Chick Embryo Neural Retina Research Articles

Effect of e-cigarette flavoring agents on the neural retina of chick embryo: histological and gene expression study.

An electronic cigarette (e-cigarette) is initially marketed as an assistant product to quit smoking or limit its use. However, recent studies suggest the opposite, describing it as a product that lacks adequate quality and user safety. The present study aimed to investigate the effect of e-cigarette flavoring agent (cinnamon flavor) on the neural retina development of chick embryos and apoptosis induction after the early and late apoptosis stages by quantitative detection of gene expression CASP-3 at both embryonic days E9 and E17. Fertilized chicken eggs were divided into two groups: control and treatment, and each group included two embryonic days; E9 and E17. For each treatment stage, two dosages of the treatment were applied, 2% and 5%. The neural retinas were dissected from the sclera and retinal pigment epithelium for subsequent RNA extraction and histological examination. This study indicated that aerosol of the subtle cinnamon flavor e-liquid causes downregulated expression of CASP3 in neural retina development. In addition, the hematoxylin and eosin (H&E)-stained sections showed multiple structural changes in the retinal layers and evidence of apoptotic cell death. Cell death was visible and abundant in E9, and E17 concludes that flavor vapor condensate treatment caused neuronal cell death. CASP-3 was downregulated, which indicates that cell death occurred independently of CASP-3.

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.

Growth Hormone Promotes Axon Growth in the Developing Nervous System

Postnatally, endocrine GH is primarily produced by pituitary somatotrophs. GH is, however, also produced in extrapituitary sites, including tissues of the developing nervous system such as the neural retina. Whereas GH roles in the nervous system are starting to emerge, they are still largely unknown. We show here that GH in the neural retina is mainly present in the axons of retinal ganglion cells (RGCs) in embryonic day (E) 4-12 chick embryos, but it is no longer present at E14-18. This temporal window corresponds to the period of RGC axon growth. GH receptor mRNA was also detected within cells of the E7 RGC layer and GH receptor protein colocalized with GH in RGC axons. The possibility that GH promotes axon growth was thus investigated. Exogenous GH induced a significant increase in axon elongation at 10(-9) and 10(-6) M in E7 RGC culture purified by immunopanning. RNA interference-mediated gene silencing was used to examine whether endogenous GH similarly alters axon outgrowth. The ability of GH small-interfering RNA to knock down GH was first tested using HEK cells on a LacZ-cGH expression plasmid and found to reach 90%. Upon transfection of GH small-interfering RNA to immunopanned RGC culture, a 63% knockdown of endogenous GH was detected and RGC axon length was found to be reduced by 40%. Taken together, these data suggest that GH acts as an autocrine or paracrine signaling molecule to promote axon growth in a developing nervous tissue, the neural retina of chick embryos.

Retinal growth hormone in the chick embryo.

The presence of GH and GH mRNA in the eyes of embryonic chicks is controversial and has, therefore, been further examined. In this study, GH cDNAs identical in size and sequence to the full-length pituitary transcript were generated by RT-PCR from mRNA extracted from the neural retinas of embryonic day (ED) 7 chick embryo eyes. GH immunoreactivity in the neural retina of embryos was primarily associated with proteins of 15 and 16 kDa, whereas only trace amounts of monomer (22-25 kDa) GH, the most abundant form in the pituitary gland, were present. GH immunoreactivity was also present in the vitreous humor, although this was associated only with the 15-kDa protein. After hatch, retinal proteins with GH immunoreactivity of 15 and 16 kDa were present neonatally but not after 42 d of age. The GH immunoreactivity in the neural retina of ED8 embryos was widespread, although GH staining was particularly abundant in retinal ganglion cells (RGCs). Full-length GH mRNA was similarly located, by in situ hybridization, throughout the neural retina and concentrated in cells in the RGC layer. The neural retina is also a site of GH action because 10-6 m chicken GH greatly increased (4- to 5-fold) the content of IGF-1 mRNA in 48-h cultured ED8 neural retinas. These results demonstrate the presence of full-length GH mRNA in the neural retina of chick embryos, in which GH immunoreactivity is primarily associated with RGCs and submonomer GH proteins of 15-16 kDa. These results also demonstrate GH action in the neural retina of embryos and suggest hitherto unsuspected roles for GH in retinal development and/or ocular function.

Calcium signaling to nucleus via store-operated system during cell cycle in retinal neuroepithelium

Intracellular Ca 2+ is a regulatory signal for cell proliferation. To reveal Ca 2+ signal dynamics during cell cycle, we applied Ca 2+ fluorescence imaging to the neural retina of chick embryo, where the soma changes its position during the cell cycle. Purinoceptors were stimulated to cause Ca 2+ release from Ca 2+ stores, since the purinoceptor activation promotes DNA synthesis. Ca 2+ rises occurred in the nucleoplasm of cells at around S-phase. The soma of S-phase cell is located in the inner layer of the retinal neuroepithelium and issues an outer process, which extends to the ventricular surface. Fluorescent probes for endoplasmic reticulum (ER) showed that the ERs in the outer process and the nuclear envelope (NE) or peri-nuclear ER formed the Ca 2+ store. Depletion of the Ca 2+ store induced capacitative Ca 2+ entry (CCE), which caused Ca 2+ rises in the terminal of outer process and soma. The store-operated Ca 2+ signaling declined in M-phase cells and postmitotic cells (retinal ganglion cells (RGCs)) with the loss of the outer process. These results suggest that the Ca 2+ signaling to nucleus via the store-operated system including the ERs in the outer process is crucial for the cell cycle progression in the retinal neuroepithelium.

Neural retina of chick embryo in organ culture: effects of blockade of growth factors by suramin.

The neural retina is a highly organized organ whose final histoarchitecture depends on the presence of diverse growth factors and on their interactions with extracellular matrix components. However, the role of growth factors on retinal development is not fully understood. Suramin has been shown to produce diverse cellular effects via the simultaneous block of the action of several growth factors. We have therefore studied the effects of suramin on organotypic culture of chick embryo neural retina in order to gain further insights into the participation of growth factors in neural retinal development. Neural retina was incubated for 24 h with suramin at 50-200 microM and then processed to determine cell proliferation, nuclear morphology, and actin distribution. Suramin provoked extensive morphological changes revealed by a decrease in BrdU incorporation, alterations in cellular organization, and disruption of the outer limiting membrane, with the emergence of cellular elements through it. All of these effects were dose-dependent and markedly attenuated by the simultaneous presence of suramin and fibroblast growth factor 2 (FGF-2) in the culture medium. These findings indicate that suramin induces pleiotropic effects on the histoarchitecture of the chicken neural retina in organ culture and suggest that FGF-2 is one of the biological modulators involved in the maintenance of the structural organization of the chicken neural retina.

Lysophosphatidic acid-induced Ca(2+) mobilization in the neural retina of chick embryo.

Lysophosphatidic acid (LPA) plays various roles in the regulation of cell growth as a lipid mediator. We studied the effect of LPA on intracellular Ca(2+) concentration ([Ca2+]i) with Fura-2 in the neural retina of chick embryo during neurogenesis. Bath application of LPA (1-100 microM) to the embryonic day 3 (E3) chick retina caused an increase in [Ca2+](i) in a dose-dependent manner, with an EC(50) value of 9.2 microM. The Ca(2+) rise was also evoked in a Ca(2+)-free medium, suggesting that release of Ca(2+) from intracellular Ca(2+) stores (Ca(2+) mobilization) was induced by LPA. U-73122, a blocker of phospholipase C (PLC), inhibited the Ca(2+) rise to LPA. Pertussis toxin partially inhibited the Ca(2+) rise to LPA, indicating that G(i)/G(o) protein was at least partially involved in the LPA response. The developmental profile of the LPA response was studied from E3 to E13. The Ca(2+) rise to LPA declined drastically from E3 to E7, in parallel with decrease in mitotic activity of retinal progenitor cells. The signal transduction pathway and developmental profile of the Ca(2+) response to LPA were the same as those of the Ca(2+) response to adenosine triphosphate (ATP), which enhances the proliferation of retinal progenitor cells. The coapplication of LPA with ATP resulted in enhancement of Ca(2+) rise in the E3 chick retina. Our results show that LPA induces Ca(2+) mobilization in the embryonic chick retina during neurogenesis.

Regulation of capacitative Ca2+ entry by tyrosine phosphorylation in the neural retina of chick embryo

Capacitative Ca2+ entry occurs in the neural retina of chick embryo during neurogenesis. We studied the effects of blockers of capacitative Ca2+ entry (SK&F 96365, Zn2+, Ni2+), genistein, a tyrosine kinase inhibitor and vanadate, a protein-phosphotyrosine phosphatase inhibitor in the embryonic chick retina with fura-2 fluorescence measurements. After incubation of the retina in a Ca2+-free solution with or without an inhibitor of Ca2+ pumps of intracellular Ca2+ stores, re-introduction of extracellular Ca2+ caused capacitative Ca2+ entry which was inhibited by SK&F 96365 (10 μM), Zn2+ (1 mM), Ni2+ (5 mM) and genistein (100 μM). On the contrary, vanadate (1 mM) enhanced the Ca2+ entry. These results suggested that tyrosine phosphorylation was involved in the activation of capacitative Ca2+ entry.

Characterization of MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) Identified by a Monoclonal Antibody Generated against Chick Embryo Neural Retina

To identify molecular markers of cell differentiation in developing nervous tissue, monoclonal antibodies against chick embryo neural retina were made. One of them, 3C3mAb, recognized a developmentally regulated antigen present in several organs of the CNS. Data from MALDI-TOF mass spectrometry and peptide sequencing of the immuno-affinity purified protein indicated identity of the antigen with MARCKS. The immunoreactive material was always found as a unique polypeptide (Mr71 kDa) in SDS–PAGE, however isoelectrofocusing revealed the existence of several bands (pIranging from 4.0 to 4.5). Interestingly some retinal cell types, as photoreceptors, exhibited an extremely significant decrease in the intensity of the immunoreactive material during the final phases of terminal differentiation while others, as some retinal neurons, maintained the immunoreactivity when fully differentiated. Taken together these results indicate that MARCKS, a protein susceptible of several posttranslational modifications as myristoylation and phosphorylation at variable extent, may act differently in neural retina cell types.

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.

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.

Compartmental organization of the synthesis of GM3, GD3, and GM2 in golgi membranes from neural retina cells.

The relationship among lactosylceramide-(LacCer), GD3- and GM2-synthases and between the two last transferases and their common GM3 acceptor was investigated in intact Golgi membrane from chick embryo neural retina cells at early (8-days) and late (14 days) stages of the embryonic development. [3H]Gal was incorporated into endogenous glucosylceramide by incubation of Golgi membranes with UDP-[3H]Gal. Conversion of the synthesized [3H]Gal-LacCer into GM3, and of the latter into GD3, GM2 and GD2 was examined after a second incubation step with unlabeled CMP-NeuAc and/or UDP-GalNAc. With CMP-NeuAc, most [3H]Gal-LacCer was converted into GM3 in either 8- or 14- day membranes. However, while about 90% of GM3 was converted into GD3 in 8-day membranes, only about 25% followed this route in 14-day membranes. With CMP-NeuAc and UDP-GalNAc, about 90% of GM3 was used for synthesis of GM2 in 14-day membranes, while in 8-day membranes about 80% followed the route to GD3, and a part to GD2. Performing the second incubation step in the presence of increasing detergent concentrations showed that conversion of GM3 to GM2 was inhibited at concentrations lower than those required for inhibition of LacCer to GM3 conversion. Taken together, results indicate that transfer steps leading to synthesis of GM3, GD3, GM2 and GD2 from LacCer are functionally coupled in the Golgi membranes, and that GD3- and GM2-synthases compete in a common compartment for using a fraction of GM3 as substrate. In this competition, the relative activities of the transferases and their relative saturation with the respective donor sugar nucleotides, are important factors influencing conversion of GM3 toward either GD3 or GM2.

Prospects for the development of validated screening tests that measure developmental toxicity potential: view of one skeptic.

Humans are exposed to a variety of potential developmental toxicants. This fact, combined with the knowledge that human development can be disrupted by "environmental" agents, has led to the development of methods designed to identify potential developmental toxicants. Currently, the principal method used to screen drugs and chemicals that are potential human developmental toxicants is the segment II study (i.e., a study in which prospective drugs and chemicals are tested in pregnant animals). Because of the cost and time involved in such studies and the pressure to reduce the number of animals used in such testing, alternative methods for developmental toxicity testing have been sought. This has resulted in a number of in vitro tests whose aim is to screen large numbers of agents quickly and inexpensively. Although numerous in vitro tests of developmental toxicity have been developed during the last 15 years, no one system or combination of tests have been validated for the purpose intended. Nonetheless, two systems--the limb bud/CNS micromass, and the chick embryo neural retina cell culture (CERC)--continue to be advanced as viable in vitro developmental toxicology tests. The purpose of this commentary is to evaluate the prospects for the development of an in vitro test system(s) that can screen the universe of drugs and chemicals and reliably identify those that require further study and those that do not. The conclusion of this investigator is that the prospects for validating such in vitro tests are not promising. This conclusion is based primarily on the lack of basic knowledge regarding the relevance of end points assayed in the micromass and CERC test systems to those end points known or thought to be critical for normal development.

S24-4 - Purinergic and muscarinic Ca2+ mobilizations in the neural retina of chick embryo.

S24-4 - Purinergic and muscarinic Ca2+ mobilizations in the neural retina of chick embryo.

Evaluation of Chick Embryo Neural Retina Cell Culture as a Screen for Developmental Toxicants

Evaluation of Chick Embryo Neural Retina Cell Culture as a Screen for Developmental Toxicants. Daston, G. P., Baines, D., Elmore, E., Fitzgerald, M.P., and Sharma, S. (1995). Fundam. Appl. Toxicol. 26, 203-210. This paper describes a study to evaluate the concordance with in vivo results of an in vitro screen for developmental toxicants. The screen is a primary culture of chick embryo neural retina cells (CERC) which undergo processes of cell-cell recognition and interaction, growth, and differentiation over a 7-day culture period. Each of these developmentally significant events is measured separately as formation of multicellular aggregates, protein content, and glutamine synthetase activity, respectively. A total of 45 chemicals, 24 of which have been shown to be teratogenic at some dosage to mammalian embryos in utero, 7 of which are embryotoxic (but not teratogenic) in utero at high dosage, and 14 of which have not produced developmental toxicity in vivo, were evaluated in this assay by investigators who were blinded to the identity of the chemicals. Chemicals were tested up to concentrations that were frankly cytolethal, or up to a maximum of 5 mg/ml. Chemicals were present only during the first 24 hr of culture. The chemicals were selected to be representative of a variety of chemical classes (e.g., solvents, metals, food additives, anticonvulsants, antineoplastics). In several cases, pairs of structurally similar compounds with different developmental toxic potencies (e.g., valproate and 2- en-valproate, formamide, and N,N-dimethylformamide) were tested. Of the 31 developmental toxicants, 25 affected at least one endpoint in the assay at concentrations which are achievable in vivo (i.e., below the systemic concentration at a lethal dose), yielding a false-negative rate of 19%. Two of the nondevelopmental toxicants, saccharin, and penicillin G, had adverse effects at concentrations below those that may be biologically achievable in vivo, giving a false-positive rate of 14%. Overall concordance with in vivo results by these criteria was 82%. Quantitative comparisons were also made between the lowest observed effect concentration (LOEC) in the assay and (i) lowest developmentally toxic dosage (mostly ip) reported in rats or mice in vivo and (ii) LOEC in rodent whole embryo culture. In the first instance, 77% of the LOECs (LOELs) were within an order of magnitude and 93% were within a factor of 30. In the second instance 81% of the LOECs were within an order of magnitude. Potency ranking of four alkoxy acids was comparable in CERC and the in vivo rodent embryo. These results indicate that the CERC assay is concordant with developmental toxic potential and potency for the diverse group of compounds selected, and that it could serve as a preliminary screen for developmental toxicity.

Evaluation of Chick Embryo Neural Retina Cell Culture as a Screen for Developmental Toxicants

This paper describes a study to evaluate the concordance with in vivo results of an in vitro screen for developmental toxicants. The screen is a primary culture of chick embryo neural retina cells (CERC) which undergo processes of cell-cell recognition and interaction, growth, and differentiation over a 7-day culture period. Each of these developmentally significant events is measured separately as formation of multicellular aggregates, protein content, and glutamine synthetase activity, respectively. A total of 45 chemicals, 24 of which have been shown to be teratogenic at some dosage to mammalian embryos in utero, 7 of which are embryotoxic (but not teratogenic) in utero at high dosage, and 14 of which have not produced developmental toxicity in vivo, were evaluated in this assay by investigators who were blinded to the identity of the chemicals. Chemicals were tested up to concentrations that were frankly cytolethal, or up to a maximum of 5 mg/ml. Chemicals were present only during the first 24 hr of culture. The chemicals were selected to be representative of a variety of chemical classes (e.g., solvents, metals, food additives, anticonvulsants, antineoplastics). In several cases, pairs of structurally similar compounds with different developmental toxic potencies (e.g., valproate and 2-en-valproate, formamide, and N,N-dimethylformamide) were tested. Of the 31 developmental toxicants, 25 affected at least one endpoint in the assay at concentrations which are achievable in vivo (i.e., below the systemic concentration at a lethal dose), yielding a false-negative rate of 19%. Two of the nondevelopmental toxicants, saccharin, and penicillin G, had adverse effects at concentrations below those that may be biologically achievable in vivo, giving a false-positive rate of 14%. Overall concordance with in vivo results by these criteria was 82%. Quantitative comparisons were also made between the lowest observed effect concentration (LOEC) in the assay and (i) lowest developmentally toxic dosage (mostly ip) reported in rats or mice in vivo and (ii) LOEC in rodent whole embryo culture. In the first instance, 77% of the LOECs (LOELs) were within an order of magnitude and 93% were within a factor of 30. In the second instance 81% of the LOECs were within an order of magnitude. Potency ranking of four alkoxy acids was comparable in CERC and the in vivo rodent embryo. These results indicate that the CERC assay is concordant with developmental toxic potential and potency for the diverse group of compounds selected, and that it could serve as a preliminary screen for developmental toxicity.

Effects of Exogenous FGFs on Growth, Differentiation, and Survival of Chick Neural Retina Cells

Fibroblast growth factors (FGFs) are known to play important roles in various processes including development and differentiation. Chick embryo neural retina cells, capable of transdifferentiation into lentoid bodies and pigmented cells, were used in vitro to examine the effects of exogenous acidic (aFGF) and basic FGF (bFGF) on proliferation and protein accumulation. We demonstrate that both factors increase the proliferation of glial cells and modulate the survival of neurons without affecting protein accumulation within these cells. Moreover, FGFs stimulate the differentiation of the photoreceptors. The rate of proliferation varies over the period of culture, with a maximum occurring after 2 weeks, followed by a decrease concommitant with the appearance of lentoid bodies. The concentration of aFGF was measured using an enzyme immuno assay and showed an accumulation of this protein only in bFGF-treated cultures, suggesting that bFGF positively modulates aFGF synthesis in neural retina cell cultures.

Purification and characterization of NCAD90, a Soluble endogenous form of N‐cadherin, which is generated by proteolysis during retinal development and retains adhesive and neurite‐promoting function

The cadherins are calcium-dependent cell adhesion molecules which regulate cell-cell interactions during morphogenesis. During development, cadherin expression is subject to dynamic patterns of regulation. We have previously demonstrated that expression of N-cadherin, the predominant cadherin of neural tissues, is sharply down-regulated during development of the retina and brain during later stages of histogenesis (Lagunowich and Grunwald, Dev Biol 135:158-171, 1989; Lagunowich et al., J Neurosci Res 32:202-208, 1992), and that this down-regulation is due to multiple factors, including decreased mRNA levels and turnover apparently mediated by endogenous metalloproteolytic activity (Roark et al., Development 114:973-984, 1992). In the present study, we describe metabolic studies which provide direct biochemical evidence for turnover of 130-kDa N-cadherin in embryonic retina tissues, yielding a soluble 90-kDa N-terminal fragment. We demonstrate that this form of N-cadherin, which we refer to as NCAD90, accumulates in vivo during development. We further demonstrate that purified NCAD90, obtained from embryonic vitreous humor, retains biological function and promotes cell adhesion and neurite growth in a dose-dependent fashion among chick embryo neural retina cells when present in a substrate-bound form. The morphology of retinal cells and neurites grown on a substrate of NCAD90 differs strikingly from that seen on a laminin substrate, in a manner similar to that described for intact 130-kDa N-cadherin. We conclude that proteolysis of N-cadherin at the cell surface during embryonic retinal histogenesis is an endogenous mechanism for regulating N-cadherin expression which generates a novel and functional form of the protein. The results further indicate that an intact cytoplasmic domain is not essential for all cadherin functions.

Lactate Dehydrogenase Activity during the Culture of Chick Embryo Neural Retina Cells in Vitro

تتحول خلايا الشبكية العصبية بعد عدة أسابيع من زراعتها إلىخلايا عدسية وذلك في بيئة F ( بيئة تحتوي على نسبة بسيطة من الجلوكوز 6mM) وتتثبط عملية الحول هذه عند زراعة نفس الخلايا في بيئة FG (بيئة تحتوي على نسبة عالية من الجلوكوز 28mM). وباستقصاء نشاط إنزيم اللاكتات دي هيدروجيناز بطرق مختلفة تبين أن نشاطه يكون عاليا في البيئة التي تحتوي على نسبة عالية من الجلوكوز (FG)، وعند استخدام طريقة الصبغ الكيميائي للأنسجة تأكد أن إنزيم اللاكتات دي هيدروجيناز يوجد بنسبة عالية ومكثفة في بيئة FG عنه في بيئة F. وتوضح نتائج التحليل بجهاز الإلكتروفورسيس أن هناك شريطين ( two bands) من الإنزيمات الشبيهة يتكونان عند زراعة هذه الخلايا في بيئة FG عند عمر 25 يوما من الزراعة ، وذلك كما هو موجود لنفس الخلايا في الطور البالغ، في حين في بيئة F، وفي نفس العمر، يتكون شريطإنزيم شبيه واحد وذلك كما هو موجود في خلايا الشبكية الجنينية. وتدل هذه النتائج على ارتفاع معدل الأيض في خلايا الشبكية مستخدمة الجلوكوز المتوافر بتراكيز عالية في بيئة FG.

Chick embryo neural retinal cell culture as a screen for developmental toxicity

An in vitro screen for developmental toxic potential of chemicals using primary cultures of chick embryo neural retina cells in described. The neural retinas of incubation Day 6.5 White Leghorn chick embryos are dissociated into single cells, which are subsequently maintained in a rotating suspension culture. Under normal circumstances, neural retina cells form spheroidal aggregates of a consistent size over the first 24 hr of culture, an event which is dependent on competent cell-cell interactions. Over the remaining 7-day period of culture, cells continue to divide and grow, and differentiation takes place. Each of these developmentally important events—aggregation, growth, and differentiation—is objectively and quantitatively measured as aggregate size and number, aggregate protein content, and glutamine synthetase (a marker of differentiation) activity, respectively. The effects on each developmental endpoint of 22 chemicals, 14 of which have been demonstrated to be developmentally toxic in one or more mammalian species in vivo, and 8 of which are not developmentally toxic, were evaluated. Chemicals were tested up to a concentration of 40 m m, or until marked cytolethality was observed. Of the known developmental toxicants, all but one, 2-methoxyethanol, affected one or more endpoints in the assay. The teratogenic metabolite of 2-methoxyethanol, 2-methoxyacetic acid, was active in the assay. None of the 8 nondevelopmental toxicants had any effect up to c concentration of 40 m m, or at biologically achievable concentrations (e.g., in vivo systemic concentrations at the LD50). Thus, the assay is 95% concordant with in vivo results for this set of chemicals. Quantitative comparisons were made (1) between developmentally toxic ip dosages in rats or mice in vivo and effective concentrations in the chick retina cell culture, and (2) between effective concentrations in chick retina cell culture and rodent whole embryo culture. In the first instance, 71% of the comparisons, and in the second instance, 89% of the comparisons, were within the same order of magnitude (and usually within a factor of two), indicating that the chick retina cell culture is also concordant with developmental toxic potency. Last, it was observed that test agents differentially affect developmental endpoints. Because the assay's endpoints are measured separately and objectively, it may be possible to use the assay to evaluate the effects of test agents on cellular development. The effects of tested chemicals on chick neural retina cells was consistent with reported effects of those chemicals. For example, chemicals which interfere with intercellular communication affected aggregation (e.g., all-trans retinoic acid, TPA), heavy metals (CdCl 2, HgCl 2) diminished growth, and chemicals which affect gene expression inhibited differentiation (e.g., bromodeoxyuridine, actinomycin D). Thus, the chick embryo retina assay appears to be capable of recapitulating in vivo developmental toxicity, and also may be useful in providing information about the mode of action of a developmental toxicant at the cellular level.