Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast

Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.     

Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct.

Populations of fibroblast-like cells from 14 day embryonic chick cornea, heart, and skin were grown in vitro as primary cultures and found to be antigenically distinct from one another. Corneal fibroblasts were obtained by dissection, whereas heart and skin fibroblast-like cells were separated from nonfibroblastic cell types by their rapid adhesion to substrata. Cultured cells were used as antigens in rabbits. Antisera were first absorbed against homogenates of embryonic chicks from which the homologous tissue was removed. Each such 1° absorbed antiserum then was absorbed against homogenates of the two respective heterologous fibroblast-like cell populations (2° and 3° absorptions). Resulting 3° absorbed antisera were tested for specificity by immunodiffusion, immune agglutination, immune cytotoxicity (trypan blue uptake and ⁵¹Cr release), and indirect immunofluorescence. Each 3° antiserum was judged tissue specific when it reacted only with the fibroblast-like cells of its own tissue, i.e., the homologous population. Unabsorbed antisera reacted with both homologous and heterologous fibroblast-like cells, as did 1° absorbed antisera. Absorption of 1° antisera with homogenates of the two heterologous fibroblast-like populations removed antibodies against the heterologous populations without significantly reducing the 3° antiserum titer against the homologous fibroblast cell type. Moreover, absorption of 1° antisera with each of the two heterologous fibroblast-like populations removed antibodies not removed by the other. Thus, the fibroblast-like cells from cornea, heart, and skin are antigenically different from one another in vitro. The stable antigenic differences detected may have arisen during the differentiation of these cells in vivo. Some of the tissue-specific antigens detected must occur on the cell surface.     

Isolation and characterization of flat cells, a subpopulation of the embryonic chick retina

When the embryonic neutral retina is dissociated into single cells which are maintained in stationary culture, the neuronal cells associate on the surfaces of a second population which we refer to as flat cells. The flat cells appear in the culture in significant numbers after 2 days and are required for neuronal cell attachment. We have been able to isolate pure flat cells from early cultures of mixed retina cells and have identified several antigens which support the concept that these cells are related to the glia. The cells have been tested by immunofluorescence for glial fibrillary acidic protein and have been found positive. Cell surfaces were labeled by transfer of tritiated galactose from UDP-galactose to endogenous acceptors in the presence of exogenous galactosyl transferase. After SDS-PAGE and fluorography, the surface glycoproteins of flat cells were seen to be significantly different from those of the original retina, and from chick fibroblasts. Immunoelectron microscope studies of detergent-extracted flat cells have demonstrated a complex network of intermediate filaments and actin fibers. We conclude that the flat cells are derived from the glia subpopulation of the retina and have adapted to the tissue culture environment by assuming this configuration. The unique surface properties of flat cells may be related to their role as an intermediate substrate between the neuronal cells and the tissue culture dish.     

Cell-substratum adhesion in chick neural retina depends upon protein- heparan sulfate interactions

Embryonic chick neural retina cells in culture release complexes of proteins and glycosaminoglycans, termed adherons, which stimulate cell-substratum adhesion when adsorbed to nonadhesive surfaces. Two distinct retinal cell surface macromolecules, a 170,000-mol-wt glycoprotein and a heparan sulfate proteoglycan; are components of adherons that can independently promote adhesion when coated on inert surfaces. The 170,000-mol-wt polypeptide contains a heparin-binding domain, as indicated by its retention on heparin-agarose columns and its ability to bind [3H]heparin in solution. The attachment of embryonic chick retinal cells to the 170,000-mol-wt protein also depends upon interactions between the protein and the heparan sulfate proteoglycan, since heparan sulfate in solution disrupts adhesion of chick neural retina cells to glass surfaces coated with the 170,000-mol-wt protein. This adhesion is not impaired by chondroitin sulfate or hyaluronic acid, which indicates that inhibition by heparan sulfate is specific. Polyclonal antisera directed against the cell surface heparan sulfate proteoglycan also inhibit attachment of retinal cells to the 170,000-mol-wt protein, which suggests that cell-adheron binding is mediated in part by interactions between cell surface heparan sulfate proteoglycan and 170,000-mol-wt protein contained in the adheron particles. Previous studies have indicated that this type of cell-substratum adhesion is tissue-specific since retina cells do not attach to muscle adherons. Schubert D., M. LaCorbiere, F. G. Klier, and C. Birdwell, 1983, J. Cell Biol. 96:990-998.     

Immunologically unique and common domains within a family of proteins related to the retina Ca2+-dependent cell adhesion molecule, NcalCAM

Rabbit polyclonal antibodies raised to gp90, a fragment of the embryonic chick neural retina Ca2+-dependent adhesive molecule, gp130, recognize gp130 and inhibit Ca2+-dependent cell-cell adhesion. When tested against a panel of 10-day embryonic tissues, one of these antisera recognizes a component with a molecular weight identical to that of gp130 in embryonic chick cerebrum, optic lobe, hind brain, spinal cord and neural retina only; the second antiserum recognizes a similar component in all of the embryonic chick tissues tested. These data imply the existence of an extended family of closely related cell surface components with immunologically distinct subgroups each of which may mediate Ca2+-dependent cell-cell adhesion. As the term CAM, or cell adhesion molecule, has become common usage we propose to refer to these molecules as calCAMs, reflecting their calcium dependence. Analysis of fragments and endoglycosidase digests of NcalCAM have allowed a comparison of its structure with similar molecules from different tissues and species that have been implicated in Ca2+-dependent cell-cell adhesion.     

Membrane associated antigens of human malignant melanoma V: Serological typing of cell lines using antisera from nonhuman primates

Summary Antisera against various melanoma cell lines were raised in nonhuman primates (Cercopithecus aethiop.). After exhaustive absorption with AB Rh + red blood cells and pooled platelets from about 200 donors the sera were still reactive to various degrees in the microimmune adherence test with other melanoma lines, with embryonic fibroblasts, and with non-melanoma lines. As proven by absorption experiments, the main-specificity of the antisera was not directed against components of the fetal calf serum used for cell culture or against mycoplasma grown from commercial fetal calf serum. In addition, no cross-reactivity was observed with Bacillus Calmette-Gérin, and in blocking experiments no reactivity against extracts of common bacterial antigens or mixed molds was detected. Absorption with embryonic fibroblasts or embryonic tissue showed that the reactivity of most antisera was directed against melanoma-associated antigens expressed also on fetal tissue. It was not possible to determine whether the remaining reactivity on some cell lines was melanoma-specific or directed against fetal antigens not contained in the fetal material used for absorption. Cross-absorption of antisera with other melanoma cells revealed that various cell lines express different patterns of tumor-associated antigens with no, or only partial, overlap. The cross-absorption experiments made it possible to type the cell lines according to their surface antigens and arrange the cell lines in order according to the degree of mutual antigenic relationship.     

Transient expression of adheron molecules during chick retinal development.

Neuritogenesis and synapse formation are transient phenomena mediated in part by filopodial attachments (Tsui, Lankford, and Klein, Proc. Natl. Acad. Sci. 82:8256-8260 1985). The attachments can be labeled by antisera against adherons, adhesive microparticles isolated from cell culture media (Tsui, Schubert, and Klein, J. Cell Biol. 106:2095-2108 1988). Here, two monoclonal antibodies raised against adherons have been found to recognize transiently expressed membrane antigens of developing avian retina. Early in development, monoclonal antibody (mAb) AD1 stained antigens that spanned the entire tissue. With time, immunoreactivity became restricted to optic fiber, ganglion cell, and inner plexiform layers. Immunoblots of embryonic day (E) 13 retina showed a broad band at 66-72 kD for particulate fractions and a fine band at 70 kD for soluble fractions. The particulate forms disappeared as retinas matured, but the soluble form did not. mAb AD2 initially labeled retina antigens of optic fiber, ganglion cell, and inner plexiform layers (IPL). Labeling in the plexiform layer showed discrete lamina. Immunoreactivity first appeared at E9, peaked at E15, and then disappeared shortly after hatching. In isolated cells, AD2 labeled small cell surface aggregates. Cytoarchitectural studies, using whole-mount transmission electron microscopy, showed AD2 antigen in cell surface microfilaments, including some that joined filopodia together. The adheron antigens recognized by mAbs AD1 and AD2 thus were (1) topographically restricted; (2) associated with cell surfaces; and (3) developmentally down-regulated. This pattern suggests a role in developmentally transient cell surface phenomena, such as neurite extension or junction biogenesis.     

Recognition of antigenic differences among neurons using antiserums to clonal neural retina hybrid cells.

Antiserums prepared against hybrid cell strains formed between freshly isolated rodent neural retina cells and a human fibroblast cell line W.I. 18, VA-2 recognize cell surface antigens that are 1) restricted to the neural retina, 2) present in both embryonic and adult retina, and 3) localized to groups of cells within the retina. Normal segregants (i.e., those lines that had retained rodent chromosomes and lost human chromosomes) were used as immunogens in rabbits to produce the antiserums. Antiserums against both whole cells and purified plasma membranes were adsorbed with human parent VA-2 cells and nonneural and neural rodent tissue to remove cross-reacting specificities. All six antiserums studied continued to react with embryonic retina after brain cross-reactivity was removed.     

Isolation of an adhesion-mediating protein from chick neural retina adherons

Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells. They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina. The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 100:56-63). This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor. The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture. The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans. Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions. The RP protein is found in neural retina, but not in other tissues such as brain and muscle. These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells.     

Immunologic detection of retina cognin on the surface of embryonic cells

The retina cell-aggregating glycoprotein, referred to as the retina cognin, has been demonstrated to be located at the surface of embryonic neural retina cells. The term cognin is used to indicate its postulated role in the mechanism of mutual recognition and morphogenetic association of embryonic cells. Antiserum was prepared to the highly purified retina cognin derived from isolated cell membranes of chick embryo retina, and it was used to detect the cognin on cells from chick embryos by means of complement-mediated cell lysis. Retina cells (from 10-day embryos) freshly dissociated with trypsin showed little—if any—lysis by the cognin antiserum; this is consistent with the sensitivity of the cognin to trypsin. However, the cells became susceptible to immunolysis after a period of incubation at 37 °C, which indicates regeneration of the cognin at the cell surface during the recovery period. This regeneration required protein synthesis. Immunofluorescence tests showed binding of the antiserum to the surface of the recovered cells, thereby further demonstrating the surface location of the cognin. The presence, availability or ability to regenerate the cognin, as assayed here, declined sharply with the embryonic age of the cells. Addition of exogenous cognin to freshly trypsin-dissociated retina cells (from 10-day embryos) markedly increased their susceptibility to immunolysis by the cognin antiserum, which indicates that the added cognin becomes associated with the surface of these cells. In contrast, addition of retina cognin to cells freshly trypsinized from 10-day embryo optic tectum and cerebrum, or from 14-day retina did not increase their susceptibility to immunolysis by the cognin antiserum. These results are consistent with earlier findings that enhancement of cell aggregation by the retina cognin is tissue-specific and stage-specific. Cells from non-neural tissues of the chick embryo were not lysed by the retina cognin antiserum. However, neural tissues, such as optic tectum, were found to contain cells which showed surface cross-reaction with the retina cognin antiserum.     

Transient expression of adheron molecules during chick retinal development

Neuritogenesis and synapse formation are transient phenomena mediated in part by filopodial attachments (Tsui, Lankford, and Klein, Proc. Natl. Acad. Sci. 82:8256–8260 1985). These attachments can be labeled by antisera against adherons, adhesive microparticles isolated from cell culture media (Tsui, Schubert, and Klein, J. Cell Biol. 106:2095–2108 1988). Here, two monoclonal antibodies raised against adherons have been found to recognize transiently expressed membrane antigens of developing avian retina. Early in development, monoclonal antibody (mAb) AD1 stained antigens that spanned the entire tissue. With time, immunoreactivity became restricted to optic fiber, ganglion cell, and inner plexiform layers. Immunoblots of embryonic day (E) 13 retina showed a broad band at 66–72 kD for particulate fractions and a fine band at 70 kD for suluble fractions. The particulate forms disappeared as retinas matured, but the soluble form did not. mAb AD2 initially labeled retina antigens of optic fiber, ganglion cell, and inner plexiform layers (IPL). Labeling in the plexiform layer showed discrete lamina. Immunoreactivity first appeared at E9, peaked at E15, and then disappeared shortly after hatching. In isolated cells, AD2 labeled small cell surface aggregates. Cytoarchitectural studies, using whole mount transmission electron microscopy, showed AD2 antigen in cell surface microfilaments, including some that joined filopodia together. The adheron antigens recognized by mAbs AD1 and AD2 thus were (1) topographically restricted; (2) associated with cell surfaces; and (3) developmentally down-regulated. This pattern suggests a role in developmentally transient cell surface phenomena, such as neurite extension or junction biogenesis. © 1992 John Wiley & Sons, Inc.     

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.     

NS-7 (nervous system antigen-7): a cell surface antigen of mature brain, kidney, and spermatozoa shared by embryonal tissues and transformed cells

Xenogeneic immunization of rabbits with cells of a mouse undifferentiated neural tumor, MUNTAD, produces antisera which after appropriate absorption bind to the cell surfaces of MUNTAD cells and to three normal adult mouse tissues: brain, kidney, and spermatozoa. Any one of these tissues quantitatively absorbs all of the activity for MUNTAD cells from the antisera, indicating that a single antigen or antigens is shared. This is designated NS-7. The adult tissues positive for NS-7 are the same as those that carry NS-6, but NS-7 is distinguished serologically from NS-6 by being present on many embryonic tissues, tumors, and cell lines which completely lack NS-6. Blocking and capping studies show that on the MUNTAD cell surface, some NS-7 antigenic sites and all NS-6 sites are physically related.     

Enzymatic dissection of embryonic cell adhesive mechanisms

In this paper we describe a kinetic assay for cell adhesion which measures the formation of cell clusters. Cluster formation is dependent on both calcium and protein synthesis, two parameters essential for the formation of histotypic aggregates. We also describe modifications of the stndard method for trypsinization of tissues which result in populations of single cells that appear to bear intact and functional cell surface adhesive systems. These modifications involve the use of chymotrypsin and the inclusion of calcium during enzyme digestion of tissues with trypsin and chymotrypsin. Using the cluster formation assay and the modified tissue dissociation techniques, we demonstrate the presence of two functionally distinct adhesive systems operating among embryonic chick neural retina cells. These two systems differ in proteolytic sensitivity, protection by calcium against proteolysis, dependence on calcium for function and morphogenetic potential. Cells possessing one of these intact adhesive systems are capable of extensive morphogenetic interactions in the absence of protein synthesis.     

A role for adherons in neural retina cell adhesion

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion; they also stimulate the rate of cell-cell aggregation. Adheron-stimulated adhesion is tissue specific, and the spontaneous aggregation of neural retina cells is inhibited by monovalent Fab' fragments prepared from an antiserum against neural retina adherons. Therefore cell surface antigenic determinants shared with adherons are involved in normal cell-cell adhesions. The particles from the heterogeneous neural retina population contain many proteins and several glycosaminoglycans. The adherons migrate as a symmetrical 12S peak on sucrose gradients and are predominantly 15-nm spheres when examined by electron microscopy. Finally, the specific activity of neural retina adherons increases from embryonic days 7 through 12 and then declines. These results suggest that glycoprotein particles may be involved in some of the adhesive interactions between neural retina cells and between the cells and their environment.     

Immunohistochemical characterization of delta crystallin-containing retina/optic nerve "boundary" cells in the chick embryo

The term "transdifferentiation" has been used to describe the apparent phenotypic conversion of chick embryo neural retina Müller glial cells into lens-like cells in vitro. This phenotypic conversion is characterized by expression of such lens-specific proteins as delta crystallin and has been viewed as an example of cells transforming from the phenotype of a given tissue to that of another. We have identified a population of neuroglia-like cells in the embryonic chick retina which express high levels of delta crystallin as a function of normal development. The position and morphology of these cells is quite distinctive in that they form a loose meshwork which defines the boundary between the neural retina and the optic nerve head. These "boundary" cells are detectable as early as Day 5 of development through hatching. However, the meshwork structure formed by the cells is only readily observed between Days 8 and 9 of development. Double-immunolabeling procedures comparing delta crystallin staining to that of glial and neuronal markers suggest that these cells are a form of retinal Müller glial cell. The results show that under appropriate microenvironmental conditions, expression of delta crystallin falls into the normal repertoire of retinoblast cells. The results also demonstrate the presence of a cellular boundary defining the junction between the neural retina and the optic nerve, tissues that are ontogenetically and structurally continuous but functionally distinct.     

A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.     

Tissue-specific distribution and variation of the channel-forming protein ductin during development of Drosophila melanogaster

Ductins represent membrane channel proteins which are supposed to form both proton channels in V-ATPases and connexon channels in gap junctions. In order to localize and characterize these proteins in different tissues of Drosophila, we applied indirect immunofluorescence microscopy and immunoblots, using antisera prepared against Drosophila ductin and against Nephrops ductin. Previously, these antisera have been shown to recognize, in ovarian follicles and young embryos of Drosophila, the ductin monomer of 16 kDa and a putative dimer of 29 kDa. Moreover, both anti-ductin sera label antigens in plasma membranes and in the cytoplasm and block, when microinjected, cell-cell communication via gap junctions. In the present study, comparing several embryonic, larval and adult tissues, the anti-ductin sera were found to recognize antigens with various locations in cells of the midgut, the salivary gland, the nervous system, the muscles and the epidermis. For example, in midgut cells, antigens were labeled mainly in apical plasma membranes and in the apical part of the cytoplasm, while in salivary-gland cells, labeling was found throughout the plasma membranes and the cytoplasm. We conclude that putative gap junctions were revealed in the salivary gland, the nervous system and the epidermis, while plasma membrane-associated putative V-ATPases were detected in the midgut, the salivary gland and the muscles. Moreover, V-ATPases associated with cytoplasmic vesicles were found in almost every tissue. On immunoblots of homogenates from various tissues, the anti-ductin sera specifically labeled bands of 16, 21 and 29 kDa. When comparing these bands using peptide mapping with V8 protease, we found that they represent closely related proteins. Therefore, either different ductins or modifications of a single ductin appear to be present in different cellular regions, cell types and developmental stages of Drosophila.     

Brain histogenesis in vitro: Reconstruction of brain tissue from dissociated cells

Summary Comparative studies of the aggregative behavior of cells dissociated from different areas of embryonic chick and mouse brains show that each of the regionally differentiated lobes (cerebrum, optic tectum, and cerebellum), and the stem areas (diencephalon and medulla), form characteristic aggregates distinctive in size and shape. Bispecific co-aggregates are produced by commingling dissociated mouse cerebrum cells with chick cells from various brain regions, or from non-nervous tissues; the size of these co-aggregates and the extent of internal sorting out of cell types is closely related to the degree of homology between the interacting cell populations, e.g. co-aggregates of the closely homologous mouse and chick cerebral cell types contain homogeneous tissue fabrics of intermingled mouse and chick cells. Cell surface constituents involved in selective recognition and association of nerve cells were sought and cell-free supernatant preparations were obtained from short-term monolayer cultures of embryonic cerebrum cells (of either mouse or chick origin) which caused a striking, specific enhancement of aggregation of homologous cerebrum cells. These materials had no such effect on heterologous tissues tested: optic tectum, cerebellum, medulla, neural retina, liver, kidney or limb bud. These findings are discussed in relation to control mechanisms governing normal brain histogenesis and to the specificity of neural associations. This work was supported by United States Public Health Service research grant HD-01253 to Aron Moscona and by the Louis Block Fund of the University of Chicago.