Uncovering the role of the subcommissural organ in early brain development through transcriptomic analysis.

BACKGROUND: The significant role of embryonic cerebrospinal fluid (eCSF) in the initial stages of brain development has been thoroughly studied. This fluid contains crucial molecules for proper brain development such as members of the Wnt and FGF families, apolipoproteins, and retinol binding protein. Nevertheless, the source of these molecules remains uncertain since they are present before the formation of the choroid plexus, which is conventionally known as the primary producer of cerebrospinal fluid. The subcommissural organ (SCO) is a highly conserved gland located in the diencephalon and is one of the earliest differentiating brain structures. The SCO secretes molecules into the eCSF, prior to the differentiation of the choroid plexus, playing a pivotal role in the homeostasis and dynamics of this fluid. One of the key molecules secreted by the SCO is SCO-spondin, a protein involved in maintenance of the normal ventricle size, straight spinal axis, neurogenesis, and axonal guidance. Furthermore, SCO secretes transthyretin and basic fibroblast growth factor 2, while other identified molecules in the eCSF could potentially be secreted by the SCO. Additionally, various transcription factors have been identified in the SCO. However, the precise mechanisms involved in the early SCO development are not fully understood. RESULTS: To uncover key molecular players and signaling pathways involved in the role of the SCO during brain development, we conducted a transcriptomic analysis comparing the embryonic chick SCO at HH23 and HH30 stages (4 and 7 days respectively). Additionally, a public transcriptomic data from HH30 entire chick brain was used to compare expression levels between SCO and whole brain transcriptome. These analyses revealed that, at both stages, the SCO differentially expresses several members of bone morphogenic proteins, Wnt and fibroblast growth factors families, diverse proteins involved in axonal guidance, neurogenic and differentiative molecules, cell receptors and transcription factors. The secretory pathway is particularly upregulated at stage HH30 while the proliferative pathway is increased at stage HH23. CONCLUSION: The results suggest that the SCO has the capacity to secrete several morphogenic molecules to the eCSF prior to the development of other structures, such as the choroid plexus.

Role of the subcommissural organ in the pathogenesis of congenital hydrocephalus in the HTx rat.

The present investigation was designed to clarify the role of the subcommissural organ (SCO) in the pathogenesis of hydrocephalus occurring in the HTx rat. The brains of non-affected and hydrocephalic HTx rats from embryonic day 15 (E15) to postnatal day 10 (PN10) were processed for electron microscopy, lectin binding and immunocytochemistry by using a series of antibodies. Cerebrospinal fluid (CSF) samples of non-affected and hydrocephalic HTx rats were collected at PN1, PN7 and PN30 and analysed by one- and two-dimensional electrophoresis, immunoblotting and nanoLC-ESI-MS/MS. A distinct malformation of the SCO is present as early as E15. Since stenosis of the Sylvius aqueduct (SA) occurs at E18 and dilation of the lateral ventricles starts at E19, the malformation of the SCO clearly precedes the onset of hydrocephalus. In the affected rats, the cephalic and caudal thirds of the SCO showed high secretory activity with all methods used, whereas the middle third showed no signs of secretion. At E18, the middle non-secretory third of the SCO progressively fused with the ventral wall of SA, resulting in marked aqueduct stenosis and severe hydrocephalus. The abnormal development of the SCO resulted in the permanent absence of Reissner's fibre (RF) and led to changes in the protein composition of the CSF. Since the SCO is the source of a large mass of sialilated glycoproteins that form the RF and of those that remain CSF-soluble, we hypothesize that the absence of this large mass of negatively charged molecules from the SA domain results in SA stenosis and impairs the bulk flow of CSF through the aqueduct.

Polarized expression of integrin beta1 in diencephalic roof plate during chick development, a possible receptor for SCO-spondin.

The roof plate of the caudal diencephalon is formed by the posterior commissure (PC) and the underlying secretory ependyma, the subcommissural organ (SCO). The SCO is composed by radial glial cells bearing processes that cross the PC and attach to the meningeal basement membrane. Since early development, the SCO synthesizes SCO-spondin, a glycoprotein that shares similarities to axonal guidance proteins. In vitro, SCO-spondin promotes neuritic outgrowth through a mechanism mediated by integrin beta1. However, the secretion of SCO-spondin toward the extracellular matrix that surrounds the PC axons and the expression of integrins throughout PC development have not been addressed. Here we provide immunohistochemical evidence to suggest that during chick development SCO cells secrete SCO-spondin through their basal domain, where it is deposited into the extracellular matrix in close contact with axons of the PC that express integrin beta1. Our results suggest that SCO-spondin has a role in the development of the PC through its interaction with integrin beta1.

Synthesis of transthyretin by the ependymal cells of the subcommissural organ.

Transthyretin (TTR) is a protein involved in the transport of thyroid hormones in blood and cerebrospinal fluid (CSF). The only known source of brain-produced TTR is the choroid plexus. In the present investigation, we have identified the subcommissural organ (SCO) as a new source of brain TTR. The SCO is an ependymal gland that secretes glycoproteins into the CSF, where they aggregate to form Reissner's fibre (RF). Evidence exists that the SCO also secretes proteins that remain soluble in the CSF. To investigate the CSF-soluble compounds secreted by the SCO further, antibodies were raised against polypeptides partially purified from fetal bovine CSF. One of these antibodies (against a 14-kDa compound) reacted with secretory granules in cells of fetal and adult bovine SCO, organ-cultured bovine SCO and the choroid plexus of several mammalian species but not with RF. Western blot analyses with this antibody revealed two polypeptides of 14 kDa and 40 kDa in the bovine SCO, in the conditioned medium of SCO explants, and in fetal and adult bovine CSF. Since the monomeric and tetrameric forms of TTR migrate as bands of 14 kDa and 40 kDa by SDS-polyacrylamide gel electrophoresis, a commercial preparation of human TTR was run, with both bands being reactive with this antibody. Bovine SCO was also shown to synthesise mRNA encoding TTR under in vivo and in vitro conditions. We conclude that the SCO synthesises TTR and secretes it into the CSF. Colocalisation studies demonstrated that the SCO possessed two populations of secretory cells, one secreting both RF glycoproteins and TTR and the other secreting only the former. TTR was also detected in the SCO of bovine embryos suggesting that this ependymal gland is an important source of TTR during brain development.

Transcription of SCO-spondin in the subcommissural organ: evidence for down-regulation mediated by serotonin.

The subcommissural organ (SCO) is a brain gland located in the roof of the third ventricle that releases glycoproteins into the cerebrospinal fluid, where they form a structure known as Reissner's fiber (RF). On the basis of SCO-spondin sequence (the major RF glycoprotein) and experimental findings, the SCO has been implicated in central nervous system development; however, its function(s) after birth remain unclear. There is evidence suggesting that SCO activity in adult animals may be regulated by serotonin (5HT). The use of an anti-5HT serum showed that the bovine SCO is heterogeneously innervated with most part being poorly innervated, whereas the rat SCO is richly innervated throughout. Antibodies against serotonin receptor subtype 2A rendered a strong immunoreaction at the ventricular cell pole of the bovine SCO cells and revealed the expected polypeptides in blots of fresh and organ-cultured bovine SCO. Analyses of organ-cultured bovine SCO treated with 5HT revealed a twofold decrease of both SCO-spondin mRNA level and immunoreactive RF glycoproteins, whereas no effect on release of RF glycoproteins into the culture medium was detected. Rats subjected to pharmacological depletion of 5HT exhibited an SCO-spondin mRNA level twofold higher than untreated rats. These results indicate that 5HT down-regulates SCO-spondin biosynthesis but apparently not its release, and suggest that 5HT may exert the effect on the SCO via the cerebrospinal fluid.

Cellular mechanisms involved in the stenosis and obliteration of the cerebral aqueduct of hyh mutant mice developing congenital hydrocephalus.

Two phases may be recognized in the development of congenital hydrocephalus in the hyh mutant mouse. During embryonic life the detachment of the ventral ependyma is followed by a moderate hydrocephalus. During the first postnatal week the cerebral aqueduct becomes obliterated and a severe hydrocephalus develops. The aim of the present investigation was to elucidate the cellular phenomena occurring at the site of aqueduct obliteration and the probable participation of the subcommissural organ in this process. Electron microscopy, immunocytochemistry, and lectin histochemistry were used to investigate the aqueduct of normal and hydrocephalic hyh mice from embryonic day 14 (E-14) to postnatal day 7 (PN-7). In the normal hyh mouse, the aqueduct is an irregularly shaped cavity with 3 distinct regions (rostral, middle, and caudal) lined by various types of ependyma. In the hydrocephalic mouse, these 3 regions behave differently; the rostral end becomes stenosed, the middle third dilates, and the caudal end obliterates. The findings indicate that the following sequence of events lead to hydrocephalus: 1) denudation of the ventral ependyma (embryonic life); 2) denudation of dorsal ependyma and failure of the subcommissural organ to form Reissner fiber (first postnatal week); 3) obliteration of distal end of aqueduct; and 4) severe hydrocephalus. No evidence was obtained that NCAM is involved in the detachment of ependymal cells. The process of ependymal denudation would involve alterations of the surface sialoglycoproteins of the ependymal cells and the interaction of the latter with macrophages.

Reissner fiber binds and transports away monoamines present in the cerebrospinal fluid.

The subcommissural organ (SCO) is a brain gland that secretes glycoproteins into the cerebrospinal fluid (CSF), where they subsequently aggregate to form Reissner fiber (RF). By addition of newly released glycoproteins to its cephalic end, RF grows constantly through the Sylvian aqueduct, fourth ventricle and central canal of the spinal cord. Disaggregation of RF-material and passage to blood occur when RF reaches the terminal ventricle at the filum. The present investigation was designed to test the hypothesis that RF binds, transports and clears away monoamines present in the CSF. Four experimental protocols were applied: (i) in vivo binding of labeled monoamines to the rat RF, studied by pulse and chase, and after perfusion for 7 days; (ii) identification of monoamines, by high-performance liquid chromatography (HPLC), naturally occurring in the bovine RF; (iii) in vitro binding of labeled and unlabeled monoamines to the isolated bovine RF; and (iv) tentative identification of the amine binding site(s) in RF-proteins by use of specific antibodies. The results obtained indicate that RF participates in the regulation of the CSF concentration of monoamines either by binding and transporting them away throughout the central canal of the spinal cord (L-DOPA, noradrenaline, adrenaline), or by transiently binding them and releasing them back to the CSF (serotonin). Furthermore, evidence was obtained that (i) adrenaline and noradrenaline share the same binding site, and that this site would correspond to a repeated sequence present in the SCO-spondin, the major protein component of RF; and (ii) serotonin has its own binding site in RF.

The floor plate cells from bovines express the mRNA encoding for SCO-spondin and its translation products.

The floor plate (FP) is a transient structure of the embryonic central nervous system (CNS) which plays a key role in development driving cell differentiation and patterning in the ventral neural tube. The fact that antisera raised against subcommissural organ (SCO) secretion immunostain FP cells and react with high-molecular-mass proteins in FP extracts, prompted us to investigate the expression of a SCO-related polypeptide in FP cells. RNA from bovine FP was analyzed by means of reverse transcriptase polymerase chain reaction (RT-PCR), using primers derived from the 3' end of SCO-spondin which revealed products of 233, 237, 519 and 783 bp. Sequence analysis of the 233 bp PCR fragment confirmed the identity between this FP product and SCO-spondin. FP-translation of the SCO-spondin encoded polypeptide(s) was demonstrated by Western blot analysis and immunocytochemistry, using antisera raised against (i) the glycoproteins secreted by the bovine SCO, and (ii) a peptide derived from the open reading frame of the major SCO secretory protein, SCO-spondin, respectively. Additional evidence pointing to active transcription and translation of a SCO-spondin related gene was obtained in long term FP organ cultures. On the basis of partial sequence homologies of SCO-spondin with protein domains implicated in cell-cell contacts, cell-matrix interactions and neurite outgrowth it is possible to suggest that the SCO-spondin secreted by the FP is involved in CNS development.

Biosynthesis and molecular biology of the secretory proteins of the subcommissural organ.

Ependymal cells are specialized in the synthesis and release of different factors into the cerebrospinal fluid (CSF). The subcommissural organ (SCO) is one of the most active areas of the ventricular walls secreting into the CSF. This gland is localized in the roof of the third ventricle covering the posterior commissure. Glycoproteins synthesized in SCO cells are released into the ventricular CSF where they aggregate, in a highly ordered fashion, forming an elongated supramacromolecular structure known as the Reissner's fiber (RF). RF grows caudally and extends along the brain aqueduct, the fourth ventricle, and the whole length of the central canal of the spinal cord. The SCO cells synthesize glycoproteins of high molecular weight. A precursor form of 540 kDa is synthesized in bovine and chick SCO cells, and a transcript of 10--14 kb is expressed selectively in the bovine SCO cells. The processing of this molecule generates at least one protein of about 450 kDa (RF-Gly-I), which, after being released, is involved in the formation of RF. Additionally, biochemical data indicate that bovine SCO cells synthesize a second precursor compound of 320 kDa, which is also detected in rat, rabbit, and dog. We postulate that RF is formed by two different complexes, one of which has a very high molecular mass (700 kDa or more) and is made up of at least six polypeptides, with the polypeptide of 450 kDa being its main component. The molecules that form RF in different species have different primary structures but they express common epitopes associated to the existence of cysteine bridges, which are probably crucial for polymerization of RF. Molecular procedures involving the use of anti-RF antibodies have led to the isolation of cDNA clones encoding two proteins known as RF-GLY-I and SCO-spondin. In the last 3 years, five partial cDNA sequences encoding SCO-spondin-like proteins have been obtained (Y08560, Y08561, AJ132107, AJ132106, AJ133488). These clones along with RF-GLY-I and SCO-spondin were computer-assembled generating a cDNA consensus sequence of 14.4 kb. Analyses of the long consensus sequence revealed an extended open reading frame (ORF-1) spanning from base 1,634 to 14,400 that encodes for a putative protein of 4,256 amino acids (approximately 450 kDa). The Mr of the predicted protein is consistent with the observed Mr of the largest protein recognized with anti-RF antibodies in SCO and RF extracts. However, the absence of consensus sequences typically present near the 5J'-end of the translation initiation site suggests the existence of a second open reading frame (ORF-2) extending from base 1 to base 14,400 in frame with the ORF-1 and probably encoding for the largest protein precursor (540 kDa). An antibody raised against a peptide sequence, deduced from the open reading frame encoded by a SCO cDNA, reacted specifically with the bovine and rat SCO-RF complex, thus indicating that the protein encoded by the cloned cDNA is part of RF. Immunoblots of bovine SCO extracts using the anti-peptide serum revealed bands of 540 kDa and 450 kDa, but it did not react with the proteins of 320 and 190 kDa. These data support the existence of two precursors for the bovine RF-glycoproteins (540 and 320 kDa) with the 450-kDa protein being a processed form of the 540-kDa precursor. We postulate that the cloned cDNAs encode for a protein that corresponds to the 540-kDa precursor and that at least part of this sequence is present in the processed form of 450 kDa that is secreted to form the RF.

Organ culture of the bovine subcommissural organ: evidence for synthesis and release of the secretory material.

The subcommissural organ (SCO) is a brain circumventricular organ formed by ependymal and hypendymal secretory cells. It secretes glycoproteins into the cerebrospinal fluid of the third ventricle where they condense into a thread-like structure known as Reissner's fiber (RF). The present study was designed to investigate whether or not the bovine SCO continues to synthesize and release glycoproteins after a long-term culture. Cultured explants of SCO survive for several months. The content of the secretory granules present in the cultured ependymocytes displayed immunoreactive and lectin-binding properties similar to those of the core glycosylated glycoproteins found in the bovine SCO. The explants actively incorporated (35)S-cysteine. In the cultured ependymocytes, the pattern of distribution of the radioactive label and that of the immunoreactive secretory material was similar, thus indicating that this material has been synthesized during culture. At the ultrastructural level, the cultured tissue exhibited a high degree of differentiation comparable to that of the bovine SCO in situ. A striking finding was the observation of similar results when cerebrospinal fluid was used as a culture medium. The addition of antibodies against RF-glycoproteins into the culture medium allowed visualization, by means of different immunocytochemistry protocols, deposits of extracellular immunoreactive secretory material on the free surface of the cultured ependymocytes, indicating that release of secretory glycoproteins into the culture medium does occur. Primary culture of dispersed SCO ependymocytes, obtained either from fresh or organ cultured bovine SCO, showed that these cells release RF-glycoproteins that aggregate in the vicinity of each cell. The present investigation has shown that: (1) two types of secretory ependymocytes become evident in the cultured SCO; (2) under culture conditions, the SCO cells increase their secretory activity; (3) explants of bovine SCO synthesize RF-glycoproteins and release them to the culture medium; (4) after release these proteins aggregate but do not form a RF; (5) a pulse of anti-RF antibodies into the culture medium blocks the secretion of RF-glycoproteins for several days.

Spatial distribution of Reissner's fiber glycoproteins in the filum terminale of the rat and rabbit.

The subcommissural organ secretes into the third ventricle glycoproteins that condense to form the Reissner's fiber (RF). At the distal end of the central canal of the spinal cord, the RF-glycoproteins accumulate in the form of an irregular mass known as massa caudalis. Antibodies against RF-glycoproteins and a set of lectins were used at the light and electron microscopic level to investigate the spatial distribution of the massa caudalis material in the rat and rabbit filum terminale. In the sacral region of the rat, the central canal presents gaps between the ependymal cells through which RF-glycoproteins spread out. The bulk of massa caudalis material, however, escapes through openings in the dorsal wall of the terminal ventricle. In the rabbit, the massa caudalis is formed within the ependymal canal, at the level of the second coccygeal vertebra, it accumulates within preterminal and terminal dilatations of the central canal, and it escapes out through gaps in the dorsal ependymal wall of the terminal ventricle. The existence of wide intercellular spaces and a large orifice (neuroporous) in the dorsal ependymal wall of the terminal ventricle, and the passage of RF-material through them, appear to be conserved evolutionary features. After leaving the terminal ventricle of the rat and rabbit, RF-glycoproteins establish a close spatial association with the numerous blood vessels irrigating the filum terminale, suggesting that in these species the blood vessels are the site of destination of the RF-glycoproteins escaping from the central canal, thus resembling the situation found in lower vertebrates. When passing from the RF stage to the massa caudalis stage, the rabbit RF-glycoproteins lose their sialic acid residues, exposing galactose as the terminal residue. Since this sialic acid-galactose modification of RF-glycoproteins had also been described in lamprey larvae, it may be regarded as a conserved evolutionary feature associated with the formation of the massa caudalis.

Functional aspects of the subcommissural organ-Reissner's fiber complex with emphasis in the clearance of brain monoamines.

Reissner's fiber (RF) extends along the cerebral aqueduct, fourth ventricle, and the entire length of the central canal of the spinal cord. It grows continuously in the caudal direction by addition of newly released glycoproteins by the subcommissural organ (SCO) to its proximal end. Several hypotheses about RF function have been advanced. One of them postulates that RF binds biogenic amines present in the CSF and clears them away. In recent years, this hypothesis has been tested in our laboratory by using several experimental protocols. Firstly, the CSF concentration of monoamines was investigated in RF-deprived rats subjected to immunological neutralization of the SCO-RF complex. Secondly, the capacity of RF to bind monoamines in vivo was studied by injecting radiolabeled serotonin or noradrenaline into the rat CSF, and by perfusing them into the CSF, during one week, using an Alzet's osmotic pump. In vitro binding studies were performed using isolated bovine RF. All the findings obtained indicate that RF binds monoamines present in the ventricular CSF and then transports them along the central canal. In the absence of RF, the CSF concentration of monoamines increases sharply.

Human subcommissural organ, with particular emphasis on its secretory activity during the fetal life.

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 glyco- protein(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.

Vertebrate floor plate transiently expresses a compound recognized by antisera raised against subcommissural organ secretion.

Located along the ventral midline of the neural tube, the floor plate (FP) performs an essential role in central nervous system development, especially in the patterning of the ventral region of the neural tube and axonal guidance. Several studies have been directed to the identification of molecules mediating some of the functions of the FP. Most of the models proposed for floor plate actions involve contact-mediated- and/or gradients of diffusible-signals acting throughout the nervous tissue. This report presents and discusses findings showing that the FP cells secrete a novel compound, which is recognized by antisera raised against secretory products of the subcommissural organ (SCO). This immunoreactive compound appears to be very similar to one of the glycoproteins secreted by the SCO. This immunoreactivity is expressed transiently during central nervous system development, and its rostro-caudal extension along the anterior-posterior axis of the FP displays some species variations. However, a constant feature in all species investigated is that this immunoreactive compound is highly expressed in the FP located in the mesencephalic-metencephalic boundary. The distribution of this compound is compatible with basal and apical pathways of release from FP cells. The former might participate in the formation of some brain commissures. The latter might involve the use of the cerebrospinal fluid as a route for performing actions on distant targets, a pathway somehow disregarded by most models accounting for morphogen actions.

Isograft and xenograft of the subcommissural organ into the lateral ventricle of the rat and the formation of Reissner's fiber.

The subcommissural organ (SCO) secretes glycoproteins into the cerebrospinal fluid (CSF) that aggregate and form Reissner's fiber (RF). The factors involved in this aggregation are not known. One factor may be the hydrodynamics of the CSF when flowing through the aqueduct. This hypothesis was tested by isografting rat SCO and xenografting bovine SCO into the lateral ventricle of rats. Xenografts were either fresh bovine SCO or explants cultured for 30 days before transplantation. The grafts were investigated by electron microscopy and immunocytochemistry using antibodies against RF glycoproteins, serotonin and the glucose transporter I. Maximal time of transplantation was 43 days for isografts and 14 days for xenografts. The isografts were not reinnervated but were revascularized; they secreted into the ventricle RF glycoproteins that became progressively packed into pre-RF and RF structures identical to those formed by the SCO in situ. RF was confined to the host ventricle and at its distal end the constituent proteins disassembled. Xenografts were neither reinnervated nor revascularized and secreted into the host ventricle a material that never formed an RF. These findings indicate that the CSF factor responsible for the formation of RF is species specific, and that this process does not depend on the hydrodynamics of the CSF. The blood vessels revascularizing the isografted SCO acquired the characteristics of the vessels irrigating the SCO in situ, namely, a tight endothelium displaying glucose transporter I, and a perivascular space containing long-spacing collagen, thus indicating that basal release of glycoproteins may also occur in the grafted SCO.

Partial sequencing of Reissner's fiber glycoprotein I (RF-Gly I).

The bulk of the secretion of the subcommissural organ is formed by glycoproteins that appear to be derived from two precursor forms of 540 and 320 kDa. Upon release into the ventricle, these glycoproteins aggregate to form Reissner's fiber. We report the isolation of three cDNA clones from a cDNA library prepared from bovine subcommissural organ RNA, by using an anti-Reissner's fiber serum for immunoscreening. Inserts of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain reaction, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a short non-homologous region was present in the 0.7-kb insert when compared with the 1. 2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showed a single mRNA species of approximately 9.5 kb present in the subcommissural organ and missing in the choroid plexus, brain cortex, and liver. In situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal antibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovine and rat subcommissural organ-Reissner's fiber complex. In immunoblots of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is concluded that the cloned cDNA encodes for the major constitutive glycoprotein of Reissner's fiber, here designated as RF-Gly I. The sequenced region of RF-Gly I displays a high degree of homology with some regions of the von Willebrand factor and certain mucins; it also displays two motifs homologous with repeats present in proteins of the spondin family and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.

Observations on the discharge of the secretion from ependymal cells of the subcommissural organ (SCO) of some South American primates.

The secretion of the subcommissural organ (SCO) is synthesized in the intrinsic cells of that structure, a neuroendocrine gland. The organelles involved in the synthesis of this secretion are rough-surfaced endoplasmic reticulum (RER) and the Golgi apparatus. It is still uncertain whether and to what extent the latter participates in the synthesis. In highly active secretory ependymal cells of the SCO, the Golgi apparatus exhibits distinct signs of intense activity. This suggests that the Golgi apparatus is involved in the preparation of the secretion, even if this is difficult to prove electron microscopically. The secretion is discharged from the optical part of the SCO-cells into the cerebrospinal fluid (CSF) of the IIIrd ventricle, where Reissner's fibre is formed. Different phases of release of the content of the secretory granula into the IIIrd ventricle are described; our findings are in agreement with previously published observations. In 2 cases delicately granulated and moderately electron-dense material was found in circumscriptly dilated vaults of the intercellular spaces. Release of secretory material into intercellular spaces appears to be possible, but is evidently infrequent. Regular occurrence of a basal (peripheral) discharge into the hypendymal capillaries is not unanimously agreed upon as yet. In this paper, a passage of granula is described from the cytoplasm of the end feet of SCO cells through the walls of the capillaries into the systemic circulation. This provides structural evidence that secretory material synthesized in the SCO is released into the capillaries. These observations must ultimately be confirmed with the aid of ultracytochemical methods, particularly using labelled material.