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.     

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.     

Aspects of fine structure and function of the subcommissural organ of the embryonic chick

The fine structure of the subcommissural organ (SCO) of the embryonic chick has been studied. No evidence of secretory release was obtained. The SCO seems not to be involved in such previously suggested roles as water regulation or response to varying light cycles. The introduction of electron dense markers into the 3rd ventricle demonstrated rapid and massive uptake into coated pits and coated vesicles of the ependymal cells and its subsequent incorporation into terminal bodies. It is suggested that the uptake of macromolecules from the cerebrospinal fluid may be an important and hitherto unsuspected function of the SCO.     

Histochemical study of the subcommissural organ in chickens during development]

Some histochemical and particularly histoenzymological tests are performed on the subcommissural organ of chick embryos. A secretory activity appears about the 7th day. In 10 days old embryos and new hatched chicken the enzyme activities are of rather low intensity. Compared with the 10 days embryos, the newborn show some increase, but compared with the adult birds the activities remain weak. However the acid phosphatase activity is higher in the subcommissural organ than in the ependyma even in 10 days embryos.     

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     

Enzymatic organization of the subcommissural organ.

In the subcommissural organ (SCO) of the guinea pig, rat, golden hamster, and mouse the activity and distribution of enzymes related to the energy-supplying metabolism and of some marker enzymes of different cell organelles have been investigated by means of mostly modified histochemical methods. The results were compared with findings in the ciliated ependyma of the ventricular wall and with those in the ependyma of the choroid plexus of the third ventricle. In the ependymal part of the SCO only a moderate activity of hexokinase is observed in its specialized columnar cells whereas a high activity is present both in the ciliated ependyma and the choroid plexus. - The staining pattern of glucose-6-phosphatase is similar to that of hexokinase but this enzyme is found is the SCO only. - Likewise hexokinase, glycogen granules and enzymes related to glycogen metabolism (phosphoglucomutase, uridine-diphosphoglucose pyrophosphorylase, glycogen synthetase and phosphorylase) are regularly found most numerous and active in the nuclear and supra-nuclear area of the ependymal part. These enzymes are less active in both the other ependymal regions. - Uridine-diphosphoglucose dehydrogenase could not be demonstrated in the SCO. The NADP-linked enzymes of the pentose phosphate shunt, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, show a moderate activity which decreases also from the nuclear towards the apical area of the ependymal cells of the SCO. Enzymes of the glycolytic pathway, such as glucosephosphate isomerase, fructose-6-phosphate kinase, fructose-I,6-diphosphate aldolase, glyceraldehyde-3-phosphate and lactate dehydrogenase, are highly active in the SCO and are located mainly in the supranuclear area, too. Fructose-1,6-diphosphatase could not be demonstrated thus indicating that in the SCO the pathway is most probably only glycolytic but not gluconeogenetic. Compared to the ependyma of the ventricular wall and of the choroid plexus, in the SCO the M type subunits of lactate dehydrogenase predominate. Glycolytic enzymes are also very active in the choroid plexus but less in the ciliated ependyma. Compared to the ciliated ependyma and especially to the ependyma of the choroid plexus, the activities of enzymes which are only present in mitochondria (NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, NAD-linked malate dehydrogenase after preextraction, cytochrome oxidase, 3-hydroxybutyrate and glycerolphosphate and glutamate dehydrogenase) are relatively low. Mitochondria are accumulated near the superior pole of the nuclei as well as in the most apical part of the ependymal cells. - The staining pattern of NADP-linked isocitrate and malate dehydrogenase as well as of NADH dehydrogenase suggests that these enzymes are localized both in and out of mitochondria. The extramitochondrial activity of the first two enzymes might be localized in the cytosol. The extramitochondrial activity of NADH dehydrogenase might be localized in the endoplasmic reticulum...     

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.     

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.     

Lectin histochemistry of the human fetal subcommissural organ

The subcommissural organ (SCO) of 7 human fetuses, 3 to 6.5 months old, was investigated by means of: (i) immunocytochemistry employing three different antisera against secretory products extracted from the bovine SCO and Reissner's fiber; (ii) lectin binding using concanavalin A (Con A; affinity: mannose, glucose), wheat-germ agglutinin (WGA; affinity: N-acetyl-glucosamine, sialic acid), and Limax flavus agglutinin (LFA; affinity: sialic acid). Sections of bovine SCO were processed simultaneously and examined for comparative purposes. The human fetal SCO displayed lectin-binding properties identical to those in the SCO of other mammals. Thus, Con A-binding sites were restricted to abundant supranuclear structures that most likely corresponded to the rough endoplasmic reticulum, but were missing from granules located in the apical cytoplasm. The latter secretory material was strongly WGA- and LFA-positive and formed a distinct zone in the most apical portion of the ependymal cells. In contrast, this type of reactivity was missing in the adjacent cells of ependyma proper. In the bovine SCO, LFA-positive granules were also aggregated in an apical layer. The secretory material in the bovine SCO, especially its apical granular component, was strongly immunoreactive with the three antisera used; the human fetal SCO, however, lacked this immunoreactivity. It is postulated that the SCO of human fetuses secretes glycoproteins with a carbohydrate chain similar to--and a protein backbone different from--the secretions elaborated by the SCO of other vertebrate species.     

Immunocytochemical investigation of the subcommissural organ in the rat

Summary [¹⁴C]2-deoxyglucose uptake by neurons located in the octavo-lateralis complex of adult flatfish is asymmetrical on the two sides of the brain. It appears that the neuronal activity on the side oriented upward is higher than that on the side of the brain facing downward. This finding may be significant with respect to the mechanisms of metamorphosis of flatfish and may account for the peculiar fact that these animals swim on one body side during adult live.     

Histochemical investigations on the human fetal subcommissural organ

Summary A histochemical and ultrastructural study was carried out on subcommissura organs from 42 human embryos and fetuses in order to characterize some large granulesTypical granules make their appearance in the rostral hypendymal region of the subcommissural organ (SCO) in fetuses of about 50 mm CRL. Although they appear in other SCO-regions later, the highest number of granules is always located towards the pineal gland.Typical granules are of spherical shape with a diameter of about 2 microns. The various histochemical reactions reveal a reactivity which differentiates the shell of the granules from the granule interior. Nucleoproteins are present in the shell together with phospholipids and/or lipoproteins. The interior of the granules can contain different materials such as glycogen or lipid or neurosecretory substance. Ultrastructural observations show that a granule consists of whorls of endoplasmic reticulum sparsely studded with ribosomes surrounding an interior containing either lipid or lipoprotein inclusions, large amounts of glycogen or simply cytoplasm.It is suggested that the concentric lamellar organelle (CLO) is a morphological entity that might be involved in secretory processes rather than being the secretory granules themselves.This work was supported by a grant from Statens almindelige Videnskabsfond, Copenhagen.     

Histochemical investigations on the human foetal subcommissural organ

Summary A histochemical investigation was carried out on subcommissural organs from 28 human foetuses with crown-rump lenths ranging from 28 mm to 167 mm. The human foetal subcommissural organ (SCO) consists of a characteristic, high columnar epithelium covering the anterior-inferior surface of the posterior commissure.Histochemical reactions provide clear cut evidence that the cells of SCO contain glycoproteins as well as abundant amounts of glycogen. A very strong activity was found for cystine, tyrosine, tryptophan and arginine. Based on reactions for nucleoproteins different cell types were described.A remarkable activity of alkaline phosphatase clearly delineated the borders of the SCO. The localization of the reaction products corresponded to the outlines of the plasma membranes. A strong nodular cytoplasmic activity of acid phosphatase and of AS-esterase was found.Special attention was paid to an accumulation of hyaluronic acid and chondroitin-4-sulphate and/or chondroitin-6-sulphate in a perivascular position. The extended perivascular space was discussed in relation to the blood-brain barrier. It was suggested that the human foetal SCO is very active and that this activity has to do with an exchange of neurohormones from the SCO to the blood and in addition that absorptive and secretory functions might be carried out between the cerebro-spinal fluid and the SCO.     

Vascular and leptomeningeal projections of the subcommissural organ in reptiles

Summary In the snake, Natrix maura, and the turtle, Mauremys caspica, the basal processes of the ependymal cells of the subcommissural organ project toward the local blood vessels and the leptomeninges. These processes and their endings were studied using aldehyde-fuchsin (AF), periodicacid Schiff (PAS), periodic-acid silver-methenamine (PASM), concanavalin A (ConA), wheat germ agglutinin (WGA), immunoperoxidase staining (employing an antiserum against bovine Reissner's fiber; AFRU), and conventional transmission electron microscopy. For the purposes of comparison, the ventricular cell pole was also analyzed. The secretory material located in the ventricular cell pole and that present in ependymal endings had only a few staining properties in common, i.e., affinity for AF, ConA, and AFRU at a dilution of 1:1000. On the other hand, PAS, PA-SM, WGA, and AFRU at a dilution of 1:200 000 stained the apical (ventricular) secretory material but not the secretory material of the ependymal processes. The histochemical features of the secretory material located in the terminals of ependymal processes, as well as the presence at these sites of numerous rough-endoplasmic-reticulum cisternae and secretory granules, suggest that secretory material may by synthesized in these terminals. The probable fate of this material, i.e., release to the perivascular and leptomeningeal spaces or transport to the ventricular cell pole, is discussed.This work was partially supported by grants from the Stiftung Volkswagenwork, Federal Republic of Germany (1/38259), from the Dirección de Investigaciones, Universidad Austral de Chile (S-85-39), and from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile (6027; all to E.M.R.)     

Electron microscopic and cytochemical studies of the mouse subcommissural organ

Summary In mice most of the ependymal cells of the subcommissural organ (SCO cells) are densely packed with dilated cisternae of the endoplasmic reticulum (ER) containing either finely granular or flocculent materials. The well developed supra-nuclear Golgi apparatus consists of stacks of flattened saccules and small vesicles; the two or three outer Golgi saccules are moderately dilated and exhibit numerous fenestrations; occasional profiles suggesting the budding of coated vesicles and formation of membrane-bound dense bodies from the ends of the innermost Golgi saccules are seen. A few coated vesicles and membrane-bound dense bodies of various sizes and shapes are also found in the Golgi region.The contents of the dilated ER cisternae are stained with periodic acid-silver methenamine techniques. In the Golgi complex the two or three inner saccules are stained as deeply as the dense bodies, and the outer saccules are only slightly stained. The stained contents of ER cisternae are more electron opaque than those of the outer but less opaque than those of the inner Golgi saccules and the dense bodies.Acid phosphatase activities are localized in the dense bodies, some of the coated vesicles in the Golgi region, and in the one or two inner Golgi saccules.On the basis of these results the following conclusions have been reached: (1) In mouse SCO cells the finely granular and the flocculent materials in the lumen of ER cisternae contain a complex carbohydrate(s) which is secreted into the ventricle to form Reissner's fiber; (2) the secretory substance is assumed to be synthesized by the ER and stored in its cisternae, and the Golgi apparatus might play only a minor role, if any, in the elaboration of the secretory material; (3) most of the dense bodies in the mouse SCO cells are lysosomal in nature instead of being so-called dark secretory granules.Sponsored by the National Science Council, Republic of China.     

The surface fine structure of the bovine subcommissural organ

The bovine subcommissural organ was studied by using scanning electron microscopy. The most prominent findings was the existence of protruded and dilated endings of the ependymal cells. The majority of these cells were ciliated with two or more cilia; only a few unciliated cells were seen. Some pore-like structures were also seen on the surface. From the functional point of view, the most interesting finding was an amorphous heterogeneous material on the subcommissural ependyma. Especially in the caudal part of the organ this material accumulated in abundance. No real filamentous structures such as Reissner's fibre could be seen, however, it was assumed that the heterogeneous material corresponds to this formation. No supraependymal neurones were demonstrated.