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Y von Boxberg

Publications and source records attributed to Y von Boxberg.

13 recordsLinked to original sources
PubMed2000

Morphofunctional plasticity in the adult hypothalamus induces regulation of polysialic acid-neural cell adhesion molecule through changing activity and expression levels of polysialyltransferases.

Polysialic acid-neural cell adhesion molecule (PSA-NCAM) expression in the adult nervous system is restricted to regions retaining a capacity for morphological plasticity. For the female rat hypothalamoneurohypophysial system (HNS), we have previously shown that lactation induces a dramatic decrease in PSA-NCAM, while leaving the level of total NCAM protein unchanged. Here, we wanted to elucidate the molecular mechanisms leading to a downregulation of PSA, thereby stabilizing newly established synapses and neurohemal contacts that accompany the increased activity of oxytocinergic neurons. First, we show that the overall specific activity of polysialyltransferases present in tissue extracts from supraoptic nuclei decreases by approximately 50% during lactation. So far, two polysialyltransferase enzymes, STX and PST, have been characterized for their capacity to transfer PSA onto NCAM in vitro. Using a competitive RT-PCR on RNA extracts from the HNS, we demonstrate furthermore a significant decrease in the expression levels of both STX and PST mRNAs in lactating versus virgin animals. Interestingly, this downregulation of NCAM polysialylation is not correlated with the post-transcriptional regulation of variable alternative spliced exon splicing, in contrast to neural development. The control of polysialylation via a regulation of both enzyme activity and expression underlines the important role of this post-translational modification of NCAM in morphofunctional plasticity in adult brain.

Animals↗
PubMed1999

The mouse homeodomain protein OTX2 regulates NCAM promoter activity.

The homeodomain transcription factor OTX2 is involved in defining regional identities in developing rostral brain. It appears to participate in morphogenetic processes leading to the formation of boundaries and substrates for early axon growth, processes which are in the end largely based on site-specific expression of cell adhesion molecules. Here, we present evidence that a candidate target of OTX2 is the gene encoding the neural cell adhesion molecule, NCAM. When Otx2 is transfected into NIH3T3 cells, NCAM protein expression is upregulated. Moreover, while mock-transfected cells display only the 140 kDa-isoform of NCAM, Otx2 transfected cells express also the two other major isoforms (NCAM-120 and -180), in agreement with the presence of the corresponding transcripts in Northern blots. In addition, transient expression of Otx2 in COS7 cells is able to dramatically enhance the transcriptional activity of the NCAM promoter. Taken together, our results argue for a regulation of NCAM expression by OTX2.

Animals↗
PubMed1998

A potential role for the OTX2 homeoprotein in creating early 'highways' for axon extension in the rostral brain.

Brain pattern formation starts with a subdivision of the neuroepithelium through site-specific expression of regulatory genes and, subsequently, the boundaries between presumptive neuromeres may provide a scaffold for early formation of axon tracts. In the mouse forebrain, the transcription factor OTX2 is strongly expressed at several such boundaries. Combining dye tracing and staining for OTX2 protein, we show that a number of early fibre tracts develop within stripes of OTX2 expression. To analyse a putative influence of OTX2 on the expression of molecules involved in neurite growth, we generated several clones of NIH3T3 cells stably expressing OTX2 protein at varying levels. As shown by immunoblotting, Otx2 transfection affects the expression of a variety of cell and substratum adhesion molecules, rendering the cells a favourable substratum in neurite outgrowth assays. Among the molecules upregulated with increasing levels of OTX2 are NCAM, tenascin-C and DSD-1-PG, which also in situ colocalize with zones of OTX2 expression at boundaries. These data suggest that Otx2 might be involved in defining local substrata for axon extension in the forebrain.

3T3 Cells↗
PubMed1997

Modulation of NCAM polysialylation is associated with morphofunctional modifications in the hypothalamo-neurohypophysial system during lactation.

Post-transcriptional modification of the neural cell adhesion molecule (NCAM) by polysialic acid significantly decreases NCAM adhesiveness and more generally modifies cell-cell interactions. Polysialic acid-NCAM (PSA-NCAM) is mainly expressed in the developing nervous system. In the adult, its expression is restricted to regions that retain morphological plasticity, such as the hypothalamo-neurohypophysial system during lactation in rats. Since cell-cell interactions and synaptic contacts in the hypothalamo-neurohypophysial system are greatly increased during lactation, we examined whether PSA-NCAM expression is modified during this period. Immunohistochemistry and immunoblotting showed that, compared with virgin rats, PSA-NCAM dramatically decreased during lactation in both the supraoptic nuclei and the neurohypophysis, and returned to its initial level only after weaning. This decrease was progressive and became significant only at the end of the first week of lactation. By contrast, modifications in the level of NCAM protein or changes in the splicing pattern of NCAM mRNAs could not be detected. The decline in polysialic acid on the NCAM molecule could strengthen membrane appositions, thereby stabilizing the newly established synapses and neurohaemal contacts in the hypothalamo-neurohypophysial system that accompany the increased neuronal activity that occurs during lactation. We also studied the regulation of the phosphorylated microtubule-associated protein-1B (MAP1B-P), whose distribution pattern largely overlaps with that of PSA-NCAM in the adult brain. Expression of MAP1B-P was greatly increased during lactation in the hypothalamic axons projecting into the neurohypophysis. Thus, the expression patterns of both PSA-NCAM and MAP1B-P may reflect the permanent structural plasticity characterizing the hypothalamo-neurohypophysial system in the adult.

Animals↗
PubMed1994

Affinity purification of monospecific antibodies from polyclonal sera as a means for the identification of differentially expressed genes and proteins.

The identification and characterization of genes and proteins that are either cell-type specific or exhibit an otherwise spatially or temporally restricted expression pattern is a crucial step toward understanding the complex interactions between the different cell types in an organism. We demonstrate here that monospecific polyclonal antibodies purified from rabbit antisera by a simple Western blotting and reelution technique can be successfully used for the detection of such proteins, as well as the corresponding genes by screening cDNA expression libraries. This strategy is discussed as a supplement to other approaches, such as the generation of tissue-specific monoclonal antibodies and the "subtractive hybridization technique."

Animals↗
PubMed1993

Guidance and topographic stabilization of nasal chick retinal axons on target-derived components in vitro.

We studied mechanisms underlying the generation of topographic order within the developing chick retinotectal connection by combining the recently introduced stripe assay with a novel membrane protein fractionation technique. Our experiments show a preference of temporal and nasal retinal fibers for growing on cell membranes prepared from their proper target area. In addition, membrane preparations from posterior tectum were found to prolong substantially the survival of nasal neurites in vitro. We conclude that tropic as well as trophic interactions contribute to the generation of topographic maps during embryogenesis, in our case to the homing of nasal axons within the posterior tectum.

Animals↗
PubMed1993

Differential reaction of crossing and non-crossing rat retinal axons on cell membrane preparations from the chiasm midline: an in vitro study.

In the rat, a small subpopulation of retinal ganglion cell axons forms a persistent projection to the ipsilateral half of the brain. These fibres originate almost exclusively from the ventrotemporal margin of the retina. In contrast to all other retinal axons they seem to be deflected from the midline of the optic chiasm and thereby led into the ipsilateral optic tract. In order to analyse the interactions between growing fibres and chiasm midline, we have developed the following in vitro model. Axons of the embryonic rat retina are grown on a carpet of tectal cell membranes used as a general growth-permissive substratum. At a certain distance from the explant (200-450 microns), the advancing fibres are confronted with two stripes of cell membranes prepared from the chiasm midline. Such chiasm membranes are shown to act as a barrier for the presumptive non-crossing axons, while they do not influence growth of fibres originating from any other regions of the retina, including the dorsotemporal part. The repulsion of non-crossing fibres by chiasm membranes is observed in vitro only when retinal explants from embryonic day (E) 17/18 and chiasm preparations from E14/15 are used. Fibres and tissue from different regions of the brain as well as from different developmental ages, and even from different species, can be combined in this assay system. In a first attempt to characterize the molecular basis of the repulsive effect of chiasm membranes on ventrotemporal fibres, similar assays were performed with membranes derived from other regions of the central nervous system midline, some of which are known to have repulsive properties against certain axon populations. Since these cell membranes did not act as a barrier for the ventrotemporal retinal axons, we suggest that the guidance cues at the chiasm are very specific. Our results are consistent with the hypothesis that certain cells at the chiasm midline (very likely radial glial cells) express 'repulsive or inhibitory' molecules, which act in a specific way on ipsilaterally projecting axons.

Animals↗
PubMed1990

Use of the biotin-avidin system for labelling, isolation and characterization of neural cell-surface proteins.

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated.

Animals↗
PubMed1990

Biochemical characterization of a putative axonal guidance molecule of the chick visual system.

Temporal retinal axons growing in vitro on carpets of tectal membranes are deflected by cell membranes of posterior tectum. The activity responsible for this deflection can be abolished by antibodies raised against tectal membranes and the corresponding Fab fragments. Analysis of tectal membranes by two-dimensional gel electrophoresis and immunoblotting reveals a 33 kd glycoprotein that has a higher concentration in posterior than in anterior tectum. Its expression is developmentally regulated, and it is sensitive to phosphatidylinositol-specific phospholipase C. These are properties expected for a molecule responsible for the phenomena observed in experiments on in vitro guidance of retinal axons.

Animals↗
PubMed1990

Structural and compositional divergencies in the extracellular matrix encountered by neural crest cells in the white mutant axolotl embryo.

The skin of the white mutant axolotl larva is pigmented differently from that of the normal dark due to a local inability of the extracellular matrix (ECM) to support subepidermal migration of neural crest-derived pigment cell precursors. In the present study, we have compared the ECM of neural crest migratory pathways of normal dark and white mutant embryos ultrastructurally, immunohistochemically and biochemically to disclose differences in their structure/composition that could be responsible for the restriction of subepidermal neural crest cell migration in the white mutant axolotl. When examined by electron microscopy, in conjunction with computerized image analysis, the structural assembly of interstitial and basement membrane ECMs of the two embryos was found to be largely comparable. At stages of initial neural crest cell migration, however, fixation of the subepidermal ECM in situ with either Karnovsky-ruthenium red or with periodate-lysine-paraformaldehyde followed by ruthenium red-containing fixatives, revealed that fibrils of the dark matrix were significantly more abundant in associated electron-dense granules. This ultrastructural discrepancy of the white axolotl ECM was specific for the subepidermal region and suggested an abnormal proteoglycan distribution. Dark and white matrices of the medioventral migratory route of neural crest cells had a comparable appearance but differed from the corresponding subepidermal ECMs. Immunohistochemistry revealed only minor differences in the distribution of fibronectin, laminin, collagen types I, and IV, whereas collagen type III appeared differentially distributed in the two embryos. Chondroitin- and chondroitin-6-sulfate-rich proteoglycans were more prevalent in the white mutant embryo than in the dark, especially in the subepidermal space. Membrane microcarriers were utilized to explant site-specifically native ECM for biochemical analysis. Two-dimensional gel electrophoresis of these regional matrices revealed a number of differences in their protein content, principally in constituents of apparent molecular masses of 30-90,000. Taken together our observations suggest that local divergences in the concentration/assembly of low and high molecular mass proteins and proteoglycans of the ECM encountered by the moving neural crest cells account for their disparate migratory behavior in the white mutant axolotl.

Ambystoma↗
PubMed1989

Factors influencing regeneration of retinal ganglion cell axons in adult mammals.

Adult retina ganglion cells regrow vigorously their lesioned axons in sciatic nerve segments transplanted at the site of optic nerve transection. In order to determine whether the transplanted peripheral nerve produces secretable substances involved in regeneration, a nerve exudate was collected from the adult sciatic nerve. Transection of the sciatic nerve in situ, implantation of silicone tubes around its proximal stump, and additional crush of the nerve further proximal permitted the reinnervation of the nerve stump inside the tube and the release of regeneration-induced substances. Biochemical analysis revealed that several proteins are secreted into the tubes. The fluid contents of the implanted tubes were removed 1 week after implantation and tested for neurotrophic activity in cultures of adult retinae. Massive regrowth of ganglion cell axons has been found in the presence of the nerve exudate. The numbers of axons were significantly higher than these obtained in the presence of nerve and fibroblast growth factors. The results suggest that the lesioned peripheral nerves deliberate during the period of reinnervation substances which also support axonal regrowth of injured central neurons.

Animals↗
PubMed1988

Local embryonic matrices determine region-specific phenotypes in neural crest cells.

Membrane microcarriers were used to determine the ability of regional extracellular matrices to direct neural crest cell differentiation in culture. Neural crest cells from the axolotl embryo responded to extracellular matrix material explanted from the subepidermal migratory pathway by dispersing and by differentiating into pigment cells. In contrast, matrix material from the presumptive site of dorsal root ganglia stimulated pronounced cell-cell association and neurotypic expression. Cell line segregation during ontogeny of the neural crest that leads to diversification into pigment cells of the skin or into elements of the peripheral nervous system appears to be controlled in part by local cell-matrix interactions.

Ambystoma mexicanum↗