Developmentally Regulated Secreted Factors Control Expression of Muscarinic Receptor Subtypes in Embryonic Chick Retina

Abstract: Two molecular mass subtypes of muscarinic receptor are expressed by the chick retina (72 and 86 kDa). During development, the ratio of subtypes changes, with the 72-kDa form becoming predominant. We have found that subtype switch can occur in retina cell culture, and have investigated factors that influence this in vitro increase in the 72-kDa receptor. Increases similar to those in vivo occurred when cells were cultured at 10⁵ cells/cm², but not at 10-fold lower density. High-density cultures, maintained on coverslips, showed no receptor development when transferred to large volumes of fresh medium, indicating that cell-cell contact alone was not responsible for induction. However, replacement of fresh medium with conditioned medium (from high-density cultures) resulted in normal induction. There were no morphological differences between cultures with high and low levels of the 72-kDa receptor. Conditioned medium also induced 72-kDa receptors in low-density cultures, consistent with a minimal role for cell-cell contact. Efficacy of conditioned medium was markedly dependent on age. Media from cells cultured 1–4 days had no effect, but media from cells cultured 5–8 and 1–8 days elicited 1.6-fold and fourfold increases in the 72-kDa subtype, respectively. The data indicate that maturing retina cells secrete developmentally regulated factors that are necessary for abundant expression of the 72-kDa muscarinic receptor subtype.     

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons¹, which has been difficult with the conventional method of injection and electroporation at E1.5².     

Changes in Insulin Binding to Developing Embryonic Chick Neural Retina Cells

Specific cell surface insulin binding to embryonic chick neural retina cells has been demonstrated in vivo. Kinetics of insulin binding as well as hormonal specificity were similar to those reported for other vertebrate cells and tissues, both neural and nonneural. When surface insulin binding to retinal cells was studied as a function of embryonic age, a developmental relationship was observed. Scatchard analysis revealed that the number of cell surface insulin receptors decreased approximately 75% between days 10 and 16 of embryonic development. Receptor affinities remained fairly constant for this period.     

Nuclear Binding of Hydrocortisone-Receptors in the Embryonic Chick Retina and its Relationship to Glutamine Synthetase Induction

Binding of hydrocortisone (HC) to cytoplasmic receptors andof the resulting receptor-hydrocortisone complex (R-HC) to nuclearacceptor sites has been studied in neural retina cells of thechick embryo, in which this hormone induces glutamine synthetase(GS). These studies were done in a cell-free system, as wellas in intact retina tissue in culture. Optimal conditions, specificity, and the quantitative aspectsof R-HC binding to nuclei in the cell-free system were determined.Isolated nuclei retained their binding specificity for R-HCprepared from retina cytosol; at saturation, the total numberof nuclear acceptor sites for R-HC was estimated to be in therange of 1500 per nucleus. These sites were resistant to RNAsebut sensitive to DNAse. When retina tissue was cultured in the presence of progressivelyhigher doses (0–90 nM) of HC, increasing amounts of receptorswere translocated from the cytoplasm to the nuclei. Followingincubation of retina with 9–90 nM HC, the cytoplasm wasalmost completely depleted of receptors and the nuclei becamesaturated with R-HC. From the amount of R-HC bound to nucleiat saturation, the estimated total number of R-HC acceptor sitesper nucleus was comparable to that derived from the cell-freesystem. Increases in the level of GS induction in the retinacorrelated well with amounts of R-HC complex bound by nuclei.     

Acetylcholine and Dopamine Promote the Production of Platelet Activating Factor in Immature Cells of Chick Embryonic Retina

We have previously shown that platelet-activating factor (PAF), a naturally occurring lipid mediator of cell-to-cell communication, was produced by 3-day-old chick retina stimulated with acetylcholine (ACh) and dopamine (DA), but not with other neurotransmitters. ACh and DA stimulated PAF synthesis via a dithiothreitol (DTT)-insensitive cholinephosphotransferase, without affecting the acetyltransferase pathway, which was stimulated only by the calcium ionophore A23187. Therefore, we attempted to study the effects of neurotransmitters on PAF production and on the activities of the DTT-insensitive cholinephosphotransferase and acetyltransferase in the developing chick embryo retina up to hatching. Our results show that PAF was produced already at 8 days of development, when retinal cells are still rather immature and ganglion and Mueller cells are the only differentiated cells. The stimulation of PAF production occurred with ACh and not with other neurotransmitters. In older stages, DA also stimulated PAF production, as already described in the chick after hatching. DTT-insensitive cholinephosphotransferase and acetyltransferase activities were present in 8-day-old embryos, the earliest stage analyzed. Both enzymatic activities increased with age; DTT-insensitive cholinephosphotransferase increased rapidly from day 12 up to day 18, whereas acetyltransferase activity increased linearly up to the time of hatching. To promote PAF production, ACh and DA activate DTT-insensitive cholinephosphotransferase, but not acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stress Protein and Crystallin Synthesis During Heat Shock and Transdifferentiation of Embryonic Chick Neural Retina Cells

Embryonic chick neural retina responds to heat shock by the synthesis of "stress" polypeptides with molecular weights of 85 and 70 kd. Both stress proteins are synthesised from newly-transcribed messenger RNA. Sodium arsenite induces an additional stress protein of MW 25 kd. The heat shock response does not change during culture and subsequent transdifferentiation, and crystallin synthesis is not coinducible with the heat-shock proteins. We have also examined the pattern of protein synthesis at various stages of culture in both monolayer and aggregate systems; although changes in the protein synthetic profine are evident, there is no stress protein induction above basal levels at any time. Whilst mammalian α crystallin (B₂ chain) exhibits considerable homology to four small Drosophila heat-shock proteins, no significant antigenic similarity is apparent between δ crystallin and the major avian heat shock proteins. Thus during transdifferentiation, (a) the crystallin proteins do not behave in a manner analogous to stress proteins; moreover (b) crystallin production is not mediated by stress proteins resulting from a culture-induced stress response.     

In Vivo and In Vitro Expression of Gangliosides in Chick Retina Müller Cells

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from the chick neural retina was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach, after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. In retinas from 6-day-old embryos (R6), most cells (approximately 80%) expressed GD3 but not GTC. After culturing for 7 days, (R6+7), the expression of GTC was found confined to neuron-like cells; flat cells derived from Müller cells expressed GD3 but were negative for GTC expression. On the other hand, postmitotic Müller cells obtained from 13-day-old embryo (R13) or 1-day-old hatched chick retina (RP1) expressed GD3, GM1, and polysialosyl-GTC but were unable to maintain the expression of these GTCs when kept in culture for several days. According to these results, retinal cells can be defined on the basis of their ganglioside expression as follows: (a) retinoblasts, by the expression of GD3; (b) postmitotic neuronal cells, by the expression of GTC; and (c) postmitotic Müller cells, by the expression of GD3 and GTC.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Influence of Hydrocortisone on Chick Embryo Retina Development

Treatment of chick embryos in ovo with hydrocortisone-21-phosphate (a single dose of 150 micrograms) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65-70%) in 8-10-day-old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 micrograms daily for 2 days) also produced an age-dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 micrograms of metopirone, a compound that prevents the 11 beta-hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the action of hydrocortisone.     

Possible Roles for Cognin in Chick Embryo Neural Retina

In this review, we discuss current information about a cell-cellrecognition protein present in chick embryo neural retina. Thisprotein, retina cognin, has cell adhesion or aggregation promotingpropertiesin vitro. We discuss five questions. First, what isretina cognin (R-cognin)? Second, what do we know about cogninin chick retina? We discuss its histological distribution inretina and how that distribution changes during embryonic andearly post-hatching development. Third, where is cognin withincells? We review light microscopy evidence for its localizationin plasma membranes of somas and neurites of selected retinalneurons as an intrinsic membrane protein. Fourth, how is cognindistributed in membranes? We summarize evidence that cogninmight not be uniformly distributed over cellsurfaces and thatit might bind to specific proteins on the surfaces of otherretina cells. From the available information, we ask what wecan deduce about cognin's biological role in the neural retina.     

Glycerol Phosphate Dehydrogenase in Developing Chick Retina and Brain

Abstract: The development of cytoplasmic glycerol phosphate dehydrogenase (GPDH) activity in chick neural retina is compared with that in brain. GPDH converts dihydroxyacetone phosphate to glycerol 3-phosphate, an intermediate in phospholipid synthesis. The enzyme is known to be under corticosteroid control in rat brain and spinal cord (but not muscle or liver) and in primary oligodendrocyte cultures. It has not been previously studied in the eye. In chick brain the GDPH specific activity rises fivefold from the early embryo to the adult, with nearly all the increase occurring between embryonic day 14 and hatching. This time course correlates well with the known maturation of chick adrenal cortex (which produces corticosteroids). On the other hand, in chick retina the GPDH specific activity remains at a low basal level throughout development. Furthermore, adult rat and beef retinas show much lower enzyme activity than do the corresponding brain tissues. GPDH can be induced precociously by hydrocortisone in embryonic chick brain from days 12 through 16, both in the intact embryo and in tissue culture; however, GPDH is not at all inducible in chick retina. The developmental increase in chick brain GPDH can be correlated qualitatively with myelin formation, as shown by luxol fast blue staining, whereas no myelin is seen in retina at any age. Our results are consistent with recent immunocytochemical studies demonstrating that GPDH in rat brain is associated with myelin-producing oligodendroglial cells, absent in retina. In comparison, another glial enzyme, glutamine synthetase (GS), known to be inducible in both chick brain and retina, is localized in brain astrocytes and retinal Müller cells.     

Effects of Light on Dopamine Metabolism in the Chick Retina

The effect of prolonged exposure to light on the activity of dopaminergic neurons and dopamine (DA) metabolism of chick retinae was investigated. alpha-Fluoromethyldopa, a potent and specific irreversible inactivator of aromatic amino acid decarboxylase, was used to assess DA turnover after inhibition of synthesis, and also to assess in vivo tyrosine hydroxylase activity by dihydroxyphenylalanine accumulation. After 48 h of light exposure, retinal DNA in 12-day-old chicks was about 30% higher (p less than 0.005) whereas dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were elevated two to three times (p less than 0.005) the level of controls kept in the dark for the same period. DA turnover was about twofold faster in the light (t 1/2 = 31 min) than in the dark (t 1/2 = 65 min). Tyrosine hydroxylase, assayed in vitro with saturating levels of cofactor and substrate, increased by about 50% after light exposure. The apparent tyrosine hydroxylase activity in vivo was approximately sixfold higher in the light than the dark. These results are interpreted and discussed in terms of the regulation of DA synthesis, and the use of DOPAC and HVA as indices of DA function in the retina.     

Developmental Expression of Transferrin Binding Protein in Oligodendrocyte Lineage Cells of the Embryonic Chick Spinal Cord

Oligodendrocytes develop from precursor cells in the neuroepithelium of the ventral ventricular zone. Oligodendrocytes in the different stages of development are characterized by expression of a number of different marker molecules such as myelin genes, growth factors, and specific antigens. We have previously identified that transferrin binding protein (TfBP), a member of heat shock protein 90 families, is a novel avian ER-associated membrane protein that is specifically localized in oligodendrocytes in adult chicken CNS. In this study we describe the developmental expression of TfBP in the embryonic chick spinal cord. A few, distinct, TfBP+ cells appeared at the lateral margin of the subventricular neuroepithelium of the spinal cord at E7. Thereafter, some TfBP+ cells, exhibited a migrative form of unipolar or bipolar shape occurred around E8 in the mantle layer, midway between the neuroepithelium and the marginal layer of the primitive spinal cord. Thereafter, the TfBP+ cells rapidly increased in number as well as their staining intensity, and overall distribution of TfBP+ cells at E15 was comparable to that of a mature spinal cord. Our observations suggest that TfBP is expressed in the subpopulation of oligodednrcyte lineage in the development and a putative role of TfBP in relation to transferrin and iron trafficking is considered. SW Park and HS Lim contributed equally to this work.     

Tetrodotoxin Desensitization in Aggregates of Embryonic Chick Heart Cells

Spontaneous beating of heart-cell aggregates from 4-day chick embryos was initially blocked by 10^-5 g/ml tetrodotoxin (TTX). With continued exposure to the drug, the fraction of blocked aggregates decreased from about 80% at 15 min to about 25% at 2–3 h, at which time, beating aggregates had become desensitized to the toxin, showing no response to a fresh dose. Aggregates from 5-day hearts were more sensitive to TTX, but fewer became desensitized in its presence. Desensitization to TTX was not seen in 6- and 7-day aggregates. Inhibition of protein synthesis by cycloheximide did not affect beating or initial sensitivity to TTX of 4-day aggregates, but desensitization failed to occur. Before TTX, the mean value of maximal upstroke velocity (V_max) of the action potentials in 4-day aggregates was 33 V/s. After desensitization V_max was 12 V/s. Activity of desensitized aggregates in the presence of TTX was augmented by elevated calcium levels, and suppressed by presumed inhibitors of slow inward current (manganese, D600). Desensitization was reversible; upon removal of TTX 10^-5 g/ml, aggregates regained their responsiveness to a fresh dose of the drug with a 2–3 h time-course similar to that of desensitization. This was prevented by continued exposure to TTX at concentrations as low as 10^-8 g/ml. These data suggest that (a) desensitization involves a change in the mode of action-potential generating from one involving Na-specific, TTX-sensitive channels to one utilizing slower Mn-sensitive channels; (b) the process of desensitization occurs over a period of 2–3 h and is dependent upon the products of protein synthesis; and (c) desensitization is reversible after removal of TTX over a 2–3 h time-course similar to its onset.     

The Spread of Excitation in the Embryonic Chick Heart

The spread of excitation in embryonic chick hearts, ranging in age from 7 to 20 days, was studied with both intracellular and extracellular electrodes. Evidence that the delay in ventricular excitation could be attributed to the cells of the entire atrioventricular (AV) ring was obtained, in part, from sagittal sections of the heart. In the intact preparation, uniform propagation occurred throughout the atrial roof at an apparent conduction velocity of 0.4 to 0.5 meter/sec. Delay of impulse propagation was localized in a very narrow band of tissue which extended across the AV ring. The apparent conduction velocity of this tissue was between 0.003 and 0.005 meter/sec. Both normal and retrograde propagation revealed the spread of conduction across the AV ring to be decremental in nature. This finding was supported by high frequency stimulation experiments which gave rise to AV block localized in the cells of the AV ring. Cardiac rhythmicity and AV transmission were responsive to acetylcholine and norepinephrine in much the same manner as in the adult mammalian heart. The present findings are in support of the hypothesis that the embryonic AV ring is the functional counterpart of the adult AV node.     

The Electrophysiological Organization of the Embryonic Chick Heart

Both intracellular and surface electrodes were employed to record electrical activity from embryonic chick hearts between the ages of 3 and 20 days. Cells from the sinus venosus, sinoatrial (SA) valves, atrium, atrioventricular (AV) ring, and ventricle were localized and characterized on the basis of shape, amplitude, rise time, and duration of transmembrane potentials. The differences in transmembrane potentials from these various regions provided the basis for a hypothesis concerned with the distribution of pacemaker potentiality and one related to the origin of the His-Purkinje system. Action potentials recorded along the entire embryonic AV ring were comparable to those of the adult rabbit AV nodal cells in both configuration and sequence of activation and were thus categorized into three functional regions (AN, N, NH). Histological sections of 7 and 14 day hearts demonstrated muscular continuity between the right atrium and ventricle across the muscular AV valve.     

Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Müller Cells of the Retina

Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.