An axon growth associated antigen is also an early marker of neuronal determination

We have previously described the generation of a monoclonal antibody (DSS-3) that binds to all neurons in cockroach embryos at 50% development and to only a small subset of interneurons in the adult nervous system. This developmental stage-specific antigen was observed to reappear in all axotomized adult neurons that were undergoing axonal regeneration. In the present study the time course of the appearance of this growth-associated antigen during embryonic development was determined. Unexpectedly, the antigen was observed to be present in embryonic neurons long before axon growth. In addition, all cells in the CNS neuronal lineage (neuroblasts, ganglion mother cells, and neurons) bind the antibody as soon as they can be morphologically identified. However, the antigen is also transiently present in all neuroepithelial cells at a stage prior to the morphological differentiation of some of them to neuroblasts. Analogous patterns of DSS-3 binding to cells involved in the development of sensory neurons and leg pioneer neurons are observed. The DSS-3 antigen is therefore a very early marker for the capacity of ectodermal epithelial cells to develop along a neuronal lineage.     

Developmental changes in unique cell surface antigens of chick embryo spinal motoneurons and ganglion cells

The monoclonal antibody technique was used to investigate neuronal heterogeneity and its developmental changes in the chick embryo trunk especially at the thoracic level. We report here four monoclonal antibodies (called SC 1, SC 2, SC 3, and SC 4) that bound to cell surface antigens. These antigens appeared to be proteins or glycoproteins because of their susceptibility to trypsin. In the spinal cord, antibody SC 3 stained all cells, but antibody SC 1 specifically stained motoneurons and ventral epithelial cells. The staining of motoneurons by antibody SC 1 was transient. It appeared at early stages (stage 16-17; Hamburger and Hamilton), but decreased markedly in intensity at older stages (stage 30-31). Antibody SC 2 did not stain cells in the spinal cord. It stained only neurons in the dorsal root and sympathetic ganglia. Antibody SC 4 stained only cells derived from the neural crest at the early stages (stage 16-20). At later stages, it stained a wider population of cells, including sensory neurons, Schwann cells, and cells in the central nervous system. In the dorsal root ganglion, antibodies SC 1 and SC 2 stained only neuronal cells whereas antibodies SC 3 and SC 4 stained both neuronal and glial cells. The dorsal root ganglionic antigens recognized by these antibodies were not expressed concurrently but appeared in a developmental sequence. Staining with antibodies SC 3 and SC 4 appeared first, then SC 1, and finally SC 2. Among these four antigens, the antigens common to both neuronal and glial cells appeared earlier than the neuron specific antigens. Thus, our monoclonal antibodies revealed heterogeneities in cell surface neuronal molecules and their transient and sequential appearance during embryonic development.     

Developmental regulation of ecdysteroid receptors in the nervous system of Manduca sexta.

The technique of steroid hormone autoradiography has been used to study the cellular distribution of ecdysteroid binding sites in the ventral nervous system of the tobacco hornworm moth, Manduca sexta. The ligand was 26-[125I]iodoponasterone. Tissue was examined from the subesophageal ganglia, thoracic ganglia, and abdominal ganglia of larvae at two times during the larval-pupal transient: the 2nd day of wandering and the prepupal stage. The patterns of neuronal binding seen were compared with those found in earlier autoradiographic studies of hormone binding in tissue sampled on the 1st day of wandering, in the pharate adult, and in the 4-day-old moth (Fahrbach and Truman, '89). The pattern of binding was reproducible but dependent upon developmental stage: whereas only a subset of neurons exhibited nuclear accumulation of radiolabeled ecdysteroids on the 1st day of wandering, less than 24 hours later nearly every neuron in the ventral nervous system was labeled. A limited pattern of binding, however, was seen again in the prepupal nervous system. Thus, the insect nervous system is able to use a single hormone both as a general cue for metamorphic development and as a single targeted to stage-specific subsets of neurons by alternating periods of ubiquitous expression of receptor with periods during which the capacity to bind the steroid hormone is highly restricted.     

The stage-specific expression of TEC-1, -2, -3, and -4 antigens on bovine preimplantation embryos

The preimplantation developmental period is associated with constant changes within the embryo, and some of these changes are apparent on the embryo cell surface. For example, during transition from maternal to embryonic genome control and the compaction and differentiation of embryonic cells, the cell surface undergoes morphologic alterations that reflect changes in gene control. In order to gain insight into the events occurring during embryonic development and cellular differentiation, monoclonal antibodies specific for cell surface antigens (TEC antigens) of embryonic cells have been generated previously and shown to recognise either the carbohydrate moiety of embryoglycan or a developmentally regulated protein epitope. The TEC antigens have been identified on mouse preimplantation embryos, and their expression is specific to particular developmental stages. To determine whether these antigens are conserved in higher mammals, we examined the expression of four TEC antigens (TEC-1 to TEC-4) on in vitro–derived bovine and murine embryos during the preimplantation stage of development. It was found that bovine oocytes and embryos derived from in vitro maturation (IVM) and in vitro fertilisation (IVF) showed stage-specific expression of each of the TEC antigens investigated, with the pattern of expression overlapping but not identical to that seen in the mouse. Immunoprecipitation together with Western blot analysis showed that the TEC monoclonal antibodies recognised a single glycoprotein band with an apparent molecular weight of 70 kDa. Confocal microscopy of immunofluorescence staining of the bovine cells showed this protein to be located on the cell surface. The apparent negative expression of these TEC antigens by immunohistochemistry and immunoprecipitation at particular stages of development appears to be due to the epitopes being inaccessible to the TEC antibodies, since Western blotting revealed the TEC antigens to be present at all stages of development examined. Antibodies identifying stage-specific antigens will provide useful markers to characterise early embryonic cells, monitor normal embryonic development in vitro, and identify cell surface structures having a function in cell-cell interactions during embryogenesis and differentiation. Mol. Reprod. Dev. 49:19–28, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of Nervana, a Drosophila melanogaster Neuron-Specific Glycoprotein Antigen Recognized by Anti-Horseradish Peroxidase Antibodies

Abstract: Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identify neurons in Drosophila and other insects. We are interested in characterizing the gene product(s) recognized by anti-HRP antibodies because it may be important for nervous system function and/or development. Here we identify and purify from adult Drosophila heads an anti-HRP-reactive M_r 42K glycoprotein that is likely to be the major contributor to neuronal specific anti-HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between M_r 38K and 42K that vary as a function of developmental age. We have collectively named these components Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti-HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti-HRP staining in adult nervous system. It should now be possible to undertake a full biochemical and functional characterization of Nervana in Drosophila .     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system.

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex alpha-galactosyl and alpha-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell-cell interactions during development and maintenance of vomeronasal connections.     

Initial appearance and regional distribution of the neuron-glia cell adhesion molecule in the chick embryo

This study represents a global survey of the times of the first appearance of the neuron-glia cell adhesion molecule (Ng-CAM) in various regions and on particular cells of the chick embryonic nervous system. Ng-CAM, originally characterized by means of an in vitro binding assay between glial cells and brain membrane vesicles, first appears in development at the surface of early postmitotic neurons. By 3 d in the chick embryo, the first neurons detected by antibodies to Ng-CAM are located in the ventral neural tube; these precursors of motor neurons emit well-stained fibers to the periphery. To identify locations of appearance of Ng-CAM in the peripheral nervous system (PNS), we used a monoclonal antibody called NC-1 that is specific for neural crest cells in early embryos to show the presence of numerous crest cells in the neuritic outgrowth from the neural tube; neither these crest cells nor those in ganglion rudiments bound anti-Ng-CAM antibodies. The earliest neurons in the PNS stained by anti-Ng-CAM appeared by 4 d of development in the cranial ganglia. At later stages and progressively, all the neurons and neurities of the PNS were found to contain Ng-CAM both in vitro and in vivo. Many central nervous system (CNS) neurons also showed Ng-CAM at these later stages, but in the CNS, the molecule was mostly associated with neuronal processes (mainly axons) rather than with cell bodies; this regional distribution at the neuronal cell surface is an example of polarity modulation. In contrast to the neural cell adhesion molecule and the liver cell adhesion molecule, both of which are found very early in derivatives of more than one germ layer, Ng-CAM is expressed only on neurons of the CNS and the PNS during the later epoch of development concerned with neural histogenesis. Ng-CAM is thus a specific differentiation product of neuroectoderm. Ng-CAM was found on developing neurons at approximately the same time that neurofilaments first appear, times at which glial cells are still undergoing differentiation from neuroepithelial precursors. The present findings and those of previous studies suggest that together the neural cell adhesion molecule and Ng-CAM mediate specific cellular interactions during the formation of neuronal networks by means of modulation events that govern their prevalence and polarity on neuronal cell surfaces.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex α-galactosyl and α-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell–cell interactions during development and maintenance of vomeronasal connections.     

Expression of Neural Antigens in Normal Xenopus Embryos and Induced Explants

Monoclonal antibodies were raised against neural tissues of Xenopus larvae. Three monoclonal antibodies, named NEU-1, NEU-3, and NEU-4, were specific for neural tissue and first bound to neural cells at stage 25 after neural tube formation (NEU-1 and NEU-3) or at stage 31 (NEU-4). These antibodies bound to differentiating neural cells, but not to germinal neuroepithelial cells. NEU-1 and NEU-3 recognized antigens in cell bodies as well as neural fibers of neural cells, and these antigens were distributed throughout the central nervous system. NEU-4 bound to antigens in granular materials in neural cells, and these antigens were present in head and trunk regions but not in the tail region.
These three antibodies were used as neural markers in two types of induction experiments, in which 1) the animal pole region and the dorsal blastopore lip from stage-10 gastrulae were combined, or 2) the animal pole region and the vegetal pole region from stage-8 blastulae were combined. In both experiments, most conjugated explants expressed the NEU-1, NEU-3, and NEU-4 antigens, although the expression of NEU-4 antigen was delayed compared with those of the NEU-1 and NEU-3 antigens. These results show that these antibodies are useful as markers in neural induction experiments.     

Differentiation capabilities of human germ cell tumors

It is well known that human germ cell tumors are an excellent model to study not only differentiation capacity of tumor cells but also human normal somatic cell differentiation. A variety of polyclonal and monoclonal antibodies were developed against cell surface antigens of murine embryos and teratocarcinomas. Accumulated data has revealed that these antigens are sequentially expressed on embryonic cells in a well-programmed manner. They have also been shown to be useful markers to investigate somatic cell differentiation in fetal and adult tissue. In humans, however, little is known about the cellular differentiation mechanism in early embryos and whether they could be studied, i.e. whether they occur in human germ cell tumors. In present review, we discussed newly established monoclonal antibodies which were raised from human embryonal carcinoma cells. We have been studying differentiation capacity of human germ cell tumor cells by using these antibodies. Some of these antibodies clearly indicates their usefulness to specify the developmental stage of normal tissue.     

Brugia pahangi: stage-specific antigens on microfilariae detected by serum from infected jirds or by monoclonal antibodies

Experiments were carried out to determine whether there are stage-specific antigens on microfilariae of Brugia pahangi, using sera from Mongolian jirds infected with B. pahangi and monoclonal antibodies against microfilariae of B. pahangi. These studies showed that microfilariae have both stage-specific and nonspecific antigens. The nonspecific antigens were also present on adult worms and on infective larvae. Among monoclonal antibodies, 6 out of 14 clones produced antibodies against the microfilarial stage-specific antigens, and 8 clones produced antibodies against nonspecific antigens. These monoclonal antibodies could not distinguish between adults, microfilariae, or infective larvae of B. malayi and B. pahangi.     

Developmental changes of glycosphingolipids and expression of glycogenes in mouse brains

Glycosphingolipids (GSLs) and their sialic acid-containing derivatives, gangliosides, are important cellular components and are abundant in the nervous system. They are known to undergo dramatic changes during brain development. However, knowledge on the mechanisms underlying their qualitative and qualitative changes is still fragmentary. In this investigation, we have provided a detailed study on the developmental changes of the expression patterns of GSLs, GM3, GM1, GD3, GD1a, GD2, GD1b, GT1b, GQ1b, A2B5 antigens (c-series gangliosides such as GT3 and GQ1c), Chol-1alpha (GT1aalpha and GQ1balpha), glucosylceramide, galactosylceramide (O1 antigen), sulfatide (O4 antigen), stage-specific embryonic antigen-1 (Lewis x) glycolipids, and human natural killer-1 glycolipid (sulfoglucuronosyl paragloboside) in developing mouse brains [embryonic day 12 (E12) to adult]. In E12-E14 brains, GD3 was a predominant ganglioside. After E16, the concentrations of GD3 and GM3 markedly decreased, and the concentrations of a-series gangliosides, such as GD1a, increased. GT3, glucosylceramide, and stage-specific embryonic antigen-1 were expressed in embryonic brains. Human natural killer-1 glycolipid was expressed transiently in embryonic brains. On the other hand, Chol-1alpha, galactosylceramide, and sulfatide were exclusively found after birth. To provide a better understanding of the metabolic basis for these changes, we analyzed glycogene expression patterns in the developing brains and found that GSL expression is regulated primarily by glycosyltransferases, and not by glycosidases. In parallel studies using primary neural precursor cells in culture as a tool for studying developmental events, dramatic changes in ganglioside and glycosyltransferase gene expression were also detected in neurons induced to differentiate from neural precursor cells, including the expression of GD3, followed by up-regulation of complex a- and b-series gangliosides. These changes in cell culture systems resemble that occurring in brain. We conclude that the dramatic changes in GSL pattern and content can serve as useful markers in neural development and that these changes are regulated primarily at the level of glycosyltransferase gene expression.     

Autoradiographic identification of ecdysteroid-binding cells in the nervous system of the moth Manduca sexta

The steroid hormone 20-hydroxyecdysone regulates many aspects of nervous system development in the moth Manduca sexta, including stage-specific neuronal morphology and stage-specific neuronal death. We have used steroid hormone autoradiography to study the distribution of cells that concentrate ecdysteroids in the ventral nervous system of this insect. The ligand was [3H]-ponasterone A, a bioactive phytoecdysone. Tissue was examined from three stages of development: the end of larval life (first day of wandering), the end of metamorphosis (pharate adult), and 4-day-old adults. In the abdominal ganglia of wandering larvae and pharate adults, a subset of neurons including both motoneurons and interneurons exhibited a nuclear concentration of radiolabeled hormone. The pattern of binding was reproducible but stage-specific, with a greater proportion of neurons showing binding in the larvae than in pharate adults. No labeled neurons were found in abdominal ganglia from mature (4-day-old) adults. In the case of the pharate adult ganglia, the ecdysteroid receptor content of specific, identified motoneurons was determined. These results are discussed in light of the responses of these neurons to physiological changes in levels of circulating ecdysteroids.     

Heterophilic interactions of DM-GRASP: GRASP-NgCAM interactions involved in neurite extension

DM-GRASP is an immunoglobulin superfamily cell adhesion molecule that is expressed in both the developing nervous and immune system. Specific populations of neurons respond to DM-GRASP substrates appears to require homophilic interactions between DM-GRASP molecules. We were interested in determining whether DM-GRASP interacts heterophilically with other ligands as well. We have found that eleven proteins from embryonic chick brain membranes consistently bind to and elute from a DM-GRASP-Sepharose affinity column. One of these proteins is DM-GRASP itself, consistent with its known homophilic binding. Another protein, at 130 kD, is immunoreactive with monoclonal antibodies to NgCAM. Other neural cell adhesion molecules were not detected in the eluate. The DM- GRASP-Sepharose eluate also contains a potent neurite stimulating activity, which cannot be accounted for by either DM-GRASP or NgCAM. To investigate the interaction of DM-GRASP and NgCAM, antibodies against DM-GRASP were added to neuronal cultures extending neurites on an NgCAM substrate. The presence of antibodies to DM-GRASP decreased neurite extension on laminin, suggesting that the antibody is not toxic or generally inhibiting motility. We present two possible models for the DM-GRASP-NgCAM association and a hypothesis for neural cell adhesion function that features the dimerization of cell adhesion molecules.     

A cofilin-like protein is involved in the regulation of actin assembly in developing skeletal muscle

An actin-binding protein of 20 kDa (called 20K protein) was purified from the sarcoplasmic fraction of embryonic chicken skeletal muscle. The properties of this protein were very similar to cofilin, which was discovered in porcine brain (Nishida et al. (1984) Biochemistry, 23, 5307-5313): it bound to both G- and F-actin, inhibited actin polymerization in a pH-dependent manner, inhibited binding of tropomyosin to F-actin, and had almost the same molecular size and pI as cofilin. A specific monoclonal antibody to 20K protein (MAB-22) was prepared to examine the expression and location of 20K protein during skeletal muscle development. When the whole protein lysates of embryonic and post-hatched chicken skeletal muscles were examined by means of immunoblotting combined with SDS-PAGE, 20K protein was detected in skeletal muscle through the developmental stages. Location of 20K protein in the cells differed between the embryonic and adult tissues; immunofluorescence staining of the cryosections of embryonic muscle with MAB-22 visualized irregular dot-like structures, but adult muscle sections were stained faintly and uniformly. 20K protein was present as a complex with actin in embryonic muscle, as judged by the ability to bind to a DNase I affinity column, while the same protein was free from actin in the cytoplasm of adult muscle. From these results, it is suggested that 20K protein regulates actin assembly transiently in developing skeletal muscle.     

Differential expression of alternate forms of a Drosophila src protein during embryonic and larval tissue differentiation

The Dsrc28C gene encodes two major proteins, p66 and p55, each of which contains a tyrosine kinase domain. Using monoclonal antibodies we have completed a detailed investigation of the spatial expression of Dsrc28C proteins during embryonic and larval development. Differentiation of a number of embryonic tissues is accompanied by the induction of Dsrc28C expression. With the exception of the developing salivary glands which express high levels of p66, developing tissues express the p55 form of Dsrc28C. Notable examples are cells of the and peripheral nervous systems which express p55 from the early stages of neurogenesis through the remainder of embryogenesis and pole cells which transiently express p55 during portions of embryonic stages 10 and 11. Nervous system expression includes the cell bodies and neuronal fibers of the central nervous system, the anterior sensory organs, and the peripheral sensory neurons. During larval development, p55 levels within the central nervous system remain high but substantial changes in the pattern of expression take place. p55 gradually disappears from the neuronal fibers of the central nervous system and from embryonic cell bodies. During the third larval instar, the birth of immature neuroblasts within the ventral and midbrain ganglia, but not within the optic ganglia, is marked by a transient high level of p55 expression. All imaginal cells that have been observed within the larva express the p66 protein. The patterns of expression that we have noted suggest that expression of the p55 form of Dsrc28C protein is an early event in the differentiation of neuronal cells, while expression of the p66 form is characteristic of cells committed to ectodermal cell differentiation.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

Idiotypic and fluorometric analysis of the antibodies that distinguish the lesion of the I-A mutant B6.C-H-2bm12

The serologic lesion of the I-A mutant mouse strain, bm12, was investigated with the use of monoclonal anti-Iab antibodies and anti-idiotypic (Id) reagents produced against these antibodies. In a fluorometric analysis, three different monoclonal anti-Iab antibodies (25-9-17, 34-5-3, 28-16-8) failed to bind bm12 cells, whereas two anti-Iab antibodies (25-5-16 and 17/227), which bound bm12 cells, showed about one-half the fluorescence intensity that they showed in binding to Iab antigens. Of the three monoclonal antibodies that failed to react with bm12 cells, two antibodies (25-9-17 and 34-5-3) were found to bind the same steric site on Iab molecules (cluster I). In contrast, the antibodies (25-5-16 and 17/227) that reacted with both Iab and Iabm12 antigens were found to bind a second distinct site (cluster II). The binding of antibody 28-16-8 to Iab antigens inhibited reciprocally the binding of cluster I and II anti-Iab antibodies, suggesting a possible third site, sterically located intermediate between the other two sites. To assess the relatedness of the antibodies defining the serologic lesion of bm12 mice, xenogeneic and syngeneic anti-Id reagents were produced against antibodies 25-9-17 and 28-16-8. By using these anti-Ids in a binding site-related inhibition assay, a cross-reactive idiotype was detected that is shared by 25-9-17 and 34-5-3 antibodies; thus these two monoclonal antibodies share several features, including 1) idiotypic determinants, 2) failure to bind bm12 cells, 3) binding the same spatial Iab site, and 4) having indistinguishable serologic fine specificity that corresponds with a previously defined predominant alloantigenic determinant recognized in the bm12 anti-Iab humoral response. Therefore, several parameters of antibody recognition of Ia can now be correlated with structural changes in Ia molecules. These findings will potentiate future studies of the T cell recognition of these same Ia epitopes.     

Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons

The identification of surface proteins restricted to subsets of embryonic axons and growth cones may provide information on the mechanisms underlying axon fasciculation and pathway selection in the vertebrate nervous system. We describe here the characterization of a 135 kd cell surface glycoprotein, TAG-1, that is expressed transiently on subsets of embryonic spinal cord axons and growth cones. TAG-1 is immunochemically distinct from the cell adhesion molecules N-CAM and L1 (NILE) and is expressed on commissural and motor neurons over the period of initial axon extension. Moreover, TAG-1 and L1 appear to be segregated on different segments of the same embryonic spinal axons. These observations provide evidence that axonal guidance and pathway selection in vertebrates may be regulated in part by the transient and selective expression of distinct surface glycoproteins on subsets of developing neurons.     

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.