Melitta Schachner

Gm1 ganglioside as a marker for neuronal differentiation in mouse cerebellum.

The distribution of GM1 ganglioside in developing mouse cerebellum was monitored by indirect immunofluorescent detection of choleragenoid receptors. In frozen sections of cerebellum from mice 5 to 10 days old, fluorescence is observed on granule cells in the inner rows of the external granular layer, in the growing molecular layer, the Purkinje cell layer, and the internal granular layer. In sections of adult mice, fluorescence is restricted to the bodies of Purkinje and internal granule neurons. The percentage of fluorescent dissociated or cultured cerebellar cells increases with the postnatal age of the mouse or the duration of time in vitro. No fluorescence is observed in the absence of choleragenoid or if the test material is extracted with chloroform:methanol. To determine whether the expression of surface GM1 ganglioside in culture is a reflection of a developmental program, mice are injected at particular times with [3H]thymidine and cerebellar cultures processed for simultaneous autoradiography and immunofluorescence. Granule cells from 8-day-old mice having cholera toxin receptors at 20 hr in vitro are a distinct population born 1 day or earlier prior to sacrifice. Cells synthesizing DNA on the day of sacrifice are not fluorescent at 20 hr in vitro. This observation correlates well with immunohistological results showing a lack of fluorescence in the outer proliferative rows of the external granular layer. Therefore GM1 ganglioside is not present on granule cell precursors but is expressed at some time after the cells become postmitotic. GM1 ganglioside is detected on growing parallel fibers in situ and neurites in vitro but not on adult axons, suggesting differential localization at a later stage of development.

Ns-4 (nervous system antigen-4), a cell surface antigen of developing and adult mouse brain and sperm.

Abstract When rabbits are injected with particulate fractions of cerebellum from 4-day-old C57BL 6J mice, an antiserum is produced that is cytotoxic for single-cell suspensions from cerebellum. After several absorptions with nonnervous system-derived tissues (liver, spleen, kidney, thymocytes) this heterologous antiserum reacts only with tissue from the developing and adult nervous system and sperm from vas deferens and epididymis. This antigen or set of antigens is present in neuroectodermally derived tissues at all ages tested: 12-day-old embryos and 12-month-old adults being the earliest and latest stages, respectively. The antigen is expressed not only on cerebellum but also on all parts of the normal central nervous system tested including retina. It is demonstrable on a murine medulloepithelioma and to a lesser degree on glioblastoma and ependymoblastoma but not on glioma G26, ependymoblastoma EPA and neuroblastoma C1300. The antigen is found in all mouse strains tested ( C57BL 6J , ICR, BALB cJ , A J and ( C57BL 6J × C3H HeJ ) F1 hybrid) and in rat.

Histochemical characterization of lectin binding in mouse cerebellum

Abstract Eight fluorescein-isothiocyanate-conjugated lectins differing from each other in carbohydrate binding specificities have been used to reveal qualitative differences in carbohydrate composition among the various cell types of the early postnatal and adult cerebellar cortex, cerebellar deep nuclei and medulla. By treating unfixed frozen tissue sections with unconjugated lectin in the presence of excess hapten sugar and subsequently staining with fluorescein-isothiocyanate-conjugated lectin in the presence of bovine serum albumin, carbohydrate specific staining was observed for all of the lectins studied. Lectin binding was selectively inhibited by appropriate hapten sugars. For several lectins (wheat germ agglutinin, Ricinus communis agglutinins and Lens culinaris agglutinins) complex carbohydrates were required for inhibition of staining. In the adult cerebellum, concanavalin A intensely stained the soma of granule cells, synaptic glomeruli and the cytoplasm of Purkinje cells, while the molecular layer and white matter were relatively unstained. Wheat germ agglutinin heavily labeled the soma of granule cells, synaptic glomeruli, parallel fibers of the molecular layer, and the nuclear membrane of Purkinje cells, and weakly labeled the white matter. Ricinus communis agglutins (m.w. 60,000 and 120,000) intensely stained the soma of granule cells, synaptic glomeruli, parallel fibers in the molecular layer, the cytoplasm of Purkinje cells, and the white matter. None of the above lectins stained Purkinje cell dendrites. Lens culinaris lectins A and B stained the soma of granule cells, synaptic glomeruli, the cytoplasm of Purkinje cells, Purkinje cell dendrites, and white matter. In the 5- and 12-day-old postnatal cerebellum, concanavalin A stained the molecular layer more strongly than in the adult. Concanavalin A, wheat germ agglutinin and Lens culinaris lectins moderately stained cell cytoplasm in the external and internal granule layers, but the external granule cell layer was heavily labeled with both Ricinus communis lectins. With the soybean agglutinin, weak staining of the molecular layer and synaptic glomeruli was detected in young cerebellum, in contrast to adult tissue where no staining was observed. No labeling of the young or adult cerebellum was observed with Ulex europaeus agglutinin I. The specific patterns of staining with different lectins probably reflect the different carbohydrate compositions of the various cell types and perhaps of the intercellular matrix.

Isolation of glial cell-enriched and -depleted populations from mouse cerebellum by density gradient centrifugation and electronic cell sorting.

Preparative amounts of populations enriched and depleted in glial cells have been isolated from 10-day-old mouse cerebella. Discontinuous density bovine serum albumin (BSA) gradients provide two distinct populations: the one derived from the 10-15% BSA interface is enriched in cyclic nucleotide phosphohydrolase (CNPase) activity, and a percentage of S-100, GFA protein, and NS-1 antigen-positive cells; the other, located as the 15-31% interface, contains a cell population depleted in these compounds. This report also describes the first use of flow microfluorimetry and electronic cell sorting techniques for the analysis and isolation of cell populations derived from the mammalian central nervous system. Glial cell-enriched and -depleted populations obtained by BSA density gradient centrifugation were analyzed for their forward angle light scattering properties and their capacity to bind anti-corpus anti-serum to the cell surface. The glial cell-enriched fraction shows a different frequency distribution of light scattering than the glial-depleted fraction. Anti-corpus callosum antiserum binds preferentially to the glial cell-rich fraction. Cells can be sorted into corpus callosum antigen-positive and -negative cell fractions and recovered with a viability of more than 95% as judged by trypan blue exclusion. Anti-corpus callosum-positive sorted cells are enriched in CNPase activity, S-100, and glial fibrillary acidic proteins.

Brain and sperm cell surface antigen (NS-4) on preimplantation mouse embryos

Abstract Antiserum prepared in rabbit against 4-day-old mouse cerebellum (anti-NS-4 serum) reacts in the complement-mediated cytotoxicity test with unfertilized and fertilized mouse eggs, cleavage stage embryos, and cells of the trophoblast and inner cell mass of the mouse blastocyst. This activity is specifically removed by absorption of antiserum with adult mouse brain and epididymal sperm but not with adult liver, spleen, kidney, and thymocytes. The antiserum reacts most strongly with cells of the trophoblast and inner cell mass and, in order of decreasing reactivity, with four- to eight-cell stage embryos, zygotes, unfertilized eggs, and two-cell stage embryos.

CELL SURFACE PROTEINS OF CULTURED BRAIN CELLS AND THEIR RECOGNITION BY ANTI-CEREBELLUM (ANTI-NS-4) ANTISERUM

Abstract— Surface proteins of cultured young postnatal mouse cerebella and embryonic mouse cerebral hemispheres were identified by Iactoperoxidase‐catalysed radioiodination and by their interaction with an anti‐mouse cerebellum antiserum (anti‐NS‐4 serum) which recognizes surface components on brain cells. Several (8 10) iodinated polypeptides are recognized by radioautography after polyacrylamide gel electrophoresis. Their surface location was confirmed by their sensitivity to mild trypsin treatment on intact cells. Iodinated polypeptides from cells of non‐nervous tissues showed a different gel pattern. Immuno‐precipitates of solubilizcd surface‐iodinated cerebellar cells with anti‐NS‐4 serum contained two prominent labeled proteins with apparent molecular weights of 200 × 103 and 145 × 103. These proteins were also biosynthetically labeled with [3H]leucine. The 145 × 103 molecular weight component was also found in immunoprecipitates prepared from embryonic cerebral cells, but the 200 × 103 molecular weight component was replaced by a broad peak with an apparent molecular weight of around 250 × 103.

Distribution of H-2 alloantigen in adult and developing mouse brain

Abstract Quantitative assays for the major histocompatibility antigen system H-2 in mouse brain were carried out by absorption technique. The representation of H-2 in brain reaches the adult level 3–4 weeks after birth and is approximately equal in various parts of the brain. A method is described for obtaining single cell suspensions by trypsinization of developing mouse brain and the cells were used in a complement mediated cytotoxic test. H-2 is stable to trypsin treatment and was detected by this method at 8 days but not at 4 days after birth. While the direct cytotoxic test is less sensitive than the quantitative absorption methods, it should serve for detection of antigenic specificities unique to brain.

Brain cell surface antigens detected by anti-corpus callosum antiserum.

When rabbits are injected with tissue homogenates of white matter from bovine corpus callosum, an antiserum is produced which reacts with the surface membrane of 20-30% of all cells obtained by trypsin-dissociation of cerebellum from 10-day-old mice. The antigen or set of antigens recognized by this antiserum is detectable on embryonic, early postnatal, and adult mouse brain, but not on liver, spleen, kidney, thymus and sperm. The antigen is expressed in different regions of the brain and also, in decreased amounts, on retina. In histological sections of cerebellum from 21-day-old mice the antigen is predominantly localized in white matter tracts. Whereas nervous tissue from chicken and rabbit does not carry detectable levels of the antigen, rat, bovine and human brains are antigen-positive.

Nervous system antigen-5, an antigenic cell surface component of neuroectodermal origin

The antigenic cell surface component NS-5 (nervous system antigen-5) is recognized by antiserum raised in C3H.SW/Sn mice against cerebellum of 4-day-old C57BL/6J mice. When analyzed in the cytotoxicity test the antiserum detects a cell surface antigen or set of antigens present not only an cerebellum but also other parts of the central nervous system, including retina, as well as on mature spermatozoa and to a lesser degree on kidney. All other non-neural tissues tested, liver, splee, thymocytes, muscle, testis, adrenal gland and epidermis do not express detectable amounts of the antigen. Among seven murine tumors of the nervous system, medulloepithelioma shows high levels of NS-5 expression, whereas neuroblastoma Cl300, glioma G26, glioblastome, ependymoblastoma, ependymoblastoma EPA and glioblastoma G26l do not carry detectable NS-5. All mouse strains tested (C57BL/6J, C3H.SW/Sn, C3H/HeDiSn, A/J, AKR/J, BALB/cJ and DBA/2) express similar levels of NS-5. The antigen is demonstrable not only on postnatal day 4 neural tissue, but also in lower amounts on adult nervous system. On embryonic day 9, the earliest stage tested, and at all subsequent stages during embryonic development, NS-K is already present in brain and spinal cord, but not in gut.

Cerebellar cell surface components detected by anti-Weaver mutant antiserum.

Of the many regions of the mammalian central nervous system, the histogenesis of the relatively simply organized cerebellar cortex has been studied in relatively great detailS,14,16. The molecular mechanisms which govern the highly specific patterns of cell migration and synapse formation have, however, remained largely unknown. To uncover these mechanisms by genetic means, several cerebellar mouse mutants have been investigated which show selective cell death, perhaps based on developmental abnormalities in cell interaction. One of these mutants, the autosomal mutation, Weaver (wv), has been a focus of anatomical studies concerning cerebellar histogenesis in homozygous and heterozygous mutant animals 6,1~,1s,ao. In the homozygous situation, the mutation gives rise to a cerebellum which is markedly reduced in size, reflecting an almost complete loss of granule cells 26 which causes behavioral abnormalities consisting of instability of gait and posture, hypotonia and tremor. When on an inbred background, the homozygous mutant animals die at the age of three weeksT. At present, the primary cellular or molecular target of the Weaver genetic locus is unknown, but it seems likely that the early postnatal loss of granule cells is caused by extensive death of already generated granule cells in the external granular layer prior to migration to the internal granular layer~6. One of the reasons for this early cell death could be the failure of the external granule cells to migrate to their final position in the internal granular layer~O. This, in turn, could result from an abnormality of the radially oriented Bergmann glial cells, the processes of which have been shown to serve as guidelines for the migrating granule cells 19. During their migration in the normal cerebellum, granule cells have been observed in close (20 nm) apposition with Bergmann glial fibers 16, and it has, therefore, been hypothesized that in the mutant an altered cell surface composition of Bergmann glial cells may underlie the granule cells inability to recognize their guidelines and thus may cause degeneration of granule