing Chick Retina: Expression Patterns, Protein Distribution, andin VitroEffects

We have investigated whether the activin family of growth factors is involved in the regulation of retinal cell differentiation. Immunocytochemistry andin situhybridization have shown that activin/inhibin subunits α, βA, and βB; receptors II and IIB; follistatin; and a follistatin-like gene are expressed in different regions of the chick embryo retina in developmentally regulated patterns. When tested in dissociated retinal cultures, activin did not appear to affect cell survival or proliferation, but it exerted marked inhibitory effects on the differentiation of photoreceptors, while stimulating the differentiation of nonphotoreceptor neurons; both effects were concentration-dependent and follistatin-sensitive. The results are consistent with the possibility that activin family members play significant roles in the regulation of retinal development.

Involvement of P2 purinoceptors in the regulation ofdna synthesis in the neural retina of chick embryo

The activation of P2 purinoceptors induces Ca2 mobilization in the earlyembryonic chick neural retina. This purinergic Ca2 response declines parallel with thedecrease in mitotic activity during retinal development. To investigate the role of P2purinoceptors in the regulation of retinal cell proliferation, we studied the effects of the P2purinoceptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2fn2fn2triphosphate ; [Ca2]i, intracellular Ca2 concentration ;DMSO, dimethyl sulfoxide ; E, embryonic day ; EC50, concentration for half maximalexcitation ; F340, fluorescence excited at 340 nm ; F380, fluorescenceexcited at 380 nm ; F340/F380, the ratio of F340/F380 ;FCS, fetal calf serum ; fura-2 AM, fura-2 acetoxymethyl ester ; IC50, concentrationfor half maximal inhibition ; PPADS, pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid ;UTP, uridine 5-triphosphate.,4-disulphonic acid (PPADS), and of the agonist ATP on DNA synthesis in retinal organcultures from embryonic day 3 (E3) chick. Suramin inhibited [3H]-thymidineincorporation in a dose-dependent manner (IC50 : ∼70 μM). PPADS alsoreduced [3H]-thymidine incorporation with maximum inhibition of 46% at 100 μM. Exogenous ATP enhanced [3H]-thymidine incorporation in adose-dependent manner to maximally 200% of control (EC50 : ∼70 μM). Indissociated retinal cultures from E7 chick, both antagonists showed similar inhibitory effects on [3H]-thymidine incorporation without affecting cell viability. In line with theseobservations, the presence of extracellular ATP was demonstrated both in vitro and in vivo. In the medium of E3 retinal organ cultures, the concentration of ATP increased25-fold within 1 h of incubation and this concentration was kept for at least 24 h. In the chickamniotic fluid, the ATP concentration was nearly 3 μM at E3 and declined to 0.15 μM at E7. The results indicate that P2 purinoceptors activated by autocrine or paracrinerelease of ATP are involved in the regulation of DNA synthesis in the neural retina at earlyembryonic stages.

Differential expression of AP-2alpha and AP-2beta in the developing chick retina: repression of R-FABP promoter activity by AP-2.

Retinal fatty acid binding protein (R-FABP) is the avian counterpart of murine brain FABP implicated in glial cell differentiation and neuronal cell migration. R-FABP is highly expressed in the undifferentiated retina and brain of chick embryos. We have previously shown by in vitro studies that the AP-2 transcription factor binds to a consensus AP-2 binding site in the R-FABP promoter region. Based on the expression pattern of AP-2 in the developing retina and on mutational analysis of the AP-2 binding site in DNA transfection experiments, we proposed that AP-2 could be involved in the down-regulation of R-FABP transcription. Here, we describe the cDNA isolation of two members of the AP-2 family expressed in the chick retina, AP-2alpha and AP-2beta. We show that R-FABP mRNA and the AP-2 factors are expressed in mutually exclusive patterns in the differentiating retina: whereas AP-2alpha and AP-2beta are selectively expressed either in amacrine, or in amacrine and horizontal cells, respectively, R-FABP mRNAis found in Muller glial cells and/or bipolar cells. Furthermore, a decrease in R-FABP-dependent expression is obtained upon cotransfection of primary retinal cultures with AP-2 expression vectors and a CAT reporter construct. The early and cell-specific expression of AP-2alpha and AP-2beta in the developing retina suggest a role for this transcription factor family in the early steps of amacrine and horizontal cell differentiation. Repression of the R-FABP gene in these cells may be an important component of their developmental program.

The Effect of Nitric Oxide on the Contractile Tone of Müller Cells

The effect of nitric oxide (NO) on the contractile tone of Müller cells was investigated. Müller cells, isolated from the retina of chick embryos, were cultured on thin sheets of silicone. S-Nitro-N-acetyl-DL-penicillamine (SNAP), an NO donor, was applied in the presence or the absence of carboxyl phenyltetramethylimidazole oxide (C-PTIO), an NO scavenger. The contractile tone of Müller cells was assessed by the extent of wrinkles created on the silicone sheets. The change of contractile tone was evaluated quantitatively by digitizing the photograph before and after the application of SNAP. Relaxation of wrinkles was induced by SNAP. C-PTIO inhibited the SNAP-induced relaxation of wrinkles. These results suggest that NO affects the contractility of embryonic Müller cells and could thus modulate ocular development.

Biochemical and morphological analysis of non-NMDA receptor mediated excitotoxicity in chick embryo retina.

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate glutamatergic neurotransmission, and when pathologically overstimulated induce excitotoxic neuronal death. Of the two families of iGluRs, the non-NMDA receptors have received less experimental attention than the NMDA receptors as mediators of neuronal death in in vitro systems. We have demonstrated that non-NMDA receptor activation is highly lethal for neurons of the chick embryo retina, and further characterize this phenomenon here. Treatment of isolated retinas with any of the non-NMDA receptor agonists glutamate, AMPA, or KA, in the presence of the NMDA receptor antagonist MK-801, led to pathomorphology and cell death. KA was the most effective toxin. All of KA-induced toxicity could be blocked by selective AMPA receptor blockers. The toxicity of both AMPA and glutamate could be greatly increased using cyclothiazide, which blocks AMPA receptor desensitization. These results indicate that KA is the most powerful toxin because it is a non-desensitizing agonist at the AMPA receptors. Glutamate exhibited a paradoxical ability to prevent KA-induced toxicity as measured by a biochemical assay of cell death. Also, histological studies indicated that glutamate selectively blocked KA-induced pathomorphological changes in bipolar cells. This protective effect of glutamate was not mimicked by AMPA, NMDA, or any of several metabotropic receptor agonists, indicating that it may be mediated by a receptor of undescribed pharmacology.

Developmental neuronal death is not a universal phenomenon among cell types in the chick embryo retina.

The goal of this study was to investigate whether all the cell types present in the chick embryo retina undergo developmental neuronal death. Apoptosis was investigated in retinal sections at different developmental stages, processed either with propidium iodide, which stains pyknotic nuclei intensely, or with terminal transferase-mediated deoxyuridine triphosphate (d-UTP)-biotin nick-end labeling (TUNEL). Internucleosomal DNA fragmentation was investigated in tissue extracts by agarose gel electrophoresis. TUNEL-positive (T+) cells and pyknotic nuclei were first detectable in the ganglion cell layer (GCL) around embryonic day (ED) 8 and peaked at ED 10. In the inner nuclear layer (INL), T+ and pyknotic cells first appeared on ED 8, reached maximum frequency on ED 11, and were largely absent after ED 14. DNA ladders were observed at all the stages, when T+ and pyknotic cells were abundant, but not on ED 4, when only scattered dead cells were observed histologically. Dying cells were virtually never detected in the outer nuclear layer (ONL) from ED 4 to postnatal day 2. After unilateral midbrain ablation on ED 5, there was a striking increase in the number of pyknotic and T+ cells in both the GCL and in the INL of the contralateral eye but not in the ONL. The absence of apoptotic cell death in the ONL during normal development and after tectal ablation shows that developmental death is not universal among the various cell populations present in the chick embryo retina and raises questions regarding mechanisms controlling both photoreceptor survival and the matching of pre- and postsynaptic elements in the outer plexiform layer of this species.

Glutamine synthetase (GS) activity and spatial and temporal patterns of GS expression in the developing chick retina: Relationship with synaptogenesis in the outer plexiform layer

The profile of glutamine synthetase (GS) activity in the neural retina of chicken embryos and adults was studied alongside the in vivo spatio-temporal patterns of generation and morphogenesis of Müller cell and of retinal synaptogenesis. The rise of GS activity during development is not related to Müller cell differentiation but to synaptogenesis in the outer plexiform layer (opl). GS expression was investigated by immunoreaction with GS-specific antiserum. Three spatial gradients of decreasing labeling were observed between embryonic (E)15 and E18, from central to peripheral retina, dorsal to ventral, and temporal to nasal, which are in spatio-temporal relationship with synaptogenesis in the opl. GS is localized in Müller cells and apparently also in a population of astrocyte-like glial cells, located in the ganglion cell layer throughout the retina. Precocious induction by hydrocortisone, in ovo, at E10, does not show the spatial pattern of GS immunoreactivity observed in control retinas at the time of natural induction (E15). We also show that dissociated (non-cultured) Müller cells of E18-20 retinas, to which only photoreceptors or photoreceptors and neurons remain joined, maintain an immunodetectable level of GS, while those in isolated state lose GS immunoreactivity rapidly. Our results suggest that the induction of GS expression might be mediated by Müller cell-neuron interactions at the opl and also perhaps at the outer nuclear layer (onl). An analysis of our results and those of previous authors suggests that the level of GS in differentiated Müller cells could be determined by conjoint cell interactions at the onl, opl, and inner plexiform layer.

Co-localization of the Insulin Receptor, Jun Protein and Choline Acetyltransferase in Embryonic Chick Retina

Previous work demonstrated that the availability of insulin to the embryonic chick retina at a critical developmental stage stimulated the activity of the acetylcholine synthetic enzyme, choline acetyltransferase (ChAT) and that this increase required the AP-1 transcription factor, c-jun. Here it is shown that immediately following a 2–5 min exposure to insulin there is, in the amacrine and ganglion cells of the chick embryo retina, a transient increase in the level of jun protein followed by a long-lasting increase in ChAT. These and previous results show that insulin receptor activation is necessary for the characteristic retina developmental increase in ChAT protein and that this increase is preceded by a transient increase in the synthesis of the transcription factor c-jun in the same retina cells. The data demonstrate an intracellular signal transduction pathway from the developmentally-activated insulin receptor through c-jun to ChAT and cholinergic differentiation.

Fenamates protect neurons against ischemic and excitotoxic injury in chick embryo retina

Three fenamates (flufenamate, meclofenamate and mefenamate) were examined for their protective effect on neurons under ischemic (glucose/oxygen deprivation) or excitotoxic conditions, using the isolated retina of chick embryo as a model. Retinal damage was evaluated by histology and lactate dehydrogenase assay. Whole-cell recording was used to examine the direct effect of the fenamates on glutamate receptor-mediated currents. The fenamates protected the retina against the ischemic or excitotoxic insult. Part of the neuroprotection by the fenamates derived from inhibition of N-methyl- d-aspartate receptor-mediated currents. However, kainate receptor-mediated currents were not blocked by the fenamates, which nonetheless reduced kainate receptor-mediated retinal damage. Our results raise the possibility that fenamates may serve as lead structures in the development of novel therapeutic agents against brain ischemia.

The effects of tgf- ; IGF, insulin-like growth factor ; DTT, dithiothreitol. EDTA, ethylenediaminetetraacetic acid ; PMSF, phenylmethylsulfonyl fluoride ; TBS, tris buffer saline. SDS-PAGE, sodium dodecyl sulfate-polyacrilamide gel electrophoresis.1 on chick embryo retina development in vitro

This paper studies the effect exerted by TGF-β1 on the development of chick embryo retina cultured in vitro. The addition of TGF-β1 to retinal explants inhibited DNA synthesis, measured as 3H-thymidine incorporation into trichloroacetic acid-insoluble fraction, while it increased both wet weight and protein content, in particular that of extracellular matrix proteins. Lastly, in explants treated with TGF-β1 an increment in the level of fibronectin was demonstrated by means of Western blotting analysis.

Insulin and IGFs induce apoptosis in chick embryo retinas deprived of L-glutamine

In chick embryo retinas, cultured in serum-free medium lacking L-glutamine, IGF-I, IGF-II and insulin induced apoptotic DNA fragmentation and cell death, IGF-I being the most efficacious compound. The apoptotic effect, which was particularly evident in retinas removed from 7-day-old chick embryos, declined with the age of the embryos and disappeared after day 11. Apoptosis appeared after a time lag of 8 h and then increased with time up to 16 h. Cycloheximide, an inhibitor of protein synthesis, was capable of entirely abolishing apoptotic cell death. The effect induced by IGFs or insulin was suppressed by the addition of glutamine. Cytokine-mediated apoptosis was also observed after withdrawal of phosphate. We suggest that IGFs or insulin may produce, in retinas cultured in medium lacking L-glutamine or phosphate, a conflict of signals that could be lethal for retinal cells.

Changes in protein composition during chick embryo retina development.

Two dimensional gel electrophoresis was employed to analyse the protein composition of chick embryo retina during "in ovo" development in order to individuate some components which are typical of different stages of growth. The study of protein composition of 7-day-old chick embryo retinas revealed the presence of about eighty different soluble components. In four of these proteins the staining intensity decreased during retina development. One of these components, of 35 KD with an isoelectric point of about 5.15, was partially purified by means of preparative isoelectric focusing. Other components, which were absent at day 7 appeared during the second week of development and were clearly visualised at day 16. These results were also compared with those obtained by means of 2DGE performed on liver and brain of chick embryo at day 7 and 16 of development.

A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate.

Two bilaterally symmetric eyes arise from the anterior neural plate in vertebrate embryos. An interesting question is whether both eyes share a common developmental origin or they originate separately. We report here that the expression pattern of a new gene ET reveals that there is a single retina field which resolves into two separate primordia, a suggestion supported by the expression pattern of the Xenopus Pax-6 gene. Lineage tracing experiments demonstrate that retina field resolution is not due to migration of cells in the median region to the lateral parts of the field. Removal of the prechordal mesoderm led to formation of a single retina both in chick embryos and in Xenopus explants. Transplantation experiments in chick embryos indicate that the prechordal plate is able to suppress Pax-6 expression. Our results provide direct evidence for the existence of a single retina field, indicate that the retina field is resolved by suppression of retina formation in the median region of the field, and demonstrate that the prechordal plate plays a primary signaling role in retina field resolution.

7 Organoculture of the Chick Embryonic Neuroretina

This chapter focuses on the chick whole-retina culture system and on the assays to evaluate some of the cellular processes occurring in the culture. The protocols can be adapted either to retinas of other vertebrates or to other embryonic organs. The chick neuroretina is a portion of the central nervous system well characterized in terms of neuroanatomic patterns and stages of development. Much larger than the rodent retina and accessible to in vivo manipulation in ovo, the chick embryo retina is suitable for a comprehensive approach that includes biochemical, molecular, and cellular studies, as well as for establishing correlation between in vitro and in vivo studies.

Capacitative Ca2+ influx in the neural retina of chick embryo.

Depletion of intracellular Ca2+ stores induces a capacitative Ca2+ influx in non-neural cells. It has been unknown whether the capacitative Ca2+ influx occurs in the cells of nervous systems. We found the capacitative Ca2+ influx in the neural retina of early embryonic chick with Fura-2 fluorescence measurements. A Ca(2+)-free medium containing thapsigargin (500 nM), an inhibitor of Ca(2+)-ATPase of intracellular Ca2+ stores, was applied to the neural retina of embryonic day 3 (E3) chick. A rise in intracellular Ca2+ concentration was evoked after the reintroduction of extracellular Ca2+, and this Ca2+ rise was suppressed by Zn2+ (1 mM) and Ni2+ (5 mM). The developmental changes in the Ca2+ rise induced by thapsigargin (250 nM) were studied from E3 to E13. The thapsigargin-induced Ca2+ rise was largest at E3, declined rapidly toward E6, and then decreased gradually until E13, when the Ca2+ rise almost disappeared. This developmental profile correlated with the decline in the mitotic activities of the retinal cells studied by Prada et al. The fluorescence imaging with the vertical slice of the E9 retina showed that the site at which the thapsigargin-induced Ca2+ rise was largest was the most outer layer of the retina, where proliferating cells are located. This spatial distribution and the above developmental profile may suggest that the capacitative Ca2+ influx occurs at the early period of neurogenesis when the cells have mitotic activities.

Ca 2+ Stores in the Chick Embryo Retina Cells

The Ca 2+ stores of digitonin permeabilized chick embryo retina cells in culture were characterized, by using the fluorescence of Fluo-3 potassium salt to follow continuously the free [Ca 2+] in the medium. After ATP dependent Ca 2+accumulation, the Ca 2+ release was induced by several agents: 10 μM cyclic-ADP-ribose (cADPR), 40 μM Ins(1,4,5)P 3 10 μM thapsigargin (Th), 25 μM ionomycin (Ion), 15 μM CCCP together with 4.5 μg/ml oligomycin (CCCP/Olig), 50 μM arachidonic acid (AA). Neither Ins(1,4,5)P 3 nor cADPR were able to mobilize Ca 2+ from internal stores in these cells, but Th and AA were effective in releasing Ca 2+. Four major Ca 2+ stores in chick embryo retina cells were distinguished: i) the thapsigargin sensitive Ca 2+ store, most likely the ER; ii) the Ca 2+ store sensitive to oligomycin and CCCP, most likely the mitochondrial Ca 2+ store, iii) an AA sensitive Ca 2+ store, which is distinct from the previous two; and, iv) the Ca 2+ store only sensitive to ionomycin. The capacities of these different Ca 2+ stores of the chick embryo retina cells, relative to the total intracellular stores, are: 63.3%, 14.1%, 8.2%, for the ER, the mitochondrial and for the AA sensitive Ca 2+ stores, respectively.

Nitric oxide enhances amino acid release from immature chick embryo retina

Nitric oxide (NO) was investigated for its ability to induce amino acid release from immature chick retina. The production of endogenous NO by activation of NO synthase after stimulation of N-methyl- d-aspartate (NMDA) subtype of glutamate receptor caused a significant increase in basal release of γ-aminobutyric acid (GABA) and glutamine, whereas a more modest increase in the glutamate release was also observed. The exposure of chick retina from 9-day-old embryos to NO-generating compounds, S-nitroso- N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) produced a dose dependent increase in GABA, glutamine, and glutamate release. This effect was reduced by about 80% by haemoglobin. These results indicate that NO has a stimulatory effect on amino acid release from chick embryo immature retina. However, this effect does not appear to involve a cGMP-related mechanism because 8-bromo-cGMP, a stable analogue of cGMP, failed to affect spontaneous amino acid release and because zaprinast did not enhance NMDA-stimulated release. In conclusion, our present observations may account for a role of NMDA-mediated events in the biochemical maturation under depolarizing conditions.

A light and electron microscopic radioautographic study on RNA synthesis in the retina of chick embryo

The incorporation of 3H-uridine into RNA of chick embryo retina was studied by light and electron microscopic radioautography. The numbers of silver grains were counted over the nucleus, nucleolus and cytoplasm of the cells in three different regions of the same retina of 2, 3, 4, and 7 day chick embryos. The results showed an increase of 3H-uridine incorporation from embryonic day 2 to 7. In every stage of development of the chick embryo retina, the number of silver grains was higher in the anterior than in the other two regions of the retina. In the three cell compartments of every embryo group, the number of silver grains was higher in the nucleus than in the nucleolus and cytoplasm. The results show further that the grains were less in the cytoplasm of the retinal cells of the day 2 embryo group and higher in the other groups especially in the day 7 embryos. Ultrastructural changes were also observed during the studied period of retina development.

Localization of cytosolic aldehyde dehydrogenase in the developing chick retina: in situ hybridization and immunohistochemical analyses.

Cytosolic aldehyde dehydrogenase (ALDH) mRNA is present at high levels in the undifferentiated chick retina. Tissue maturation is accompanied by a 20-25x decrease in transcript levels. To determine the spatial and temporal distribution pattern of the ALDH transcript and its encoded protein in the developing retina, in situ hybridization and immunohistochemical analyses were carried out using chick embryos at different stages of development. The ALDH transcript and protein were detected at the earliest stage tested, in the inner layer of the optic cup of stage 14 (day 2) embryos. Both the ALDH transcript and protein were found in the dorsal retina of chick embryos from stage 18 (day 3) to day 16 of incubation. Accumulation of the ALDH protein in the neurites of ganglion cells could readily be detected at early developmental stages. Staining of this ganglion fiber layer was strong in the dorsal retina and could be followed up to and into the optic nerve. By day 11, ALDH mRNA was located primarily in the ciliary margin and in the inner nuclear layer of the dorsal retina. In addition to these areas, the ALDH protein was also found in the inner plexiform and optic nerve fiber layers. These results suggest that environmental or transcriptional factors involved in the regulation of the ALDH gene are restricted to the dorsal retina at early developmental stages and that there is a requirement for the compartmentalization of the ALDH transcript/protein in the undifferentiated chick retina.

Expression of the chicken cysteine-rich fibroblast growth factor receptor (CFR) during embryogenesis and retina development.

The expression of the chicken cysteine-rich fibroblast growth factor receptor (CFR) during organogenesis and specifically during retina formation was studied by Northern blotting and a sensitive in situ hybridization. At days 2 and 4 of embryonic development (E2 and E4), CFR mRNA was present in a wide variety of developing organs; it was abundantly expressed in nervous structures, particularly in the retina. The levels of CFR transcripts were high during the proliferation and the subsequent differentiation phases of retinal neurogenesis, reached a maximum around E11 during the onset of the major period of retinal cell death, and then declined progressively. CFR mRNA was not detected at late stages when the final arrangement of retinal cell layers has been established. In prolonged primary cell cultures of chicken embryo retina, CFR expression showed a similar down-regulation to that seen with increasing age in vivo. It was up-regulated either directly or indirectly by its ligands. The CFR expression pattern in the developing retina was complementary to that of two other fibroblast growth factor (FGF) receptors, namely FGF-R1 and FGF-R2. In regard to a progressive increase in the expression of their ligands during retinal development, we suggest that CFR may have a role distinct from that of the tyrosine kinase FGF receptors during retinogenesis. Finally, the comparison of CFR expression with those of the other high affinity receptors indicates a regulation of the FGF function at the receptor level during neural retina development.