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.

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.

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.

Appearance and distribution "in situ" of nicotinic acetylcholine receptors in cervical myotomes of young chick embryos. Radioautographic studies by light and electron microscopy.

Localization of the acetylcholine (nicotinic) receptor sites was investigated in the developing cervical myotomes of the early chick embryo by radioautography at the light and electron microscope level, using 125I-alpha-bungarotoxin. The presence of cholinergic receptor sites was detected in situ as early as 60 hours of incubation (stage 17); their relative density increased in the myotome during the differentiation of the somite. Specific labeling of these receptor sites was detected in the myotomal tissue but not in the notochord, spinal cord or periaxial mesenchyme. The distribution of the receptor sites was uniform in the myotome at 3 days in ovo. An anterior-posterior asymmetry of the density appeared at 4 days in ovo and developed up to the 6th day. The highest density of these toxin-binding receptor sites was observed near the spinal motor nerve bundle as revealed by silver staining. These observations, made in situ, are discussed with respect to the possible neurotrophic or physical effects of the early motor innervation.

[Neuroactive compounds and vertebral teratogenesis in the bird embryo (author's transl)]. / Neuroactifs et tératogenèse vertébrale chez l'embryon d'oiseau.

Anticholinesterasic and depolarizing (analogues of acetylcholine) agents administered to quail embryos after 3 days of incubation give rise to vertebral fusions in addition to neck deformities. Antagonists of acetylcholine (gallamine and hexamethonium) produce only vertebral joint fusions. The incidence of all these compounds in the vertebral defects by the way of there neuroactive properties is proposed.

[Anticholinesterase agents and axial teratogenesis in quail embryos]. / Agents anticholinestérasiques et tératogénèse axiale chez l'embryon de Caille.

The results of an investigation made with organo-phosphorous compounds (OP), carbamates (C), and a compound including oxamide groups (GO), indicate that spine anomalies in Japanese quail embryos are linked with cholinesterase inhibition. The blocking effect of these various compounds on cholinesterases has been examined both in vitro and in vivo.In vitro, the embryonic cholinesterases are particularly sensitive to carbamates, whereas high concentrations of organo-phosphorous compounds-and especially those which need to be activated at the level of metabolism-block only part of the relevant activities of the enzyme.In vivo, among the products examined, only malathion (OP) and ambenonium (GO) were found to be inactive (or having little effect) so far as cholinesterases are concerned. Although they appeared rather strongly embryotoxic, these two poisons did not prove teratogenic. A strong inhibition of cholinesterase activities was noticed in all cases, when applying the other compounds at teratogenic doses.The close relationship between the inhibition of cholinesterases and the appearance of axial abnormalities is confirmed by using increasing doses of parathion (OP) and neostigmine (C).Beak and leg malformations were brought about only by dicrotophos and eserine, and did not prove to be linked with cholinesterase inhibition, since they were not observed with compounds having a high activity towards these enzymes, such as parathion (OP), neostigmine (C) and demecarium (C).

Neuromuscular blocking agents and axial teratogenesis in the avian embryo. Can axial morphogenetic disorders by explained by pharmacological action upon muscle tissue?

A comparative qualitative and quantitative study of the embryotoxic and teratogenic activities of various chemical agents known to have neuromuscular blocking properties was done on the quail embryo. Intrinsic embryotoxic activity and teratogenicity were evaluated for each agent, and the qualitative nature of the teratogenic effects was correlated with the pharmacologic properties of the different effectors. Gross malformations (contorted posture) were found only with agents which directly (cholinergic agonists) or indirectly (cholinesterase inhibitors) favor muscle membrane depolarization, suggesting that such malformations may be due to muscle contraction following depolarization. All of the agents studied produced variable degrees of vertebral fusion, apparently due to paralysis resulting either from sustained depolarization (cholinesterase inhibitors and cholinergic agonists) or directly from cholinergic blockade (cholinergic antagonists and neurotoxins).

[Accumulation of exogenous monoamines in the notochord of lampreys: fluorescence microscopy and radiography study]. / Accumulation de monoamines exogènes dans la corde de la lamproie de Planer: étude en microscopie de fluorescence et en radioautographie.

Storage of biogenic amines was studied in the notochord of the lamprey (Lampetra planeri) after injection of cold or tritiated amines or their precursors. After injection of dopamine, noradrenaline or 5-hydroxytryptophane, an intense formaldehyde-induced fluorescence was observed in the cytosol of chordal cells and in the perichordal sheat. A similar location occurred after injection of the same tritiated products. No qualitative or significant quantitative differences could be distinguished between the larval and the metamorphosing forms. RO4-4602, a decarboxylase inhibitor, and Nialamide, a monoamine-oxydase inhibitor, did not modify significantly the incorporation of 3H-5-hydroxytryptophan. Using histochemical technique a monoamine oxydase activity could be detected in the nervous system and in the muscle but was not shown in the notochord.

[Pralidoxime prevents certain teratogenic effects induced by bidrin in quail embryos]. / Prévention par la pralidoxime de certains effets tératogènes induits par le bidrin chez l'embryon de caille

Bidrin treatment of quail embryos results in axial anomalies as well as malformations of the beak and the limbs. Whereas the administration of pralidoxime to teratogen-treated embryos prevents the appearance of the axial anomalies, the morphogenesis of the beak and limbs remains profoundly altered.

[Parathion axial teratogenesis after giving several compounds with known anti-teratogenic or antitoxic properties in vertebrate adults or embryos after exposure to organic phosphates. Study in the quail embryo (Coturnix coturnix japonica)]. / Tératogenèse axiale du parathion après action de divers composés connus chez l'adulte ou l'embryon de vertébrés pour posséder un pouvoir antitoxique ou antitératogène aprés des expositions aux phosphates organiques. Etude chez l'embryon de caille (Coturnix coturnix japonica)

Axial abnormalities, highly specific of parathion, are prevented by pyridine aldoximes P2S, 2-PAM and TMB4. Atropine, well known as an antimuscarinic agent is ineffective and so it is for tryptophan and DMSO, compounds having protective effect on some morphogenetic disturbances induced by organic phosphates. These results point out the specificity of pyridine aldoximes as alleviating agents on the characteristic axial deformities induced by parathion.

[Prevention of abnormalities induced by 2 organophosphate insecticides (parathion and bidrin) in quail embryos]. / Prévention des anomalies induites par deux insecticides organophosphorés (parathion et bidrin) chez l'embryon de caille

When Quail embryos, injected with bidrin at the unincubated stage, are treated with nicotinamide, beak and leg abnormalities are prevented, but niacin has no beneficial effect on the axial deformities caused by both parathion and bidrin. In contrast, vertebral defects are greatly reduced by giving pralidoxim, an antidote known and used in organophosphorus intoxications. But this compound has no effect on beak and leg damage caused by bidrin. Two other cholinesterase reactivators, diacetylmonoxime and monoisonitrosoacetone have any effect on organophosphorus abnormalities and have no antiteratogenic action either on the beak and legs or on the vertebral column. From these observations the multiple causes of teratogenic effects induced by organophosphorus compounds are evident. Both aspects of teratogenesis, one related to the nicotinamide level, and the other related to the physiology of the cholinergic system are discussed.

[Ultrastructural studies of the evolution of the notochord and dorsal embryonic constituents derived from the mesoderm in the chicken and Japanese quail during teratogenic exposures to parathion]. / Etudes ultrastructurales de l'évolution de la notocorde et des constituants embryonnaires dorsaux dérivés du mésoderme chez le poulet et la caille japonaise au cours des expositions tératogènes au parathion

After experimental expositions to parathion--an organophosphorous insecticide which, in birds, gives rise to elective axial abnormalities--the evolution of the notochord and the different dorsal embryonic tissues issued from the mesoderm was analysed with the chicken and the japanese quail embryos. The abnormalities being settled, the notochord, which has normally developed, exhibited no ultrastructural change. Sclerotomic cells had normally migrated and, at the time when the provertebrae were formed, it was not possible to detect any histological and cytological alterations at their level. At later stages (8-9 days of incubation), the cartilaginous cells of the vertebral body did not show particular sign of an organophosphorous cytotoxicity but their secretory function was weakened, fact that may explain growth retardation. On these observations, it is evident that the tardive cartilaginous injuries cannot determine the axial abnormalities occurring earlier. The main results of this study deal with the cytological alterations of the myotomic elements which appeared to be the earliest and the more important ones. At the beginning (5th day), the myopathic changes were relatively located but they extended to the all cervical area. At 8-9 days, the cervical muscular cells are especially characterised by a lack of myofibrills. In showing only muscular lesions at the first step of teratogenesis, the present investigation set aside a chordal or sclerotomic cells intervention in the teratism induced by parathion. On the other hand, fromthe involvement of the contractile tissue in the formation and in the maintenance of the articulations, it appears that the muscular lesions can offer an embryologic interpretation to the parathion axial abnormalities. The possible effect of parathion in the myopathic evolution by the way of its anticholinesterasic properties is proposed.

[Plurality in the determinism of organophosphorus teratogenic effects (author's transl)]. / Pluralité dans le déterminisme des effets tératogènes des composés organophosphorés

In Quail embryos, nicotinamide prevents beak and legs abnormalities produced by bidrin but remains inefficient against vertebral defects induced by bidrin and parathion. In contrast, the vertebral deficiencies are greatly alleviated or abolished by pralidoxim, an antidote known and used in organophosphorus intoxications. From these observations, a plurality in the determinism of teratogenic effects induced by organophosphorus compounds is evident.

[Cholinesterase activity and expression of axial teratogenesis in quail embryo exposed to organophosphates]. / Activités cholinestérasiques et expression de la tératogenèse axiale chez l'embryon de Caille exposé aux organophosphorés.

The expression of the axial terotogenesis and the cholinesterasic activities have been studied simultaneously in the Quail embryo. High concentration of malathion (1 000 microgram) slightly reduces the cholinesterasic activities and does not produce morphogenetic disturbance. Bidrin (250 microgram) and parathion (100 microgram), strongly inhibit the cholinesterasic activities producing drastic axial deformities. The use of graduated concentrations of parathion shows that these axial abnormalities take place with a high level of cholinesterase inhibition. With organophosphorus, there is a good correlation between the axial teratogenesis and the cholinesterase inactivation.

Lectin binding sites on the outer segment membranes of photoreceptor cells in the pineal organ of Lampetra planeri (Cyclostomata).

The glycoproteins of the outer segment membrane of photoreceptor cells in the pineal organ of Lampetra planeri were studied using the affined properties of lectins to sugar moieties. Among five different lectins, labelled with fluorescein-isothiocyanate, Concanavalin A (Con A) and wheat germ agglutinin (WGA) induced an intense fluorescence on the large outer segments protruding into the lumen of the pineal organ. The complementary sugars, alpha-D-mannopyranosyl (for Con A) and N-acetyl-D-glucosamine (for WGA), inhibit the binding of both lectins. By means of electron microscopy, after incubation in Con A and labelling with peroxidase, and intense enzymatic activity was revealed in the diskal spaces by the technique of Graham and Karnovsky (1966). These results suggest the presence of glycoproteins rich in mannose and N-acetyl-D-glucosamine thus implying the presence of a photopigment of similar composition and structure to that of rhodopsin or rhodopsin itself.

Analysis of the secretions of the subcommissural organs of several vertebrate species by use of fluorescent lectins.

The glycoprotein secretions of the subcommissural organ were analyzed with the use of nine fluorescent lectins, specific to different sugar moieties. After exposure to Concanavalin A a bright fluorescence was observed in the ependymal cells of the subcommissural organs of all vertebrates studied (Lampetra planeri, Ameiurus nebulosus, Bufo bufo, Lacerta vivipara, Gallus gallus, Rattus norvegicus, Ovis aries). The fluorescence is abolished by the competitive sugar, alpha-D-mannopyranosyl. The intensity of the lectin fluorescence decreases from the phylogenetically lower to the higher forms, paralleled by a change in polarity of the secretion from a vascular (lower vertebrates) to a ventricular (higher vertebrates) direction. The strong affinity for Concanavalin A suggests the presence of a glycoprotein rich in mannosyl residues in the ependymal cells and a similarity of composition of this glycoprotein among the vertebrates. Lens culinaris agglutinin and wheat germ agglutinin revealed fluorescent "rosettes" in the hypendymal cells of the sheep. Binding of both these lectins suggests the presence of a glycoprotein rich in N-acetyl-D-glucosamine. In the underlying ventricular cavity, no fluorescence could be observed, suggesting that the Reissner's fiber does not possess the same carbohydrate constitution as the ependymal secretion of the subcommissural organ.

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.

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.