Effect of dissociative methods on cortisol binding and glutamyltransferase inducibility in chick embryo retina

The ability of the isolated embryonic chick retina (12 days) to bind a steroid (cortisol) decreases when the tissue is dissociated; the extent of this decrease depends upon the method of dissociation. Trypsin and mechanical dissociation decreased cortisol binding slightly; papain dissociation essentially eliminated it. Cortisol binding decreased with time in culture in both whole retina and monolayer cultures; this decrease may reflect, in part, a similar development decrease in ovo. Inducibility of glutamine synthetase in whole retinas and retinal monolayers prepared with either trypsin or papain also decreased with time in culture. For whole and trypsin-dissociated retinas, the drop in inducibility correlates with the drop in cortisol-binding capacity. This was not the case for monolayer cultures prepared by papain dissociation.     

Effect of dissociative methods on cortisol binding and glutamyltransferase inducibility in chick embryo retina

The ability of the isolated embryonic chick retina (12 day) to bind a steroid (cortisol) decreases when the tissue is dissociated; the extent of this decrease depends upon the method of dissociation. Trypsin and mechanical dissociation decreased cortisol binding slightly; papain dissociation essentially eliminated it. Cortisol binding decreased with time in culture in both whole retina and monolayer cultures; this decrease may reflect, in part, a similar developmental decreasein ovo. Inducibility of glutamine synthetase in whole retinas and retinal monolayers prepared with either trypsin or papain also decreased with time in culture. For whole and trypsin-dissociated retinas, the drop in inducibility correlates with a drop in cortisol-binding capacity. This was not the case for monolayer cultures prepared by papain dissociation.     

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.     

Interaction of beta-carboline with chick embryo spontaneous motility.

The effect of beta-carboline (beta-CCE) on spontaneous motility and its development was studied in chick embryos between the 11th and 19th day of incubation. 1. Acutely administered beta-CCE (7.5 mg/kg e.w.) already induced significant activation of motility in 11-day-old embryos. From the 17th day of incubation activation acquired a paroxysmal character. 2. In spinal embryos (decapitated on the second day of incubation) there was no such activating effect, demonstrating that it is associated with supraspinal components of the CNS. 3. In chronic administration from the fourth day of incubation (1.55 +/- 0.24 mg/kg e.w./24h), beta-CCE led to reduced development of spontaneous motility. The effect was concentrated in the period between the fourth and eighth day of incubation. The chronic administration of beta-CCE augmented the activating effect of metrazol and weakened GABA-inhibition of spontaneous motility. 4. On the basis of their findings, the authors express the hypothesis that the benzodiazepine beta-CCE-sensitive component of the complex GABA receptor evidently already functions from the beginning of the second half of incubation of chick embryos.     

Proteome profiling of embryo chick retina

Background  

Little is known regarding the molecular pathways that underlie the process of retinal development. The purpose of this study was to identify proteins which may be involved in development of retina. We used a proteomics-based approach to identify proteins that are up- or down-regulated during the development of the embryo chick retina.     

Culture of chick embryo neural retina in serum-free medium : Expression of cholinergic proteins

Dissociated retinal cells from 8-day-old chick embryos were cultivated in serum-containing and in defined serum-free media. Under the latter conditions, and using polylysine as a substrate, the proliferation of glial cells was almost completely prevented, and pure (>90%) neuronal cultures could be maintained for up to 7 days in vitro. The specific but not the total activities of choline acetyltransferase and of the nicotinic and the muscarinic acetylcholine receptor were increased under serum-free culture conditions. Autoradiographic studies with [¹²⁵I]α-bungarotoxin, a selective ligand for nicotinic cholinergic receptors, showed that serum-free culture conditions may constitute a useful tool for identifying biochemically different types of retinal neurons in tissue culture.     

Expression of cone-like properties by chick embryo neural retina cells in glial-free monolayer cultures

We report here that cells present in embryonic chick retinal monolayer cultures express differentiated properties characteristic of chick cones developing in vivo. Cell suspensions from 8-d chick embryo retina (a stage when photoreceptor differentiation has not yet started) were cultured for up to 7 d in low density, glial-free monolayers. Under these conditions, monopolar cells represent approximately 40% of the total number of process-bearing neurons. After 6 d in vitro, most of these monopolar cells showed morphological features reminiscent of developing chick cones. These features could be detected with phase-contrast microscopy, lectin cytochemistry, and transmission and scanning electron microscopy. Characteristic cone traits expressed by cultured monopolar cells included the following: (a) a highly polarized organization; (b) a single, short, usually unbranched neurite; (c) the polarized position of the nucleus close to the origin of the neurite; (d) characteristic cone inner segment features such as abundant free ribosomes, a polarized Golgi apparatus, a cluster of mitochondria distal to the nucleus, a big, membrane-bound, pigment-containing vacuole reminiscent of the "lipid droplet" characteristic of chick cones, and at least in some cases, a well-developed paraboloid; (e) the presence of a complex of apical differentiations including abundant microvilli and in some cases also a cilium-like process; and (f) the staining of the apical region of the cell with peanut lectin, which has been shown to be selective for chick embryo cones (Blanks, J.C., and L.V. Johnson, 1983, J. Comp. Neurol., 221:31-41; and Blanks, J.C., and L.V. Johnson, 1984, Invest. Ophthalmol. Visual Sci., 25:546-557). This pattern of differentiation achieved by 8-d chick retina cells after 6 d in vitro is similar to that shown by 14-d-old chick embryo cones in vivo. Outer segments are not present at this stage of development either in vivo or in vitro. This experimental system is now being used to search for cellular and molecular signals controlling survival and differentiation of cone cells.     

Lysophosphatidic acid‐induced Ca2+ 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 Ca²⁺ concentration ([Ca²⁺]_i) with Fura‐2 in the neural retina of chick embryo during neurogenesis. Bath application of LPA (1–100 μM) to the embryonic day 3 (E3) chick retina caused an increase in [Ca²⁺]_i in a dose‐dependent manner, with an EC₅₀ value of 9.2 μM. The Ca²⁺ rise was also evoked in a Ca²⁺‐free medium, suggesting that release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) was induced by LPA. U‐73122, a blocker of phospholipase C (PLC), inhibited the Ca²⁺ rise to LPA. Pertussis toxin partially inhibited the Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ response to LPA were the same as those of the Ca²⁺ response to adenosine triphosphate (ATP), which enhances the proliferation of retinal progenitor cells. The coapplication of LPA with ATP resulted in enhancement of Ca²⁺ rise in the E3 chick retina. Our results show that LPA induces Ca²⁺ mobilization in the embryonic chick retina during neurogenesis. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 495–504, 1999     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially single cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4--6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4 degrees C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially sigle cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4–6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4°C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Ligatin from embryonic chick neural retina

Ligatin, a filamentous protein previously found in suckling rat ileum, has been purified from plasma membranes of embryonic chick neural retina. The isolated plasma membranes are covered in part by 4.5-nm filaments that can be released from the membranes by treatment with Ca++. Subsequent dialysis against EGTA followed by sieve chromatography results in purification of the 10,000-dalton ligatin monomer. When labeled either with radioisotopes or with fluorescamine, the monomer is shown to electrophorese as a single discrete band in polyacrylamide gels. However, during standard fixing and staining procedures it diffuses from the gels and thus is not visualized. Ligatin's amino acid composition is distinguished by its high content of polar residues, especially Glx and Asx, and by the presence of phosphorylated serine. Upon re-addition of Ca++, purified ligatin monomers polymerize to form filaments 3 nm in Diam, identical to those formed by purified ileal ligatin. However, in both retina and ileum, the filaments observed on plasma membranes are greater than 3 nm in Diam. In ileum, this enlargement results from ligatin's function as a baseplate for the attachment of another protein, a beta-N-acetylhexosaminidase, to the cell surface. In retina, a corresponding difference in diameter between filaments seen in vivo and those formed from repolymerized ligatin alone and the co-solubilization of other proteins with ligatin suggest that ligatin may also function there as a baseplate for other cell surface proteins. The proteins associated with ligatin in retina differ morphologically from beta-N-acetylhexosaminidase and do not possess this enzymatic activity.     

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture. II.

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.