Microscopic anatomy of the subcommissural organ in the brain of the adult greater cane rat (Rodentia: Thryonomyidae).

The subcommissural organ (SCO) is a well-developed gland present in the brain of vertebrates. The SCO secretes glycoproteins into the circulating cerebrospinal fluid and these assemble to form Reissner's fibre. It also plays an important function in neurogenesis and axonal guidance during embryogenesis. This study delves into the microscopic anatomy of the SCO in the adult greater cane rat (GCR), shedding light on its histoarchitectural characteristics. By utilizing histological techniques and microscopic analysis, we investigated the SCO's location and cellular composition within the brain of adult GCR. Our findings showed that the SCO in this species is located ventrally to the posterior commissure (PC) and dorsally to the third ventricle. The SCO consists of specialized ependymal or nuclear cell layer and apical processes lining the third ventricle. Moreover, the SCO's proximity to the PC and the third ventricle highlights its strategic position within the brain's ventricular system. With immunohistochemical analyses, the SCO cells expressed glial fibrillary protein when immunolabelled with Glial fibrillary acid protein (GFAP) antibody, a marker for astrocytes/astrocytic-like cells. Few microglia-like cells were immuno-positive for Ionized calcium-binding adapter molecule 1 (Iba1) antibody, that are existing within the SCO. However, the SCO in the GCR showed a negative immunostaining to NeuN antibody. This study contributes to our understanding of the microscopic anatomy of the SCO in a lesser-studied mammalian species. Further research into the SCO's functional significance especially during development in the GCR, may hold promise for more insights into neurological health and pathology.

The subcommissural organ and the Reissner fiber: old friends revisited.

The subcommissural organ (SCO) is an ancient and conserved brain gland secreting into cerebrospinal fluid (CSF) glycoproteins that form the Reissner fiber (RF). The present investigation was designed to further investigate the dynamic of the biosynthetic process of RF glycoproteins prior and after their release into the CSF, to identify the RF proteome and N-glycome and to clarify the mechanism of assembly of RF glycoproteins. Various methodological approaches were used: biosynthetic labelling injecting 35S-cysteine and 3H-galactose into the CSF, injection of antibodies against galectin-1 into the cerebrospinal fluid, light and electron microscopical methods; isolated bovine RF was used for proteome analyses by mass spectrometry and glycome analysis by xCGE-LIF. The biosynthetic labelling study further supported that a small pool of SCO-spondin molecules rapidly enter the secretory pathways after its synthesis, while most of the SCO-spondin molecules are stored in the rough endoplasmic reticulum for hours or days before entering the secretory pathway and being released to assemble into RF. The proteomic analysis of RF revealed clusterin and galectin-1 as partners of SCO-spondin; the in vivo use of anti-galectin-1 showed that this lectin is essential for the assembly of RF. Galectin-1 is not secreted by the SCO but evidence was obtained that it would be secreted by multiciliated ependymal cells lying close to the SCO. Further, a surprising variety and complexity of glycan structures were identified in the RF N-glycome that further expands the potential functions of RF to a level not previously envisaged. A model of the macromolecular organization of Reissner fiber is proposed.

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.

Continuous delivery of a monoclonal antibody against Reissner's fiber into CSF reveals CSF-soluble material immunorelated to the subcommissural organ in early chick embryos.

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.

Novel distribution of calcitonin gene-related peptide in rodent subcommissural organs.

For the first time we showed the presence of CGRP immunoreactivity in the SCO of golden hamsters, Wistar rats and Mongolian gerbils. In hamsters, the intense CGRP-like immunoreactivity was detected in the whole SCO, including Reissner's fiber- and granule-like structures at the ventricular surface of the SCO. The CGRP-positive hypendymal cells were frequently in contact with local blood vessels and some of them were situated intimately to the leptomeningeal space. In the SCOs of rats and gerbils, only the supranuclear area and apical cytoplasm of the ependymal cells were positive for CGRP, whereas the basal pole of the cells and the hypendyma were CGRP-negative. The existence of CGRP in rodents SCO was confirmed by the expression of CGRP mRNA in rat SCO by RT-PCR. The present results indicate that the SCOs of rodent species contain CGRP that may be in part synthesized by ependymocytes themselves. A species difference in the CGRP distributions of rodents SCOs may therefore imply its different synthesis rates or release pathways.

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.

Subcellular distributions of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the subcommissural organ of the golden hamster (Mesocricetus auratus).

Immuno-electron microscopy specifically enhanced with silver staining has been used to demonstrate the localization of calcitonin gene-related peptide (CGRP) in the ependymocytes of the hamster subcommissural organ (SCO). Hamster SCO consists of the ependymal and hypendymal cell layers, the latter being arranged as rosette-like structure across the posterior commissure (PC) and often arranged with longitudinal axis parallel to the ventricle. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with CGRP. In the hypendymal layer, the CGRP positive hypendymal cells were frequently in contact with local blood vessels and arranged in-groups traversing the thick portion of the PC. Ultrastructurally, the CGRP-immunoreaction products were distributed at the dilated cisternae of the rough endoplasmic reticulum (rER) and secretory granules of the ependymal and hypendymal cells. The dilated cisterna of the rER was usually concentrated in the basal region of the ependymal cells and irregular in shape. These dilated cisternae were filled with a flocculent material or finely granular substance, but hardly studded with ribosomes. Labelled secretory granules were abundant in the apical pole of the ependymal cells and discharged their contents into the third ventricle in the form of a thin layer of secretion. This CGRP positive material appeared to constitute the pre-Reissner's fiber (RF). On the basis of the present ultrastructural evidences, we proposed that ependymocytic CGRP in SCO may be synthesized and stored in the cisternae of rER, then released and incorporated into the RF in the third ventricle through the secretory granules. The abundant amount of CGRP in ependymocytes of SCO and RF in the third ventricle suggests a significant endocrine function of CGRP in hamster SCO.

Transcription factor CREB and its stimulus-dependent phosphorylation in cell and explant cultures of the bovine subcommissural organ.

The subcommissural organ (SCO) is an ependymal brain gland that synthesizes and secretes glycoproteins. Very little is known about the signal transduction cascades operating in this organ and their impact on gene expression. An important transcription factor that regulates gene expression in glial cells and neurons is the cyclic-AMP-responsive element binding protein (CREB), which is activated by phosphorylation of the serine residue 133. Here, we analyzed the presence of CREB in bovine SCO cells and its phosphorylation by drugs that activate cyclic-AMP-dependent or calcium-dependent signal transduction pathways. We also investigated the effects of three natural signaling molecules, serotonin (5HT), substance P (SP) and ATP, on CREB phosphorylation and on the second messengers cyclic AMP and calcium. Investigations were performed with cell and explant cultures by using immunocytochemistry, immunoblot, enzyme-linked immunosorbent assay, and the Fura-2 technique. A strong immunosignal for total (phosphorylated and unphosphorylated) CREB was found in virtually all SCO cells. Total CREB levels did not change upon stimulation. Phosphorylated (p)CREB levels were low in unstimulated cells and significantly elevated by drugs that increase the levels of cyclic AMP or free calcium ions. pCREB was also induced by SP and ATP; both substances increased the intracellular calcium concentration but did not affect the formation of intracellular cyclic AMP. 5HT did not influence the phosphorylation of CREB, the intracellular calcium concentration, or the formation of cyclic AMP. Our data identify CREB as an SCO transcription factor that can be activated by the second messengers cAMP and calcium. SP and ATP stimulate the phosphorylation of CREB apparently via a calcium-dependent mechanism and are thus involved in the control of gene expression in the bovine SCO.

A programmed ependymal denudation precedes congenital hydrocephalus in the hyh mutant mouse.

Hydrocephalic hyh mice are born with moderate hydrocephalus and a normal cerebral aqueduct. At about the fifth postnatal day the aqueduct becomes obliterated and severe hydrocephalus develops. The aim of the present investigation was to investigate the mechanism of this hydrocephalus, probably starting during fetal life when the cerebral aqueduct is still patent. By use of immunocytochemistry and scanning electron microscopy, mutant (n = 54) and normal (n = 61) hyh mouse embryos were studied at various developmental stages to trace the earliest microscopic changes occurring in the brains of embryos becoming hydrocephalic. The primary defect begins at an early developmental stage (E-12) and involves cells lining the brain cavities, which detach following a well-defined temporo-spatial pattern. This ependymal denudation mostly involves the ependyma of the basal plate derivatives. There is a relationship between ependymal denudation and ependymal differentiation evaluated by the expression of vimentin and glial fibrillary acidic protein. The ependymal cells had a normal appearance before and after detachment, suggesting that their separation from the ventricular wall might be due to abnormalities in cell adhesion molecules. The process of detachment of the ventral ependyma, clearly visualized under scanning electron microscope, is almost completed before the onset of hydrocephalus. Furthermore, this ependymal denudation does not lead to aqueductal stenosis during prenatal life. Thus, the rather massive ependymal denudation appears to be the trigger of hydrocephalus in this mutant mouse, raising the question about the mechanism responsible for this hydrocephalus. It seems likely that an uncontrolled bulk flow of brain fluid through the extended areas devoid of ependyma may be responsible for the hydrocephalus developed by the hyh mutant embryos. The defect in these embryos also includes loss of the hindbrain floor plate and a delayed in the expression of Reissner fiber glycoproteins by the subcommissural organ.

Aspects of the ultrastructure and function of the subcommissural organ in the Mongolian gerbil (Meriones unguiculatus).

The subcommissural organ (SCO) of the Mongolian gerbil has been studied by transmission electron microscopy. The presence of extensive rough endoplasmic reticulum replete with flocculent material, well-developed Golgi complexes, condensing vesicles, electron-opaque and dense granules in the cytoplasm of SCO cells suggests that these cells are involved in protein synthesis. The secretory granules are released at the apical surface of the ependymal cells by exocytosis into the ventricular lumen, where their product condenses to form the Reissner's fiber. From the fact that the intercellular spaces of the ependymal cells of the gerbil SCO are sealed from the ventricular lumen by tight junctions, the accumulation of secretory material within the enlarged intercellular spaces provides ultrastructural evidence of basal secretion. The secretory material observed in the prominent hypendemal intracellular canaliculi, also appears to reach the intercellular spaces. The lack of perivascular spaces and external basal lamina in the gerbil SCO apparently permits basal secretions to reach vasculature and/or leptomeningeal spaces. The presence of smooth pinocytotic and coated vesicles, multivesicular bodies and lysosomes in the cycoplasm of ependymal cells suggests that the uptake of ions and macromolecules from the cerebrospinal fluid may be an additional function of the SCO.

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.

Analysis and quantification of the secretory products of the subcommissural organ by use of monoclonal antibodies.

Bovine Reissner's 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.

Neural input and neural control of the subcommissural organ.

The neural control of the subcommissural organ (SCO) has been partially characterized. The best known input is an important serotonergic innervation in the SCO of several mammals. In the rat, this innervation comes from raphe nuclei and appears to exert an inhibitory effect on the SCO activity. A GABAergic innervation has also been shown in the SCO of the rat and frog Rana perezi. In the rat, GABA and the enzyme glutamate decarboxylase are involved in the SCO innervation. GABA is taken up by some secretory ependymocytes and nerve terminals, coexisting with serotonin in a population of synaptic terminals. Dopamine, noradrenaline, and different neuropeptides such as LH-RH, vasopressin, vasotocin, oxytocin, mesotocin, substance P, alpha-neoendorphin, and galanin are also involved in SCO innervation. In the bovine SCO, an important number of fibers containing tyrosine hydroxylase are present, indicating that in this species dopamine and/or noradrenaline-containing fibers are an important neural input. In Rana perezi, a GABAergic innervation of pineal origin could explain the influence of light on the SCO secretory activity in frogs. A general conclusion is that the SCO cells receive neural inputs from different neurotransmitter systems. In addition, the possibility that neurotransmitters and neuropeptides present in the cerebrospinal fluid may also affect the SCO activity, is discussed.

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.

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.

The ultrastructure of the subcommissural organ ependyma of the goat.

The cells of pseudostratified columnar ciliated ependyma of the subcommissural organ in the goat were classified into two types on the basis of the distribution of chromatin material and nuclear clefts. Amongst the cell organelles the endoplasmic reticulum was highly developed and formed three types of Nebenkerne systems. The type-I Nebenkerne had spiral concentric lamellae and was associated with round lipid droplets. The type-II Nebenkerne, with widely spaced coils, was expanded towards its central and peripheral parts. The type-III Nebenkerne, composed of a meshwork of lamellae, was modified into a vacuolated form. The concentration of mitochondria was greatly increased towards the basal processes of the ependymal cells. The inclusion bodies included small electron-dense bodies, osmiophilic asteroid droplets, large round to spherical bodies and large round osmiophilic bodies with inner structures.

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.

Spontaneous congenital hydrocephalus in the mutant mouse hyh. Changes in the ventricular system and the subcommissural organ.

The subcommissural organ is an ependymal gland located at the entrance of the cerebral aqueduct. It secretes glycoproteins into the cerebrospinal fluid, where they aggregate to form Reissner's fiber. This fiber grows along the aqueduct, fourth ventricle, and central canal. There is evidence that the subcommissural organ is involved in the pathogenesis of congenital hydrocephalus. This organ was investigated in the mutant mouse hyh developing a congenital hydrocephalus. The central nervous system of normal and hydrocephalic hyh mice, 1 to 40 days old, was investigated using antibodies recognizing the subcommissural organ secretory glycoproteins, and by transmission and scanning electron microscopy. At birth, the affected mice displayed open communications between all ventricles, absence of a central canal in the spinal cord, ependymal denudation of the ventricles, stenosis of the rostral end of the aqueduct, and hydrocephalus of the lateral and third ventricles and of the caudal end of the aqueduct. Around the 5th postnatal day, the communication between the caudal aqueduct and fourth ventricle sealed, and hydrocephalus became severe. It is postulated that the hyh mice carry a genetic defect affecting the ependymal cell lineage. The subcommissural organ showed signs of increased secretory activity; it released to the stenosed aqueduct a material that aggregated, but it did not form a Reissner's fiber. A large area of the third ventricular wall differentiated into a secretory ependyma synthesizing a material similar to that secreted by the subcommissural organ. It is concluded that the subcommissural organ changes during hydrocephalus; whether these changes precede hydrocephalus needs to be investigated.