The cell adhesion molecule retina cognin is a cell surface protein disulfide isomerase that uses disulfide exchange activity to modulate cell adhesion.

The retina cell adhesion molecule, R-cognin, shares cDNA sequence with protein disulfide isomerase (PDI) but has a different molecular size and subcellular location. We asked whether R-cognin originated from a unique PDI gene transcript or was a product of posttranscriptional processing. The 3'-terminal partial cDNA clone for R-cognin was extended by both 5' RACE and by PCR from sequence near the 5' end of the PDI-translated region. The cDNA sequence was compared to those of chicken, bovine, and human PDI. The R-cognin cDNA sequence was identical to that of chicken PDI and differed by less than 10% from mammalian PDI proteins. The role of the disulfide exchange activity characteristic of both proteins was studied by assessing the cell-aggregation-enhancing ability and tissue specificity of R-cognin and recombinant human PDI and its derivatives. Chicken and normal human PDI proteins showed tissue- and developmental-specific enhancement of cell aggregation identical to R-cognin, and this activity was blocked by inactivation of the -WCGHC- motifs which function in disulfide exchange. Dependence of retina cell aggregation on disulfide exchange activity was shown by blocking that activity with the inhibitor, DTNB, or with a recombinant human PDI with the -WCGHC- motif cysteines mutated. The results suggest that one -WCGHC- motif in R-cognin is sufficient and that the more C-terminal motif is most active. We conclude that R-cognin is a tissue-specific protein product of the standard PDI chicken gene. The -WCGHC- motif in mature R-cognin is necessary, but not sufficient, for cell adhesion.

Thioreductase activity of retina cognin and its role in cell adhesion.

Retina cognin (R-cognin) is a 50-kDa protein on the surface of embryonic chick retina cells that mediates cell-cell recognition and neuronal differentiation. It is developmental stage- and tissue-specific in its expression. The partial cDNA clone for R-cognin is nearly identical to that of chicken protein disulfide isomerase (chicken PDI) and enzyme with thioreductase activity. The R-cognin clone extends from beyond the 3' polyadenylation site up to the boundary between PDI exons 1 and 2, with the putative R-cognin equivalent of PDI exon 1 remaining uncloned. The question posed here was whether the sequence-specific properties of PDI were significant in the action of R-cognin. We show that R-cognin, like PDI, has thioreductase activity as revealed by RNase renaturation enzymatic assays. We then asked if this thioreductase activity was involved in the mediation of cell adhesion and recognition in developing chick retina. We show, through cell aggregation assays, that both R-cognin and chicken PDI enhance chick retina cell aggregation but not that of cells from other CNS tissues. We also show that treating R-cognin and chicken PDI with the thioreductase inhibitor 5,5'-dithio-bis (2-nitrobenzoic acid), which covalently binds to the functional cysteines of the thioreductase active sites, reduces the enhancement of cell aggregation. Thus R-cognin acts, in part, by catalyzing a covalent protein-protein linkage at the cell surface.

Co-localization of the insulin receptor, jun protein and choline acetyltransferase in embryonic chick retina.

Previous work demonstrated that the availability of insulin to the embryonic chick retina at a critical developmental stage stimulated the activity of the acetylcholine synthetic enzyme, choline acetyltransferase (ChAT) and that this increase required the AP-1 transcription factor, c-jun. Here it is shown that immediately following a 2-5 min exposure to insulin there is, in the amacrine and ganglion cells of the chick embryo retina, a transient increase in the level of jun protein followed by a long-lasting increase in ChAT. These and previous results show that insulin receptor activation is necessary for the characteristic retina developmental increase in ChAT protein and that this increase is preceded by a transient increase in the synthesis of the transcription factor c-jun in the same retina cells. The data demonstrate an intracellular signal transduction pathway from the developmentally-activated insulin receptor through c-jun to ChAT and cholinergic differentiation.

Causal role for jun protein in the stimulation of choline acetyltransferase by insulin in embryonic chick retina.

Previous work showed that the availability of insulin to the embryonic chick retina at a critical developmental stage stimulated the activity of the acetylcholine synthetic enzyme, choline acetyltransferase (ChAT) (R. E. Hausman et al., 1991, Dev. Brain Res. 59, 31-37). Here we show that a 2- to 5-min exposure to insulin results in a greater than 24 hr elevation in ChAT protein. Immediately following exposure to insulin there is a transient increase in the level of jun protein followed by an increase in ChAT. The stimulation of ChAT protein is not the result of an overall stimulation of protein synthesis as other proteins are not affected. Exposure of the cells to antisense oligonucleotide to jun, but not to sense oligonucleotide, reduces the increase in both jun and ChAT. These and previous results suggest that insulin is necessary for the characteristic increase in ChAT protein during retina development and that this increase requires the transient synthesis of jun.

Antisense inhibition of R-cognin expression modulates differentiation of retinal neurons in vitro.

PURPOSE: Retina cognin (R-cognin) is a 50 kDa membrane-associated polypeptide expressed during retinogenesis where it is involved in mediating tissue-specific cell-cell interactions. In addition to its intercellular role in aggregation, R-cognin may act as a cell surface signaling molecule. An antisense oligonucleotide was used to inhibit R-cognin expression and to investigate the effects of this inhibition on subsequent neuronal differentiation. METHODS: Cultures of retina cells were prepared from 6 day (E6) and 8 day (E8) chicken embryos and were incubated with a deoxyoligonucleotide complimentary to 20 bases of the sequence encoding R-cognin or random oligonucleotides. The levels of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD), markers of cholinergic and GABAergic differentiation, respectively, were detected by Western blots on protein extracts from treated cultures. RESULTS: The antisense treatment inhibited ChAT levels at E6 and GAD levels at E8. The treatment resulted in no decrease in the level of the enzyme glyceraldehyde 3-phosphate dehydrogenase. A random oligonucleotide did not affect the levels of any of the proteins. CONCLUSIONS: These results confirm the cell recognition role of R-cognin and suggest that it is important in intracellular signaling cascades necessary for normal retina development.

Developmental localization of retina cognin synthesis by in situ hybridization.

Retina cognin (R-cognin) is a 50 kDa protein involved in cell recognition and neuronal differentiation during development of the embryonic chick retina. Initial characterization of a partial cDNA encoding R-cognin revealed a striking similarity to the cDNA encoding protein disulfide isomerase (PDI), a 57 kDa multifunctional protein. The exact nature of the relationship between R-cognin and PDI is not known; however, both proteins appear to be encoded by the same gene. In the present study, we developed cRNA probes to examine the expression of R-cognin and PDI transcripts in embryonic chick retina and liver. In the retina, the amount of transcript decreased with embryonic age, in parallel to a similar decrease in R-cognin protein. In the liver, where PDI is prominently expressed, the amount of transcript was not developmentally regulated. The spatial and temporal pattern of expression of the R-cognin-encoding retinal transcript was examined by in situ hybridization. R-cognin mRNA was expressed in cells across the retina early in retinogenesis, but became restricted to the cells of the inner retina later in development. This pattern of expression was the same as the developmental pattern of R-cognin protein [Dobi et al., Invest. Ophthalmol. Vis. Sci. 27, (1986) p. 323-329], thus, demonstrating that this secreted protein functions at the surface of the cells where it is transcribed.

Expression of aldolase A steady-state mRNA is delayed relative to other muscle-specific genes during differentiation of chicken myoblasts.

Expression of several muscle-specific genes was monitored during chicken muscle development and myoblast differentiation in primary cultures. The individual patterns of expression for many muscle-specific genes are well documented in ovo and in other model systems of muscle development. However, comparison of aldolase A to other muscle-specific genes in one system has not been reported. Both sarcomeric and cytosolic genes important for the adult muscle fiber were examined in order to elucidate their timing of expression and its relationship to cell fusion. Steady-state mRNA expression was measured using RNase protection assays with cRNA probes generated from cDNA clones for muscle creatine kinase, fast skeletal troponin-T, embryonic myosin heavy chain, and aldolase A. Nonmuscle genes expressed largely in the embryo, aldolase C and beta-actin, were used as controls. The expression of all six genes revealed differences in temporal expression patterns between limb and axial muscle. The temporal expression patterns of all six genes were also monitored in primary myoblast cultures relative to myoblast fusion. In both axial and limb myoblast cultures most of the muscle-specific genes were expressed prior to fusion. During the differentiation of myoblasts to myotubes there was a biphasic pattern in the expression of the muscle-specific genes. The appearance of measurable mRNA was detected by 16 hr in culture, prior to appreciable fusion of the cells. During further differentiation the expression increased gradually and then more rapidly at 96 hr, once fusion was complete. Meanwhile, the nonmuscle embryonic gene expression declined only slightly. For one gene, aldolase A, expression was delayed relative to the other muscle-specific genes, both in the appearance of measurable mRNA and in the later rapid increase in mRNA.

Determination of fractal dimension of physiologically characterized neurons in two and three dimensions.

Although there is a growing interest in the application of fractal analysis in neurobiology, questions about the methodology have restricted its wider application. In this report we discuss some of the underlying principles for fractal analysis, we propose the cumulative-mass method as a standard method and we extend the applicability of fractal analysis to both 2 and 3 dimensions. We have examined the relationship between the method of log-log Sholl analysis and fractal analysis and have found that they correlate well. Measurements of physiologically characterized retinal ganglion cells indicate that different cell types can have significantly different fractal dimensions. Such differences may allow the correlation of the physiological type of a neuron with its morphological fractal dimension.

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 micrograms/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.

Effect of insulin on GABAergic development in the embryonic chick retina.

We investigated the role of insulin in GABAergic differentiation in the embryonic chick retina at different embryonic ages using glutamate decarboxylase (GAD) and high-affinity GABA uptake as developmental markers. Both these GABAergic markers exhibit developmentally programmed increases in activity during retinogenesis that also occur in culture. Insulin stimulated GABA uptake in retina neurons at all embryonic ages in a dose-dependent manner and GAD activity by 30% in embryonic retina neurons after 11 days of development. The stimulation of GABA uptake by insulin was blocked by addition of ouabain suggesting a role for the Na+,K+ ATPase. The same concentration of insulin caused a 76% stimulation of protein synthesis in these retinal cells, and previous work demonstrated that insulin also stimulates cholinergic differentiation in the chick retina (Hausman et al., Dev. Brain. Res. 59, (1991) 31-37). Thus, there was no selective stimulation of GABAergic differentiation by insulin but likely a neurotrophic effect. The increase in GAD activity in neurons from post-11-day embryonic neurons contrasts with our previous findings at embryonic days 6-7 where there is little change in GAD activity after addition of insulin. It is possible that the failure of insulin to stimulate GAD activity during early retina development is due to the increased accumulation of GABA in the presence of insulin. GABA levels were increased more than two-fold by 100 ng/ml insulin.

cDNA for R-cognin: homology with a multifunctional protein.

Retina cognin (R-cognin) is a developmentally regulated 50-kDa protein that was isolated from chicken embryo retina cell membranes. It mediates the adhesion and reaggregation in vitro of retina cells from chicken and mouse embryos, but not of cells from other tissues, and may be involved in neuronal differentiation. We report here the cloning of a cDNA for R-cognin. A chicken embryo retina cDNA library was constructed in lambda gt11 vector and was screened with polyclonal R-cognin antiserum, yielding several immunoreactive clones. Antiserum prepared to the R-cognin-beta-galactosidase fusion protein produced by one recombinant lysogen recognized the 50-kDa R-cognin protein derived from retina cell membranes. This antiserum inhibited the reaggregation of dissociated retina cells and immunostained chicken embryo retina tissue in a pattern similar to that obtained with R-cognin antiserum. In vitro translation of RNA from a cDNA subclone yielded a 50-kDa protein that was recognized by R-cognin antiserum on a Western blot. By these criteria we identify the cDNA clone as representative of the gene encoding R-cognin. This cDNA is nearly identical to a major portion of the cDNA for the multifunctional protein that is the beta subunit of prolyl 4-hydroxylase and has both protein disulfide isomerase activity and thyroid hormone-binding activity. These findings demonstrate that R-cognin differs from other cell adhesion molecules and suggest possible mechanisms for its action in cell adhesion and neuronal differentiation.

Role of the cell recognition molecule, cognin, in GABAergic differentiation in chick retina.

Previous work showed that GABAergic differentiation in developing chick retina depends on insulin and cell interactions. Here, we investigated whether it depended on cell signaling mediated by retina cognin, a 50 kDa cell recognition molecule. Cognin mediates cell adhesion in vitro and occurs on retinal neurons that become both GABAergic and cholinergic. We investigated two markers of GABAergic differentiation: glutamate decarboxylase (GAD) activity and high-affinity GABA uptake. Both increase during differentiation of retinal neurons in culture and can be easily measured. We blocked cognin-mediated cell signaling with cognin antibody and found a reduction of the developmental increase in GAD activity in cultures of retinal neurons from 7 and 11 day chick embryos. There was no reduction of high-affinity GABA uptake. This suggested that cognin-mediated signaling was necessary for the normal developmental increase in GAD but not for high-affinity GABA uptake. These results contrasted with our previous observations on cholinergic differentiation in cultured retinal neurons. We found that cognin antibody blocked the normal developmental increase in choline acetyltransferase (ChAT) only if the cells were exposed before embryonic day 7. Thus, while both GAD and ChAT activity appear to be controlled by cell signaling involving cognin, the periods of developmental sensitivity for the two differentiation markers are different. Antibodies to other adhesion molecules, Ng-CAM, and N-cadherin, did not similarly affect GAD activity. Antibodies to laminin at a 10-fold higher concentration inhibited GAD activity only in early embryonic retina. Tests for protein synthesis and "housekeeping" enzyme activity demonstrated that the cognin antibody effect was selective for neuronal differentiation pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

The cell recognition molecule, cognin, mediates choline acetyltransferase activity in embryonic chick retina.

Cell signaling and cell-cell interactions play an important role in neuronal differentiation in the embryonic CNS. Previous work (Hausman, R.E., Vivek Sagar, G.D. and Shah, B.H., Dev. Brain Res., 59 (1991) 31-37) had shown that cholinergic differentiation in the embryonic chick retina depends on insulin and neuron-neuron interactions. Here, we pursued the molecular nature of that dependence on cell interactions. The embryonic chick retina is known to contain several cell adhesion or recognition molecules. We asked if retina cognin, a 50 kDa cell surface-associated protein, played a role in controlling cholinergic differentiation in the developing chick retina. As previously, cholinergic differentiation was measured by two markers: choline acetyltransferase (ChAT) activity and high-affinity choline uptake. We used polyclonal antibody to cognin to determine if blocking cognin-mediated cell interactions would affect the normal embryonic increases in these cholinergic markers. We demonstrated a 40% inhibition of the normal developmental appearance of ChAT activity in retina neuronal cultures from early development, but no effect in cultures from more differentiated retina. The inhibition was selective for retina, since it was not seen in neural tissues like cerebrum and cerebellum that also express ChAT. In contrast to the effect of insulin, choline uptake was not affected by treatment with cognin antibody. Antibodies to two other cell recognition molecules present in the retina (Ng-CAM and N-cadherin) did not block the normal developmental appearance of ChAT. These results suggest that cognin-mediated interactions play a unique role in the control of one aspect of cholinergic differentiation in the developing chick retina.

Effect of viscosity on neurite outgrowth and fractal dimension.

The growth mechanism by which neurons achieve their characteristic ramified morphology has long been of interest, but determining whether physical parameters, such as viscosity, are important has been difficult due to a lack of useful hypotheses and standard reproducible techniques. We have recently shown that neurons exhibit fractal behavior and that their fractal dimension (df) is consistent with a physical process called diffusion-limited aggregation (DLA). We suggested that this DLA behavior might stem from viscosity differences, chemical gradients or electrical fields (Caserta et al., Phys. Rev. Lett., 64 (1990) 95-98). DLA is a model for a large family of growth processes. In order for a process to fit the DLA model, the growth rate must be proportional to the gradient of a field at a point on the growing structure (Feder, Plenum, New York, 1988, Ch. 4). Chemical, electrical, or fluid pressure fields can fit the model depending on the particular physical system under study. Here, we studied growth of retinal neurons from chick embryos in culture media of various fluid viscosities. Thus, we test whether DLA in this system was based on a fluid pressure field. As viscosity was increased from 1 to 4.3 cps, the number of neurite branches decreased 98%. However, there was no effect on df. Over this range of viscosities, total cellular protein synthesis decreased only 17%. The results indicate that, while differences in viscosity between the interior and exterior of the cell affect neurite outgrowth, they do not affect the fractal behavior of neurons. Thus, viscosity differences are not the basis for the DLA pattern of neuronal arborization.

Initial cholinergic differentiation in embryonic chick retina is responsive to insulin and cell-cell interactions.

Previous work [Kyriakis et al., Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7463-7467] had shown that insulin, when added during a window of binding from embryonic days 9-11, stimulates the normal developmental increase in choline acetyltransferase (ChAT) activity (a marker for cholinergic differentiation) in cultured embryonic chick retinal neurons. Here, we investigated the effect of insulin and IGF 1 on embryonic chick retinal neurons at the stage of development (embryonic day 6) when ChAT activity is first expressed. We investigated insulin peptide effects in retinal tissue developing in vitro as well as in cultures of retinal cells. We show that insulin also stimulated the initial embryonic increase in ChAT activity but had no stimulatory effect on glutamic acid decarboxylase activity (a marker for GABAergic differentiation), an enzyme whose activity also increases developmentally in the same retinal neurons. In fact, insulin inhibited the expression of GAD activity in the retina. The insulin-mediated increase in ChAT activity was independent of normal cell-cell interactions but could not replace them. Insulin also stimulated choline uptake but only after a two day delay, suggesting that the normal program for cholinergic differentiation in the chick retina was induced by insulin. IGF 1 did not have any effect on either cholinergic or GABAergic differentiation. We conclude that cholinergic differentiation in chick embryo retinal neurons is dependent on both insulin- and cell contact-mediated signals.

Prostaglandin-dependent phosphatidylinositol signaling during embryonic chick myogenesis.

Previous investigations suggested that binding of prostaglandin to a myoblast membrane receptor initiates a second messenger cascade which is essential for subsequent myogenesis. Initial evidence of the sensitivity of myogenesis to lithium suggested the involvement of inositol phosphate metabolism. That possibility is investigated here. The accumulation of inositol monophosphate in response to prostaglandin binding was studied in aggregate cultures of chick embryo myoblasts in vitro. At 22 or 28 h in culture mononucleated myoblasts were labeled with [3H]inositol, which was then incorporated into phosphoinositides. After experimental manipulations of prostaglandin metabolism and the addition of Li+ prior to prostaglandin binding at 33 h, [3H]inositol monophosphate accumulation was measured by anion-exchange chromatography between 33 and 37 h. Inositol monophosphate was found to accumulate rapidly following 33 h. However, after 36 h of myogenesis, no inositol monophosphate accumulation was observed. The accumulation was dependent on prostaglandin as indomethacin, which also blocks subsequent membrane events in myogenesis, blocked inositol phosphate accumulation. Like subsequent myogenesis, inositol phosphate accumulation was restored by the addition of exogenous prostaglandin. Finally, the accumulation of inositol phosphate began only after the binding of prostaglandin. The results demonstrate that an inositol phosphate signal transduction mechanism connects prostaglandin binding to membrane events in embryonic chick myogenesis.

Requirement for G protein activity at a specific time during embryonic chick myogenesis.

Signaling between embryonic myoblasts involves prostaglandin metabolism, the activation of a membrane receptor and changes in polyphosphatidyl inositol metabolism. Many of these membrane-localized events occur between 33 to 35 h of differentiation, concomitant with a dramatic change in membrane organization, in myoblast aggregates in culture. Since many receptors affect inositol phosphate metabolism by activating a GTP-binding protein (G protein), we asked if there was evidence for such a protein in myogenic signaling. We show that during the period of differentiation in culture when prostaglandin is needed to bind to a transient receptor, a pertussis toxin-sensitive but cholera toxin-insensitive G protein must act. If this activation is blocked, the characteristic change in myoblast cell adhesion and subsequent membrane fusion do not occur. We suggest that a G protein couples the activated prostaglandin receptor and the change in polyphosphatidyl inositol metabolism and that this membrane transduction step is necessary for subsequent membrane differentiation events during myogenesis.