Tissue-specific cell-surface antigens in embryonic cells.

With the use of antisera prepared in rabbits against suspensions of live embryonic chick tissue cells, qualitative differences in cell surface antigens were demonstrated on cells from different embryonic chick tissues by immune agglutination and immunofluorescence. Unabsorbed antisera reacted with both homologous and nonhomologous cells; thorough absorption of the antisera with heterologous tissues removed cross-reacting antibodies, and the antisera acquired a high degree of tissue specificity. Thus, antiretina cell serum absorbed with nonretina cells or tissues, agglutinated only neural retina cells, and was shown by immunofluorescence tests to react specifically with the surface of retina cells, both in cell suspensions and in frozen tissue sections. Comparable results with antisera against cells from embryonic liver and other tissues demonstrated the existence of tissue-specific, phenotypic disparities in the antigenicities of embryonic cell surfaces, in addition to the presence of cell-surface antigens shared by certain classes of cells, and of antigens common to all cells in the embryo. The results are discussed in terms of the possible involvement of such phenotypic determinants in the specification of cell surfaces, in relation to cell recognition and developmental interactions.

Effects of cytosine arabinoside on differential gene expression in embryonic neural retina. I. Accumulation of glutamine synthetase with suppression of macromolecular synthesis.

The analogue of cytidine, cytosine arabinoside (Ara-C), elicited a significant increase in the level of glutamine synthetase (GS) in embryonic chick neural retina in the absence of the steroid inducer of the enzyme. The increase was due to de novo synthesis of GS and was mediated by RNA which accumulated in the presence of the effective concentration of Ara-C. Accumulation of GS did not result from the inhibition of DNA synthesis for which Ara-C is best known. This new effect of Ara-C involves differential suppression of macromolecular synthesis in this system: the concentration of Ara-C which caused maximum GS accumulation suppressed overall protein and RNA syntheses 65-75% without inhibiting the transcription and translation of templates essential for GS synthesis. Withdrawal of Ara-C resulted in restoration of RNA synthesis and cessation of GS accumulation, even though preformed templates for the enzyme were present; however, if all RNA synthesis was arrested with actinomycin D at the time of Ara-C withdrawal, GS continued to accumulate. The results are consistent with the hypothesis that Ara-C differentially affects the activity of structural and regulatory genes involved in the regulation of GS levels in the retina: Ara-C allows transcription of the enzyme-specific templates, but reversibly inhibits the expression of regulatory genes which limit the accumulation of GS.

Effects of cytosine arabinoside on differential gene expression in embryonic neural retina. II. Immunochemical studies on the accumulation of glutamine synthetase.

Cytosine arabinoside (Ara-C) elicits a significant increase in the level of the enzyme glutamine synthetase (GS) while it markedly reduces overall RNA and protein synthesis in cultures of embryonic chick neural retina. This increase was analyzed by radioimmunochemical procedures and compared with the induction of GS by hydrocortisone (HC). Accumulation of GS in Ara-C-treated retinas was found to be due to de novo synthesis of the enzyme; however, unlike the induction of GS by HC, Ara-C caused no measurable increase in the rate of GS synthesis. The results indicate that Ara-C facilitates GS accumulation largely by preventing degradation of the enzyme. Even though Ara-C inhibits the bulk of RNA synthesis in the retina, it does not stop the formation of GS-specific RNA templates. However, the progressive accumulation of these templates does not result in an increased rate of GS synthesis unless Ara-C is withdrawn from such cultures under suitable experimental conditions. Thus, it is suggested that the continuous presence of Ara-C imposes a reversible hindrance at the translational level which limits the rate of GS synthesis. The results demonstrate that the increase in retinal GS elicited by Ara-C is achieved through mechanisms which are quite different from those involved in the hydrocortisone-mediated induction of this enzyme.

The fine structure of embryonic chick skeletal muscle cells differentiated in vitro.

Dissociated myoblasts from 12-day chick embryos were cultured in monolayer, and the differentiation of skeletal muscle cells was studied by electron microscopy. The results have revealed a striking ultrastructural similarity between the in vivo and the in vitro developing muscle, particularly with respect to the myofibrils and sarcoplasmic reticulum. This study demonstrates that all the characteristic organelles of mature skeletal muscle can develop in vitro in the absence of nerves.

Embryonic and neoplastic cell surfaces: availability of receptors for concanavalin A and wheat germ agglutinin.

Embryonic tissue cells dissociated with ethylenediaminetetraacetate are readily agglutinated by the carbohydrate-binding protein concanavalin A. In this property, they resemble transformed, neoplastic cells; and they differ from untransformed adult cells, which are agglutinated by concanavalin A only after their receptors are unmasked by proteolytic treatment. Receptor sites for wheat germ agglutinin are also present on the surface of embryonic cells, but in a masked form, as on untransformed adult culture cells; they can be unmasked by treatment of the cells with trypsin. Concanavalin A binding sites on embryonic cells may function in cell contact and cell organization during embryonic morphogenesis and differentiation and later become masked in adult cells. The unmasking of these sites in neoplastic cells may represent a return, in this respect, to a condition resembling that of embryonic cells and may be related to cell mobility associated with infiltration and metastasis.

Enzyme induction by corticosteroids in embryonic cells: steroid structure and inductive effect.

Glutamine synthetase in the developing retina of the chick embryo can be induced to increase by certain corticosteroids. The inductive effectiveness of various natural corticosteroids has been examined in organ cultures of embryonic retina and correlated with specific groupings on the steroid molecules.

Induction of glutamine synthetase in embryonic retina: its dependence on cell interactions.

A relation between enzyme induction in embryonic cells and cellular organization is indicated by the finding that the levels of glutamine synthetase induced by hydrocortisone in the embryonic neural retina in vitro are dependent on the associations between the retina cells. Intact retina tissue, aggregates of dissociated cells, and cells in monolayer culture showed a decreasing response, in this order, to glutamine synthetase induction. With time of culture, the enzyme activity continued to rise in the intact retina and in cell aggregates, but activity declined in monolayer cultures even though the inducer was continuously present. Dispersed cells cultured in monolayer without the inducer showed after 24 hours a loss of inducibility which could not be reversed by reaggregating such modified cells but could be prevented by maintaining the freshly dispersed cells at a low temperature.

Embryonic morphogenesis: role of fibrous lattice in the development of feathers and feather patterns.

In the morphogenesis of embryonic feather germs the formation of dermal cell groupings is associated with the development of a highly regular pattern of birefringence in the dermis. This birefringence is due to a lattice-like system of collagenous tracts along which dermal cells become progressively aligned and grouped in regularly spaced sites. The experimental results suggest that this fibrous lattice is of major significance in the morphogenesis of feather germs and in their characteristic pattern of distribution.

Cell aggregation: its enhancement by a supernatant from cultures of homologous cells.

A supernatant medium has been prepared from living embryonic neural retina cells which specifically promotes their histogenetic aggregation. Its function is dependent upon at least two experimentally separable steps: selective uptake and functional utilization.

Accumulation of c-src mRNA is developmentally regulated in embryonic neural retina.

Accumulation of c-src mRNA gradually increased during early development of the neural retina in chicken embryos and reached a peak by days 11 to 13 of embryonic life. Thereafter, its amount declined to a low level which persisted also in adult retina. The early increase in c-src mRNA correlated inversely with the decrease in the amount of H3.2 replication histone mRNA and with the decline in the rate of cell growth. The accumulation profile of c-src mRNA corresponded to that of pp60c-src protein, suggesting that the latter is regulated at the level of transcription.

Expression of v-src in embryonic neural retina alters cell adhesion, inhibits histogenesis, and prevents induction of glutamine synthetase.

Using Rous sarcoma virus as the vector, v-src or c-src genes were introduced into 6-day chicken embryo retina tissue in organ culture and their effects on retina development were investigated. Overexpression of c-src in many of the cells had no noticeable effect on retina development. In contrast, infection with v-src resulted in abnormal histogenesis and inhibition of differentiation. Although only a portion of the cells in infected tissue expressed the oncogene and displayed the transformation phenotype, the other cells were also hindered from becoming normally positioned and organized. Therefore, presence of oncogene-transformed cells within the tissue hindered organization and development of adjacent nontransformed cells. Failure of normal cell relationships impeded induction by cortisol of glutamine synthetase in Muller glia, which requires contact associations of the glia cells with neurons. The transformed cells tended to assemble into chaotic clusters, suggesting that their adhesiveness and contact affinities had become altered. This was confirmed by aggregation experiments with dissociated cells which showed that adhesiveness of transformed cells was greatly reduced and that they had lost the ability to cohere with nontransformed cells. In binary mixtures of transformed and nontransformed cells, the two sorted out into separate aggregates. Transformed cells formed loose clusters devoid of tissue architecture; aggregates of nontransformed cells became organized into retinotypic structures, and glutamine synthetase was inducible. Our findings suggest that the mechanisms of cell adhesion and cell affinities are a key target of v-src activity in infected cells and that modification of the cell surface may be a leading factor in other cellular changes characteristic of the v-src transformation phenotype.

Variable CA II compartmentalization in vertebrate retina.

We have generated a series of polyclonal and monoclonal antibodies to mammalian, avian, and osteichthian CA II for the purpose of studying its distribution in vertebrate nervous systems. In mature chicken retina, CA II is immunohistochemically detectable only in Müller glial cells. However, during embryonic development, CA II expression is suddenly "switched-on" early as a general constituent of all retinoblasts, later becoming restricted to Müller cells and transiently to a distinct type of amacrine neuron. A similar developmental pattern occurs in mouse. However, at maturity high CA II levels remain in certain amacrine neurons in addition to Müller cells. Comparative analyses of mature retinas of lower vertebrates show that reptiles parallel chicken with high CA II only in Müller cells, certain amphibians show CA II staining in Müller cells, amacrine neurons as in mouse, and in horizontal neurons, teleost and elasmobranch fish possess high CA II in Müller cells and the horizontal neurons, and lamprey eel shows CA II staining primarily in horizontal cells. An evolutionary sequence that will be discussed is thus suggested.

Gangliosides in postmitotic retina of chick embryo: changes in vivo and in cell cultures.

Developmental changes in ganglioside composition of postmitotic neural retina of chick embryo were analyzed by thin-layer chromatography. Gangliosides were identified by comparing their chromatographic mobilities with reference standards. The outstanding changes are decrease in the concentration of GD3L and increase in GD1a and GM1 concentrations. By depleting Müller glia cells from retina tissue of 13- and 16-day embryos (R13, R16) we determined that the bulk of the major gangliosides is associated with the neurons. Analysis of gangliosides in monolayer cultures of R13 and R16 cells highly enriched for Müller cell-derived gliocytes indicated that these cells express the same types of gangliosides as neurons, but in somewhat different concentrations and relative proportions; however, after time in culture these cells showed ganglioside types and changes in ganglioside profile that are not characteristic of normal retina. The latter observation is consistent with other evidence that the phenotype of Müller glia cells becomes altered in monolayer culture. In contrast to cultures of early embryonic retina, in organ cultures of later postmitotic retina, ganglioside composition did not continue to change as in normal development. This suggests that in postmitotic retina, normal developmental progression of ganglioside changes requires systemic and/or other conditions which are missing or altered when this tissue is isolated and cultured in vitro.

Induction of glutamine synthetase in embryonic neural retina: its suppression by the gliatoxic agent alpha-aminoadipic acid.

Competence for cortisol-mediated induction of glutamine synthetase (GS) is a differentiation marker of embryonic neural retina. Earlier work has indicated that the induction and accumulation of GS is localized in the Müller glia cells. This localization was presently confirmed by the finding that the gliatoxin D,L-alpha-amino-adipic acid (AAA) reduces responsiveness to GS induction by 60--90% due to preferential damage to Müller cells. The tests were performed on organ cultures of retina tissue from chick embryos, and on retina cell aggregates in which there is tissue reconstruction. The presence of GS-inducible Müller cells was monitored by immuno-staining of tissue sections with anti-GS antiserum. Reduction of GS inducibility due to pretreatment with AAA resulted in virtual absence of cells that immunostained for GS. The preferential toxicity of AAA for Müller cells was also demonstrated by cell viability tests; it was further corroborated by the finding that treatment with AAA greatly reduced the level of carbonic anhydrase activity, another enzyme localized predominantly in Müller cells, but did not affect gamma-aminobutyric acid transaminase and choline acetyl transferase, neuronal enzymes. Susceptibility of Müller cells to AAA was found to increase with embryonic development of the retina. We suggest that acquisition of susceptibility for AAA represents another differentiation marker of embryonic Müller cells.

Cellular compartmentalization of carbonic anhydrase-C and glutamine synthetase in developing and mature mouse neural retina.

Using immunohistochemical methods, we have determined the cellular localization of the enzymes, glutamine synthetase (GS) and carbonic anhydrase-C (CA-C), in mouse neural retina during development and in the mature tissue. GS is always confined exclusively to the Müller glial cells; it is first detectable in these cells post-natally on about day 12, i.e. shortly before the eyes open. Also CA-C in the mature retina is localized in the Müller cells but, in addition, it is found in certain amacrine neurons as well. CA-C is first detectable in the retina already several days before birth; at that time it is found in most of the cells, with the exception of the emerging ganglion cells. However, with advancing differentiation, CA-C becomes progressively restricted to Müller cells and to a sub-category of amacrine neurons, and persists only in these cells in the mature retina. The present results extend our previous studies on these enzymes in the avian retina; they demonstrate that also in mammalian retina, different temporal and cellular patterns of GS and CA-C expression and localization earmark distinct phases of structural and functional differentiation of the retina. The striking developmental changes in the cellular localization of CA-C, and the finding of this enzyme in certain amacrine neurons as well as in Müller cells, raise questions about the role of CA-C in the retina, and about mechanisms regulating its expression in specific cell types.

Changes in patterns and synthesis of proteins in embryonic neural retina studied by 2-dimensional gel electrophoresis.

Changes in protein patterns during early differentiation of embryonic neural retina (chick) were studied by 2-dimensional gel electrophoresis. The procedures employed here made it possible to visualize the overall population of proteins present in the tissue at a given time and, on the same gel to distinguish labeled from unlabeled proteins. 2-Dimensional gels were stained by a highly sensitive silver stain to visualize, map and quantitate proteins (and polypeptides) resolved by electrophoresis; the same gels were then autoradiographed in order to differentiate between actively synthesized and pre-existing proteins at each development stage. The effectiveness of this combinative analysis was first verified by identifying and localizing glutamine synthetase, an inducible enzyme marker of retina differentiation. Next, protein patterns in retina tissue at 2 embryonic ages were compared. Of the large number of spots visualized by the above methods approximately 10% showed distinct qualitative-quantitative developmental changes; these were grouped into 7 classes representative of major modes of alteration of protein patterns during cell differentiation.

Changes in ganglioside profile in chick embryo retina: Studies on tissue and cell cultures.

The developmental profile of gangliosides in the neural retina of the chick embryo is characterized by a progressive decrease in the concentration of GD3 complex from a high level on day 6; by a continuous increase in GD1a concentration; and by less striking increases in GD1b and GT1b concentrations during the growth phase; GM1 increases in the post-mitotic retina. Gangliosides were analyzed by thin layer chromatography and by densitometry of the TLC plates. (Ganglioside nomenclature is according to Svennerholm.(37)) We have examined comparatively ganglioside changes in organ cultures of retina tissue from 6 day embryos (R(36)), in cell aggregates and in primary monolayer cultures of R(26) cells, all maintained for 6 days in vitro. In all cases, the pattern of ganglioside changes was qualitatively similar to that in the retina in vivo. These results suggest that, unlike some other aspects of retina differentiation, the progression of ganglioside changes in the 6-12 day embryonic retina is not critically dependent on histotypic cell organization or on specific contact-dependent cell interactions; these changes appear to be largely preprogrammed in the cells at some earlier phase of development.