Ontogenetical development of the chick and duck subcommissural organ

Summary The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day of 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 i) blood vessels penetrated the SCO, ii) scarce hypendymal cells appeared, iii) the first signs of ventricular release of IRM were noticed, iv) 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 i) hypendymal cells proliferated, developed processes and migrated dorsally, ii) 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.Supported by Grant I/60 935 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile. M.H. was recipient of a personal grant from JNO (29-5-54), which is gratefully acknowledged     

Light- and electron-microscopic immunocytochemical investigation of the subcommissural organ using a set of monoclonal antibodies against the bovine Reissner's fiber

Ten monoclonal antibodies (Mabs) against glycoproteins of the bovine Reissner's fiber (RF) have been used in a structural and ultrastructural immunocyto-chemical investigation of the bovine subcommissural organ (SCO) and RF. The SCO of other vertebrate species has also been studied. For comparison, polyclonal antibodies against bovine RF (AFRU) were used. The SCO and RF of ox, pig and dogfish and the SCO of dog, rabbit, rat and frog were submitted to light-microscopic immunocytochemistry using AFRU and Mabs. Postembedding ultrastructural immunocytochemistry was applied to sections of bovine SCO using AFRU and Mabs. Bovine SCO consists of ependymal and hypendymal cell layers, the latter being arranged as cell strands across the posterior commissure, or as hypendymal rosette-like structures. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with AFRU. Six Mabs showed the same staining pattern as AFRU, one Mab stained RF strongly and SCO weakly, two Mabs stained RF but not SCO, and, finally, one Mab (3B1) exclusively stained the apices of the ependymal and hypendymal cells. All Mabs recognized the SCO and RF of the pig. Two Mabs bound to the SCO of the dog. One Mab stained the SCO of the rabbit and another the SCO of the rat. The SCO of frog and dogfish were totally negative. Bovine SCO stained with AFRU, showed label in the rough endoplasmic reticulum (RER) and the secretory granules (SG) of the ependymal and hypendymal cells. The former, in the form of parallel cisternae, reticulum or concentric rings, was seen throughout all cytoplasmic regions. SG were abundant in the apical pole of the ependymal and hypendymal cells. Only one Mab showed a staining pattern similar to AFRU. Five Mabs showed strong reactions in the SG but weak labeling of the RER. Mab 3B1 showed the label confined to the SG only. Our results suggest that: (i) in the bovine tissue, some epitopes are present in both precursor and processed materials, whereas others are characteristic of mature glycoproteins present in SG and the RF; (ii) the bovine SCO secretes at least two different compounds present in ependymal and hypendymal cells: (iii) both compounds coexist in the same secretory granule; (iv) there are conserved, class-specific, and species-specific epitopes in the glycoproteins secreted by the SCO of vertebrates.     

Comparative immunocytochemical study of the subcommissural organ

Summary The subcommissural organs (SCO) of 76 specimens belonging to 25 vertebrate species (amphibians, reptiles, birds, mammals) were studied by use of the immunoperoxidase procedure. The primary antiserum was obtained by immunizing rabbits with bovine Reissner's fiber (RF) extracted in a medium containing EDTA, DTT and urea. Antiserum against an aqueous extract of RF was also produced. The presence of immunoreactive material in cell processes and endings was regarded as an indication of a possible route of passage. Special attention was paid to the relative development of the ventricular, leptomeningeal and vascular pathways established by immunoreactive structures.The SCO of submammalian species is characterized by (i) a conspicuous leptomeningeal connection established by ependymal cells, (ii) scarce or missing hypendymal cells, and (iii) a population of ependymal cells establishing close spatial contacts with blood vessels.The SCO of most mammalian species displays the following features: (i) ependymal cells lacking immunoreactive long basal processes, (ii) hypendymal secretory cells occurring either in a scattered arrangement or forming clusters, (iii) an occasional leptomeningeal connection provided by hypendymal cells, and (iv) in certain species numerous contacts of secretory cells with blood vessels. In the hedgehog immunoreactive material was missing in the ependymal formation of the SCO, but present in hypendymal cells and in the choroid plexuses. The SCO of several species of New-and Old-World monkeys displayed immunoreactive material, whereas that of anthropoid apes (chimpanzee, orangutan) and man was completely negative with the antisera used.Supported by Grant I/38 259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RR-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile.The authors wish to thank Mrs. Elizabeth Santibánez and Mr. Genaro Alvial for valuable technical cooperation, and Dr. P. Fernandez-Llebrez, University of Malaga, for providing the specimens of Natrix maura.     

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.C. Hoyo-Becerra and M.D. López-ávalos contributed equally to this study and should be considered as first authors. C. Hoyo-Becerra was the recipient of a predoctoral fellowship (PFPI) from the Ministerio de Educacion y Cultura (Spain). This work was supported by grants from DGICYT (BFI2003-03348; Spain) and FIS (01/0948; Spain), FIS (01-0948, PI021517; Spain) and ISCIII (red CIEN, nodo Fundación Carlos Haya).     

Reissner’s fiber formation depends on developmentally regulated factors extrinsic to the subcommissural organ

Reissners fiber (RF) is a threadlike structure present in the third and fourth ventricles and in the central canal of the spinal cord. RF develops by the assembly of glycoproteins released into the cerebrospinal fluid (CSF) by the subcommissural organ (SCO). SCO cells differentiate early during embryonic development. In chick embryos, the release into the CSF starts at embryonic day 7 (E7). However, RF does not form until E11, suggesting that a factor other than release is required for RF formation. The aim of the present investigation was to establish whether the factor(s) triggering RF formation is (are) intrinsic or extrinsic to the SCO itself. For this purpose, SCO explants from E13 chick embryos (a stage at which RF has formed) were grafted at two different developmental stages. After grafting, host embryos were allowed to survive for 6–7 days, reaching E9 (group 1) and E13 (group 2). In experimental group 1, the secretion released by the grafted SCOs never formed a RF; instead, it aggregated as a flocculent material. In experimental group 2, grafted SCO explants were able to develop an RF-like structure, similar to a control RF. These results suggest that the factor triggering RF formation is not present in the SCO itself, since E13 SCO secretion forms an RF in E13 brains but never develops RF-like structures when placed in earlier developmental environments. Furthermore, the glycoproteins released by implanted SCOs bind specifically to several structures: the apical portion of the mesencephalic floor plate and the choroid plexus of the third and fourth ventricles.C. Hoyo-Becerra and M. D. López-Avalos contributed equally to this study and should be considered as joint first authors. C. Hoyo-Becerra was the recipient of a predoctoral fellowship (PFPI) from the Ministerio de Ciencia y Tecnología (Spain). This work was supported by grants from DGICYT (BFI2003-03348; Spain) and FIS (01/0948; Spain), FIS (01–0948, PI021517; Spain) and ISCIII (red CIEN, nodo Fundación Carlos Haya).     

Complex-type glycoproteins synthesized in the subcommissural organ of mammals

Summary The secretory activity in the subcommissural organ (SCO) of the sheep and cow was examined by means of lectin histochemistry and cytochemistry. Among the various lectins tested, Concanavalin A (Con A) revealed glycoproteins rich in mannosyl residues in the rough endoplasmic reticulum of ependymal and hypendymal cells. One of these Con A-positive glycoproteins may represent the precursor of the specific secretory component elaborated in the SCO, giving rise to Reissner's fiber. Lens culinaris agglutinin (LCA) and Phaseolus vulgaris hemagglutinins (E-PHA and L-PHA), known to bind to oligosaccharides, as well as wheat-germ agglutinin (WGA) revealing neuraminic acid, labeled secretory granules located in the apical part of ependymal and hypendymal cells of ruminants, and also Reissner's fiber. Electron-microscopic visualization of WGA-positive material in the Golgi complex shows that complex-type glycoproteins are synthesized in the subcommissural organ of mammals. The electron-dense material is mainly secreted into the ventricular cavity and gives rise to Reissner's fiber. On the basis of lectin affinity for oligosaccharides, a structure of the complex-type oligosaccharide is proposed.     

The subcommissural organ expresses D<Subscript>2</Subscript>, D<Subscript>3</Subscript>, D<Subscript>4</Subscript>, and D<Subscript>5</Subscript> dopamine receptors

Dopamine receptors have been found in certain populations of non-neuronal cells in the brain, viz., discrete areas of ciliated ependyma and the ependymal cells of the choroid plexus. We have studied the presence of both tyrosine-hydroxylase-immunoreactive nerve fibers and dopamine receptors in the subcommissural organ (SCO), an ependymal brain gland that is located in the roof of the third ventricle and that secretes, into the cerebrospinal fluid, glycoproteins that aggregate to form Reissners fiber (RF). Antibodies against D₂, D₃, D₄, and D₅ dopamine receptors were used in immunoblots of bovine striatum, fresh SCO, and organ-cultured SCO, and in immunocytochemistry of the bovine, rat, and mouse SCO. Only a few tyrosine-hydroxylase fibers appeared to reach the SCO. However, virtually all the secretory ependymal and hypendymal cells of the SCO immunoreacted with antibodies against D₂, D₄, and D₅ receptors, with the last-mentioned rendering the strongest reaction, especially at the ventricular cell pole of the secretory ependymocytes, suggesting that dopamine might reach the SCO via the cerebrospinal fluid. The antibodies against the four subtypes of receptors revealed corresponding bands in immunoblots of striatum and fresh SCO. Although the cultured SCO displayed dopamine receptors, dopamine had no apparent effect on the expression of the SCO-spondin gene/protein or on the release of RF-glycoproteins (SCO-spondin included) by SCO explants, suggesting that dopamine affects the function(s) of the SCO differently from the secretion of RF-glycoproteins.Financial support was provided by grants PI 030756 and Red CIEN, Instituto de Salud Carlos III, Spain (to J.M.P.F.), and 1030265 from Fondecyt, Chile (to E.M.R.)     

Reduced subcommissural organ glycoprotein immunoreactivity precedes aqueduct closure and ventricular dilatation in H-Tx rat hydrocephalus

The H-Tx rat has fetal-onset hydrocephalus associated with closure of the cerebral aqueduct and a reduction in the secretory cells of the subcommissural organ (SCO), a circumventricular organ situated in the dorsal wall of the cerebral aqueduct. The objective of this study was to determine the role of the SCO in hydrocephalus pathogenesis. Serial brain sections through aqueduct regions containing the SCO from H-Tx rats, together with non-hydrocephalic Fischer F344 rats, were studied at E16, before hydrocephalus onset, at E17, the beginning of onset, and at P0 when the hydrocephalus was overt. Tissues were immunostained by AFRU, an antibody against the SCO glycoprotein, and for the intermediate filament nestin. The area of SCO cells with AFRU immunostaining and the severity of lateral ventricle dilatation were quantified by image analysis. At E16 all fetuses had distinct SCO ependymal cells, open aqueducts and normal lateral ventricles. The H-Tx fetuses fell into two groups with large areas and small areas of AFRU immunoreactivity, all with a full complement of SCO cells. By E17, fetuses with small areas of immunoreactivity had reduced numbers of tall SCO secretory cells, and most had aqueducts closed posteriorly and dilated ventricles. Three additional fetuses with small areas of immunoreactivity had narrow but patent aqueducts and normal ventricles, and another had an open aqueduct and dilated ventricles. At P0, pups previously identified as hydrocephalic had small areas of AFRU immunoreactivity, an aqueduct that was closed anteriorly but open posteriorly, ventricular dilatation, and an absence of SCO secretory cells. The aqueduct even when closed was lined by typical ependymal cells throughout. Decreased nestin immunostaining accompanied the SCO changes. It is concluded that reduced SCO glycoprotein immunoreactivity precedes both aqueduct closure and expansion of the lateral ventricles in the H-Tx rat.Funding was provided by the National Institutes of Health (NS40359). K.C.S. was supported by the University of Florida Scholars Program and Sigma Xi Grants-in-Aid     

Spatial and structural interrelationships between secretory cells of the subcommissural organ and blood vessels

Summary In 76 specimens (amphibians, reptilians, mammals) belonging to 25 different vertebrate species, the region of the subcommissural organ (SCO) was investigated with the use of a primary antiserum raised against an extract of bovine Reissner's fiber+the immunoperoxidase procedure according to Sternberger et al. (1970).In the SCO of a toad (Bufo arenarum) and several species of reptiles (lacertilians, ophidians, crocodilians), the ependymal cells were the only type of secretory cell displaying vascular contacts, whereas in mammals ependymal and hypendymal cells established intimate spatial contacts with blood vessels. In Bufo arenarum, but especially in the reptilian species examined, the ependymo-vascular relationship was exerted by a population of ependymal cells having a rather constant location within the SCO and projecting to capillaries that showed a remarkably constant pattern of anatomical distribution. In the SCO of mammals the modality and degree of the structural relationships between secretory cells and blood vessels varied greatly from species to species. In the SCO of the armadillo and dog the secretory tissue was organized as a thick, highly vascularized layer with most of the cells oriented toward the capillaries. A rather opposite situation was found in the SCO of New-and Old-World monkeys, where vascular contacts were restricted to a few ependymal cells.Supported by Grant I/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidal Austral de Chile     

Light- and electron-microscopic investigation of the rat subcommissural organ grafted under the kidney capsule,with particular reference to immunocytochemistry and lectin histochemistry

Summary There is increasing evidence that, in the rat, a serotonin-mediated neural input may have an inhibitory influence on the secretory activity of the subcommissural organ (SCO). In the present investigation the rat SCO was studied 7, 30 and 90 days after transplantation under the kidney capsule, an area devoid of local serotonin-containing nerves. The grafted tissue was examined by use of immunocytochemistry employing a series of primary antisera, lectin histochemistry and transmission electron microscopy. The grafted SCO survived transplantation and contained, in addition to secretory ependymal and hypendymal SCO-cells, also elements immunoreactive with antisera against glial fibrillary acidic protein or S-100 protein. In transplants, SCO-cells produced a material displaying the characteristic immunocytochemical and lectin-binding properties of SCO-cells observed under in-situ conditions. The ependymal cells lined 1–3 small cavities, which contained secretory material. A fully developed structural equivalent of Reissner's fiber was, however, never found. The immunocytochemical and ultrastructural study of the grafted SCO showed an absence of nerve fibers within the graft and suggested a state of enhanced secretory activity. A network of protruding basal lamina structures connected the secretory cells to the newly formed capillaries revascularizing the SCO. One week after transplantation, long-spacing collagen started to appear in expanded areas of such laminar networks and also in the perivascular space. It is suggested (i) that the formation of long-spacing forms of collagen is triggered by factors provided by the SCO-secretory cells, and (ii) that secretory material of the ependymal and hypendymal cells may reach the reticular extensions of the basal lamina. In contrast to the SCO in situ, the grafted SCO-cells showed a positive immunoreaction for neuron-specific enolase. They became surrounded by a S-100-immunoreactive glial sheath that separated them from other transplanted cell types and the adjacent kidney tissue of the host.Supported by Grant I/63 476 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grants 187 and 0890/88 from Fondo Nacional de Desarrollo Cientifico y Tecnológico, Chile, and Grant S-85-39 from the Directión de Investigaciones, Universidad Austral de Chile. The authors wish to acknowledge the valuable help of Ms. Elizabeth Santibañez and Mr. Genaro Alvial (Valdivia) and Ms. Inge Lyncker (Giessen)     

The rat SCO responsiveness to prolonged water deprivation: implication of Reissner's fiber and serotonin system

The osmotic stress is a potent stimulus that can trigger several peripheral as well as central impairments. The brain is a vulnerable target of the osmotic stress and particularly circumventricular organs (CVOs) regarding their strategic localization as sensory organs of biochemical changes in the blood and cerebrospinal fluid circulations. The subcommissural organ (SCO) is a CVO which releases doubly in the CSF and blood circulation a glycoprotein called Reissner's fiber (RF) that has been associated to several functions including electrolyte and water balances. The present work was aimed on the assessment of the secretory activity of the SCO and its serotoninergic innervation following 2 weeks of total water restriction in Wistar rat. Using the immunohistochemistry of RF and serotonin (5HT), our data showed a significant overall reduction of RF immunoreactivity within both ependymal and hypendymal cells of the SCO of dehydrated rats compared to their corresponding controls, this decrease was concomitant with an enhancement of fibers 5HT immunoreactivity in the SCO as well as in the classical ependyma and in the dorsal raphe nucleus (DRN), constituting the origin of this innervation. The present findings support the possible involvement of the SCO in the response to prolonged water deprivation by decreasing its secretory materials which may result from either a direct peripheral hormonal control and/or the consequence of the enhanced 5HT innervation of the SCO.     

Concanavalin A-binding glycoproteins in the subcommissural and the pineal organ of the sheep (Ovis aries)

Summary Glycoproteins rich in mannosyl or glucosyl residues were analyzed in the subcommissural organ (SCO) and the pineal organ of the sheep (Ovis aries). By use of concanavalin A labelled with fluorescein isothiocyanate, fluorescent material was found both in ependymal and hypendymal cells of the SCO. In the pineal organ, either isolated or grouped parenchymal cells showed a marked fluorescence. These cells may correspond to ependymal elements also called interstitial cells or supporting cells. In addition, scarce slender, fluorescent processes were observed in the pineal parenchyma. The techniques of electrophoresis and electrotransfer on nitrocellulose paper have been applied to analyze the glycopeptide content of the SCO and the pineal organ in comparison to cerebellar and cerebral fractions solubilized by use of Triton X 100. Approximately 30 different concanavalin A-reactive glycopeptides were revealed in each fraction. In the SCO extract four glycopeptides (30, 54, 72, 100 kd) might correspond to subunits of the glycoprotein(s) characteristically stored in the ependymal cells of the SCO. In addition, two glycopeptides (32/33, 115 kd) are specific to the pineal organ extract. The possible similarity of the concanavalin A-reactive material in both organs is discussed and a putative secretory activity of the pineal ependymal cells is postulated.     

Studies on the subcommissural organ of Salmo gairdneri

The light microscopic analysis of serial sections of the subcommissural organ (SCO) of the rainbow trout (Salmo gairdneri) shows that the form of the groove-like (in cross section) organ varies over its total length. Its rostral origin is a tunnel-like structure anterior to the orifice of the hollow pineal stalk. The SCO forms the dorsal wall of the brain. Caudally the SCO is increasingly displaced from the surface of the brain by the fibers of the posterior commissure; the organ ends in a tabular area beyond the latter. The orifice of the pineal stalk is surrounded by the ependyma of the SCO that invaginates like a funnel and joins with the ependyma of the pineal stalk after a considerable narrowing. The rudimentary parapineal organ is located on the left side of the brain and is connected with the left habenular ganglion through the parapineal tract. It contacts the third ventricle with a short channel within the ependyma of the SCO. The histological organization of the ependymal and hypendymal cells of the SCO is typical of teleosts. Secretory material is located basally and apically in relation to the nucleus, but there is no indication of a basal secretory release. Basal ependymal processes terminate with broadened endings at the membrana limitans externa. The apical product is discharged into the third ventricle, where it aggregates into the thread-like structure of Reissner's fibre. The SCO cells have no direct contact with cerebral or meningeal blood vessels.     

The secretion of the subcommissural organ

Summary The secretion of the subcommissural organ (SCO) has been studied immunocytochemically by use of the following antisera: (1) antiserum against an aqueous extract of bovine Reissner's fiber (RF), (anti-RF-DC antiserum); (2) antiserum against the protein fraction F₁ obtained by gel electrophoresis of the aqueous RF-extract (RF-DF₁-antiserum); (3) antiserum against the protein fraction F₂ prepared in the same manner (RF-DF₂-antiserum). As shown by immunological and/or immunocytochemical experiments in bovines and rats, the three antisera are of high specificity, i.e., react exclusively with the secretion of the SCO, which appears to be a unique product of the vertebrate organism. Concerning the distribution of the reaction products within the SCO-cells, no differences were found light microscopically after use of the RF-DC-antiserum, the RF-DF₁-antiserum, or the RF-DF₂-antiserum.Comparative studies were carried out with the RF-DC-antiserum only. A positive immunocytochemical reaction of the SCO-secretion was obtained in many vertebrate species (mammals, birds, reptiles, amphibians, bony fishes, sharks, and cyclostomes). RF gives a positive reaction in mammals only; to date RF of non-mammalian vertebrates did not react immunocytochemically with the present antiserum. Comparative immunocytological studies have shown that (1) the SCO-cells of the ependymal layer as well as the SCO-cells of the hypendymal layer contain immunoreactive material, (2) in the majority of vertebrates hypendymal structures are more common than has been previously supposed, and (3) RF or constituents of this structure are produced by the SCO. The immunocytochemical studies have led to the impression that the SCO-secretion is not only discharged into the cerebrospinal fluid, but also in hypendymal vessels and/or leptomeningeal spaces, as has been postulated previously by Oksche and others (for review, see Oksche 1969).Supported by grants from the Ministry for Science and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. M. Eigenbrod, Mrs. D. Naumann and Mrs. B. Wolff is gratefully acknowledgedDedicated to Professor Berta Scharrer on the occasion of her 75th birthday     

The subcommissural organ and the development of the posterior commissure in chick embryos

The subcommissural organ (SCO) is an ependymal differentiation located in the diencephalon under the posterior commissure (PC). SCO-spondin, a glycoprotein released by the SCO, belongs to the thrombospondin superfamily and shares molecular domains with axonal pathfinding molecules. Several lines of evidence suggest a relationship between the SCO and the development of the PC in the chick: (1) their close location to each other, (2) their differentiation at the same developmental stage in the chick, (3) the abnormal PC found in null mutants lacking an SCO and (4) the release by the SCO of SCO-spondin. By application of DiI crystals in the PC of chick embryos, we have identified the neurons that give rise to the PC. Labelling is confined to the magnocellular nucleus of the PC (MNPC). To gain insight into the role of the SCO in PC development, coculture experiments of explants of the MNPC region (MNPCr) from embryos at embryonic day 4 (E4) with SCO explants from E4 or E13 embryos have been performed and the neurite outgrowth from the MNPCr explants has been analysed. In the case of coculture of E4 MNPCr with E4 SCO, the number of neurites growing from the MNPCr is higher at the side facing the SCO. However, when E4 MNPCr and E13 SCO are cocultured, the neurites grow mostly at the side opposite to the SCO. These data suggest that, at early stages of development, the SCO releases some attractive or permissive molecule(s) for the growing of the PC, whereas at later stages, the SCO has a repulsive effect over neurites arising from MNPCr.     

Intraneural vascular response to defective development of the chick mesencephalon

The development of intraneural vessels was studied in response to an induced hypermorphosis of neural tissue inthe midbrains of 38 chick embryos ranging in age from three days through 14 days of incubation. The pattern of vascularization was compared with that of normal chick embryos at comparable stages of development. In the experimental embryos, the increase in mitotic figures along the ventricular borders of the mesencephalon is accompanied by the establishment of an endoneural plexus approximately one day earlier than is the case during normal vascularization of the midbrain. This plexus also penetrates more deeply and extensively into the ependymal layer. Surface vessels and intraneural vascular elements are dilated, and the cerebrospinal fluid contains varying amounts of blood released from large intraneural vessels which protrude into the ventricle. The most prominent cerebrovascular effects seem to occur between the fourth and eighth days of incubation. Thereafter, the cerebrovascular pattern becomes more normal except for relatively few isolated hemorrhagic areas.     

Analysis of the efferent projections of the lateral geniculate nucleus with special reference to the innervation of the subcommissural organ and related areas

In order to define central neurons projecting to the subcommissural organ (SCO) and to related areas in the postero-medial diencephalon, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the lateral geniculate nucleus of the rat. PHA-L-labelled neurons send axonal processes medially through the posterior thalamic nuclei and the posterior commissure to the other hemisphere. Branches of fibres originating from this projection form a plexus of nerve terminals in the underlying precommissural nucleus and in the nucleus of the posterior commissure. A small number of PHA-L-immunoreactive nerve fibres penetrate from the precommissural nucleus into the lateral part of the SCO. A few labelled fibres penetrate directly from the posterior commissure into the medial part of the caudal SCO. Most of the PHA-L-immunoreactive fibres occur in the hypendymal layer, although a few terminate near the ependymal cells of the organ. Many labelled fibres are found in the ventricular ependyma adjacent to the SCO, some fibres lying close to the ventricular lumen. These results were obtained only if the tracer was delivered into the intergeniculate leaflet of the lateral geniculate nucleus (IGL). The IGL innervates both the suprachiasmatic nucleus and the pineal organ; the connections between the IGL and the midline structures, including the SCO, suggest that these areas are influenced by the circadian system.     

Chick embryo culture using duck egg shell--first successful hatch

The suitability of duck egg shell (DES) for chick embryo culture was investigated. Chick embryos were transferred into DESs with all egg contents after 3 days of normal incubation and cultured. The vessels made of polyethylene cling film were used for shell-less control. Among 35 embryos cultured in DESs, 21 survived until 16 days of incubation (13 days after transfer) and finally 3 newly hatched chicks were obtained at 22 days of incubation. One of them died 4 days later, but remaining two became full-grown cocks showing normal body weight and production of fertile sperms. Among 37 embryos cultured in polyethylene vessels, none survived over the period of 19 days of incubation. It is suggested that DES culture system may be useful for the various experiments using chick embryos.     

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 Reissners 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. Financial support was provided by grants 1030265 from Fondecyt, Chile, to E.M.R. and 201.035.002-1.0 DIUC to H.M.