Subcommissural organ/Reissner's fiber complex: characterization of SCO-spondin, a glycoprotein with potent activity on neurite outgrowth.

In the developing vertebrate nervous system, several proteins of the thrombospondin superfamily act on axonal pathfinding. By successive screening of a SCO-cDNA library, we have characterized a new member of this superfamily, which we call SCO-spondin. This extracellular matrix glycoprotein of 4,560 amino acids is expressed and secreted early in development by the subcommissural organ (SCO), an ependymal differentiation located in the roof of the Sylvian aqueduct. Furthermore, SCO-spondin makes part of Reissner's fiber (RF), a thread-like structure present in the central canal of the spinal cord. This novel protein shows a unique arrangement of several conserved domains, including 26 thrombospondin type 1 repeats (TSR), nine low-density lipoprotein receptor (LDLr) type A domains, two epidermal growth factor (EGF)-like domains, and N- and C-terminal von Willebrand factor (vWF) cysteine-rich domains, all of which are potent sites of protein-protein interaction. Regarding the huge number of TSR, the putative function of SCO-spondin on axonal guidance is discussed in comparison with other developmental molecules of the CNS exhibiting TSR. To correlate SCO-spondin molecular feature and function, we tested the effect of oligopeptides, whose sequences include highly conserved amino acids of the consensus domains on a neuroblastoma cell line B 104. One of these peptides (WSGWSSCSRSCG) markedly increased neurite outgrowth of B 104 cells and this effect was dose dependent. Thus, SCO-spondin is a favorable substrate for neurite outgrowth and may participate in the posterior commissure formation and spinal cord differentiation during ontogenesis of the central nervous system.

SCO-spondin and RF-GlyI: two designations for the same glycoprotein secreted by the subcommissural organ.

SCO-spondin and RF-GlyI are two designations for cDNAs strongly expressed in the bovine subcommissural organ (SCO), characterized, respectively, in 1996 and 1998 by two different research groups. Because both cDNAs were partial sequences and exhibited close similarities in their nucleotide and deduced amino acid sequences, it was thought that they might be part of the same encoding sequence. To find out, we performed 3'RACE using a SCO-spondin-specific upstream primer. From the RT-PCR product generated and by nested PCR techniques, we amplified both SCO-spondin and RF-GlyI specific products with the expected length. Also, probes generated from both PCR products hybridized to the same major 14 kb transcript in Northern blot analyses, clearly showing that SCO-spondin and RF-GlyI cDNAs do belong to the same encoding sequence. In addition, we amplified, cloned, and sequenced a PCR product of 3 kb spanning both the known SCO-spondin and RF-GlyI sequences. The deduced amino acid sequence contains nine thrombospondin type 1 repeats that alternate with sequences sharing similarities with the D-domain of von Willebrand factor. Taken together, these findings show that SCO-spondin and RF-GlyI are two designations of the same gene encoding proteins secreted by the bovine SCO and forming Reissner's fiber. In addition, compared to the sequence provided by Nualart et al. (1998), we extended the reading frame and identified new conserved domains in the 3' end of SCO-spondin. The putative function of SCO-spondin on axonal pathfinding is discussed regarding the presence of a great number of thrombospondin type 1 repeats.

Use of a heterologous monoclonal antibody for cloning and detection of glial fibrillary acidic protein in the bovine ventricular ependyma.

From protozoans to vertebrates, ciliated cells are characterized by well-developed cytoskeletal structures. An outstanding example is the epiplasm, a thick, submembranous skeleton that serves to anchor basal bodies and other cell surface-related organelles in ciliated protozoans. An epiplasm-like cytoskeleton has not yet been observed in metazoan ciliated cells. In a previous study, we reported on MAb E501, a monoclonal antibody raised against epiplasmin-C, the major membrane skeletal protein in the ciliate Tetrahymena pyriformis. It was shown that MAb E501 cross-reacts with glial fibrillary acidic protein (GFAP), the class III intermediate filament protein found in astrocytes and other related glial elements. Here we used a post-embedding immunogold-staining method to localize MAb E501 cross-reactive antigens in ciliated cells from the ventricular ependyma in bovine embryos. When ependymocytes were treated with MAb E501, the ciliated region of the cell cortex was devoid of significant labeling. Instead, a gold particle deposit was evident around the nucleus, with only conventional ependymocytes being immunostained. Similar results were obtained by utilizing a rabbit antiserum against GFAP, revealing glial filaments and indicating an astroglial lineage of conventional bovine ependymocytes. In contrast, secretory ependymocytes of the subcommissural organ (SCO) were not stained by either of the two antibodies. Using MAb E501 as a heterologous probe, we cloned bovine GFAP cDNA. In situ hybridization experiments failed to detect GFAP transcripts in SCO ependymocytes, confirming the abscence of immunoreactivity in these cells.

SCO-spondin is evolutionarily conserved in the central nervous system of the chordate phylum.

Bovine SCO-spondin was shown to be a brain-secreted glycoprotein specifically expressed in the subcommissural organ, an ependymal differentiation located in the roof of the Sylvian aqueduct. Also, SCO-spondin makes part of Reissner's fiber, a phylogenetically and ontogenetically conserved structure present in the central canal of the spinal cord of chordates. This secretion is a large multidomain protein probably involved in axonal growth and/or guidance. As Reissner's fiber is highly conserved in the chordate central nervous system, we sought genes orthologous to the bovine SCO-spondin gene by Southern blot analysis in several members of the chordate phylum: urochordates, cephalochordates, cyclostomes, and lower and higher vertebrates, including humans. In addition, conserved glycoproteins present in the subcommissural organ and Reissner's fiber were revealed by immunohistochemistry using antibodies raised against bovine Reissner's fiber. Variation in the sites of Reissner's fiber production according to chordate subphylum, presence of this structure in the spinal cord, and conservation of the SCO-spondin gene are discussed in the context of chordate central nervous system development. These results indicate that SCO-spondin is an ancient ependymal secretion, making part of Reissner's fiber, that may have had an important function during the evolution of the central nervous system in chordates, including that of the spinal cord.

Complex expression pattern of the SCO-spondin gene in the bovine subcommissural organ: toward an explanation for Reissner's fiber complexity?

Bovine SCO-spondin is a glycoprotein secreted by the subcommissural organ (SCO), an ependymal derivative located in the roof of the third ventricle. It shows homology with developmental molecules involved in directional axonal growth. Using SCO-spondin cDNAs as probes, we analysed the specific expression of the corresponding gene in the bovine SCO by Northern blot and in situ hybridization (ISH). A strong expression was detected in the secretory ependymal and hypendymal cells of the SCO and the main transcripts showed a large size 14 kb. A single copy gene was revealed by Southern blot analysis of bovine genomic DNA. The presence of additional transcripts suggested a transcriptional regulation of the SCO-spondin gene. A comparative analysis of the results obtained by molecular and immunological techniques (immunoblotting and immunopurification) pointed to the presence of several SCO-spondin related proteins in the SCO encoded by the same gene. The presence in the cerebral hemispheres (CH) of a 54-kDa glycoprotein with a common epitope is discussed as a putative cleaved SCO-spondin product carried by the cerebrospinal fluid, that may act on neuronal development.

SCO-spondin: a new member of the thrombospondin family secreted by the subcommissural organ is a candidate in the modulation of neuronal aggregation.

A number of cues are known to influence neuronal development including growth factors, cell-adhesion molecules, components of the extracellular matrix and guidance molecules. In this study, we present molecular and functional evidence that SCO-spondin, a novel relative of the thrombospondin family, could also be involved in neuronal development by modulating cell aggregative mechanisms. SCO-spondin corresponds to glycoproteins secreted by the subcommissural organ (SCO), an ependymal differentiation of the vertebrate brain located at the entrance to the Sylvian aqueduct. A cDNA clone of 2.6 kb, isolated from a bovine SCO cDNA library, was shown to be specifically and highly expressed in the bovine SCO by in situ hybridization and was subsequently sequenced. Analysis of the deduced amino acid sequence reveals the presence of four conserved domains known as thrombospondin (TSP) type I repeats. To account for the homology with thrombospondins and F-spondin, this secreted glycoprotein was called SCO-spondin. Two potent binding sites to glycosaminoglycan (BBXB) and to cytokine (TXWSXWS) are also found in the TSP type I repeats. The deduced amino acid sequence exhibits three other conserved domains called low density lipoprotein (LDL) receptor type A repeats. The possibility of SCO-spondin involvement in neuronal development as a component of the extracellular matrix is discussed regarding these molecular features. The idea of a modulation of cell-cell and/or cell-matrix interaction is further supported by the anti-aggregative effect observed on cultured neuronal cells of material solubilized from Reissner's fiber. That Reissner's fiber, the condensed secretory product of the SCO present along the whole spinal cord can be a potent morphogenetical structure is an important concept for the analysis of the molecular mechanisms leading to spinal cord differentiation.

Specific transcripts analysed by in situ hybridization in the subcommissural organ of bovine embryos.

The subcommissural organ (SCO) secretes specific glycoproteins into the cerebrospinal fluid that aggregate to constitute Reissner's fiber (RF), a thread-like structure running along the central canal of the spinal cord. For further identification of the gene(s) encoding these secretions, we have prepared a cDNA library in the vector IGT11 from bovine embryonic SCO. The screening of this library was performed using a polyclonal antibody raised against bovine RF. Three positive clones were isolated and purified and one of these lambda RF101 comprising an insert of #400 nucleotides was undercloned into pBluescript plasmid and mapped. After labeling with 35S (ATP) this cDNA fragment served as a probe to analyse the presence of specific transcripts in the subcommissural organ of the embryonic bovine by in situ hybridization. A labeling signal was observed in the embryonic SCO both in the secretory ependymal and hypendymal cells. This labeling is specific since the ependymal layer bordering the ventricular cavity as well as the surrounding nervous tissue remained negative. Thus, the embryonic SCO contains specific transcripts that are colocalized with the specific glycoproteins as shown after the use of a specific monoclonal antibody C1B8A8. In addition, the pattern of labeling with the specific SCO cDNA is different from those of beta actin cDNA and tear lipocalin cDNA, which, respectively, served as positive and negative controls. In a subsequent set of experiments the expression pattern was compared in embryos at two different stages of development (4-month-old and 8-month-old embryos).(ABSTRACT TRUNCATED AT 250 WORDS)

The bovine subcommissural organ: cytochemical and immunochemical characterization of the secretory process.

Specific glycoproteins of the bovine subcommissural organ (SCO) were studied by means of various techniques: light and electron microscopy, immunoaffinity chromatography, electrophoresis and Western blotting. Use of lectins (Con A, WGA, PHA-E and -L, LCA) allowed to specify the synthesis and release of complex-type glycoproteins that bear high-mannose-carbohydrate chains in their precursor forms and probably triantennary carbohydrate chains in their mature forms. Antibodies raised against SCO extracts were characterized by means of various tests and used to purify specific compounds. Immunopurified fractions using A99 polyclonal antibody contained numerous polypeptides reactive with Con A, their apparent molecular weight (MW) ranging from 240 to 50 kDa. Only two glycopeptides were strongly labeled with WGA (98 and 52/54 kDa MW). Immunopurified fractions using C1B8A8 monoclonal antibody, specific of the complex-type glycoproteins at different steps of glycosylation, showed three specific Con A-reactive polypeptides at 88, 54 and 34 kDa MW. Only the 34 kDa glycopeptide was strongly labeled with WGA. The latter could correspond to the monomeric form of the secreted compound. Electrophoretical analyses of Reissner's fiber material allowed the detection of a WGA-positive smear in the upper part of the blots, suggesting that the complex-type glycoproteins, when released into the CSF, constitute a stable polymer.

Monoclonal antibodies as probes for the analysis of the secretory ependymal differentiation in the subcommissural organ of the chick embryo.

Monoclonal antibodies directed against components of the subcommissural organ (SCO) of the chick embryo were produced by immunizing mice with SCO homogenate. In three series of production, 788 hybridomas were screened by immunofluorescence microscopy. Four hybridoma cell lines producing antibodies that specifically recognize both SCO cells and Reissner's fiber (RF) were selected and cloned. Using these immunological probes, the ontogenetic development of the SCO and RF was investigated in the chick embryo. Immunoreactive material could be detected in the SCO anlage from stage 17 on and RF was first observed in the central canal of the thoracal part of the spinal cord in 10-day-old embryos. Monoclonal antibodies can be useful as markers for analyzing molecular mechanisms involved in the specific function of these ependymal cells.

A radioautographic study of [3H]-A 23187 (calcimycin) in components of the brain. Distribution in different cell types of the diencephalo-mesencephalic roof.

The distribution of the ionophore [3H]-A 23187 was examined by means of light and electron microscopy in elements of the central nervous system located in the diencephalo-mesencephalic roof. A 23187 is not evenly distributed in the components studied (ependyma, secretory ependyma of the subcommissural organ and neurons of the mesencephalon). At the cellular level, A 23187 appears preferentially associated with the cytoplasmic membrane as well as with the internal membranous system.

The complex-type glycoprotein secreted by the bovine subcommissural organ: an immunological study using C1B8A8 monoclonal antibody.

The secretory pathway of the complex-type glycoprotein specific to the subcommissural organ (SCO) was examined using the monoclonal antibody (Mab) C1B8A8. Immunoreactive material was revealed in various compartments of the secretory ependymocyte, i.e., the endoplasmic reticulum, the Golgi area and the secretory vacuoles. In addition, immunoreactive material was also observed in the ventricular cavity. Evidence of a release both at the apical lining and at the basal process of the SCO ependymocytes suggests that the same protein could be secreted into the cerebrospinal fluid and the perivascular spaces. After immunoaffinity chromatography of soluble extracts of the SCO on Mab C1B8A8 immunoadsorbent columns, three glycopeptides were identified on Western blots; they were concanavalin A (Con A)-positive (88, 54 and 34 kDa) and wheat-germ agglutinin (WGA)-positive (54 and 34 kDa). The Con A-positive glycopeptide (88 kDa) is probably related to the high-mannose-type glycoprotein, the precursor form of the secreted compound, whereas the 54 kDa-glycopeptide that is both Con A- and WGA-positive could represent an intermediate form. The 34 kDa-glycopeptide that is strongly WGA-positive could be related to the monomeric form of the secreted compound. These three glycopeptides were not revealed in eluted fractions of soluble extracts of the ependyma that served as control.

A comparative immunocytochemical and immunochemical analysis of glycoproteins synthesized in the bovine subcommissural organ.

To extend our previous immunochemical investigations in the chick embryo (Karoumi et al., 1990 b), we raised antibodies in the rabbit against crude extracts of the subcommissural organ (SCO) of the bovine. The antiserum labeled A99 was absorbed by crude brain extracts and its specificity was tested by different techniques. Comparison of crude SCO and cerebral hemispheres supernatants after immunoblotting allow to identify specific 98, 60, 52, 42, 38, and 32 kDa polypeptides in the SCO profile. Immunoaffinity chromatography on A99 immunoadsorbent of crude SCO, cerebral hemispheres (CH) and classical ependyma (CE) supernatants was followed by electrophoretical analysis and electrotransfer. Concanavalin A (Con A) and wheat germ agglutinin (WGA) labeling procedures demonstrated the presence of numerous glycopeptides specific of crude SCO supernatants and having an apparent molecular weight ranging from 240 to 50 kDa. In the CH-eluted fraction, 50 and 52 kDa glycopeptides were revealed by ConA and WGA, whereas in the CE-immunopurified fraction no band was visualized. The similarity of the chick embryo and bovine electrophoretic pattern corresponding to the SCO eluted fractions speaks in favour of a high degree of conservation of the SCO secretory material and an evolutionary stability of the antigens recognized by A99IgG.

Ontogenesis of the secretory epithelium of the bovine subcommissural organ. A histofluorescence study using lectins and monoclonal antibodies.

A spatio-temporal analysis of the differentiation of a group of specialized (secretory) ependymal cells in the subcommissural organ (SCO) of the brain was undertaken in the bovine using a monoclonal antibody (C1B8A8) which is specific of the secretory process in this organ. In addition, lectins (concanavalin agglutinin (Con A), Lens culinaris agglutinin (LCA), wheat germ agglutinin (WGA), and Phaseolus vulgaris agglutinin (PHA] were used to analyse the maturation of the carbohydrate moieties of the secretory product (subcommissuralin). Monoclonal antibody NC-1 specific to a complex carbohydrate epitope including a terminal 3-sulfoglucuronyl residue similar to HNK-1 was also tested to compare the reactivity of the SCO with that of other brain structures. These cells express a specific antigen related to the known secretory activity of the SCO during early embryogenesis (2 months). This antigen is recognized by C1B8A8 antibody and by Con A suggesting that high mannose-type glycoproteins are synthesized at this stage. Later on (approximately 3.5 months), appearance of C1B8A8, WGA, LCA, L- and E-PHA-positive material in the apical lining of the ependymal cells, close to the ventricular cavity, suggests that maturation of the complex-type glycoproteins (Asn-linked) occurs at this stage. Presence of secretory material in the CSF and Reissner's fibre could be detected using the same probes at a stage of 4 months. As early as 2 months NC-1-positive material was detected in the ependyma of the mesencephalic roof, while no reaction occurred in the SCO epithelium. This suggests that the carbohydrate moieties of subcommissuralin is different from that of ependymins beta and gamma. Using specific monoclonal antibodies, molecular characterization of subcommissuralin and experimental analyses on its accurate role in brain development will further our tentative comparison with ependymins. The secretory ependymal cells in the SCO express a particular phenotype and could represent an increasing model to study cell differentiation in the brain.

Glycoprotein synthesis in the subcommissural organ of the chick embryo. II. An immunochemical study.

In the chick embryo, A74 immunoaffinity chromatography allowed to purify specific glycoproteins relevant to the SCO ventricular secretory process. The eluted fractions of the subcommissural organ (SCO), the cerebral hemispheres (CH) and the medulla oblongata (MO) were compared using the Concanavalin A (Con A) and wheat germ agglutinin (WGA) staining procedures after western-blotting. Analysis of the optical density of the reactive bands allowed to estimate the relative concentration of the various glycopeptides in the eluted fractions. In the SCO-eluted fractions at least ten Con A-positive glycopeptides were identified, their apparent molecular weight ranging from 240 to 42 kD. Only three of these appeared to be WGA-positive (98, 88, and 52 kD). In the CH-eluted fractions only a 52 kD Con A- and WGA-positive glycopeptide was revealed, while in the MO-eluted fractions a 32 kD glycopeptide was also Con A- and WGA-positive. These results are discussed in regard to the known biosynthesis pathway of complex type glycoproteins.

Glycoprotein synthesis in the subcommissural organ of the chick embryo. I. An ontogenetical study using specific antibodies.

Antibodies were raised in rabbit against crude subcommissural organ (SCO) extract of 19 day old chick embryos. After absorption with crude brain extract, the IgG fraction was purified by ion exchange chromatography. The specificity of the antibodies was controlled by immunostaining and by a competition test between lectins (Concanavalin A-Con A- and wheat germ agglutinin-WGA-) and antibodies (A74 IgG). Using A74 IgG, some ependymal cells containing immunoreactive material (IRM) could be detected in the SCO anlage at 4 days of incubation. During the following stages (5 to 12 days), the immunostaining extended caudal-ward in the SCO epithelium according to a rostro-caudal gradient of differentiation. The appearance of IRM in the secretory ependymal cells of the SCO parallel that of Concanavalin A-positive glycoproteins (Bruel et al., 1987). Secretion of IRM into the ventricular cavity, contributing to the formation of Reissner's fiber (RF) occurred during the 7th day of incubation. The formation of RF was examined at different levels of the spinal cord using A74 IgG, WGA and aldehyde fuchsin (AF) staining. The appearance of SCO specific glycoproteins was observed at 11 days in the central canal but the presence of a non-immunoreactive material at 10 days suggests that the formation of RF probably happens inside a guidance material.

Complex-type glycoproteins synthesized in the subcommissural organ of mammals. Light- and electron-microscopic investigations by use of lectins.

The secretory activity in the subcommissural organ (SCO) of the sheep and cow was examined by means of lectin histochemistry and cytochemistry. Among the various lectins tested. Concanavalin A (Con A) revealed glycoproteins rich in mannosyl residues in the rough endoplasmic reticulum of ependymal and hypendymal cells. One of these Con A-positive glycoproteins may represent the precursor of the specific secretory component elaborated in the SCO, giving rise to Reissner's fiber. Lens culinaris agglutinin (LCA) and Phaseolus vulgaris hemagglutinins (E-PHA and L-PHA), known to bind to oligosaccharides, as well as wheat-germ agglutinin (WGA) revealing neuraminic acid, labeled secretory granules located in the apical part of ependymal and hypendymal cells of ruminants, and also Reissner's fiber. Electron-microscopic visualization of WGA-positive material in the Golgi complex shows that complex-type glycoproteins are synthesized in the subcommissural organ of mammals. The electron-dense material is mainly secreted into the ventricular cavity and gives rise to Reissner's fiber. On the basis of lectin affinity for oligosaccharides, a structure of the complex-type oligosaccharide is proposed.

Monoclonal antibody C1B8A8 recognizes a ventricular secretory product elaborated in the bovine subcommissural organ.

To obtain specific immunological probes for investigation of the cellular and molecular aspects of the subcommissural organ (SCO), we produced monoclonal antibodies directed against extracts from the bovine SCO. An hybridoma cell line (C1A8B8) was isolated by screening the culture media by means of the immunofluorescence method. This clone produces an IgG1 that recognizes the ventricular secretory material of the SCO including Reissner's fiber. A competition test using C1B8A8 immunoglobulin and lectins (concanavalin A and wheat-germ agglutinin) was applied to demonstrate that both the immature and mature forms of the glycoprotein were recognized. This antibody will offer a good tool for immunocytochemical localization and immunoaffinity purification of the antigen and for isolation of cDNA clones encoding it.

Ontogenetical study of the chick embryo subcommissural organ by lectin-histofluorescence and electronmicroscopy.

The secretory activity of the subcommissural organ (SCO) was studied during embryogenesis of the chick (Gallus gallus) using two lectins labelled with fluorescein isothiocyanate, concanavalin A (Con A) and wheat-germ agglutinin (WGA). While WGA labels the apical or ventricular border of the organ, Con A labels both, the apical and vascular poles of the cells. Glycoproteinaceous secretory products, visualized by Con A appear early, at 5 days, in the ependymal epithelium and expand progressively in a rostrocaudal direction. A correlation is established between histofluorescence and the ultrastructural aspects of the ependymocytes. This throws light on the role of the endoplasmic reticulum in the synthesis, storage and transport of the material produced by the SCO, and points to the existence of two poles of exchange between the secretory cells and the extracellular medium, i.e., the ventricular and the vascular one. WGA reactivity at the apical border is linked up with the formation of Reissner's fibre and shows that the secretory product of the SCO cells undergoes at least partly modifications during its intracytoplasmic transport preceding apical discharge.