Identification of a high molecular weight polypeptide in the subcommissural organ of the chick embryo (original) (raw)
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Cell and Tissue Research, 2006
The subcommissural organ (SCO) is an ependymal differentiation located in the dorsal midline of the caudal diencephalon under the posterior commissure. SCO cells synthesize and release glycoproteins into the cerebrospinal fluid (CSF) forming a threadlike structure known as Reissner’s fiber (RF), which runs caudally along the ventricular cavities and the central canal of the spinal cord. Numerous monoclonal antibodies have been raised against bovine RF and the secretory material of the SCO. For this study, we selected the 4F7 monoclonal antibody based on its cross-reactivity with chick embryo SCO glycoproteins in vivo. E4 chick embryos were injected with 4F7 hybridoma cells or with the purified monoclonal antibody into the ventricular cavity of the optic tectum. The hybridoma cells survived, synthesized and released antibody into the CSF for at least 13 days after the injection. E5 embryos injected with 4F7 antibody displayed precipitates in the CSF comprising both the monoclonal antibody and anti-RF-positive material. Such aggregates were never observed in control embryos injected with other monoclonal antibodies used as controls. Western blot analysis of CSF from E4-E6 embryos revealed several immunoreactive bands to anti-RF (AFRU) antibody. We also found AFRU-positive material bound to the apical surface of the choroid plexus primordia in E5 embryos. These and other ultrastructural evidence suggest the existence of soluble SCO-related molecules in the CSF of early chick embryos.
Cerebrospinal Fluid Research, 2008
Background The subcommissural organ (SCO) is a highly conserved brain gland present throughout the vertebrate phylum; it secretes glycoproteins into the cerebrospinal fluid (CSF), where they aggregate to form Reissner's fiber (RF). SCO-spondin is the major constituent protein of RF. Evidence exists that the SCO also secretes proteins that remain soluble in the CSF. The aims of the present investigation were: (i) to identify and partially characterize the SCO-secretory compounds present in the SCO gland itself and in the RF of the Sprague-Dawley rat and non-hydrocephalic hyh mouse, and in the CSF of rat; (ii) to make a comparative analysis of the proteins present in these three compartments; (iii) to identify the proteins secreted by the SCO into the CSF at different developmental periods. Methods The proteins of the SCO secreted into the CSF were studied (i) by injecting specific antibodies into ventricular CSF in vivo; (ii) by immunoblots of SCO, RF and CSF samples, using specific antibodies against the SCO secretory proteins (AFRU and anti-P15). In addition, the glycosylated nature of SCO-compounds was analysed by concanavalin A and wheat germ agglutinin binding. To analyse RF-glycoproteins, RF was extracted from the central canal of juvenile rats and mice; to investigate the CSF-soluble proteins secreted by the SCO, CSF samples were collected from the cisterna magna of rats at different stages of development (from E18 to PN30). Results Five glycoproteins were identified in the rat SCO with apparent molecular weights of 630, 450, 390, 320 and 200 kDa. With the exception of the 200-kDa compound, all other compounds present in the rat SCO were also present in the mouse SCO. The 630 and 390 kDa compounds of the rat SCO have affinity for concanavalin A but not for wheat germ agglutinin, suggesting that they correspond to precursor forms. Four of the AFRU-immunoreactive compounds present in the SCO (630, 450, 390, 320 kDa) were absent from the RF and CSF. These may be precursor and/or partially processed forms. Two other compounds (200, 63 kDa) were present in SCO, RF and CSF and may be processed forms. The presence of these proteins in both, RF and CSF suggests a steady-state RF/CSF equilibrium for these compounds. Eight AFRU-immunoreactive bands were consistently found in CSF samples from rats at E18, E20 and PN1. Only four of these compounds were detected in the cisternal CSF of PN30 rats. The 200 kDa compound appears to be a key compound in rats since it was consistently found in all samples of SCO, RF and embryonic and juvenile CSF. Conclusion It is concluded that (i) during the late embryonic life, the rat SCO secretes compounds that remain soluble in the CSF and reach the subarachnoid space; (ii) during postnatal life, there is a reduction in the number and concentration of CSF-soluble proteins secreted by the SCO. The molecular structure and functional significance of these proteins remain to be elucidated. The possibility they are involved in brain development has been discussed.
1995
Ten monoclonal antibodies (Mabs) against glycoproteins of the bovine Reissner's fiber (RF) have been used in a structural and ultrastructural immunocytochemical investigation of the bovine subcommissural organ (SCO) and RE The SCO of other vertebrate species has also been studied. For comparison, polyclonal antibodies against bovine RF (AFRU) were used. The SCO and RF of ox, pig and dogfish and the SCO of dog, rabbit, rat and frog were submitted to light-microscopic immunocytochemistry using AFRU and Mabs. Postembedding ultrastructural immunocytochemistry was applied to sections of bovine SCO using AFRU and Mabs. Bovine SCO consists of ependymal and hypendymal cell layers, the latter being arranged as cell strands across the posterior commissure, or as hypendymal rosette-like structures. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with AFRU. Six Mabs showed the same staining pattern as AFRU, one Mab stained RF strongly and SCO weakly, two Mabs stained RF but not SCO, and, finally, one Mab (3B 1) exclusively stained the apices of the ependymal and hypendymal cells. All Mabs recognized the SCO and RF of the pig. Two Mabs bound to the SCO of the dog. One Mab stained the SCO of the rabbit and another the SCO of the rat. The SCO of frog and dogfish were totally negative. Bovine SCO stained with AFRU, showed label in the rough endoplasmic reticulum (RER) and the secretory granules (SG) of the ependymal and hypendymal cells. The former, in the form of parallel cisternae, reticulum or concentric rings, was seen throughout all cytoplasmic regions. SG were abundant in the apical pole of the ependymal and hypendymal cells. Only one Mab showed a staining pattern similar to AFRU. Five Mabs J.
Cell and Tissue Research, 1998
The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner’s fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.
Human subcommissural organ, with particular emphasis on its secretory activity during the fetal life
Microscopy Research and Technique, 2001
The subcommissural organ (SCO) is a conserved brain gland present throughout the vertebrate phylum. During ontogeny, it is the first secretory structure of the brain to differentiate. In the human, the SCO can be morphologically distinguished in 7-to 8-week-old embryos. The SCO of 3-to 5-month-old fetuses is an active, secretory structure of the brain. However, already in 9-month-old fetuses, the regressive development of the SCO-parenchyma is evident. In 1-year-old infants, the height of the secretory ependymal cells is distinctly reduced and they are grouped in the form of islets that alternate with cuboid non-secretory ependyma. The regression of the SCO continues during childhood, so that at the ninth year of life the specific secretory parenchyma is confined to a few islets of secretory ependymal cells. The human fetal SCO shares the distinct ultrastructural features characterizing the SCO of all other species, namely, a well-developed rough endoplasmic reticulum, with many of its cisternae being dilated and filled with a filamentous material, several Golgi complexes, and secretory granules of variable size, shape, and electron density. The human fetal SCO does not immunoreact with any of the numerous polyclonal and monoclonal antibodies raised against RF-glycoproteins of animal origin. This and the absence of RF in the human led to the conclusion that the human SCO does not secrete RF-glycoproteins. Taking into account the ultrastructural, lectin-histochemical, and immunocytochemical findings, it can be concluded that the human SCO, and most likely the SCO of the anthropoid apes, secrete glycoprotein(s) with a protein backbone of unknown nature, and with a carbohydrate chain similar or identical to that of RF-glycoproteins secreted by the SCO of all other species. These, as yet unidentified, glycoprotein(s) do not aggregate but become soluble in the CSF. Evidence is presented that these CSF-soluble proteins secreted by the human SCO correspond to (1) a 45-kDa compound similar or identical to transthyretin and, (2) a protein of about 500 kDa.
Acta Histochemica, 2001
The glial subcommissural organ (SCO) is a conserved structure of the vertebrate brain that secretes a glycoprotein-rich product into both the extracellular matrix and the cerebrospinal fluid of the third ventricle that forms Reissner's fibre (RF). In order to identify specific secretory proteins of the subcommissural organ, a panel of antigen-and epitope-specific monoclonal antibodies was raised against bovine RF to study the distribution of epitopes in Western blots of bovine RF. Six groups of epitopes that were specific for SCO secretion were distinguished on the basis of their phylogenetic conservation and their different grades of resistance against chemical denaturation. The monoclonal antibody aRFME 4 recognised a carbohydrate-containing epitope that was strongly conserved in vertebrates and unique for SCO secretion. All epitopes showed essentially the same distribution pattern over 15 bovine RF glycoprotein fractions of different molecular masses in immunoblots indicating that the different RF fractions are closely related. They may represent multiple forms of SCO spondin.
Microscopy Research and Technique, 2001
Bovine ReissnerЈs fiber (RF) glycoproteins were used as antigen for the production of polyclonal and monoclonal antibodies (Mabs). We also produced Mabs against intracellular secretory glycoproteins of the bovine subcommissural organ (SCO). These Mabs were used for immunodetection of secretory proteins in situ (structural and ultrastructural immunocytochemistry), in blots, and in solutions. Three different antigen-mediated ELISA were designed to evaluate the affinity of the Mabs, to study the nature of the epitopes, and for competition test among Mabs. Two double antibody sandwich ELISA were designed to detect and quantify soluble secretory materials in different samples, to study coexistence of epitopes, and to elucidate whether epitopes for Mabs are repeated or not in the RF-glycoproteins. Twenty-three Mabs recognizing the bovine RF-and SCO-glycoproteins in solutions (ELISA) as well as in tissue sections, were obtained. Nineteen of these Mabs also recognized the pig SCO, 11 the rabbit SCO, 6 the dog SCO, and 5 the rat SCO. None of the Mabs recognized the SCO of non-mammalian species. The different types of ELISA demonstrated that: (1) the epitopes reside in the proteinaceous moiety of the secretion, (2) they coexist in the same molecular forms and, with few exceptions, they did not overlap, (3) they were not repeated in the secretory molecule(s). Three Mabs were used for immunoblotting of RF; one of them revealed the same band pattern as that shown by an anti-RF serum. It is concluded that all Mabs raised in our laboratory are directed against non-repeated sequences of RF-glycoproteins that have not been conserved in vertebrate phylogeny.
Histochemistry and Cell Biology, 1990
Two experimental protocols were used to investigate the secretory glycoproteins of the subcommissural organ (SCO). Protocol I: Lectins, specific exoglycosidases and immunocytochemistry were sequentially applied to the same section or to adjacent semithin sections of the rat SCO fixed in Bouin's fluid and embedded in methacrylate. Lectins used: concanavalin A (con A), wheat germ agglutinin, Limulus polyphemus agglutinin, Ricinus communis agglutinin and Arachis hypogeae agglutinin. Glycosidases used: neuroaminidase, β-galactosidase, α-mannosidase, α-glucosidase and β-N-acetyl-glucosaminidase. For immunocytochemistry an antiserum against bovine Reissner's fiber (AFRU) was used. Lectins and glycosidases were used in sequences that allowed the cleaved sugar residue to be identified as well as that appearing exposed as a terminal residue. This approach led to the following conclusions: (1) the terminal sugar chain of the secreted glycoproteins has the sequence sialic acid-galactose-glucosamine-; (2) the con A-binding material present in the rough endoplasmic reticulum corresponds to mannose; (3) the apical secretory granules and Reissner's fibers displayed a strong con A affinity after removing sialic acid, thus indicating the presence of internal mannosyl residues in the secreted material; (4) after removing most of the sugar moieties the secretory material continued to be strongly immunoreactive with AFRU. Protocol II: Rats were injected into the lateral ventricle with Tunicamycin and killed 12, 24, 50 and 60 h after the injection. The SCO of rats from the last two groups showed a complete absence of con A binding sites. The results from the two experiments confirm that the secretory glycoproteins of the rat SCO are N-linked complex-type glycoproteins with the conformation previously suggested (Rodríguez et al. 1986).