Kunihiko Obata

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.

Subclasses of olfactory receptor cells and their segregated central projections demonstrated by a monoclonal antibody

A library of monoclonal antibodies (MAbs) was generated against a homogenate of the rabbit olfactory bulb. One of them immunohistochemically distinguished a subgroup of olfactory nerves. Both in the olfactory bulb and the epithelium, this MAb labeled most olfactory receptor axons in the lateral but only a small fraction in the medial portion. These findings demonstrate a molecular heterogeneity among olfactory receptor cells and suggest a functional division between the lateral and the medial portions of the epithelium and the bulb.

Immunochemical homology of 3 developmentally regulated brain proteins and their developmental change in neuronal distribution

Proteins S5, S6, and S54 (mol. wts. 95,000, 100,000, and 110,000 Da) appear characteristically at certain developmental stages in the chick brain (Shirao, T. and Obata, K., J. Neurochem., 44 (1985) 1210-1216). In the present study polyclonal and monoclonal antibodies were developed against electrophoretically purified S5 and S6 proteins. Each polyclonal and monoclonal antibody specifically recognized all 3 proteins, S5, S6, and S54, by immunoblot analysis. The tissue specificities of these proteins were examined by immunoblot analysis with these antibodies. Proteins S5 and S6 were found in the neural tissue and in some non-neural tissues of chick embryo. In the adult chicken, however, they were detected neither in neural nor in non-neural tissues with the exception of the spinal ganglion. Protein S54, on the other hand, was found both in late embryonic and adult neural tissues. It was detected neither in embryonic nor in adult non-neural tissues. Immunohistochemical analysis of adult nervous system showed that S54 protein was present only in neurons. Therefore it is concluded that S54 protein is a neuron-specific protein. Developmental changes of localization of these proteins were then examined by immunohistochemistry. In the developing brain, immunostaining was first observed in newly differentiated neurons, later becoming localized in the neuronal processes. In the adult brain, the immunoreactivity was mainly localized in certain types of synaptic regions, but it was also observed in a small population of neuronal somata.

Identification of a synaptic vesicle-specific 38,000-dalton protein by monoclonal antibodies

Synaptic vesicles were purified from the guinea pig cerebrum by sucrose density gradient centrifugation, and monoclonal antibodies (MAbs) were produced against this vesicle fraction. Seven MAbs (171B5, 171E8, 174D12, 174H11, 177A2, 177H11 and 178D4) recognized a novel acidic protein of about 38,000 daltons which was specific to synaptic vesicles. In immunofluorescence microscopy, the staining pattern of these MAbs corresponded to the distribution of the synapses in the guinea pig central nervous system. These MAbs appeared to stain all synaptic regions, irrespective of their synaptic function or type of neurotransmitters. MAb 171B5 and 174H11 stained the rat, rabbit and bovine synapses similarly to the guinea pig. Two other MAbs (171E8 and 177H11) stained other mammals weakly but the remaining 3 MAbs reacted only with the guinea pig. In immunoelectron microscopy of both the cerebellar tissue and isolated vesicle fraction, these MAbs selectively labeled the synaptic vesicles but not other structures. Immunoblot analysis was performed on electrophoretically separated proteins in vesicle fraction and brain homogenate. All of 7 MAbs reacted with a band at a molecular weight of about 38,000 from the guinea pig. Isoelectric focussing disclosed that this protein was acidic (pI 4.5-5).

Immunochemical identification of subgroups of vomeronasal nerve fibers and their segregated terminations in the accessory olfactory bulb

Vomeronasal nerve (VNN) fibers and their terminations in the accessory olfactory bulb (AOB) were studied immunohistochemically using 3 monoclonal antibodies (MAbs). One MAb (R2D5) labeled all VNN fibers. Another MAb (R4B12) labeled a subgroup of the VNN fibers which terminated in the rostrolateral glomeruli in the AOB. The third MAb (R5A10) recognized a complementary subgroup of the VNN which terminated in the caudomedial portion of the AOB. These results for the first time show occurrence of subtypes in the VNN axons with segregated terminations in the AOB.

Two Acidic Proteins Associated with Brain Development in Chick Embryo

Abstract: The developmental changes in protein composition of the chick optic tectum were analyzed by twodimensional gel electrophoresis. Staining with Coomassie Brilliant Blue R revealed 54 major proteins, eight of which remarkably changed their abundance during development: Four of these proteins (S8, S14, S30, and S54) increased and two of them (S7 and S37) decreased in the course of the brain development. The other two proteins (S5 and S6) appeared at specific embryonic stages and were not detected in the adult. The abundance of S5 protein was highest at day 7, and that of S6 protein at days 9–18. The two proteins were present in other regions of the embryonic brain but were not detected in the embryonic liver. The proteins were purified from the soluble fraction of embryonic chick brains by pH 5.5 precipitation, DEAE‐Sepharose column chromatography, ammonium sulfate precipitation, and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The molecular weights of S5 and S6 proteins were 95,000 and 100,000, respectively, and their isoelectric points were about 4.5. They were compared by peptide mapping using V8 protease and found to share 11 common peptides out of 17 distinct ones. This indicates a strong degree of structural homology between these two proteins.

Projections of two subclasses of vomeronasal nerve fibers to the accessory olfactory bulb in the rabbit

The organization of the projections of subclasses of vomeronasal nerve fibers to the accessory olfactory bulb was analysed using monoclonal antibodies generated against a homogenate of the rabbit olfactory bulb. Monoclonal antibody R2D5 labels all the somata of vomeronasal receptor cells in the vomeronasal organ as well as all their axons (vomeronasal nerve fibers). Another monoclonal antibody (R4B12), which has been shown to selectively bind and thus identify a subclass of olfactory nerve fibers, also labels a subclass of vomeronasal nerve fibers. The R4B12-positive subclass of vomeronasal nerve fibers project to the glomeruli in the rostrolateral part of the accessory olfactory bulb. The third monoclonal antibody (R5A10) recognizes a complementary subclass of vomeronasal nerve fibers projecting to the glomeruli in the caudomedial part of the accessory bulb. In contrast to the clearly segregated terminations in the accessory bulb, the two subclasses of vomeronasal nerve fibers are intermingled with each other in the vomeronasal nerve bundles. Retrograde labeling of vomeronasal receptor cell somata following injection of horseradish peroxidase within the rostrolateral (R4B12-positive) part of the accessory bulb indicates that vomeronasal receptor cells of this subtype are widely distributed in the vomeronasal sensory epithelium. These results demonstrate the heterogeneity of vomeronasal receptor cells and the specificity of projections arising from subclasses of vomeronasal nerve fibers to the accessory olfactory bulb.

Conditioned medium promotes neurite growth from both central and peripheral neurons

Explants or dissociated neurons of spinal cord and other parts from chick embryos and rat fetuses were cultured for 1-4 days. Extensive outgrowth of neurites from the spinal cord was observed in the medium which had been conditioned by skeletal and heart muscle cells. In the fresh medium, the neurites were short and accompanied by the migrating non-neuronal cells. The conditioned medium promoted also the neurite outgrowth from retina, optic tectum, ciliary ganglion, superior cervical ganglion and dorsal root ganglion.

Four synaptic vesicle-specific proteins: identification by monoclonal antibodies and distribution in the nervous tissue and the adrenal medulla

Synaptic vesicles from the guinea-pig cerebrum were isolated and administered to mice for the production of monoclonal antibodies (MAb). Four vesicle-associated proteins in the guinea-pig nervous tissue were specifically and differentially recognized by MAbs thus obtained. These proteins had molecular weights of 30,000, 36,000, 38,000 and 65,000 Da and were named SVPs (synaptic vesicle proteins) 30, 36, 38 and 65, respectively. Immunohistochemistry demonstrated that all SVPs were localized in the synaptic regions throughout the central nervous system and in the adrenal medulla. Nerve terminals in skeletal muscle, smooth muscle and sympathetic ganglion contained SVPs 36 and 38. Immunoelectron microscopy of the cerebellar cortex confirmed the localization of SVPs in the synaptic vesicles and the adjacent membranes of the presynaptic nerve terminals. Fractionation of the cerebral tissue and treatment with various agents showed that SVPs were localized in the synaptic vesicles and the synaptic plasma membrane and that SVPs were integrated within the membrane and liberated only after solubilization of the membrane.

Localization of a developmentally regulated neuron-specific protein S54 in dendrites as revealed by immunoelectron microscopy

We sought to determine the ultrastructural localization of the developmentally regulated neuron-specific protein S54 in the chicken cerebellar cortex and optic tectum. The brains were fixed by perfusion with paraformaldehyde and glutaraldehyde. Frozen sections were immunocytochemically labeled with a monoclonal antibody to S54 protein. The immunoreactivity for S54 protein was localized in dendrites. No immunoreactivity for S54 protein was detected in axons and their presynaptic terminals.