In Vivo Expression of the PTB-deleted Odin Mutant Results in Hydrocephalus.

Odin has been implicated in the downstream signaling pathway of receptor tyrosine kinases, such as the epidermal growth factor and Eph receptors. However, the physiologically relevant function of Odin needs to be further determined. In this study, we used Odin heterozygous mice to analyze the Odin expression pattern; the targeted allele contained a ß-geo gene trap vector inserted into the 14th intron of the Odin gene. Interestingly, we found that Odin was exclusively expressed in ependymal cells along the brain ventricles. In particular, Odin was highly expressed in the subcommissural organ, a small ependymal glandular tissue. However, we did not observe any morphological abnormalities in the brain ventricles or ependymal cells of Odin null-mutant mice. We also generated BAC transgenic mice that expressed the PTB-deleted Odin (dPTB) after a floxed GFP-STOP cassette was excised by tissue-specific Cre expression. Strikingly, Odin-dPTB expression played a causative role in the development of the hydrocephalic phenotype, primarily in the midbrain. In addition, Odin-dPTB expression disrupted proper development of the subcommissural organ and interfered with ependymal cell maturation in the cerebral aqueduct. Taken together, our findings strongly suggest that Odin plays a role in the differentiation of ependymal cells during early postnatal brain development.

Congenital hydrocephalus and abnormal subcommissural organ development in Sox3 transgenic mice.

Congenital hydrocephalus (CH) is a life-threatening medical condition in which excessive accumulation of CSF leads to ventricular expansion and increased intracranial pressure. Stenosis (blockage) of the Sylvian aqueduct (Aq; the narrow passageway that connects the third and fourth ventricles) is a common form of CH in humans, although the genetic basis of this condition is unknown. Mouse models of CH indicate that Aq stenosis is associated with abnormal development of the subcommmissural organ (SCO) a small secretory organ located at the dorsal midline of the caudal diencephalon. Glycoproteins secreted by the SCO generate Reissner's fibre (RF), a thread-like structure that descends into the Aq and is thought to maintain its patency. However, despite the importance of SCO function in CSF homeostasis, the genetic program that controls SCO development is poorly understood. Here, we show that the X-linked transcription factor SOX3 is expressed in the murine SCO throughout its development and in the mature organ. Importantly, overexpression of Sox3 in the dorsal diencephalic midline of transgenic mice induces CH via a dose-dependent mechanism. Histological, gene expression and cellular proliferation studies indicate that Sox3 overexpression disrupts the development of the SCO primordium through inhibition of diencephalic roof plate identity without inducing programmed cell death. This study provides further evidence that SCO function is essential for the prevention of hydrocephalus and indicates that overexpression of Sox3 in the dorsal midline alters progenitor cell differentiation in a dose-dependent manner.

Postnatal development of the secretory activity of the goat subcommissural organ. A Reissner's fibre and angiotensin II immunohistochemical study.

Subcommissural organ (SCO) secretory activity of the goat (variations of Capra hircus, that live in arid conditions) was examined during the postnatal development, using specific antibodies against the Reissner's fibre (AFRU) and angiotensin II (AAGII). The SCO was strongly stained with the anti-glycoproteins that form the Reissner's fibre and lightly marked with the anti-angiotensin II. The AFRU-immunoreactivity (ir) was found in the ependymal and hypendymal cells and in the ventricular and peripheral secretory routes of the goat SCO. The amount AFRU increases at 6 months and decreases at adult age. In contrast, the anti-angiotensin II-ir was mainly found in the adult age, not being practically observed at one postnatal month. The AAGII-ir was mainly found in ependymal cells in which AFRU-ir was downregulated. In addition, we detected the presence of double immunostained for AFRU and AAGII in ependymocytes of the pre-commissural and subcommissural parts. In conclusion the present results may suggest a functional interrelation between AAGII and the secretory activity of the SCO of this kind of goat.

Ontogenic development of the human subcommissural organ

The structure of the human subcommissuralorgan during its ontogenic development in 24human embryos and foetuses ranging from 6 to40 weeks of gestation (WG), and three adulthuman brains from 27-, 65- and 70-year old subjectswas investigated using both qualitative andquantitative methods. Concurrently, the appearanceof the subcommissural organ, pineal glandand mesocoelic recess was determined by studyingtheir structure, length and volume. Thehuman SCO appears at the beginning of 8th WG,which confirms previous results; the completematuration of the SCO occurs at the 15th WG andthe following three parts can be distinguished:the precommissural part, located in the rostralzone of the posterior commissure (PC) andextending to the pineal recess; the subcommissuralpart, located under the PC, and the retrocommissuralpart, located in the caudal zone ofthe PC, in the mesocoelic recess and at thebeginning of the Sylvian aqueduct. The reductionin size of the SCO begins after the 17th WGand this decrease in size begins in the precommissural,continues in the subcommissural, andfinishes in the retrocommissural part. The regressionand atrophies of the SCO begin after birth,and the SCO disappears completely after the ageof 30. The mesocoelic recess starts to form at thebeginning of the 10th WG, and is completely formedby the 14th WG and this is where the retrocommissuralpart of the SCO is located. In the 40th WG the regression of the mesocoelic recessbegins and this takes place at the same time asthe regression of the SCO. A parallel developmentbetween the SCO and the pineal wasfound. Thus, we observed the first appearance ofthe pineal recess in the 7-8th WG; during the 10thWG a compact mass of cells appeared in the rostralpart of pineal recess and by the 15th WG thepineal gland (PG) had acquired an almost definitiveaspect

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The thrombospondin type 1 repeat (TSR) and neuronal differentiation: roles of SCO-spondin oligopeptides on neuronal cell types and cell lines.

SCO-spondin is a large glycoprotein secreted by ependymal cells of the subcommissural organ. It shares functional domains called thrombospondin type 1 repeats (TSRs) with a number of developmental proteins expressed in the central nervous system, and involved in axonal pathfinding. Also, SCO-spondin is highly conserved in the chordate phylum and its multiple domain organization is probably a chordate innovation. The putative involvement of SCO-spondin in neuron/glia interaction in the course of development is assessed in various cell culture systems. SCO-spondin interferes with several developmental processes, including neuronal survival, neurite extension, neuronal aggregation, and fasciculation. The TSR motifs, and especially the WSGWSSCSVSCG sequence, are most important in these neuronal responses. Integrins and growth factor receptors may cooperate as integrative signals. We discuss the putative involvement of the subcommissural organ/Reissner's fiber complex in developmental events, as a particular extracellular signaling system.

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.

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.

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.

Postnatal secretion of the subcommissural organ of the Meriones shawi: control of serotonin innervation.

The postnatal development of the subcommissural organ (SCO) glycoprotein secretion in form of Reissner's fiber and the putative control of the serotonin innervation upon the SCO activity were examined by immunohistochemistry in the semi-desert rodent, Meriones shawi. Abundant SCO secretory material and numerous serotoninergic fibers reaching the SCO were observed in newborns meriones. An increase of both secretory material and serotonin fibres density inside the SCO was observed during postnatal period and into adulthood. Neurotoxic destruction with 5,7-dihydroxytryptamine of the SCO serotonin input in the adult or the inhibition of serotonin synthesis by para-chlorophenylalanine at different postnatal ages, resulted in a decrease of the intensity of SCO Reissner's fiber immunolabelling suggesting a reduction in the SCO secretory material. This result might reflect either an inhibition of the synthesis or a stimulation of release of secretory material. These data suggest that serotonin innervation could be precociously involved in the regulation of the merione SCO secretion.

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.

GABA uptake and phenotypic characteristics of the subcommissural ependymocytes of the semi-desertic rodent, Meriones shawi: correlation with serotoninergic innervation.

Many studies have emphasized species differences in the serotoninergic innervation and phenotypic characteristics of the subcommissural organ in mammals. The post-natal distribution patterns of serotonin-containing fibers, the onset of gamma-aminobutyric acid uptake, and glial markers have been studied in the subcommissural organ of the semi-desertic rodent, Meriones shawi, by using immunohistochemical and autoradiographic techniques. Abundant serotoninergic fibers can be observed in the subcommissural organ of the newborn Meriones, some of them running among the ependymocytes and reaching the apical part of this organ. During the first 2 post-natal weeks of development, the subcommissural organ displays a progressive increase of serotonin fiber density throughout the organ, including the apical part. The existence of a dense serotonin-containing basal plexus concomitantly with a high apical innervation in this organ is a specific characteristic of Meriones. Ependymocytes of this organ have the ability to take up gamma-aminobutyric acid at birth. This uptake decreases and completely disappears from the 2nd week. The reappearance of gamma-aminobutyric acid accumulation in ependymocytes of the adult subcommissural organ after destruction of the serotonin innervation by a neurotoxin (5-7 dihydroxytryptamine) suggests an inhibitory effect of the serotonin innervation on this accumulation. Immunohistochemical studies of the phenotype of the ependymocytes with respect to glial markers during ontogeny show the transitory expression of glial fibrillary acidic protein, the presence of vimentin and the absence of S100 protein expression. No correlation has been found between the serotonin innervation and the expression of the glial markers.

Skeletal deformities of the gilthead sea bream (Sparus aurata, L.): study of the subcommissural organ (SCO) and Reissner's fiber (RF).

A high incidence of lordotic curvatures has been detected in commercial cultures of Sparus aurata. We have studied juvenile and adult lordotic specimens to elucidate whether the subcommissural organ and its secretion, the Reissner's fiber, play any role in the development of this syndrome. Animals were X-radiographed and then the brain and spinal cord dissected out and processed for light microscopy. Adult lordotic fishes had a well developed swim-bladder whereas juvenile did not. The central canal of the spinal cord showed dramatic alterations, and an altered Reissner's fiber was always present. Our histochemical results suggested a hyperactivity of the subcommissural organ in lordotic fishes.

Tissue culture of bovine subcommissural organ.

Tissue of the secretory, glial subcommissural organ (SCO) of adult, male cattle was cultured in serum-free medium for 70 days in vitro. Only minor alterations in the histoarchitecture and the cytology of the explanted SCOs could be observed by light and electron microscopy. Light- and electron-microscopic immunocytochemical investigations with an antiserum raised against bovine SCO secretory proteins revealed intra- and extra-cellularly localized immunoreactive material in tissue sections of SCO explants cultured up to 69 days in vitro. An indirect competition enzyme-linked immunosorbent assay (ELISA) was developed to detect minor quantities of SCO secretory products. By means of this assay, approximately 35 ng RF protein per ml was detected in culture medium supernatants conditioned for 3 days in SCO tissue cultures at 3, 38, and 69 days in vitro. These studies demonstrate that the bovine SCO can maintain its secretory activity throughout long periods in vitro.

Developmental neuron-glia interactions: role of serotonin innervation upon the differentiation of the ependymocytes of the rat subcommissural organ.

The rat subcommissural organ (SCO), which forms the roof of the third ventricle is an adequate model to study certain mechanisms of neuron-glia interactions in vivo. The ependymocytes, the main component of the SCO, have a glial origin. They possess particular phenotypic characteristics: they accumulate [3H]GABA by a specific uptake mechanism, contain transitory GFAP during ontogenesis and do not express PS100; on the other hand they receive a 5HT input which forms typical synaptic contacts. This innervation is of particular interest to approach neuron-glia interactions during the differentiation. Studies of GABA uptake carriers during ontogenesis in SCO ependymocytes show a correlation between the onset of the 5HT innervation and the advent of the GABA uptake. Moreover, destruction of the 5HT innervation by a neurotoxin (5-7-dihydroxytryptamine), before its arrival at the SCO in newborn rat, inhibits the formation of the GABA uptake system and causes the expression of PS100 in adult SCO cells. On the other hand, the SCO of newborn rats transplanted to the fourth ventricle of an adult host rat had no capacity to take up GABA and expressed PS100 3 months after its transplantation. Finally, the SCO ependymocytes of species devoid of 5HT innervation (rabbit, mice) were unable to take up GABA and contain PS100. These data suggest that neuron-glia interactions are necessary for the advent of GABA uptake carriers and can control the expression of glial markers during ontogenesis in SCO ependymocytes.

Developmental expression of glial markers in ependymocytes of the rat subcommissural organ: role of the environment.

The rat subcommissural organ (SCO), principally composed of modified ependymocytes (a type of glial cell), is a suitable model for the in vivo study of glial differentiation. An immunohistochemical study of the ontogenesis of rat SCO-ependymocytes from embryonic day 13 to postnatal day 10 shows that these cells express transitory glial fibrillary acidic protein (GFAP) from embryonic day 19 until postnatal day 3. However, S100 protein (S100) is never expressed in the SCO-cells, contrasting with the ventricle-lining cells of the third ventricle, which contain S100 as early as embryonic day 17. Environmental factors could be responsible for the repression of GFAP and S100 in adult rats, because GFAP and S100 are observed in ependymocytes of SCO 3 months after being grafted from newborn rat into the fourth ventricle of an adult rat. Neuronal factors might be involved in the control of the expression of S100, since after the destruction of serotonin innervation by neurotoxin at birth, S100 can be observed in some SCO-ependymocytes of adult rats. On the other hand, GFAP expression is apparently not affected by serotonin denervation, suggesting the existence of several factors involved in the differentiation of SCO-cells.

Immunohistochemical demonstration of serotonergic and peptidergic nerve fibers in the subcommissural organ of the dog.

The distributional patterns of serotonin-, luteinizing hormone-releasing hormone (LHRH)-, oxytocin (OXT)- and vasopressin (VP)-immunoreactive nerve fibers were studied in the subcommissural organ (SCO) of the dog by use of the peroxidase-antiperoxidase technique. Abundant serotonergic and moderate numbers of peptidergic nerve fibers running toward the ventricular surface were observed among the cylindrical ependymal cells in the SCO of the dog. Concerning the distributional density of the peptidergic nerve fibers, VP-immunoreactive fibers displayed the highest and LHRH-immunoreactive fibers the lowest values. Most serotonergic and peptidergic fibers returned to the basal portion of the SCO after forming loops immediately beneath the ventricular surface of the ependymal layer. Serotonin-immunoreactive fibers often established a perivascular plexus around the blood vessels in the SCO. At the electron-microscopic level, after use of antiserum to serotonin dark immunoprecipitate was observed in large granular vesicles and the matrix surrounding small and large, clear vesicles and mitochondria; VP immunoreactivity was localized in the large granular vesicles. Serotonergic nerve fibers could be detected in the SCO of the newborn dog. Although the distributional density was in principle not different from that in the adult animal, individual fibers showed immature features such as growth cones and insufficiently swollen varicosities. After penetrating into the ventricle, in the newborn dog, a few serotonin-immunoreactive fibers ran for a relatively long distance on the ependymal surface.

Formation and maturation of axo-glandular synapses and concomitant changes in the target cells of the rat subcommissural organ.

Synapse formation and maturation in the subcommissural organ (SCO) of Wistar rats were studied from birth to the end of the first month. Modifications of the secretory ependyma were analyzed over the same period. On the 1st postnatal day, the large varicosities in contact with the SCO ependymocytes appeared immature (absence or low density of vesicular population, no synaptic membrane differentiation). The synaptic contacts were formed from the 3rd postnatal day, near the glandular cell nuclei (0.1 micron distance); progressively, the content of the axonal boutons and the pre- and post-synaptic specializations became similar to those of adults. From the 21st day on, the axo-glandular innervation was considered analogous to that in the adult. Using immunocytochemistry, it was found that the increase in the serotonin-immunoreactive fiber density in the whole organ was time-dependent. Light and electron microscopy demonstrated changes in the morphology of SCO ependymocytes during the first postnatal weeks, notably in the endoplasmic reticulum and content ot apical protrusions. On postnatal day 14, two types of ependymal cells, neonatal-like and adult-like, coexisted. The evolution of SCO ependymocytes coincided with the progressive onset and maturation of axo-glandular innervation taking place after birth.

Ontogenetical development of the chick and duck subcommissural organ. An immunocytochemical study.

The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day in incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Reissner's fiber (RF) serum as the primary antibody was the principal method used. In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferentiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 blood vessels penetrated the SCO, scarce hypendymal cells appeared, the first signs of ventricular release of IRM were noticed, appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct, day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 hypendymal cells proliferated, developed processes and migrated dorsally, ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. the duck SCO appears to follow a similar pattern of development.

Quantitative development of the subcommissural organ in hypothyroid mice.

The postnatal development of the global volume of the subcommissural organ (SCO) and of the karyometric changes of the ependymocytes in the SCO and the adjacent ventricle is studied in male albino mice aged from 25 to 160 days, and in a hypothyroid group treated with propylthiouracil with and without interruption of the treatment at 35 days. Hypothyroidism produces a decrease of the global volume of the SCO and of the nuclear size of the ependymocytes in the SCO and the adjacent ventricle.