Monoclonal antibody (M2) to glial and neuronal cell surfaces

A monoclonal antibody designated M2 arose from the fusion of mouse myeloma cells with splenocytes from a rat immunized with particulate fraction from early postnatal mouse cerebellum. Expression of M2 antigen was examined by indirect immunofluorescence on frozen sections of developing and adult mouse cerebellum and on monolayer cultures of early postnatal mouse cerebellar cells. In adult cerebellum, M2 staining outlines the cell bodies of granule and Purkinje cells. A weaker, more diffuse staining is seen in the molecular layer and white matter. In sections of newborn cerebellum, M2 antigen is weakly detectable surrounding cells of the external granular layer and Purkinje cells. The expression of M2 antigen increases during development in both cell types, reaching adult levels by postnatal day 14. At all stages of postnatal cerebellar development, granule cells that have completed migration to the internal granule layer are more heavily stained by M2 antibodies than are those before and in process of migration. In monolayer cultures, M2 antigen is detected on the cell surface Of all GFA protein-positive astrocytes and on more immature oligodendrocytes, that express 04 antigen but not 01 antigen. After 3 days in culture, tetanus toxinpositive neurons begin to express M2 antigen. The same delayed expression of M2 antigen on neurons is observed in cultures derived from mice ranging in age from postnatal day 0 to 10.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.     

Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion.

Monoclonal and polyclonal L1 antibodies react by indirect immunofluorescence with the cell surface of cultured tetanus toxin-positive neurons from post-natal cerebella of mice, but not with glial fibrillary acidic protein-positive astrocytes, O4 antigen-positive oligodendrocytes or fibronectin-positive fibroblasts or fibroblast-like cells. During cerebellar development L1 antigen is detectable on tetanus toxin-positive cells as early as embryonic day 13 after 3 days in culture. In sections of the early post-natal cerebellum, L1 antigen is found on pre-migratory neurons in the internal, but not in the external part of the external granular layer. In the adult cerebellum, L1 antigen is predominantly localized in the molecular layer and around Purkinje cells. Fibers in white matter and the granular layer are also L1 antigen-positive. Granule cell bodies and synaptic glomeruli are weakly antigen-positive. Several cell lines derived from neuroblastoma C1300 also express L1 antigen. The antigen is not detectable by enzyme-linked immunosorbent assay in tissue homogenates of liver, kidney, lung, heart, sperm or thymus. With polyclonal L1 antibodies, cross-reactive determinants are found in brains of rat, guinea pig, hamster, chicken, rabbit and man, but not in frog, while monoclonal antibody reacts detectably only with mouse brain. The molecular species recognized by both monoclonal and polyclonal antibodies display two prominent bands by SDS-PAGE under reducing and non-reducing conditions with apparent mol. wts. of 140 and 200 kd. L1 antigen isolated from cultured cerebellar cells consists mainly of a band in the 200-kd range and a faint one at 140 kd. L1 antigen from neuroblastoma N2A shows two bands with slightly higher apparent mol. wts. All molecular forms of L1 antigen can be labeled by [3H]fucose and [3H]glucosamine. Ca2+-independent re-aggregation of cerebellar cells from early post-natal C57BL/6J mice and of the continuous cell line N2A derived from the murine neuroblastoma C1300 is inhibited by Fab fragments of the polyclonal, but not of monoclonal antibody, both of which are known to react with the surface membrane of these cells.     

Extracellular matrix of cultured glial cells: selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors

We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes (recognized by the monoclonal antibody LB1 at early stages of their development) synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.     

Monoclonal antibodies as neural cell surface markers

A comparison is made of the immunohistochemistry at the ultrastructural level of three monoclonal antibodies directed against surface components of CNS cells. Hybridomas secreting these antibodies were obtained from two cell fusions of a rat myeloma cell line and immune splenocytes derived from rats immunized either with primary mouse brain cultured cells or membrane components. In cultures one antibody, anti-BSP-2 (Brain Surface Protein-2), was preferentially directed against neurones while another, anti-BSP-3 (Brain Surface Protein-3), preferentially labeled astrocytes. In mouse cerebellar sections, both labeled the surface of Purkinje cells, granule cells and astrocytes. In addition a cytoplasm localization was apparent in granule cells and astrocytes. Another antibody anti-MESA-1 (Mouse Endothelial Surface Antigen-1) reacted exclusively with the surface of endothelial cells lining blood vessels. These data are discussed with reference to the biochemical nature of the corresponding antigens and to known glycoproteins of neural cell membranes.     

Recognition of Bergmann glial and ependymal cells in the mouse nervous system by monoclonal antibody

A monoclonal antibody designated anti-Cl was obtained from a hybridoma clone isolated from a fusion of NS1 myeloma with spleen cells from BALB/c mice injected with homogenate of white matter from bovine corpus callosum. In the adult mouse neuroectoderm, C1 antigen is detectable by indirect immunohistology in the processes of Bergmann glial cells (also called Golgi epithelial cells) in the cerebellum and of Muller cells in the retina, whereas other astrocytes that express glial fibrillary acidic protein in these brain areas are negative for C1. In addition, C1 antigen is expressed in most, if not all, ependymal cells and in large blood vessels, but not capillaries. In the developing, early postnatal cerebellum, C1 antigen is not confined to Bergmann glial and ependymal cells but is additionally present in astrocytes of presumptive white matter and Purkinje cell layer. In the embryonic neuroectoderm, C1 antigen is already expressed at day 10, the earliest stage tested so far. The antigen is distinguished in radially oriented structures in telencephalon, pons, pituitary anlage, and retina. Ventricular cells are not labeled by C1 antibody at this stage. C1 antigen is not detectable in astrocytes of adult or nearly adult cerebella from the neurological mutant mice staggerer, reeler, and weaver, but is present in ependymal cells and large blood vessels. C1 antigen is expressed not only in the intact animal but also in cultured cerebellar astrocytes and fibroblastlike cells. It is localized intracellularly.     

The proteoglycan chondroitin sulfate is present in a subpopulation of cultured astrocytes and in their precursors

We have used an antibody raised against the bovine nasal cartilage proteoglycan chondroitin sulfate (CS) digested with chondroitinase ABC (anti-CS serum) to stain cerebellar glial cells maintained in culture. In cultures grown in the presence of serum, the antibody stained a subclass of GFAP+ astrocytes which we have previously shown to selectively bind the monoclonal antibodies A2B5 and LB1. Also the direct bipotential precursors of these cells, capable of differentiating into GFAP+ astrocytes or into Gal-C+, O1+ oligodendrocytes depending on the culture conditions, were stained, but stopped to produce CS when they differentiated into oligodendrocytes.     

Cell type specificity of a neural cell surface antigen recognized by the monoclonal antibody A2B5

Summary The monoclonal antibody A2B5 reacts with the surface membrane of most neurons in monolayer cultures of cerebellum, retina, spinal cord, and dorsal root ganglion of embryonic and early postnatal C57BL/6J mice maintained in vitro for culture periods of 2 to 10 days. A small percentage of astroglial cells also expresses A2B5 antigen in murine, chicken and rabbit cerebellum, in chicken retina, and in murine spinal cord and dorsal root ganglion. Less mature astroglial cells are stained for A2B5 antigen to a greater extent than the more mature astrocytes. Astrocytes from rat cerebellum and mouse retina were not found to express A2B5 antigen under the present culture conditions. Some of the less mature oligodendrocytes recognized by 04 antibodies express A2B5 antigen, while the more mature 01 antigen and galactocerebroside-positive oligodendrocytes were not found to be A2B5 antigen-positive. Fibroblasts or fibroblast-like cells do not express detectable levels of A2B5 antigen. After fixation of the cells with paraformaldehyde and ethanol, all cell types present in culture are labeled by the A2B5 antibody intracellularly.     

In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Immuno-electron-microscopic identification of O-antigen-bearing oligodendroglial cells in vitro

Summary Monoclonal antibodies to cell-surface antigens of oligodendrocytes (Sommer and Schachner 1980; Schachner et al. 1980) were used to identify this cell type by immuno-electron microscopy in monolayer cultures of fetal and early postnatal mouse cerebellum. The ultrastructural features of antigen-positive cells confirm that they are immature and mature oligodendrocytes, but not neurons, astrocytes or fibroblasts or fibroblast-like cells. Type I oligodendrocytes are the immature ones with a relatively large amount of moderately electron-lucent cytoplasm, clusters of ribosomes and complex networks of rough endoplasmic reticulum. Large numbers of mitochondria and microtubules, but not intermediate-sized filaments are seen in these cells. They comprise more than 90% of all 0-antigen-positive cells. Type II cells comprise only approximately 5% of all 0-antigen-positive cells. They are characterized by a limited amount of electron-dense cytoplasm, which appears more compact and granular than in type I cells. The rough endoplasmic reticulum is distributed evenly throughout the cytoplasm. Microtubules and mitochondria are present, but more difficult to distinguish due to the compactness of the cytoplasm. Type II cells display the more mature ultrastructural features of oligodendrocytes.     

Interaction of astrochondrin with extracellular matrix components and its involvement in astrocyte process formation and cerebellar granule cell migration

We have recently characterized a chondroitin sulfate proteoglycan from the murine central nervous system which is expressed by astrocytes in vitro and carries the L2/HNK-1 and L5 carbohydrate structures. In the present study, we provide evidence that its three core proteins of different size are similar in their proteolytic peptide maps and thus designate this group of structurally related molecules astrochondrin. During development, astrochondrin and the L5 carbohydrate were hardly detectable in the brain of 14-d-old mouse embryos by Western blot analysis. Expression of astrochondrin and the L5 epitope was highest at postnatal day 8, the peak of cerebellar granule cell migration and Bergmann glial process formation, and decreased to weakly detectable levels in the adult. Immunocytochemical localization of astrochondrin in the cerebellar cortex of 6-d-old mice showed association of immunoreactivity with the cell surface of astrocytes, including Bergmann glial processes and astrocytes in the internal granular layer or prospective white matter. Endfeet of astrocytes contacting the basal lamina of endothelial and meningeal cells and contact sites between Bergmann glial processes and granule cells also showed detectable levels of astrochondrin. Furthermore, granule cell axons in the molecular layer were astrochondrin immunoreactive. In the adult, astrochondrin immunoreactivity was weakly present in the internal granular layer and white matter. Both Fab fragments of polyclonal antibodies to astrochondrin and monovalent fragments of the L5 monoclonal antibody reduced the formation of processes of mature GFAP- positive astrocytes on laminin and collagen type IV, but not on fibronectin as substrata. Interestingly, the initial attachment of astrocytic cell bodies was not disturbed by these antibodies. Antibodies to astrochondrin also reduced the migration of granule cells in the early postnatal mouse cerebellar cortex. In a solid phase radioligand binding assay, astrochondrin was shown to bind to the extracellular matrix components laminin and collagen type IV, being enhanced in the presence of Ca2+, but not to fibronectin, J1/tenascin or other neural recognition molecules. Furthermore, astrochondrin interacted with collagen types III and V, less strongly with collagen types I, II, and IX, but not with collagen type VI. The interaction of astrochondrin with collagen types III and V was saturable and susceptible to increasing ionic strength, and could be competed by chondroitin sulfate, heparin, and dextran sulfate, but not by hyaluronic acid, glucose-6-phosphate, or neuraminic acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pnn and SR family proteins are differentially expressed in mouse central nervous system

Pinin (pnn) is an SR-related protein that is ubiquitously expressed in most cell types and functions in regulating pre-mRNA splicing and mRNA export. Previously, we demonstrated that pnn is expressed in all tissues during mouse embryonic development with highest levels of expression in the central nervous system (CNS). Here we show that pnn and other SR proteins including SC35 are differentially expressed in the adult mouse CNS, displaying cell type-specific distribution patterns. Immunohistochemical analysis of whole-brain sections showed that levels of pnn and SR proteins expression were very low or nonexistent in the corpus callosum and white matter of cerebellum and spinal cord. Double-immunostaining with antibodies specific to neuron or glial cells showed that most astrocytes and microglia expressed neither pnn nor SR proteins. In contrast, oligodendrocytes and neurons expressed moderate and high levels, respectively, of both pnn and SR proteins. These results suggest that astrocytes are unique among cell types of neuroblast origin in terms of expression SR family proteins. Our results pave the way for future studies of the functional roles of pnn and SR family proteins in adults.     

Development and expression of cytoplasmic antigens in Purkinje cells recognized by monoclonal antibodies

Summary Five monoclonal antibodies reacting with intracellular constituents of Purkinje cells were investigated by means of indirect immunofluorescence on fresh-frozen sections of the cerebellum and retina from developing and adult normal and mutant mice. Antibodies PC1, PC2 and PC3, which recognize Purkinje cells, but no other cerebellar neuron type, label these cells from day 4 onward. PC4 antigen is expressed in addition to Purkinje cells also in granule cells and neurons of deep cerebellar nuclei and appears in Purkinje cells at day 4. M1 antigen (Lagenaur et al. 1980) is first detectable in Purkinje cell bodies by day 5; it is also detectable in deep cerebellar neurons. In the adult retina, only PC4 antigen is detectably expressed and is localized in the inner segments of photoreceptor cells.The neurological mutants weaver, reeler,jimpy and wobbler show detectable levels of these antigens in Purkinje cells. However, the mutants staggerer and Purkinje cell degeneration are abnormal in expression PC1, PC2, PC3, and M1 antigens. Staggerer never starts to express the antigens during development, whereas Purkinje cell degeneration first expresses the antigens, but then loses antigen expression after day 23. PC4 antigen is detectable in the remaining Purkinje cells in staggerer and Purkinje cell degeneration mice at all ages tested in this study. Deep cerebellar neurons are positive for both antigens, PC4 and M1, in all mutants and at all ages studied. In retinas of staggerer and Purkinje cell degeneration mutants, PC4 antigen is normally detectable in the inner segments of photoreceptor cells, even when these have started to degenerate in the case of Purkinje cell degeneration.     

Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca²⁺ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons.     

Glial-Cell Cultures from Brains of Carbonic Anhydrase II-Deficient Mutant Mice: Delay in Oligodendrocyte Maturation

Carbonic anhydrase II (CAII) is a multifunctional enzyme found in oligodendrocytes and astrocytes in normal mouse brains. We have begun to compare the glial cells in primary cultures from neonatal genetically CAII-deficient (Car) mice to those from normal (con) mice in order to detect developmental defects, if any, in Car glial cells. In con cultures intensely CAII-positive cells costained with antibodies against the oligodendrocytic markers, O4 and myelin basic protein (MBP), respectively. Most (82%) of the CAII-positive cells were O4-positive, but only 60% were MBP-positive. Some clumps of GFAP-positive cells were CAII-positive. At each respective number of days in vitro (DIV) total numbers of O4-positive cells were similar in Car and con cultures, and total numbers of galactocerebroside-positive cells also were similar in Car and con cultures. However, compared to cells in con cultures at 7 DIV, a lower percent of Car cells in the oligodendrocyte lineage expressed MBP, and morphological differentiation also was subnormal in that the Car cells showed fewer processes and membrane sheets. Car and con cultures expressed similar numbers of MBP-positive cells by 10 DIV. The results suggest a temporary delay in the maturation of Car oligodendrocytes.     

A neuropeptide precursor in cerebellum: proenkephalin exists in subpopulations of both neurons and astrocytes.

The adult rat cerebellum has minimal enkephalin immunoreactivity and is devoid of opiate-binding activity. Using novel monoclonal antibodies to the mammalian enkephalin precursor, we describe the immunofluorescent detection of proenkephalin, in the absence of mature enkephalin peptides, in subpopulations of rat cerebellar neurons and astrocytes. In cryostat sections, neurons that express proenkephalin include Golgi cells, macroneurons within deep cerebellar nuclei and a subpopulation of Purkinje cells. Proenkephalin messenger RNA and protein are present in subpopulations of both grey and white matter astrocytes, but not Bergmann glia. In dissociated glial culture, proenkephalin is expressed in process-bearing astrocytes, apparently in association with a subset of intermediate filaments. Proenkephalin within astrocytes is not seen until the second postnatal week and increases through to adulthood. Neuropeptide gene expression adds to the growing range of neuronal-type properties glial cells can display.     

Two proliferative stages of the oligodendrocyte lineage (A2B5+O4- and O4+GalC-) under different mitogenic control

Cell proliferation during successive stages of oligodendrocyte development was delineated in the rat brain and optic nerve. Surface antigens, A2B5, O4, and galactocerebroside (GalC) identified three cell populations emerging in sequence; the incorporation of bromodeoxyuridine into newly synthesized DNA identified the proliferative cells. In vivo, progenitor cells with phenotypes A2B5+O4- and A2B5+O4+GalC- were both proliferative, whereas differentiated GalC+ oligodendrocytes were not. Under basal conditions of culture, the proliferation of both progenitor cell types of the optic nerve was nearly abolished. Activity was restored for A2B5+O4- precursor cells with medium conditioned by either type-1 astrocytes, meningeal cells, or cerebellar interneurons. In contrast, intermediate O4+GalC- cells (proligodendrocytes) were refractory to the astroglial and meningeal signals, but remained as responsive as their precursor cells to the neuronal stimulus. These data further characterize the O4+GalC- proligodendrocyte as a distinct developmental stage, one that specifies a changing response of the cell to environmental mitogens.     

Serological characterization of a nervous system/sperm antigen (NS-52): demonstration of its presence on murine preimplantation embryos.

Anti-NS-5 antiserum raised in C3H.SW/Sn mice against cerebellum of 4-day-old C57BL/6J mice could be shown to recognize two cell surface antigens on cerebellar cells, NS-5₁ and NS-5₂, the latter antigen being shared with mouse and rat but not rabbit sperm. An antigen operationally identical to NS-5₂ was detected using indirect immunofluorescence staining on mouse preimplantation stages of development. While the unfertilized ova did not express detectable antigen on the cell surface, the fertilized egg expressed antigen shortly before the first cleavage division. From that stage onward, the anti-NS-5 antiserum stained the blastomeres of all stages, including the trophoblast cells and inner cell mass cells of the blastocyst. No difference in staining activity was observed for preimplantation embryos of various mouse strains analyzed: C57BL/6J, BALB/c, 129/J, C3H/DiSn, CKB × BALB.K, C3H.SW/Sn, and Swiss Webster mice. The staining activity was removed when the antiserum was preabsorbed with cerebellum or sperm from any of these mouse strains or with cerebellum and sperm of rats. Lymphocytes, thymocytes, liver, kidney, and skeletal muscle from early postnatal and adult mice and heart from early postnatal mice did not absorb the staining activity and neither did rabbit sperm nor cerebellum.     

Development of cerebellar astroglia: Transitions in form and cytoskeletal content

The forms, disposition, and cytoskeletal contents of astroglia in immature mouse cerebellum were studied by immunocytochemical staining with antisera against two intermediate filament proteins, vimentin (Vim) (58,000 daltons) and glial filament protein (GF) (51,000 daltons). From embryonic (E) Day 15 to postnatal (P) Day 2, Vim is expressed in cells throughout the cerebellar anlage, including radial glia and Bergmann fibers, cells with amorphous shapes and 2–3 processes, and thick longitudinal elements oriented parallel to axons within axon tracts. GF is not expressed during the first few postnatal days, but by P7, there is a dramatic increase in GF-positive astrocyte-like cells in the putative white matter that are more densely stained and more crowded than at any other age. Between P7 and P14 all astrocytes throughout the cerebellum express both Vim and GF. From P21 on, Vim expression is progressively rarer in all astrocytes except for Bergmann fibers, and GF-positive astrocytes become less numerous. These findings raise two issues: (a) the lineage and relationships of cells expressing Vim and GF; (b) Since GF-positive cells appear as axon ingrowth ceases, axons must grow in a terrain comprised of glial cells that have a different cytoskeletal composition (vimentin), reflecting a less differentiated state, than mature astrocytes or than the GF-rich astrocytes that proliferate after injury in adult CNS.     

Immunocytochemical localization of cell type-specific markers in reaggregating cell cultures of mouse cerebellum

Summary Reaggregate cultures were obtained from single-cell suspensions of fetal and early postnatal cerebellum, and fetal telencephalon and mesencephalon from C57BL/6J and NMRI mice and maintained in suspension under constant rotation as described previously (Seeds 1971). The percentage of dead cells in the aggregates as measured by the uptake of the fluorescent dye propidium iodide was always less than 5% of all cells. During the initial phase of reaggregation up to 20 h in vitro (hiv) several immunocytochemically defined cell types had a random distribution within the aggregate. Astrocytes were identified by indirect immunofluorescence by the use of the markers glial fibrillary acidic protein (GFAP), C1 and M1 antigens; neurons by NS-4 antigen and tetanus-toxin receptors; fibroblasts or fibroblast-like cells by fibronectin and laminin; and oligodendrocytes by myelin basic protein (MBP). Choleratoxin receptors and M 2 antigen served to distinguish the more mature from the less mature neurons. In reaggregates of early postnatal cerebellar cells neurons had started to redistribute after 40 hiv, forming an outer region containing more immature neurons and a core with more mature neurons. After 5 days in vitro (div) immature neurons were no longer detectable. From 3–8 div M1-and GFAP-positive astrocytic processes in the outer region showed a tendency for radial orientation. At later stages the processes appeared more randomly distributed and formed a dense glial network. Few oligodendrocytes and fibronectin-positive cells were present in the reaggregates. When reaggregates were prepared from 15 day-old embryonic cerebella, formation of radially oriented astrocytic processes and redistribution of neurons proceeded more slowly, but in a similar pattern as described for early postnatal cerebellum. GFAP was detectable at earlier ages than in situ. In reaggregates of 15 to 17 day old embryonic telencephalic anlage or midbrain, radially oriented astrocytic processes were not detectable. Similar to cerebellar reaggregates, accumulation of neurons in the inner region was observed.