Antibodies to alpha-fetoprotein disrupt histogenesis in cultured chick retinae

alpha-Fetoprotein (AFP) is an oncofetal antigen believed to play an important role in normal development and carcinogenesis but very little is known about such a role. We have investigated here the role of AFP in neural retina development by selectively neutralising AFP in vitro. AFP has been immunohistochemically located in different cells and layers of the retina during its development, 4-day-old embryos being the earliest developmental stage when AFP is detected in the growing ganglion cell layer. Seven-day-retinae treated with antibodies to AFP in organ culture for 3 days did not continue to develop in the same way that they do in the egg. Neither the plexiform layers nor the buds of photoreceptor cells were observed after this culture period. In contrast, 7-day retinae cultured in the presence of non-immune serum developed in a manner similar to retinae in ovo. We present here the first evidence, derived by selectively blocking AFP in vitro with specific antibodies for an essential role of AFP in the normal development of the chick retina.     

Immunohistochemical Localization of Glycogen Synthase and GSK3β: Control of Glycogen Content in Retina

Glycogen has an important role in energy handling in several brain regions. In the brain, glycogen is localized in astrocytes and its role in several normal and pathological processes has been described, whereas in the retina, glycogen metabolism has been scarcely investigated. The enzyme glycogen phosphorylase has been located in retinal Müller cells; however the cellular location of glycogen synthase (GS) and its regulatory partner, glycogen synthase kinase 3β (GSK3β), has not been investigated. Our aim was to localize these enzymes in the rat retina by immunofluorescence techniques. We found both GS and GSK3β in Müller cells in the synaptic layers, and within the inner segments of photoreceptor cells. The presence of these enzymes in Müller cells suggests that glycogen could be regulated within the retina as in other tissues. Indeed, we showed that glycogen content in the whole retina in vitro was increased by high glucose concentrations, glutamate, and insulin. In contrast, retina glycogen levels were not modified by norepinephrine nor by depolarization with high KCl concentrations. Insulin also induced an increase in glycogen content in cultured Müller cells. The effect of insulin in both, whole retina and cultured Müller cells was blocked by inhibitors of phosphatidyl-inositol 3-kinase, strongly suggesting that glycogen content in retina is modulated by the insulin signaling pathway. The expression of GS and GSK3β in the synaptic layers and photoreceptor cells suggests an important role of GSK3β regulating glycogen synthase in neurons, which opens multiple feasible roles of insulin within the retina.     

In vivo regulation of cell death by embryonic (pro)insulin and the insulin receptor during early retinal neurogenesis

Programmed cell death is an established developmental process in the nervous system. Whereas the regulation and the developmental role of neuronal cell death have been widely demonstrated, the relevance of cell death during early neurogenesis, the cells affected and the identity of regulatory local growth factors remain poorly characterized. We have previously described specific in vivo patterns of apoptosis during early retinal neurogenesis, and that exogenous insulin acts as survival factor (Díaz, B., Pimentel, B., De Pablo, F. and de la Rosa, E. J. (1999) Eur. J. Neurosci. 11, 1624-1632). Proinsulin mRNA was found to be expressed broadly in the early embryonic chick retina, and decreased later between days 6 and 8 of embryonic development, when there was increased expression of insulin-like growth factor I mRNA, absent or very scarce at earlier stages. Consequently, we studied whether proinsulin and/or insulin ((pro)insulin) action in prevention of cell death has physiological relevance during early neural development. In ovo treatment at day 2 of embryonic development with specific antibodies against (pro)insulin or the insulin receptor induced apoptosis in the neuroretina. The distribution of apoptotic cells two days after the blockade was similar to naturally occurring cell death, as visualized by TdT-mediated dUTP nick end labeling. The apoptosis induced by the insulin receptor blockade preferentially affected to the Islet1/2 positive cells, that is, the differentiated retinal ganglion cells. In parallel, the insulin survival effect on cultured retinas correlated with the activation of Akt to a greater extent than with the activation of MAP kinase. These results suggest that the physiological cell death occurring in early stages of retinal development is regulated by locally produced (pro)insulin through the activation of the Akt survival pathway.     

The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos

Undissociated tissue explants of the retina and retinal pigment epithelium (RPE) of 3,5-, 4-, 5- and 8-day-old chick embryos were cultured in vitro. After 7 days in culture, lentoids were observed in explants of either retina or RPE from 3,5-, 4- and 5-day-old embryos. As demonstrated by immunohistochemistry, these lentoids contained specific chick lens proteins (alpha-, beta- and delta-crystallins). No crystallin-containing cells were found in eye tissue explants from 8-day-old embryos. However, when 5-bromo-deoxyuridine (25 microM) was introduced into the medium at the beginning of culturing (for 12 h), large eosinophilic cells containing alpha-, beta- and delta-crystallins were detected in retinal explants of the 8-day old embryos. Thus, retina and RPE of 3,5-5-day-old chick embryos are capable of lens differentiation after explantation in vitro without dissociation into individual cells. This capacity is lost during development.     

A novel gene (retinovin) expressed selectively in the early stage of chick retinal development

To understand molecular mechanisms of retinal development, genes expressed selectively only in the early stage of retinal development were isolated by subtractive hybridization based on suppression polymerase chain reaction. The retina has no layered structure in 7-day chick embryos, in contrast with the fully developed multilayered structure of neurons in 15-day embryos. The subtraction between cDNA derived from retinal tissues at these different stages, followed by repeat rounds of 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE, led to isolation of a novel gene with an open reading frame encoding a putative protein with 753 amino acids. Its specific expression in the 7-day embryonic retina was confirmed by Northern blot analysis. The gene, named "retinovin," would be used as a marker for identifying retinal stem cells present at the early stage of retinal development.     

A diabetic-like condition of turkey embryos maintained in shell-less culture

Serum insulin concentration and pancreatic insulin content were determined for turkey embryos incubated in ovo and in long-term shell-less culture (ex ovo). Insulin was undetectable (less than 10 pg) in serum from 87% of the ex ovo embryos compared with their in ovo counterparts. This was evident at all incubation ages, although insulin was detectable in more of the ex ovo embryos on Day 24. Insulin increased in the embryos incubated in ovo from 122 (Day 15) to levels exceeding 2000 pg/ml at hatching. Total pancreatic insulin content was greater in the cultured embryos on Days 15, 17, and 22 compared with their in ovo counterparts. Serum glucose was significantly greater (P less than 0.05) in the ex ovo embryos at all ages. In response to an infusion of L-arginine, serum insulin increased from 566 to 1256 pg/ml in the in ovo embryos, whereas no change was evident in the ex ovo embryos (233 vs 257 pg/ml). When embryos incubated in ovo were injected with insulin, a significant (P less than 0.05) reduction of serum glucose was observed at 60 min after injection. Serum glucose concentrations remained elevated in the embryos incubated ex ovo despite the insulin injection. Liver glucose 6-phosphatase activity, assessed on Days 15 and 22 of incubation, was found to be significantly (P less than 0.05) lower in the ex ovo embryos. Turkey embryos incubated in shell-less culture exhibited chronic hyperglycemia in concert with extremely low circulating levels of insulin. The pancreatic beta cells of these embryos were not responsive to arginine or elevated glucose. Taken together these findings suggest the occurrence of a diabetic-like condition in the ex ovo embryos. This defect in insulin secretion may, in part, be responsible for some of the developmental abnormalities characteristic of the turkey embryo cultured ex ovo.     

Effects of cell signaling on the development of GABA receptors in chick retina neurons

R-cognin, a cell recognition molecule, and insulin are known to play significant roles in GABAergic differentiation in the developing chick retina. In the present study, the effects of insulin and R-cognin on post-synaptic (GABAceptive) differentiation were investigated. In ovo binding of [³H]GABA and [³H]flunitrazepam ([³H]Flu) to the GABA and benzodiazepine (BZD) receptors, respectively, remained at low levels during early embryogenesis but increased sharply from mid-embryogenesis through hatching, increases which also occur in cultured neurons from early-embryonic (E7) and mid-embryonic (E11) chick retina. E7 neurons respond to insulin treatment (100 ng/ml) with increased [³H]Flu binding but no change in [³H]GABA binding. Cognin antibody (10 g/ml) treatment of E7 neurons caused no significant inhibition of the developmental increases in binding of either radioligand. Insulin in E11 cultures led to greater developmental increases in binding sites for both radioligands, but exposure to cognin antibody was without significant effect. These data, along with previous studies, indicate that GABAergic differentiation in developing chick retina is regulated, in part, by insulin and cognin-mediated cell signaling. Insulin also regulates post-synaptic (GABAceptive) differentiation whereas cognin-mediated interactions are relatively insignificant.Abbreviations BZD benzodiazepine - ChAT choline acetyltransferase - Flu flunitrazepam - GABA -aminobutyric acid - GAD glutamate decarboxylase (glutamic acid decarboxylase)     

Biosynthesis and Expression of Gangliosides During Differentiation of Chick Embryo Retina Cells In Vitro

Cells from neural retina from 7-day chick embryos were cultured on polylysine-coated dishes up to 7 days. The small, round-shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GD1a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was found. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl-N-acetyl-galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.     

Excessive dietary salt promotes neuroinflammation to worsen retinopathy in mice with streptozotocin-induced diabetes

ObjectiveDiabetic retinopathy (DR) includes vascular and neural tissue injury. Persistent low-grade inflammation may contribute to DR. Increased salt intake has been shown to promote autoimmunity in the brain. This study determined the role of salt intake in DR development.MethodsEight-week-old C57BL/6 J male mice received streptozotocin to induce diabetes. Diabetic or non-diabetic mice were fed a diet containing normal, low and high amounts of salt. The retinal function, structure and inflammatory response were determined 8 weeks after the establishment of diabetes. Interleukin (IL)-1β or a NLR family pyrin domain containing 3 (NLRP3) inhibitor was injected intravitreally and the retinal changes were evaluated.ResultsA high salt diet worsened the functional and structural damage of retinal cells and increased IL-1β in the retina of diabetic mice. IL-1β injection impaired the function of photoreceptors and retinal structure in the diabetic mice. Blocking NLRP3 inhibited IL-1β increase in the mouse bone marrow macrophages cultured in high sodium medium. NLRP3 inhibition attenuated retinal injury of diabetic mice on high salt diet. A low-salt diet also triggered inflammation and cell damage in the retina of diabetic mice but at a lower grade than those induced by high salt diet. A low or high salt diet for 8 weeks did not induce inflammation or cell injury in the retina of mice without diabetes.ConclusionThese results indicate that high salt intake has deleterious effects in DR development through NLRP3 inflammasome activation and the subsequent production of IL-1β. Limiting salt intake may not attenuate DR development.     

Characterization of insulin signaling in rat retina in vivo and ex vivo

Insulin receptor (IR) signaling cascades have been studied in many tissues, but retinal insulin action has received little attention. Retinal IR signaling and activity were investigated in vivo in rats that were freely fed, fasted, or injected with insulin by phosphotyrosine immunoblotting and by measuring kinase activity. A retina explant system was utilized to investigate the IR signaling cascade, and immunohistochemistry was used to determine which retinal cell layers respond to insulin. Basal IR activity in the retina was equivalent to that in brain and significantly greater than that of liver, and it remained constant between freely fed and fasted rats. Furthermore, IR signaling increased in the retina after portal vein administration of supraphysiological doses of insulin. Ex vivo retinas responded to 10 nM insulin with IR beta-subunit (IRbeta) and IR substrate-2 (IRS-2) tyrosine phosphorylation and AktSer473 phosphorylation. The retina expresses mRNA for all three Akt isoforms as determined by in situ hybridization, and insulin specifically increases Akt-1 kinase activity. Phospho-AktSer473 immunoreactivity increases in retinal nuclear cell layers with insulin treatment. These results demonstrate that the retinal IR signaling cascade to Akt-1 possesses constitutive activity, and that exogenous insulin further stimulates this prosurvival pathway. These findings may have implications in understanding normal and dysfunctional retinal physiology.     

Development of chicken epidermis cultured with embryo extract

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.     

Differential Binding to Glycotopes Among the Layers of Three Mammalian Retinal Neurons by Man-Containing N-linked Glycan, Tα (Galβ1–3GalNAcα1-), Tn (GalNAcα1-Ser/Thr) and Iβ/IIβ (Galβ1–3/4GlcNAcβ-) Reactive Lectins

Carbohydrate structures between retinal neurons and retinal pigment epithelium (RPE) play an important role in maintaining the integrity of retinal adhesion to underlying RPE, and in retinal detachment pathogenesis. Since relevant knowledge is still in the primary stage, glycotopes on the adult retina of mongrel canines (dog), micropigs and Sprague-Dawley rats were examined by lectino-histochemistry, using a panel of 16 different lectins. Paraffin sections of eyes were stained with biotinylated lectins, and visualized by streptavidin-peroxidase and diaminobenzidine staining. Mapping the affinity profiles, it is concluded that: (i) all sections of the retina reacted well with Morniga M, suggesting that N-linked glycans are present in all layers of the retina; (ii) no detectable human blood group ABH active glycotopes were found among retinal layers; (iii) outer and inner segments contained glycoconjugates rich in ligands reacting with T _α (Galβ1–3GalNAcα1-Ser/Thr) and Tn (GalNAcα1-Ser/Thr) specific lectins; (iv) cone cells of retina specifically bound peanut agglutinin (PNA), which recognizes T _α residues and could be used as a specific marker for these photoreceptors; (v) the retinas of rat, dog and pig, had a similar binding profile but with different intensity; (vi) each retinal layer had its own binding characteristic. This information may provide useful background knowledge for normal retinal physiology and miscellaneous retinal diseases, including retinal detachment (RD) and age-related macular degeneration (ARMD).     

γ-Aminobutyric Acid System in Developing and Degenerating Mouse Retina

Freeze-dried sections (14 microns thick) of retinal layers were prepared from mice with retinal degeneration (C3H strain) and control mice (C57BL strain). The weighed sections (2-30 ng dry weight) were analyzed using our microassay methods. In the control retina, gamma-aminobutyric acid (GABA) concentration and glutamate decarboxylase (GAD) activity, on a dry weight basis, increased from birth to 9 weeks of age and decreased slightly at 20 weeks. In the degenerated retina, the levels of GABA and GAD activity were higher at birth than in the control retina, and continued to increase until 20 weeks of age, at which time the GAD activity reached a markedly high level. This increase was found when the total GABA and GAD levels per retina were determined. In the normal retinal layers, GABA and GAD were confined primarily to the inner plexiform layer. In the degenerated retina, GAD activity gradually increased in the inner layers during postnatal development, but by 20 weeks the increase was most prominent in the inner part of inner nuclear layer and in the outer part of inner plexiform layer. GABA transaminase activity and its distribution were not much different in both normal and degenerated retinas during development.     

Variation of nitric oxide content regulates the development of apoptosis in the retina

It was shown that retinal ischemia entailed apoptosis in the inner layers of the retina. Administration of an NO-synthase inhibitor suppressed the development of ischemic apoptosis. To ascertain whether nitric oxide could induce the retinal apoptosis by itself, a nontoxic NO donor—dinitrosyl iron complex (DNIC) with glutathione—was injected into the vitreous body. DNIC at low concentrations induced apoptosis in the same retinal layers as in ischemia. However, with increasing DNIC doses, the number of apoptotic nuclei decreased markedly. Simultaneous administration of excess glutathione prevented apoptosis at any DNIC dose. The obtained data demonstrate the neurotoxic properties of the excess of nitric oxide in the retina.     

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.     

Involvement of neuronal cell surface molecule B2 in the formation of retinal plexiform layers.

The B2 molecule is a 220 kd neuronal cell surface protein of Xenopus, recognized by monoclonal antibody B2 (MAb B2). Immunohistochemistry using MAb B2 revealed that the B2 molecule was expressed in both the inner and outer plexiform layers within the neural retina. During development of the neural retina, the B2 molecule first appeared at stages 35/36 in the newly formed plexiform layers. When embryonic eyes were cultured in the presence of anti-B2 antiserum (Fab fragments), the formation of the retinal plexiform layers was impeded. These data suggest that the cell surface molecule B2 plays a role in the development of retinal plexiform layers.     

Effects of corticosterone on protein, RNA, DNA and tubulin contents in developing male and female rat brains

The effects of corticosterone treatment on chemical components and tubulin content were studied in the cerebrum, cerebellum and hypothalamus from male and female rats during early life. A dual effect of corticosterone treatment was observed in the cerebellum during the course of growth. In the cerebellum from 10-day-old rats, total soluble protein. DNA, and tubulin content (mg per g wet tissue) increased in the hormone-treated male organ, but RNA, DNA, and tubulin content (mg per g wet tissue) increased in the hormone-treated female. On the other hand, the cerebellum from 20-day-old rats, RNA and tubulin content (mg per g wet tissue) and relative tubulin content (mg per g total protein) decreased in the hormone-treated male organ, but the female cerebellum exhibited a decrease in total protein and tubulin content (mg per g wet tissue), and relative tubulin content after corticosterone administration. Only a few effects of the corticosterone treatment were observed in the cerebrum and hypothalamus from both sexes. It is likely that corticosterone has marked effects on the cerebellum among the three brain-regions in early life, and the dual effect of the hormone in the cerebellum appears to be due to the different responsiveness in the developmental stages of nerve cells, at which the treatment was started.     

CNS Targets Support and Sustain Differentiation of Cultured Neuronal and Retinal Progenitor Cells

Superior colliculus (SC) is the target of retinal neurons, allowing them to form connections. Cultured stem cells/progenitors can potentially be used as donor tissue to reconstruct degenerated retina including perhaps replacing lost ganglion cells in glaucoma. In which case, it will be essential for these cells to integrate with the central nervous system targets. Here, we have investigated if the mid-brain region containing superior colliculus (SC) provides a permissive environment for the survival and differentiation of neural progenitors, including retinal progenitor cells propagated in cultures. Neural (NPCs) and retinal progenitor cells (RPCs) from green fluorescent protein (GFP) transgenic mice were cultured. Passage two through four neural and retinal progenitor cells were subsequently cocultured with the SC organotypic slices and maintained in culture for 17 and eight days respectively. Differentiation of the neurons was studied by immunocytochemistry for retinotypic neuronal markers. Retinal progenitor cells cocultured with SC slices were able to differentiate into various neuronal morphologies. Some cocultured progenitor cells differentiated into neurons as suggested by class III β tubulin immunoreactivity. In addition, specific retinotypic neuronal differentiation of RPC was detected by immunoreactivity for calbindin and PKC. SC provides a permissive environment that supports survival and differentiation of the progenitor cells.     

5-溴脱氧尿嘧啶核苷对鸡胚神经视纲膜染色质、DNA的园二色性光谱与热变性的影响

使用5-溴脱氧尿嘧啶核苷(Brdu),对鸡胚神经视纲膜的形态发生与生化分化进行研究,以探讨基因的调节作用。 发育六天的鸡胚视纲膜经Brdu短期培养后,形态发生不可逆地抑制。但Brdu对发育七天的视纲膜的抑制作用是可逆的,发育九天的鸡胚视纲膜却不受其影响。 从发育六、七、九天的鸡胚视纲膜中提取DNA,进行CD谱的研究,与对照相比,经Brdu处理的视纲膜DNA的CD谱发生兰移,而且在260—280nm及290—300nm区域的吸收强度发生改变。Brdu还使发育六、七天的视纲膜染色质的CD谱发生明显变化,但发育九天的视纲膜染色质的CD谱的变化则不明显。对经Brdu作用的视纲膜DNA和染色质进行热稳定性分析结果也说明发育六、七、九天的视纲膜DNA的值和增色效应都有所增加,而染色质热稳定性的变化却不完全一致。