Effects of SCO-spondin thrombospondin type 1 repeats (TSR) in comparison to Reissner's fiber material on the differentiation of the B104 neuroblastoma cell line

SCO-spondin is a newly identified protein that is strongly expressed in the subcommissural organ (SCO), an ependymal differentiation of the brain. When released into the cerebrospinal fluid at the entrance to the Sylvian aqueduct, the glycoproteins condense and form a thread-like structure, Reissner's fiber (RF). To analyze the role of SCO-spondin on neuronal development, we studied the effects induced by an oligopeptide derived from a thrombospondin type 1 repeat (TSR) of SCO-spondin on neuroblastoma B104 cells and compared them with the effects of soluble RF material containing complete SCO-spondin proteins. In low density cell culture, the TSR peptide first induced a notable flattening of cells accompanied by increased neurite outgrowth. Grouping of these differentiated B104 cells, which later formed dense aggregates, was then observed with increasing time in culture. Soluble RF material induced similar morphological changes and neurite-promoting effects on B104 cells, although the cells remained evenly distributed throughout the culture time and no aggregates were visible. In high-density cell culture, both TSR peptide and RF material induced prominent neurite outgrowth and subsequent rapid cell aggregation. Whereas soluble RF material inhibited cell proliferation, no respective effect was observed in the presence of the TSR peptide. A direct interaction of TSR peptide and soluble RF material with a B104 cell binding site was revealed by increased B104 cell metabolic activity by flow cytometry.     

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.     

The Lengthening of a Giant Protein: When, How, and Why?

Subcommissural organ (SCO)-spondin is a giant glycoprotein of more than 5000 amino acids found in Vertebrata, expressed in the central nervous system and constitutive of Reissner’s fiber. For the first time, in situ hybridization performed on zebrafish (Danio rerio) embryos shows that the gene encoding this protein is expressed transitionally in the floor plate, the ventral midline of the neural tube, and later in the diencephalic third ventricle roof, the SCO. The modular organization of the protein in Echinodermata (Strongylocentrotus purpuratus), Urochordata (Ciona savignyi and C. intestinalis), and Vertebrata (Teleostei, Amphibia, Aves and Mammalia) is also described. As the thrombospondin type 1 repeat motifs represent an increasingly large part of the protein during Deuterostomia evolution, the duplication mechanisms leading to this complex organization are examined. The functional significance of the particularly well-preserved arrangement of the series of SCO-spondin repeat motifs and thombospondin type 1 repeats is discussed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.     

Regulation of the neural patterning activity of sonic hedgehog by secreted BMP inhibitors expressed by notochord and somites

The secretion of Sonic hedgehog (Shh) from the notochord and floor plate appears to generate a ventral-to-dorsal gradient of Shh activity that directs progenitor cell identity and neuronal fate in the ventral neural tube. In principle, the establishment of this Shh activity gradient could be achieved through the graded distribution of the Shh protein itself, or could depend on additional cell surface or secreted proteins that modify the response of neural cells to Shh. Cells of the neural plate differentiate from a region of the ectoderm that has recently expressed high levels of BMPs, raising the possibility that prospective ventral neural cells are exposed to residual levels of BMP activity. We have examined whether modulation of the level of BMP signaling regulates neural cell responses to Shh, and thus might contribute to the patterning of cell types in the ventral neural tube. Using an in vitro assay of neural cell differentiation we show that BMP signaling markedly alters neural cell responses to Shh signals, eliciting a ventral-to-dorsal switch in progenitor cell identity and neuronal fate. BMP signaling is regulated by secreted inhibitory factors, including noggin and follistatin, both of which are expressed in or adjacent to the neural plate. Conversely, follistatin but not noggin produces a dorsal-to-ventral switch in progenitor cell identity and neuronal fate in response to Shh both in vitro and in vivo. These results suggest that the specification of ventral neural cell types depends on the integration of Shh and BMP signaling activities. The net level of BMP signaling within neural tissue may be regulated by follistatin and perhaps other BMP inhibitors secreted by mesodermal cell types that flank the ventral neural tube.     

The metencephalic floor plate of chick embryos expresses two secretory glycoproteins homologous with the two glycoproteins secreted by the subcommissural organ

The nature and the function of the compounds secreted by the floor plate (FP) of the metencephalon are little known. The FP cells of the hindbrain react with antibodies (AFRU) against the glycoproteins secreted by the subcommissural organ (SCO). One of the these proteins, RF-Gly I, is a 540-kDa core glycosylated protein. The aims of the present investigation were to identify by immunoblot the AFRU-immunoreactive compound secreted by the FP of chick embryos, to establish temporal and regional patterns of this secretory activity, and to obtain information about the fate of these compounds. It was established that the SCO and FP of chick embryos secrete two AFRU-immunoreactive compounds of 540 and 230 kDa. The two compounds secreted by the FP have been designated as FP-Gly I and FP-Gly II. The expression of these proteins was circumscribed to the metencephalic FP, and occurred from HH 29 to HH 36. Within the FP cells, FP-Gly I and FP-Gly II were confined to the supranuclear and apical regions, which under the electron microscope displayed numerous cisternae of the rough endoplasmic reticulum and granules. Aggregates of AFRU-immunoreactive material appeared on the free surface of the FP. The possibility that FP-Gly I and FP-Gly II are released into the ventricle to reach distant targets is discussed.     

A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube.

Cell pattern in the ventral neural tube is organized by Sonic hedgehog (Shh) secreted by floor plate cells. To assay the range of direct Shh action, we developed a general method for blocking transduction of Hedgehog (Hh) signals through ectopic expression of a deleted form of the Hh receptor Patched (Ptc), termed Ptc(Deltaloop2). We validated this method in Drosophila and used mouse Ptc1(Deltaloop2) (mPtc1(Deltaloop2)) to block Shh transduction in the chick neural tube. mPtc1(Deltaloop2) expression caused cell-autonomous ventral-to-dorsal switches in progenitor identity and neuronal fate throughout the ventral neural tube, supporting a gradient mechanism whereby Shh acts directly and at long range. mPtc1(Deltaloop2) expression also caused the abnormal spread of Shh to more dorsal cells, indicating that Shh in the neural tube, like Hh in Drosophila, induces a feedback mechanism that limits its range of action.     

Immunocytochemical detection of Reissner's fiber-like glycoproteins in the subcommissural organ and the floor plate of wildtype and cyclops mutant zebrafish larvae

The subcommissural organ (SCO) and the floor plate (FP) secrete high molecular weight glycoproteins that polymerize in the form of the Reissner's fiber (RF). To study to what extent the absence of the FP affects the expression of these glycoproteins, we have investigated the brain and spinal cord of 48-h and 72-h wildtype and cyclops (cyc) mutant zebrafish larvae by using a polyclonal antiserum against bovine RF. Wildtype larvae showed immunoreactivity in the SCO at the dorsal forebrain-midbrain boundary. In the ventricle, over the SCO surface, thin immunoreactive fibers aggregated into an RF that ran along the third and fourth ventricles and the central canal of the spinal cord until, at its caudal end, the fiber disintegrated and formed a strongly immunoreactive massa caudalis that left the neural tube and invaded the surrounding tissues of the tail fin. The rostral end of the FP, lining the pontine flexure, was also strongly immunoreactive, as was the caudal third of the FP. Cyc mutants showed an immunoreactive SCO and fibrous material in the ventricle, but an RF was missing. There was no label in the ventral midline of the neural tube except in some specimens in which the caudal FP persisted and was immunoreactive. It is concluded that the product of the cyc gene is not required for the expression of SCO glycoproteins but for their polymerization into an RF in the brain ventricles.     

Discontinuous organization and specification of the lateral floor plate in zebrafish

The floor plate is a signaling center in the ventral neural tube of vertebrates with important functions during neural patterning and axon guidance. It is composed of a centrally located medial floor plate (MFP) and a bilaterally positioned lateral floor plate (LFP). While the role of the MFP as source of signaling molecules like, e.g., Sonic Hedgehog (Shh) is well understood, the exact organization and function of the LFP are currently unclear. Based on expression analyses, the one cell wide LFP in zebrafish has been postulated to be a homogenous structure. We instead show that the zebrafish trunk LFP is discontinuously arranged. Single LFP cells alternate with p3 neuronal precursor cells, which develop V3 interneurons along the anteroposterior (AP) axis. Our mutant analyses indicate that both, formation of LFP and p3 cells require Delta-Notch signaling. Importantly, however, the two cell types are differentially regulated by Hedgehog (HH) and Nkx2.2 activities. This implicates a novel mechanism of neural tube patterning, in which distinct cell populations within one domain of the ventral neural tube are differently specified along the AP axis. We conclude that different levels of HH and Nkx2.2 activities are responsible for the alternating appearance of LFP and p3 neuronal progenitor cells in the zebrafish ventral neural tube.     

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.     

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.     

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.)     

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.     

Metacercarial cyst formation in vitro of Echinostoma paraensei

Cercariae of Echinostoma paraensei Lie and Basch 1968 encysted normally in the presence of Biomphalaria glabrata embryo (Bge) cells in culture, partially in culture conditioned medium, and not at all in fresh culture medium alone. At the ultrastructural level the cyst is composed of 2 well defined regions. The outer cyst wall (OCW) is particulate to fibrous in nature, formed from secretory granules released from the cercarial tegument. Membranous scrolls or rodlets secreted from the subtegumental cystogenous gland cells are then added to this layer, forming the inner cyst wall (ICW). After 24 hr the cultured cyst is enclosed by a thin cellular capsule similar to that found around cysts in the snail host. The capsule also contains collagen fibers, not found elsewhere in Bge cell cultures.     

Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co-culture, large numbers of tyrosine hydroxylase (TH)-immunoreactive, catecholaminergic cells could be found underneath individual striatal slices. Cell counting revealed that up to 25.3% (average 16.1%) of the total number of cells in these areas were TH-positive, contrasting a few TH-positive cells (<1%) in non-induced areas. The presence of dopamine in the conditioned culture medium was confirmed by HPLC analysis. Interestingly, not all striatal slice cultures induced TH-expression in underlying hNS1 cells. Common to TH-inductive cultures was, however, the presence of degenerating, necrotic areas, suggesting that factors released during striatal degeneration were responsible for the dopaminergic induction of the hNS1 cells. Ongoing experiments aim to identify such factors by comparing protein profiles of media conditioned by degenerating (necrotic) versus healthy striatal slice cultures.     

Enhanced secretion of tissue plasminogen activator by simultaneous use of retinoic acid and ascorbic acid from tissue cultured gastroepiploic artery

Tissue-type plasminogen activator (tPA) is a key enzyme in the fibrinolysis system and the regulation of its expression has been extensively studied in cultured vascular endothelial cells. Many kinds of supplements including growth factors are needed, however, to keep endothelial cells viable, which leads the culture condition far from the physiological milieu. Using a new device of amorphous calcium phosphate coated culture plate, we succeeded in culturing ring-cut gastroepiploic artery in a basic medium of RPMI 1640 containing 10% fetal calf serum. The overall normal vessel architecture and the antigenicity of von Willebrand factor, tPA and plasminogen activator inhibitor type 1 (PAI-1) were retained for at least 9 days. tPA was constantly secreted into the conditioned medium at least up to day 12. Employing this organ culture technique, we analyzed the effects of two well-known profibrinolytic vitamins of retinoic acid (Vit. A) and ascorbic acid (Vit. C) on the release of tPA and PAI-1. The cultured artery responded well and the tPA secretion was enhanced by factors of 1.5 fold by Vit. A, 1.7 fold by Vit C and 3.2 fold by their combination, whereas none of these stimuli increased PAI-1 secretion. These results suggested that the cultured ring-cut artery retained functional endothelial cells for at least 9 days and was suitable in analyzing the regulatory mechanism of protein synthesis and secretion from the vascular wall. Using this method, vitamins A and C were shown to lead the intravascular condition to a profibrinolytic state.     

Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis

In vertebrates the neural tube, like most of the embryonic organs, shows discreet areas of programmed cell death at several stages during development. In the chick embryo, cell death is dramatically increased in the developing nervous system and other tissues when the midline cells, notochord and floor plate, are prevented from forming by excision of the axial-paraxial hinge (APH), i.e. caudal Hensen's node and rostral primitive streak, at the 6-somite stage ( Charrier, J. B., Teillet, M.-A., Lapointe, F. and Le Douarin, N. M. (1999). Development 126, 4771-4783). In this paper we demonstrate that one day after APH excision, when dramatic apoptosis is already present in the neural tube, the latter can be rescued from death by grafting a notochord or a floor plate fragment in its vicinity. The neural tube can also be recovered by transplanting it into a stage-matched chick embryo having one of these structures. In addition, cells engineered to produce Sonic hedgehog protein (SHH) can mimic the effect of the notochord and floor plate cells in in situ grafts and transplantation experiments. SHH can thus counteract a built-in cell death program and thereby contribute to organ morphogenesis, in particular in the central nervous system.     

Sonic hedgehog signaling at gastrula stages specifies ventral telencephalic cells in the chick embryo

A secreted signaling factor, Sonic hedgehog (Shh), has a crucial role in the generation of ventral cell types along the entire rostrocaudal axis of the neural tube. At caudal levels of the neuraxis, Shh is secreted by the notochord and floor plate during the period that ventral cell fates are specified. At anterior prosencephalic levels that give rise to the telencephalon, however, neither the prechordal mesoderm nor the ventral neural tube expresses Shh at the time that the overt ventral character of the telencephalon becomes evident. Thus, the precise role and timing of Shh signaling relevant to the specification of ventral telencephalic identity remains unclear. By analysing neural cell differentiation in chick neural plate explants we provide evidence that neural cells acquire molecular properties characteristic of the ventral telencephalon in response to Shh signals derived from the anterior primitive streak/Hensen's node region at gastrula stages. Exposure of prospective anterior prosencephalic cells to Shh at this early stage is sufficient to initiate a temporal program of differentiation that parallels that of neurons generated normally in the medial ganglionic eminence subdivision of the ventral telencephalon.     

Notochord grafts do not suppress formation of neural crest cells or commissural neurons.

Grafting experiments previously have established that the notochord affects dorsoventral polarity of the neural tube by inducing the formation of ventral structures such as motor neurons and the floor plate. Here, we examine if the notochord inhibits formation of dorsal structures by grafting a notochord within or adjacent to the dorsal neural tube prior to or shortly after tube closure. In all cases, neural crest cells emigrated from the neural tube adjacent to the ectopic notochord. When analyzed at stages after ganglion formation, the dorsal root ganglia appeared reduced in size and shifted in position in embryos receiving grafts. Another dorsal cell type, commissural neurons, identified by CRABP and neurofilament immunoreactivity, differentiated in the vicinity of the ectopic notochord. Numerous neuronal cell bodies and axonal processes were observed within the induced, but not endogenous, floor plate 1 to 2 days after implantation but appeared to be cleared with time. These results suggest that dorsally implanted notochords cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest-derived dorsal root ganglia.