Distinct effects of recombinant tenascin-C domains on neuronal cell adhesion, growth cone guidance, and neuronal polarity.

Using a set of recombinantly expressed proteins, distinct domains of the mouse extracellular matrix glycoprotein tenascin-C, hereafter called tenascin, have been identified to confer adhesion, anti-adhesion, and changes in morphology of neuronal cells. In short-term adhesion assays (1 hr), cerebellar and hippocampal neurons adhered to several domains, encompassing the fibronectin type III-like (FN III) repeats 1-2 and 6-8, as well as to the alternatively spliced FN III repeats and to tenascin itself. Although no short-term adhesion to the EGF repeats containing fragment could be detected under the conditions used, it was anti-adhesive for neuronal cell bodies and repellent for growth cone advance and neuritogenesis. FN III repeats 3-5 were repellent only for growth cones but not for neuronal cell bodies. Neurite outgrowth promoting activities at early stages and induction of a polarized neuronal morphology at later stages of differentiation were associated with the EGF repeats and the FN III repeats 6-8. These observations suggest differential effects of particular domains of the tenascin molecule on distinct cellular compartments, i.e., cell body, axon and dendrite, and existence of multiple neuronal receptors with distinct intracellular signaling features.

Tenascin demarcates the boundary between the myelinated and nonmyelinated part of retinal ganglion cell axons in the developing and adult mouse.

The molecular determinants controlling the topographically restricted distribution of neural cells in the mammalian CNS are largely unknown. In the mouse, myelin-forming oligodendrocytes are differentially distributed along retinal ganglion cell axons. These axons are myelin free intraretinally and in the most proximal (i.e., retinal) part of the optic nerve, but become myelinated in the distal (i.e., chiasmal) part of the optic nerve. Tenascin protein and mRNA are detectable in increased amounts at the retinal end of the developing optic nerve before the arrival of oligodendrocyte progenitor cells and are restricted to this region in the adult optic nerve. Tenascin is a nonadhesive substrate for oligodendrocytes and their progenitor cells in vitro when offered as a substrate in choice with polyornithine. These observations suggest that tenascin is critical for the establishment and maintenance of the restricted distribution of myelin-forming oligodendrocytes along retinal ganglion cell axons of the mouse.

Expression of the extracellular matrix glycoprotein tenascin in the somatosensory cortex of the mouse during postnatal development: an immunocytochemical and in situ hybridization analysis.

Layer 4 of the rodent somatosensory cortex contains the barrel field which is the cortical representation of the whisker pad located on the contralateral side of the face. Each barrel within the barrel field is related one to one to its corresponding whisker both anatomically and physiologically. The astrocyte-derived extracellular matrix glycoprotein tenascin has been shown by immunocytochemistry to delineate the boundaries between barrels during their formation until the end of the second postnatal week. The present study describes the anatomical localization of tenascin mRNA expressing cells in the somatosensory cortex of the mouse from birth to postnatal day 15. During this time, a general down-regulation of tenascin-specific message was observed as a function of the state of maturation, with layers 5 and 6 down-regulating the message earlier than layers 1 and 2/3. Tenascin (as detected by immunocytochemistry) also revealed this gradual down-regulation with maturation. Layer 4 of the somatosensory cortex was different in that, with the onset of formation of barrel field boundaries at postnatal day 3, tenascin protein and mRNA were down-regulated more in layer 4 than in the upper and the lower layers of the somatosensory cortex and, interestingly, not in layer 4 of adjacent cortical areas. At postnatal day 6 tenascin immunoreactivity was most clearly distinguished in the barrel field boundaries while tenascin-specific mRNA was no longer detectable in layer 4. Down-regulation of tenascin message was also seen at P6 at the level of the enlarged barrel corresponding to an early postnatal lesioned row of whiskers. At postnatal day 15, tenascin protein and mRNA were no longer detectable in the somatosensory cortex. Distribution of glial fibrillary acidic protein immunoreactivity did not reveal any preferential accumulation of GFAP-positive radial glial processes in barrel field hollows versus barrel field boundaries at any stage.

Tenascin mRNA isoforms in the developing mouse brain.

The extracellular matrix glycoprotein tenascin is expressed in the developing mouse cerebellum as a group of four protein species of different molecular weights. The difference is most likely due to alternative splicing which is known to occur in tenascin mRNA within the region of the fibronectin type III repeats. In order to systematically analyze tenascin mRNA isoforms that would account for this heterogeneity, tenascin splice variants were isolated from mouse brain by the polymerase chain reaction (PCR). In agreement with Northern blot analysis, amplification by PCR revealed a general decrease in tenascin mRNA expression during development from embryonic and early postnatal to adult stages. This decrease was more pronounced for isoforms of high molecular weight compared to those of low molecular weight. In accord with the observations at the protein level, four splice variants were found to be predominantly expressed, containing insertions of either six, five, or one fibronectin type III repeat, or comprising no insertion. In addition, a minor splice variant with an insertion of four fibronectin type III repeats was isolated. Three of the isolated mRNA splice variants have not yet been described for mouse tenascin. Among them, an isoform containing six alternatively spliced repeats was found to include a novel fibronectin type III repeat. The sequence of this repeat displays 96.7% similarity to a corresponding type III repeat in human tenascin, revealing a strict evolutionary conservation between tenascin molecules from different species in the region of alternative splicing. Southern blot analysis of the amplified mRNA isoforms showed that the novel mouse type III repeat is confined to splice variants with an insertion of six fibronectin type III repeats. Furthermore, in situ hybridization on sections from mouse embryos indicated that tenascin-specific mRNAs containing the novel type III repeat are predominantly expressed in the central nervous system.

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies.

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a 35S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascin-specific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.

Enhanced expression of the developmentally regulated extracellular matrix molecule tenascin following adult brain injury.

Tenascin is an extracellular matrix molecule synthesized and released by young astrocytes during embryonic and early postnatal development of the nervous system, and it is concentrated in boundaries around emerging functional neuronal units. In the adult nervous system, tenascin can be detected only in very low levels. Distinct spatial and temporal distributions of tenascin during developmental events suggest a role in the guidance and/or segregation of neurons and their processes within incipient functional patterns. We show here, using in situ hybridization and immunocytochemistry, that stab wounds of the adult mouse cerebellar and cerebral cortices result in an enhanced expression of tenascin in a discrete region around the lesion site that is associated with a subset of glial fibrillary acidic protein-positive astrocytes. Tenascin up-regulation in the lesioned adult brain may be directly involved in failed regeneration or indirectly involved through its interactions with other glycoconjugates that either inhibit or facilitate neurite growth.

Expression of tenascin in the developing and adult cerebellar cortex.

Since tenascin may influence neuronal cell development, we studied its expression pattern using immunocytochemistry, in situ hybridization, Northern blot analysis, and immunochemistry in the developing and adult mouse cerebellar cortex. Tenascin immunoreactivity was detectable in all layers of the developing cerebellar cortex. In the external granular layer, only the radially oriented processes of Golgi epithelial cells were immunoreactive, whereas the densely packed cell bodies were immunonegative. Tenascin was hardly detectable at contact sites between migrating granule cells and processes of Golgi epithelial cells. Axons of granule cells in the molecular layer were immunoreactive, whereas their cell bodies in the internal granular layer lacked detectable levels of tenascin. By in situ hybridization, only Golgi epithelial cells and astrocytes of the internal granular layer and prospective white matter, but not nerve cells, could be shown to synthesize detectable levels of tenascin mRNA in the developing mouse cerebellar cortex. Thus, tenascin in the cerebellar cortex seems to be a glia-derived molecule that becomes adsorbed to neuronal surfaces in a topographically restricted pattern in situ. Levels of tenascin protein and mRNA decreased significantly with increasing age. In the adult, tenascin immunoreactivity was weak and mainly restricted to the molecular layer and tenascin mRNA was confined to Golgi epithelial cells, indicative for a functional heterogeneity in differentiated cerebellar astrocytes. Quantitative immunoblot analysis revealed that the 225 and 240 kDa components of tenascin were developmentally downregulated at a faster rate than the 190 and 200 kDa components, corresponding to the faster downregulation of the 8 kilobase (kb) mRNA species compared to the 6 kb mRNA species as revealed by Northern blot analysis. These observations indicate a differentially regulated expression of the tenascin components. We hypothesize that glia-derived tenascin modifies the functional properties of nerve cell surfaces and that tenascin is involved in such different morphogenetic events as neurite growth and oligodendrocyte distribution.