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).     

Reissner's fiber and the wall of the central canal in the lumbo-sacral region of the bovine spinal cord

Summary Reissner's fiber (RF) of the subcommissural organ (SCO), the central canal and its bordering structures, and the filum terminale were investigated in the bovine spinal cord by use of transmission electron microscopy, histochemical methods and light-microscopic immunocytochemistry. The primary antisera were raised against the bovine RF, or the SCO proper. Comparative immunocytochemical studies were also performed on the lumbo-sacral region of the rat, rabbit, dog and pig.At all levels of the bovine spinal cord, RF was strongly immunoreactive with both antisera. From cervical to upper sacral levels of the bovine spinal cord there was an increasing number of ependymal cells immunostainable with both antisera. The free surface of the central canal was covered by a layer of immunoreactive material. At sacral levels small subependymal immunoreactive cells were observed. From all these structures sharing the same immunoreactivity, only RF was stained by the paraldehyde-fuchsin and periodicacid-Schiff methods.At the ultrastructural level, ependymal cells with numerous protrusions extending into the central canal were seen in the lower lumbar segments, whereas cells displaying signs of secretory activity were principally found in the ependyma of the upper sacral levels. A few cerebrospinal fluid-contacting neurons were observed at all levels of the spinal cord; they were immunostained with an anti-tubulin serum.The lumbo-sacral segments of the dog, rat and rabbit, either fixed by vascular perfusion or in the same manner as the bovine material, did not show any immunoreactive structure other than RF.The possibilities that the immunoreactive ependymal cells might play a secretory or an absorptive role, or be the result of post-mortem events, are discussed.Supported by Grant I/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de ChileThe authors wish to thank Dr. Enrique Romeny from the Valdivia abattoir for kindly providing the bovine spinal cords     

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.     

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,massa caudalis and ampulla caudalis in the spinal cord of lamprey larvae (Geotria australis)

Summary The subcommissural organ (SCO), Reissner's fiber (RF) and its massa caudalis of lamprey larvae (Geotria australis) were investigated immunocytochemically by use of an antiserum raised against bovine RF as primary antibody. The affinities of RF and massa caudalis for Ricinus communis agglutinin I (RCA) with and without previous acid hydrolysis, concanavalin A (Con A), wheat-germ agglutinin (WGA), aldehyde fuchsin, and PAS reaction were also studied.SCO and massa caudalis were strongly immunoreactive, whereas RF proper was distinctly negative. RF did not react with Con A and RCA. Only the periphery of RF was WGA-positive. RCA showed affinity for RF only after acid hydrolysis. RF was homogeneously stained by the aldehyde-fuchsin and PAS-methods. At variance with RF proper, the periphery of the massa caudalis reacted with RCA without previous acid hydrolysis, but its core was WGA-positive and reacted with RCA only after hydrolysis. It is suggested that (i) RF has a coat of glycoproteins containing sialic acid as terminal residue, whereas the massa caudalis possesses a coat with galactose as terminal residue; (ii) in RF proper and the massa caudalis the spatial arrangement of glycoproteins might be different.Routine transmission electron-microscopic observations indicate that in larvae of Geotria australis an open communication exists between the ampulla caudalis and blood capillaries via large cavities or lacunae.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile, and Grant 6027 from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile     

The terminal organ of the subcommissural complex of chordates: definition and perspectives

The subcommissural organ (SCO) exhibits anatomical characteristics of an endocrine organ: The secretion is released either into the blood (hypendymal capillaries) or the CSF of the 3rd ventricle; excretory ducts are absent; the active secretory activity of the ependymal cells can be regulated by humorally transmitted messages or by neural input. The rate of production of the Reissner's fibre (RF) by the SCO is rather fast, and the secretory material is stored in the ampulla caudalis (AC) and must be continuously discharged accordingly. Structures jointly involved in depletion of the AC and the decomposition and removal of the massa caudalis (MC) are collectively called the terminal organ (TO). The TO of the SCO-complex is formed by an assemblage of different structures in the caudal segment of the spinal cord (neurogenic part) and in the tissues (non-neurogenic part) which encompass this part of the cord. The different parts of the TO are characterized, even at the cellular level, by specializations which support the discharge as well as the dissolution of the material of the MC. The RF may be a detoxicator for the CSF, but also a carrier of hormonally active substances. In this case the TO is a site of release of hormones. The function of the entire complex is still under discussion, particularly its role in endocrine integration.     

Alterations of the cerebrospinal fluid proteins and subcommissural organ secretion in the arterial hypertension and ventricular dilatation. A study in SHR rats

The aim of this work was to analyze the proteins in the cerebrospinal fluid (CSF) of spontaneously hypertensive rats, to study their possible role in the relationship between hydrocephalus, arterial hypertension and alterations in the subcommissural organ. Brains from control Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) sacrificed with chloral hydrate were used. Antiserums against some cerebrospinal fluid protein bands and Reissner's fiber (RF) were used for immunohistochemical study of the SCO. Ventricular dilation was observed in the lateral and third ventricle of the SHR. Third ventricle ependyma showed immunoreactive material (IRM) for antibody against 141 kDa protein band anti-B1 and 117 protein band anti-B2 and the SCO of the SHR showed a decrease of the IRM when compared with WKY rats. An alteration in the expression of anti-RF was found to compare the SCO of the WKY and SHR groups. Our results demonstrate that hydrocephalus and hypertension are interconnected in this kind of rat which produce alterations in SCO secretions and some proteins of the CSF.     

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.     

Discontinuity of primary and secondary neural tube in spina bifida induced by retinoic acid in mice

This report shows by light microscopy the appearance of secondary neurulation separated from primary neurulation and its developmental fate in the spinal cord of mice exposed to retinoic acid in utero. The embryos and fetuses were derived from pregnant mice (ICR strain) given 60, 40, or 0 mg/kg of retinoic acid in olive oil on day 8 of gestation orally and killed 1, 2, or 10 days later. Separation of the primary neural fold from the secondary neural tube was seen in 9- and 10-day-old embryos: the caudal part of the neuroepithelium of the primary neural fold was disarranged with non-closed posterior neuropore, and underneath it the secondary neural tissue extended caudally with abnormal notochord. At term, fetuses showed spina bifida, including myeloschisis, myelocele, and diplomyelia (diastematomyelia) with abnormal distribution of ganglionic cells. These cord lesions were located between the third lumbar and second coccygeal levels. The former two cord anomalies were associated with diplomyelia and split the dorsal and ventral portions of the spinal cord with an overlapping zone between the third lumbar and third sacral levels. These findings suggest that the separation from primary neurulation is due to the lesions in both primary neural folds and notochord induced by retinoic acid and that the spinal cord caudal to the third lumbar level originates from both neuroectoderm and mesenchyme-like cells while that caudal to the third sacral level originates from mesenchyme-like cells only.     

Neuroblastoma B104 cell line as a model for analysis of neurite outgrowth and neuronal aggregation induced by Reissner's fiber material

The subcommissural organ (SCO) is a specialized ependymal structure of the brain that secretes glycoproteins into the cerebrospinal fluid (CSF), which condense to form a thread-like structure - Reissner's fiber (RF). The effects of soluble material released by RF were examined on neuroblastoma B104 cells grown in serum-free medium, using "low-density" and "high-density" culture systems. In the presence of soluble RF material, low-density cultures were suitable for analysis of the enhanced neurite outgrowth of B104 cells, while high-density cultures allowed the increased B104 cell aggregation to be examined. RF-induced neuronal aggregation and neuritic outgrowth were restricted to a perimeter around the RF. This standardized cell culture system reproduced in part the effects observed previously with primary cortical and spinal cord cell cultures and may serve the analysis of the mechanisms leading to aggregation and neurite outgrowth. In the present study, we analyzed variations in the rate of neural cell adhesion molecules, such as N-CAM and N-cadherin, induced by soluble RF material in high-density cultures.     

Bovine Reissner’s fiber (RF) and the central canal of the spinal cord: an immunocytochemical study using a set of monoclonal antibodies against the RF-glycoproteins

The subcommissural organ secretes N-linked complex-type glycoproteins into the cerebrospinal fluid. These glycoproteins condense to form Reissner’s fiber (RF), which extends along the fourth ventricle and central canal of the spinal cord. A set of three monoclonal antibodies (Mabs 3E6, 3B1, and 2A5) has been obtained using these glycoproteins as immunogens. Competitive and sandwich enzyme-linked immunoassay methods have demonstrated that the three monoclonal antibodies are directed against different epitopes, and that there is no competition among them for their binding to glycoproteins of RF. Mab 3E6 displays immunoblotting properties that are similar to those of a polyclonal antibody against the pool of glycoproteins from RF, but that are different from those of Mabs 3B1 and 2A5. All three antibodies immunostain the bovine subcommissural organ and RF. A population of ependymal cells is stained by the polyclonal antibody, and Mabs 2A5 and 3E6, but not by Mab 3B1. The material present in a population of ependymal cells of the central canal, and the glycoproteins secreted by the subcommissural organ thus probably have partial chemical identity. Some evidence suggests that the immunoreactive ependymal cells are secretory cells. The luminal surface of the central canal is coated by a thin layer of material with immunocytochemical characteristics different from those of the ependymal cells; such a coat may correspond to material released from RF. Received: 19 December 1995 / Accepted: 30 April 1996     

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.     

Distribution of FMRFamide-like immunoreactivity in the brain and neurohypophysis of the lamprey,Lampetra japonica

Summary Distribution of molluscan cardio-excitatory tetrapeptide Phe—Met—Arg—Phe—NH₂ (FMRFamide) was determined by means of immunohistochemistry in the brain and neurohypophysis of the lamprey, Lampetra japonica. Many FMRFamide-like immunoreactive neurons were found in the periventricular nuclear region and in a region near the mammillary recess. Neurons situated in the former region were larger. The immunoreactive cell groups were shown to be located at sites differing from those of the AF-positive cell groups. The fibers of immunoreactive neurons extended in all directions within the brain and towards the spinal cord, some reaching the third ventricle and capillaries. Thus, FMRFamide-like immunoreactive peptides appear to function as neurotransmitters or neuromodulators and possibly also as neurohormones. FMRFamide-like immunoreactive material was rarely observed in the posterior neurohypophysis (neural lobe), but was noted to be present to a limited extent in the caudal part of the anterior neurohypophysis (median eminence). It would thus follow that FMRFamide-like immunoreactive neurons may not necessarily be related to the hypothalamo-neural lobe system, but may possibly be associated with the hypothalamoadenohypophysial system. The pineal body showed no FMRFamide-like immunoreactivity.     

The developmental potentials of the caudalmost part of the neural crest are restricted to melanocytes and glia

The avian spinal cord is characterized by an absence of motor nerves and sensory nerves and ganglia at its caudalmost part. Since peripheral sensory neurons derive from neural crest cells, three basic mechanisms could account for this feature: (i) the caudalmost neural tube does not generate any neural crest cells; (ii) neural crest cells originating from the caudal part of the neural tube cannot give rise to dorsal root ganglia or (iii) the caudal environment is not permissive for the formation of dorsal root ganglia. To solve this problem, we have first studied the pattern of expression of ventral (HNF3beta) and dorsal (slug) marker genes in the caudal region of the neural tube; in a second approach, we have recorded the emergence of neural crest cells using the HNK1 monoclonal antibody; and finally, we have analyzed the developmental potentials of neural crest cells arising from the caudalmost part of the neural tube in avian embryo in in vitro culture and by means of heterotopic transplantations in vivo. We show here that neural crest cells arising from the neural tube located at the level of somites 47-53 can differentiate both in vitro and in vivo into melanocytes and Schwann cells but not into neurons. Furthermore, the neural tube located caudally to the last pair of somites (i.e. the 53rd pair) does not give rise to neural crest cells in any of the situations tested. The specific anatomical aspect of the avian spinal cord can thus be accounted for by limited developmental potentials of neural crest cells arising from the most caudal part of the neural tube.     

Circulation of marker substances in the cerebrospinal fluid of an amphibian,Rana pipiens

Summary Solutions of fluorescein-labelled dextran or Evans blue-albumin were infused into the lateral cerebral ventricle of Rana pipiens. The subsequent distribution in the cerebrospinal fluid (CSF) was investigated between 2 and 24 h after infusion by freezing and examination of the cut blocks of the head and vertebral column of the stage of a freezing microtome. These marker substances move out of the ventricles into the subarachnoid space at the caudal end of the fourth ventricle and spread rapidly along the subarachnoid space of the spinal cord. The spreading of marker substances is slower into the brain subarachnoid space. When the marker is infused into the subarachnoid space of the forebrain, it becomes distributed throughout the subarachnoid space of the brain and spinal cord but not in the ventricles.Partial clearance of markers from the ventricles takes place within 5 h and total clearance within 8 h. Clearance from the brain and cord subarachnoid space is somewhat slower and can only be detected in experiments lasting 10 h or more. Absorption of the markers from the CSF occurs via the intervertebral foramina of the spinal cord. Fluorescence microscopy of sections of the cord show that the fluorescence leaves the subarachnoid space at the point where the spinal nerves traverse the arachnoid membrane.     

Secretory glycoproteins of the rat subcommissural organ are N-linked complex-type glycoproteins. Demonstration by combined use of lectins and specific glycosidases, and by the administration of Tunicamycin

Two experimental protocols were used to investigate the secretory glycoproteins of the subcommissural organ (SCO). Protocol I: Lectins, specific exoglycosidases and immunocytochemistry were sequentially applied to the same section or to adjacent semithin sections of the rat SCO fixed in Bouin's fluid and embedded in methacrylate. Lectins used: concanavalin A (con A), wheat germ agglutinin, Limulus polyphemus agglutinin, Ricinus communis agglutinin and Arachis hypogeae agglutinin. Glycosidases used: neuroaminidase, beta-galactosidase, alpha-mannosidase, alpha-glucosidase and beta-N-acetyl-glucosaminidase. For immunocytochemistry an antiserum against bovine Reissner's fiber (AFRU) was used. Lectins and glycosidases were used in sequences that allowed the cleaved sugar residue to be identified as well as that appearing exposed as a terminal residue. This approach led to the following conclusions: (1) the terminal sugar chain of the secreted glycoproteins has the sequence sialic acid-galactose-glucosamine-; (2) the con A-binding material present in the rough endoplasmic reticulum corresponds to mannose; (3) the apical secretory granules and Reissner's fibers displayed a strong con A affinity after removing sialic acid, thus indicating the presence of internal mannosyl residues in the secreted material; (4) after removing most of the sugar moieties the secretory material continued to be strongly immunoreactive with AFRU. Protocol II: Rats were injected into the lateral ventricle with Tunica-mycin and killed 12, 24, 50 and 60 h after the injection. The SCO of rats from the last two groups showed a complete absence of con A binding sites. The results from the two experiments confirm that the secretory glycoproteins of the rat SCO are N-linked complex-type glycoproteins with the conformation previously suggested (Rodríguez et al. 1986).     

Ectopic Wnt/beta-catenin signaling induces neurogenesis in the spinal cord and hindbrain floor plate

The most ventral structure of the developing neural tube, the floor plate (FP), differs in neurogenic capacity along the neuraxis. The FP is largely non-neurogenic at the hindbrain and spinal cord levels, but generates large numbers of dopamine (mDA) neurons at the midbrain levels. Wnt1, and other Wnts are expressed in the ventral midbrain, and Wnt/beta catenin signaling can at least in part account for the difference in neurogenic capacity of the FP between midbrain and hindbrain levels. To further develop the hypothesis that canonical Wnt signaling promotes mDA specification and FP neurogenesis, we have generated a model wherein beta-catenin is conditionally stabilized throughout the FP. Here, we unambiguously show by fate mapping FP cells in this mutant, that the hindbrain and spinal cord FP are rendered highly neurogenic, producing large numbers of neurons. We reveal that a neurogenic hindbrain FP results in the altered settling pattern of neighboring precerebellar neuronal clusters. Moreover, in this mutant, mDA progenitor markers are induced throughout the rostrocaudal axis of the hindbrain FP, although TH+ mDA neurons are produced only in the rostral aspect of rhombomere (r)1. This is, at least in part, due to depressed Lmx1b levels by Wnt/beta catenin signaling; indeed, when Lmx1b levels are restored in this mutant, mDA are observed not only in rostral r1, but also at more caudal axial levels in the hindbrain, but not in the spinal cord. Taken together, these data elucidate both patterning and neurogenic functions of Wnt/beta catenin signaling in the FP, and thereby add to our understanding of the molecular logic of mDA specification and neurogenesis.     

The Reissner's Fiber Termination in Some Lower Chordates

The caudal end of the neural tube of the tunicate Oikopleura, the cephalochordate Branchiostoma and newly hatched fry of the clupeiform teleosts Clupea, Engraulis and Sardinops was studied by means of the electron microscope. In Oikopleura and the teleost larvae either Reissner's fiber or an amorphous mass of fiber substance leaks out of the neural tube into the surrounding tissue spaces. In Branchiostoma the disintegrated fiber material is apparently engulfed by the caudal ependymal cells. A relationship seems to exist between the degree of fiber disintegration within the neural tube and the degree of specialization of the caudal neural tube ependymal cells, the two extremes being represented by Branchiostoma with a “closed” Reissner's fiber system with highly specialized caudal ependymal cells and a teleost fry with the intact fiber leaving the neural tube between almost undifferentiated ependymal cells. These observations on lower chordates are in accordance with the hypothesis that Reissner's fiber acts as a detoxicator for the neural tube fluid.     

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.