Neuroretinal Cells Research Articles

Lectin-induced differentiation of transformed neuroretinal cells in vitro

The orderly course of chick neuroretinal cell differentiation was disrupted in vitro by infection with a temperature-sensitive strain of the Rous sarcoma virus (LA29). The resulting cell culture LA29NR remained mitotically active at 42 °C, yet rapidly adopted a transformed phenotype upon activation of the pp60 v-arc oncogene product at 37 °C. As a further indication of metabolic state, LA29NR cells expressed the protooncogene product c-Fos, as shown by Western blot analysis. Highly proliferative LA29NR cells proved refractory to standard differentiation agents such as cAMP, and prostaglandin E1. In our novel approach, succinylated concanavalin A (SCA), a nontoxic derivative of the lectin concanavalin A, induced dramatic, reversible morphological changes in LA29NR cells, including neurite outgrowth and increased cell-to-cell adhesion. Fluoresceinated SCA appeared to localize to Golgi and lysosomal structures. Cellular response to SCA treatment included decreased growth rate, reversible decrease in the phosphorylation state of a 41-kDa phosphoprotein, and induction of neuron-specific enolase. The glial marker vimentin was also evident in these cultures. These data suggest that SCA is an effective differentiation agent for cells of neuroectodermal origin, permitting neuronal as well as glial phenotypic expression Within these cell populations.

Photoreceptor differentiation in retinal xenografts of fetal monkey retina

Although the potential of retinal grafts to provide the host eye with rod cells is presently well established, the possibility of grafting cone photoreceptors has not been documented. In this study, the neural retinas of two Cebus apella monkey fetuses were xenografted into immunosuppressed Fischer 344 adult rats. Histological analysis showed intimate apposition between the grafted donor cells and the neighboring host rat retina. The transplanted cells survived well and often overgrew the boundaries of the host retina, expanding into the host vitreous cavity. These cells formed histogenetically differentiated structures predominantly populated by the photoreceptors. When transplanted into a foreign environment, donor cells formed inner segments which exhibited the basic morphology of cells developed in situ. This study demonstrates that embryonic monkey neural retina is a viable source of xenograft material. It also indicates that an advanced embryonic stage is not a deterrent to survival and differentiation of grafted primate neuroretinal cells. The successful transplantation of these cells, especially under the relatively adverse conditions of a xenograft, raises the hope that retinal transplantation may in fact be a useful technique for repairing both rod and cone function in damaged retinas of higher animals including humans.

Glucose-6-phosphate Dehydrogenase Activity and Glycogen Content during Chick Neuroretinal Cell Culture

تتحول خلايا الشبكية العصبية بعد عدة أسابيع من زراعتها إلى خلايا عدسية وذلك في بيئة F (بيئة تحتوي على نسبة بسيطة من الجلوكوز 6mM) وتتثبط عملية التحول هذه في بيئة FG ( بيئة تحتوي على نسبة عالية من الجلوكوز 28mM). تبين من الدراسة أن نشاط إنزيم جلوكوز -6- فوسفات دي هيدروجيناز يكون عاليا في بيئة FG مقارنة بيئة F. وكذلك فإن تكوين الجليكوجين وتراكمه يزيد أثناء زراعة الخلايا الجنينية للشبكية العصبية في بيئة FG ويقل في بيئة F. وتفسر زيادة تراكم الجليكوجين بجود خلايا مولر وخاصة الموجودة تحت الخلايا العصبية المتجمعة ( المتكتلة ). وهكذا ، فإن النسبة العالية للجلوكوز في بيئة FG يمكن أن تعمل ، مع تأثيرات الخلايا العصبية، على تحسين التمايز الطبيعي لخلايا مولر التي من خصائصها تكوين الجليكوجين تخزين داخل الكائن الحي (invivo).

Stimulatory effects of retinal extract and fibroblast growth factor on lentoidogenesis in cultures of chick embryo neuroretinal cells

A crude extract prepared from embryonic chick retina stimulates growth and particularly transdifferentiation into lens when added as a supplement to neuroretinal (NR) cultures in vitro. This effect is especially marked when using a medium (H) containing 5% horse serum, where growth factors are likely to be limiting. The level of δ-crystallin (lens marker) production in such cultures increases with the concentration of extract. Using extracts from earlier and later stages of retinal development, there is an age-dependent decline in the extent to which transdifferentiation is stimulated. However, such extracts have little effect on the activity of CAT, a neuronal marker enzyme. These effects are most probably mediated by growth factors present in the retinal extract acting upon Müller glial cells or their precursors in the NR cultures. In support of this suggestion, we show that purified fibroblast growth factor (but not epidermal growth factor) exerts similar effects on both culture growth and δ-crystallin accumulation.

In vivo staining of the mammalian retina by means of a simple fluorescent method

Intraocular injections of microliter volumes of a solution containing the fluorescent stain Fluoro-Gold produces an intense and long-lasting in vivo stain of the cells forming the neural retina of rats and mice. The label is incorporated by the neuro-retinal cells. while other intraocular structures such as the lens, iris, ciliary processes and even the optic nerve head exclude it. The fluorophore can be easily demonstrated under epi-illumination by means of commonly used fluorescence filter combinations. The stain has proven to be compatible with cryostat sectioning and with plastic embedding. Furthermore, electron dense lysosomal and lamellar bodies in retinal neurons which have incorporated the stain may provide a means for identification of Fluoro-Gold labeled cells at the electron microscopic level which is consistent with previous results. Intraocular injection of Fluoro-Gold fills the need for a reliable, simple, and robust in vivo and in toto stain of the neural retina.

Two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, cooperate to induce transformation of chicken neuroretina cells.

Several studies have shown that full transformation of primary rodent fibroblasts can be achieved in vitro through the cooperation of two oncogenes (usually one nuclear and one cytoplasmic) classified on the basis of different complementation groups. We have shown previously that cooperation between v-mil (cytoplasmic, serine-threonine kinase product), and v-myc (nuclear, DNA-binding product) is required to transform 7-day-old chicken neuroretina cells, which in usual culture medium do not rapidly proliferate. v-mil induces sustained growth of chicken neuroretina cells without transformation; v-myc fails to stimulate the proliferation of chicken neuroretina cells but is required to achieve transformation of the proliferating cells. Here, we present results indicating that the P135gag-myb-ets nuclear protein of avian erythroblastosis virus E26 is able to induce proliferation but not transformation of chicken neuroretina cells. v-myc is required in addition to P135gag-myb-ets to achieve chicken neuroretina cell transformation. In contrast, we found that the P135gag-myb-ets and P100gag-mil proteins are not able to cooperate in this system.

Intraretinal Transplantation for Rod‐Cell Replacement in Light‐Damaged Retinas

Blindness from retinal disease is often the consequence of extensive damage to the photoreceptor cell population, while other cell types which form the neural retina are relatively spared. In this setting, transplantation of photoreceptor cells could offer hope for the restoration of some degree of visual function. We testd the feasibility of this approach by transplanting immature retinal cells into the eyes of adult rats affected by late stage phototoxic retinopathy, which are almost totally devoid of photoreceptor cells.Dissociated neuroretinal cells from newborn rats were injected into the hosts' retinas. These cells were labelled with the fluorescent tracer Fast-blue for identification within the host eye. Survival time ranged from 3 to 100 post-transplantation days.Fundus examination of light-irradiated eyes showed pallor caused by a considerable reduction of the retino-choroidal vascular bed after light irradiation. Histologically the hosts exhibited decimation of the elements forming the outer layers.throughout the entire retina.As visualized by light and electron microscopic procedures, we report the differentiation of clusters of transplanted photoreceptor cells, and the integration of these cells within the adjacent areas of the host retina.Fluorescence microscopy showed these clusters to be formed by fluorescently labelled cells developing in intimate contact with the unlabelled host retina. Electron microscopically it was possible to determine that these photoreceptors had established synaptic contacts. These observations indicate that successful transplantation of immature retinal cells is feasible into adult eyes that have suffered extensive retino-choroidal damage. These findings also support the concept that retinal transplantation is a procedure which may open new avenues into the study of retinal repair.

Activation and transduction of c-mil sequences in chicken neuroretina cells induced to proliferate by infection with avian lymphomatosis virus.

We report that nondividing neuroretina cells from chicken embryos can be induced to proliferate following infection with Rous-associated virus type 1 (RAV-1), an avian lymphomatosis retrovirus lacking transforming genes. Multiplication of RAV-1-infected neuroretina cells is observed after a long latency period and takes place initially in a small number of cells. We also show that serial virus passaging onto fresh neuroretina cultures leads to the generation of novel mitogenic viruses containing the mil oncogene. DNA analysis indicated that RAV-1 is the only provirus detected in cells infected at virus passage 1, whereas neuroretina cells infected at subsequent virus passages harbor mil-containing proviruses. Three viruses, designated IC1, IC2, and IC3, were molecularly cloned. Restriction mapping indicated that in each virus, truncated c-mil sequences were inserted within different portions of the RAV-1 genome. In addition, IC1 and IC2 viruses have transduced novel sequences that belong to the 3' noncoding portion of the c-mil locus. All three viruses induce neuroretina cell multiplication and direct the synthesis of mil-specific proteins. Proliferation of neuroretina cells infected at passage 1 of RAV-1 was not associated with any detectable rearrangement of c-mil, when a v-mil probe was used. However, these cells expressed high levels of an aberrant 2.8-kilobase mRNA hybridizing to mil but not to a long terminal repeat probe. Therefore, transcriptional activation of a portion of c-mil could represent the initial events induced by RAV-1 infection and lead to retroviral transduction of activated c-mil sequences.

Cellular src Gene Expression Associated with Lentoidogenesis in Transdifferentiating Cultures.: (pp60c-src /retina, tapetum/transdifferentiation/lens).

We have shown (9) that elevated pp60c-src kinase activity accompanies the transdifferentiation of chick embryo neuroretinal (NR) cells into lens in vitro; moreover, most immunologically-detectable pp60c-src protein is confined to lentoid bodies in permissive cultures (FH; 6). By contrast, pp60c-src expression is low in non-permissive cultures where lentoid formation is blocked by high glucose (FHG; 6) or medium 199 (11). We now extend these findings in several respects. Firstly, glial-enriched cultures in both FH and FHG media form small but sparse lentoid bodies at around 20 days, accompanied by increases in both δ crystallin and pp60c-src expression. In later FHG cultures, these lentoids increase neither in number/size nor in δ/pp60c-src expression, in contrast to permissive (FH) cultures. Thus the high glucose block on transdifferentiation is only partly mediated by neuronal influences (19). Secondly, transdifferentiating cultures of tapetal cells show higher levels of pp60c-src relative to redifferentiated or dedifferentiated (16, 17) cultures of these cells. Thirdly, we find no evidence that c-src oncogene expression directly signals transdifferentiation. Thus v-src expression in RSV-transformed NR cells inhibits δ crystallin accumulation (29; this study), while a c-src-substituted RSV variant has little effect on NR transdifferentiation. Late cultures of NR cells in medium 199 fail to accumulate pp60c-src protein or c-src transcripts, even though previous studies (2) showed that δ crystallin transcripts are localised within the nuclei of many cells in such cultures.

Mapping by in vitro constructs of the P100gag-mil region, accounting for induction of chicken neuroretina cell proliferation

The v-mil oncogene of the avian retrovirus MH2 is expressed as a fusion protein with viral gag determinants in infected cells. This P100gag-mil protein accounts for the proliferation of chicken embryo neuroretina cells (CNR) induced by MH2 in vitro. We constructed a series of mutants by in-frame deletions in different parts of the gag and mil domains and tested their ability to induce CNR growth. We show that gag sequences, as well as 200-base-pair 5' mil sequences, were not required to induce such a proliferation. However, gag sequences seem to contribute to a full proliferation of growing CNR. In contrast, deletions in the kinase domain abolish this induction. In particular, by deleting only 9 nucleotides localized around the unique SphI site of v-mil, we produced a totally inactive mutant (BalSp). This mutant directs the synthesis of a v-mil protein lacking the dipeptide Tyr-Leu, which is conserved in almost all the members of the large protein kinase family, and a histidine residue highly conserved in Ser-Thr protein kinase members.

Chapter 16 Retinal transplants into adult eyes affected by phototoxic retinopathy

Publisher Summary This chapter discusses retinal transplants into adult eyes affected by phototoxic retinopathy. This chapter also discusses : (1) whether, and to what extent, the adult retina would support the growth and differentiation of the immature retina beyond the implantation point; (2) whether the photically damaged host retina would provide vascularization sufficient to allow extended growth of the transplant; (3) to what extent the transplant would integrate with the host neural retina; and (4) whether eyes with extensively damaged retinas would also be able to incorporate transplanted cells and to support their growth. The results indicate that successful retinal transplantation is feasible into the extensively damaged adult eye, and that to some extent, the procedure permits the repopulation of neuroretinal cells. There are some similarities and fundamental differences between the experimental designs used and the results described in the present report. The main similarity is the ability for the transplanted retina to survive, grow and differentiate into the adult host eye, while the most significant difference is that in the present study, the host's eyes are not normal, rather suffer from extensive neural and vascular damage to retina and choroid. However, the use of labeled, dissociated cells, in addition to tissue strips, added a technical advantage to the experimental protocol.

Glucose metabolism in transdifferentiating and glucose-blocked cultures of chick embryo neuroretinal cells: an inverse relationship between glycogen and δ-crystallin accumulation

Chick embryo neuroretinal (NR) cells transdifferentiate extensively into lens when cultured for several weeks in low-glucose (FH) medium, but fail to do so when high levels of supplementary glucose (FHG) are present. We show here that most aspects of glucose metabolism are promoted in high-glucose cultures, including lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities, 2-deoxyglucose uptake, pentose shunt activity and lactate production. Continuous supplementation of high-glucose cultures with low levels of ouabain (FHGO) significantly lowers 2-deoxyglucose uptake, from FHG levels down towards FH levels, especially during the early stages of NR culture. Much later, extensive transdifferentiation into lentoids (with concomitant δ-crystallin accumulation) occurs in these FHGO cultures, which thus resemble FH rather than FHG controls. Another parameter strongly affected by ambient glucose levels is the accumulation of glycogen. Both glycogen itself and glycogen synthetase activity increase steadily in FHG cultures, but decrease slightly under FH conditions. Glycogen accumulation in FHG cultures is largely confined to glial-like cells, particularly those underlying clusters of neurones. Other studies have shown that glial differentiation in vitro is promoted by histotypic interactions with retinal neurones. Thus high glucose may act in concert with neuronal influences to stimulate or stabilize the normal differentiation of retinal glial cells, whose characteristic features in vivo include glycogen synthesis and storage. Furthermore, we show that supplementation of high-glucose cultures with forskolin or dibutyryl cyclic AMP (both of which promote glycogenolysis) results in a slower rate of glycogen accumulation and in enhanced transdifferentiation into lens. In both respects, the forskolin- and dibutyryl cAMP-supplemented FHG cultures are intermediate between FH and FHG controls. Thus the enhancement of normal glial differentiation in NR cultures by high glucose may inhibit or preclude subsequent transdifferentiation into lens.

Infection of neuroretinal cells in vitro by avian sarcoma viruses UR 1 and UR 2: Transformation, cell growth stimulation, and changes in transducin levels

Infection in vitro of differentiating chick embryo neuroretinal cells with avian sarcoma viruses UR 1 and UR 2 results in mitogenic stimulation and morphologic conversion of both support and neuronal cells. This was shown by the continuous propagation of transformed cells for over 4 months and growth of reaggregated colonies in liquid medium as well as in soft agar. Production of the transforming proteins p150 gag-fps and p68 gag-ros of UR 1 and UR 2, respectively, was similar to that of transformed chick embryo fibroblasts, as judged from in vitro kinase activity assays. The two protein subunits, T β and T γ, but not T α of the GTP binding protein transducin, found in the retina of many animal species, were present in control neuroretinal cells. Infection with Rous sarcoma virus or UR 2 resulted in an inhibition of T γ synthesis and enhancement of T β-like protein production.

Induction of proliferation of neuroretina cells by long terminal repeat activation of the carboxy-terminal part of c-mil.

Expression of the P100gag-mil protein of avian retrovirus MH2 in cultured chicken embryo neuroretina cells was previously shown to result in the proliferation of normally quiescent cell populations. We show here that long terminal repeat activation of the carboxy terminus of the c-mil gene is sufficient to induce neuroretina cell proliferation.

Induction of proliferation of neuroretina cells by long terminal repeat activation of the carboxy-terminal part of c-mil.

Expression of the P100gag-mil protein of avian retrovirus MH2 in cultured chicken embryo neuroretina cells was previously shown to result in the proliferation of normally quiescent cell populations. We show here that long terminal repeat activation of the carboxy terminus of the c-mil gene is sufficient to induce neuroretina cell proliferation.

Interference of v-mil and v-myc oncogenes with 6 day old quail embryo neuroretina cell differentiation: [formula omitted], [formula omitted], [formula omitted] and [formula omitted]. INSERM U186/CNRS UA1160 Institut Pasteur, 1 rue Calmette, 59019 Lille, FRANCE

Interference of v-mil and v-myc oncogenes with 6 day old quail embryo neuroretina cell differentiation: [formula omitted], [formula omitted], [formula omitted] and [formula omitted]. INSERM U186/CNRS UA1160 Institut Pasteur, 1 rue Calmette, 59019 Lille, FRANCE

Heat Shock May Relieve a Posttranscriptional Block on σ Crystallin Synthesis in Cultures of Chick Embryo Neuroretinal Cells: (neural retina/transdifferentiation/posttranscriptional control/δ crystallin/heat shock).

The lens protein δ crystallin is expressed at high levels in lentoids fromed by the transdifferentiation of chick embryo neuroretinal cells in long-term culture under permissive conditions. Previous work has shown that one non-permissive medium (F199) allows transcription of δ crystallin RNA in late neural retinal cultures, but that these δ transcripts remain confined to the nuclei and are neither translated nor indeed detectable in the cytoplasm. We show here that F199 cultures maintained at 43°c throughout transdifferentiate extensively into lentoids, in contrast to similar cultures at 37°c. Following transfer of late (30 day) F199 cultures from 37°c to 43°c, δ crystallin synthesis becomes increasingly prominent over a period of at least 5 days, suggesting that recovery from heat shock (rather than heat shock per se) may be important in this phenomenon. A similar response can be elicited by chemical agents (sodium arsenite, azetidine 2-carboxylic acid) which induce heat-shock (stress) proteins at 37°c. Treatment of late F199 cultures with actinomycin D does not prevent this increase in δ crystallin synthesis, provided the drug is not added until after the initiation of a heat-shock response (6 hr after temperature shift). This suggests that the observed increase in δ synthesis following heat shock involves primarily the export and/or stabilisation of δ mRNAs already present in the nuclei in late F199 cultures. Possible wider implications of this posttranscriptional level of control over δ crystallin synthesis are discussed.

Lack of Induction of Neuroretinal Cell Proliferation by Rous Sarcoma Virus Variants That Carry the c-src Gene

Expression of p60v-src of Rous sarcoma virus in cultured chicken embryo neuroretinal cells was previously shown to result in the transformation and sustained proliferation of normally quiescent cell populations. We show here that Rous sarcoma virus variants that encode p60c-src, the cellular homolog of p60v-src, lack the ability to induce morphological transformation and cell proliferation of cultured neuroretinal cells. Neuroretinal cells infected with c-src-containing viruses, however, possess no less p60 protein kinase activity assayed in the immune complex than those infected with the transformation-defective Rous sarcoma virus mutants PA101 or PA104, which do stimulate the growth of these cells.

Lack of induction of neuroretinal cell proliferation by Rous sarcoma virus variants that carry the c-src gene.

Expression of p60v-src of Rous sarcoma virus in cultured chicken embryo neuroretinal cells was previously shown to result in the transformation and sustained proliferation of normally quiescent cell populations. We show here that Rous sarcoma virus variants that encode p60c-src, the cellular homolog of p60v-src, lack the ability to induce morphological transformation and cell proliferation of cultured neuroretinal cells. Neuroretinal cells infected with c-src-containing viruses, however, possess no less p60 protein kinase activity assayed in the immune complex than those infected with the transformation-defective Rous sarcoma virus mutants PA101 or PA104, which do stimulate the growth of these cells.

Role of the mitogenic property and kinase activity of p60src in tumor formation by Rous sarcoma virus.

Expression of the src gene of Rous sarcoma virus in chicken embryo neuroretinal cells results in morphological transformation and sustained proliferation of this normally resting cell population. PA101 and PA104 are two mutants of Rous sarcoma virus which induce neuroretinal cell proliferation in the absence of morphological transformation. Their mitogenic property is temperature sensitive, and they both encode p60src proteins with low kinase activity. To study the role of the mitogenic function and protein kinase activity of p60src in tumorigenesis, we investigated the oncogenicity of PA101 and PA104. Both mutants were less tumorigenic than wild-type virus when injected into chicks. Tumorigenicity was further assayed by inoculating infected chicken embryo fibroblasts and neuroretinal cells onto the chorioallantoid membrane of embryonated duck eggs. This system provides a nonpermissive and immunodeficient environment for xenogenic cell grafting and allows the study of cell tumorigenicity within a temperature range of 37 to 39.5 degrees C. Chicken embryo fibroblasts and neuroretinal cells infected with PA101 were as tumorigenic as wild type-infected cells at 37 degrees C, but tumor development was significantly reduced at 39.5 degrees C. In contrast, both cell types infected with PA104 displayed sharply reduced tumorigenicity. Cell cultures derived from PA101 tumors induced on the chorioallantoid membrane were similar to the corresponding cells maintained in vitro in terms of morphology, production of plasminogen activator, relative amounts of phosphotyrosine in total cellular proteins, and phosphorylation of 34,000-molecular-weight protein. These results indicate that the expression of the mitogenic function of src does not account per se for cell tumorigenicity and that tumor formation is compatible with low levels of p60src protein kinase activity.