Uncovering the role of the subcommissural organ in early brain development through transcriptomic analysis.

BACKGROUND: The significant role of embryonic cerebrospinal fluid (eCSF) in the initial stages of brain development has been thoroughly studied. This fluid contains crucial molecules for proper brain development such as members of the Wnt and FGF families, apolipoproteins, and retinol binding protein. Nevertheless, the source of these molecules remains uncertain since they are present before the formation of the choroid plexus, which is conventionally known as the primary producer of cerebrospinal fluid. The subcommissural organ (SCO) is a highly conserved gland located in the diencephalon and is one of the earliest differentiating brain structures. The SCO secretes molecules into the eCSF, prior to the differentiation of the choroid plexus, playing a pivotal role in the homeostasis and dynamics of this fluid. One of the key molecules secreted by the SCO is SCO-spondin, a protein involved in maintenance of the normal ventricle size, straight spinal axis, neurogenesis, and axonal guidance. Furthermore, SCO secretes transthyretin and basic fibroblast growth factor 2, while other identified molecules in the eCSF could potentially be secreted by the SCO. Additionally, various transcription factors have been identified in the SCO. However, the precise mechanisms involved in the early SCO development are not fully understood. RESULTS: To uncover key molecular players and signaling pathways involved in the role of the SCO during brain development, we conducted a transcriptomic analysis comparing the embryonic chick SCO at HH23 and HH30 stages (4 and 7 days respectively). Additionally, a public transcriptomic data from HH30 entire chick brain was used to compare expression levels between SCO and whole brain transcriptome. These analyses revealed that, at both stages, the SCO differentially expresses several members of bone morphogenic proteins, Wnt and fibroblast growth factors families, diverse proteins involved in axonal guidance, neurogenic and differentiative molecules, cell receptors and transcription factors. The secretory pathway is particularly upregulated at stage HH30 while the proliferative pathway is increased at stage HH23. CONCLUSION: The results suggest that the SCO has the capacity to secrete several morphogenic molecules to the eCSF prior to the development of other structures, such as the choroid plexus.

Congenital hydrocephalus associated with abnormal subcommissural organ in mice lacking huntingtin in Wnt1 cell lineages.

Huntingtin (htt) is a 350 kDa protein of unknown function, with no homologies with other known proteins. Expansion of a polyglutamine stretch at the N-terminus of htt causes Huntington's disease (HD), a dominant neurodegenerative disorder. Although it is generally accepted that HD is caused primarily by a gain-of-function mechanism, recent studies suggest that loss-of-function may also be part of HD pathogenesis. Huntingtin is an essential protein in the mouse since inactivation of the mouse HD homolog (Hdh) gene results in early embryonic lethality. Huntingtin is widely expressed in embryogenesis, and associated with a number of interacting proteins suggesting that htt may be involved in several processes including morphogenesis, neurogenesis and neuronal survival. To further investigate the role of htt in these processes, we have inactivated the Hdh gene in Wnt1 cell lineages using the Cre-loxP system of recombination. Here we show that conditional inactivation of the Hdh gene in Wnt1 cell lineages results in congenital hydrocephalus, implicating huntingtin for the first time in the regulation of cerebral spinal fluid (CSF) homeostasis. Our results show that hydrocephalus in mice lacking htt in Wnt1 cell lineages is associated with increase in CSF production by the choroid plexus, and abnormal subcommissural organ.

Subdivisions of chick diencephalic roof plate: implication in the formation of the posterior commissure.

The subcommissural organ (SCO) is a roof plate differentiation located in the caudal diencephalon under the posterior commissure (PC). A role for SCO and its secretory product, SCO-spondin, in the formation of the PC has been proposed. Here, we provide immunohistochemical evidence to suggest that SCO is anatomically divided in a bilateral region positive for SCO-spondin that surrounds a negative medial region. Remarkably, axons contacting the lateral region are highly fasciculated, in sharp contrast with the defasciculated axons of the medial region. In addition, lateral axon fascicles run toward the midline inside of tunnels limited by the basal prolongations of SCO cells and extracellular SCO-spondin. Our in vitro data in collagen gel matrices show that SCO-spondin induces axonal growth and fasciculation of pretectal explants. Together, our findings support the idea that SCO-spondin participates in the guidance and fasciculation of axons of the PC.

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.

Polarized expression of integrin beta1 in diencephalic roof plate during chick development, a possible receptor for SCO-spondin.

The roof plate of the caudal diencephalon is formed by the posterior commissure (PC) and the underlying secretory ependyma, the subcommissural organ (SCO). The SCO is composed by radial glial cells bearing processes that cross the PC and attach to the meningeal basement membrane. Since early development, the SCO synthesizes SCO-spondin, a glycoprotein that shares similarities to axonal guidance proteins. In vitro, SCO-spondin promotes neuritic outgrowth through a mechanism mediated by integrin beta1. However, the secretion of SCO-spondin toward the extracellular matrix that surrounds the PC axons and the expression of integrins throughout PC development have not been addressed. Here we provide immunohistochemical evidence to suggest that during chick development SCO cells secrete SCO-spondin through their basal domain, where it is deposited into the extracellular matrix in close contact with axons of the PC that express integrin beta1. Our results suggest that SCO-spondin has a role in the development of the PC through its interaction with integrin beta1.

Neuropeptide signaling and hydrocephalus: SCO with the flow.

Congenital hydrocephalus affects 0.1-0.3% of live births, with a high mortality rate (approximately 50%) in the absence of surgical intervention. Although the insertion of shunts alleviates the symptoms of the majority of congenital cases, the molecular basis of hydrocephalus and the mechanisms of cerebrospinal fluid (CSF) circulation remain largely unknown. Two important players are the subcommissural organ/Reissner's fiber (SCO/RF) complex and the ventricular ependymal (vel) cells that together facilitate the flow of the CSF through the narrow canals of the ventricular system. In this issue of the JCI, Lang et al. demonstrate that overexpression of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC1) receptor gene results in abnormal development of the SCO and vel cells, leading to congenital hydrocephalus (see the related article beginning on page 1924). The ligand for the PAC1 receptor is the neuropeptide PACAP, which uncovers what the authors believe to be a novel role for this signaling cascade in the regulation of CSF circulation.

The secretory material of the subcommissural organ of the chick embryo. Characterization of a specific polypeptide by two-dimensional electrophoresis.

The subcommissural organ (SCO) is a cerebral gland that releases into the cerebrospinal fluid a carbohydrate-rich glycoprotein which condenses to form Reissner's fiber (RF). Western blots from two-dimensional gel electrophoresis were stained with lectins (Concanavalin-A, wheat germ agglutinin) and anti-bovine RF serum to identify the secretory products of the chick embryo SCO. Immunohistochemical investigations showed that the anti-bovine RF serum reacted exclusively with the secretion of the SCO. Comparative protein patterns of SCO, pineal organ and cerebral hemisphere extracts allowed us to characterize a specific polypeptide in the SCO electrophoretic profiles. The polypeptide was a highly acid compound (isoelectric point of 4.7) with a high molecular weight (390 kDa). On Western blots only this component was immunoreactive with the RF antiserum and it exhibited an affinity for the two lectins. On the basis of these results, this polypeptide may be considered as a specific component of the secretory material synthesized by the SCO cells of the chick embryo.

Placental expression of SCO-spondin during mouse and human development.

During mammalian development, the placenta is a transitory but indispensable structure for a harmonious gestation involving several biological processes, such as adhesion, differentiation, apoptosis or cellular guidance. Nevertheless, the molecular pathways implicated during the placentation are still not totally understood. We previously described, the subcommissural organ (SCO)-spondin, a member of the 'thrombospondin' super-family, which is strongly expressed during mammalian central nervous system development. This extra-cellular matrix glycoprotein shows a unique arrangement of several conserved domains, including thrombospondin type 1 repeats, low-density lipoprotein receptor type A domains, two epidermal growth factor-like domains, and N- and C-terminal von Willebrand factor cysteine-rich domains. The presence of these domains strongly suggests the SCO-spondin involvement in cellular events occurring during placental development and physiology. In order to define this new role of SCO-spondin during development, we demonstrated its expression at relevant steps of gestation in human and mouse placenta, using RT-PCR, immunohistochemistry and Western-blot experiments. These data initiate further insights into the molecular and genetic functions of the neuronal gene SCO-spondin during trophoblastic and more globally during placental physiology and development.

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.

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.

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.

Ontogenesis of the secretory epithelium of the bovine subcommissural organ. A histofluorescence study using lectins and monoclonal antibodies.

A spatio-temporal analysis of the differentiation of a group of specialized (secretory) ependymal cells in the subcommissural organ (SCO) of the brain was undertaken in the bovine using a monoclonal antibody (C1B8A8) which is specific of the secretory process in this organ. In addition, lectins (concanavalin agglutinin (Con A), Lens culinaris agglutinin (LCA), wheat germ agglutinin (WGA), and Phaseolus vulgaris agglutinin (PHA] were used to analyse the maturation of the carbohydrate moieties of the secretory product (subcommissuralin). Monoclonal antibody NC-1 specific to a complex carbohydrate epitope including a terminal 3-sulfoglucuronyl residue similar to HNK-1 was also tested to compare the reactivity of the SCO with that of other brain structures. These cells express a specific antigen related to the known secretory activity of the SCO during early embryogenesis (2 months). This antigen is recognized by C1B8A8 antibody and by Con A suggesting that high mannose-type glycoproteins are synthesized at this stage. Later on (approximately 3.5 months), appearance of C1B8A8, WGA, LCA, L- and E-PHA-positive material in the apical lining of the ependymal cells, close to the ventricular cavity, suggests that maturation of the complex-type glycoproteins (Asn-linked) occurs at this stage. Presence of secretory material in the CSF and Reissner's fibre could be detected using the same probes at a stage of 4 months. As early as 2 months NC-1-positive material was detected in the ependyma of the mesencephalic roof, while no reaction occurred in the SCO epithelium. This suggests that the carbohydrate moieties of subcommissuralin is different from that of ependymins beta and gamma. Using specific monoclonal antibodies, molecular characterization of subcommissuralin and experimental analyses on its accurate role in brain development will further our tentative comparison with ependymins. The secretory ependymal cells in the SCO express a particular phenotype and could represent an increasing model to study cell differentiation in the brain.

Radial secretory glia conserved in the postnatal vertebrate brain: a study in the rat.

Secretory glial cells in the roof of the last diencephalic prosomer, ependymocytes and hypendymocytes, form the subcommissural organ. The cells of this complex were labelled immunocytochemically, using an antiserum against their specific secretory products. The study aims at the characterization of this cell type in the rat as an anatomical model situation. Radially oriented secretory glial cells remain after birth behind the posterior commissure in the mesencephalic aqueduct. At about postnatal day 10, the cell bodies descend into the conventional ependyma and at postnatal day 25 they assume a compact, rounded appearance. The secretory product they release is involved in the formation of Reissner's fiber. This differentiation in phenotype is not accompanied by a change of the intermediate filament expression. In the adult rat these cells had been labelled immunopositive for cytokeratins 8 and 18 as well as vimentin but not for glial fibrillary acidic protein. DiI-marking from the third ventricle and from the dorsal surface of the brain shows that the basal processes of ependymocytes and hypendymocytes project to the external and internal glial limiting membrane, respectively, through the commissural fiber bundles. Also the subependymal located hypendymocytes have apical processes with contacts to the cerebrospinal fluid. When this secretory cell population is studied with respect to cyto-architectonical changes during ontogeny the results lead to a new understanding of the subcommissural cells. They are not specialized ependymal cells in a regionally restricted and secondary differentiated ependymal area, but rather descendants of an ontogenetically ancient, specific type of radial glia. Characteristic features for all subcommissural cells are that they: (1) appear very early during ontogeny, (2) are derived from a radial oriented glial cell type, (3) carry at least one kinocilium, (4) possess an original intermediate filament pattern, (5) release a secretory product.

The subcommissural organ and the development of the posterior commissure.

Growing axons navigate through the developing brain by means of axon guidance molecules. Intermediate targets producing such signal molecules are used as guideposts to find distal targets. Glial, and sometimes neuronal, midline structures represent intermediate targets when axons cross the midline to reach the contralateral hemisphere. The subcommissural organ (SCO), a specialized neuroepithelium located at the dorsal midline underneath the posterior commissure, releases SCO-spondin, a large glycoprotein belonging to the thrombospondin superfamily that shares molecular domains with axonal pathfinding molecules. Several evidences suggest that the SCO could be involved in the development of the PC. First, both structures display a close spatiotemporal relationship. Second, certain mutants lacking an SCO present an abnormal PC. Third, some axonal guidance molecules are expressed by SCO cells. Finally, SCO cells, the Reissner's fiber (the aggregated form of SCO-spondin), or synthetic peptides from SCO-spondin affect the neurite outgrowth or neuronal aggregation in vitro.

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.

Molecular cloning and early expression of chick embryo SCO-spondin.

SCO-spondin is a multidomain glycoprotein secreted by the subcommissural organ (SCO). It belongs to the thrombospondin type 1 repeat superfamily and has been identified in several vertebrate species. We report the cloning of the chick SCO-spondin ortholog and examine its temporal and spatial expression during early embryogenesis from Hamburger and Hamilton (HH) stage 12 to HH stage 21. Chick SCO-spondin cDNA contains a long open reading frame encoding a predicted protein of 5255 amino acids. Northern blot analysis has revealed SCO-spondin mRNA as a band of about 15 kb. Many conserved domains have been identified, including 27 thrombospondin type 1 repeats, 13 low-density lipoprotein receptor type A domains, one EMI domain (a cysteine-rich domain of extracellular proteins), three von Willebrand factor type D domains, and one cystine knot C-terminal domain. Whole-mount in situ hybridization enabled the first signal of mRNA expression to be detected at HH stage 17, exclusively in a thin area of the prosencephalon roof plate. During the following stages of development, SCO-spondin expression remained restricted to this region. The multidomain structure of SCO-spondin and its early expression suggest that it plays a role in developmental processes in the central nervous system.

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

Reissner's 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 E 9 (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.

The early development of the human subcommissural organ.

The human subcommissural organ appears in the second month of intrauterine life, in a 27 mm embryo, later than the posterior commissure and concurrently with the pineal gland.