Bovine floor plate explants secrete SCO-spondin

SCO-spondin is a large-molecular mass glycoprotein, secreted by the subcommissural organ (SCO), which has been implicated in neuronal development during ontogeny of the central nervous system. The expression of SCO-spondin is not restricted to the SCO but it also occurs in the floor plate, a key structure participating in neuronal differentiation and patterning of the neural tube. It has been postulated that SCO-spondin detected in the floor plate is released into the lumen of the neural tube, but this new route of secretion of floor plate cells needs to be further substantiated. For this purpose, we standardized long-term organ culture of bovine floor plate and performed morphological, immunological, biochemical, and gene expression analyses. The study of floor plate explants and their conditioned media allowed us to demonstrate that: (1) organ-cultured floor plate cells are actively secretory for up to 25 days; (2) SCO-spondin gene is actively transcribed and translated by the cultured floor plate cells; (3) SCO-spondin is released into the culture medium via the apical cell pole; and (4) upon release, SCO-spondin does not aggregate in the conditioned medium but remains soluble. Furthermore, in the cultured floor plate cells, SCO-spondin may be secreted through a route bypassing the Golgi apparatus.     

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).     

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.     

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).     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos

Undissociated tissue explants of the retina and retinal pigment epithelium (RPE) of 3,5-, 4-, 5- and 8-day-old chick embryos were cultured in vitro. After 7 days in culture, lentoids were observed in explants of either retina or RPE from 3,5-, 4- and 5-day-old embryos. As demonstrated by immunohistochemistry, these lentoids contained specific chick lens proteins (alpha-, beta- and delta-crystallins). No crystallin-containing cells were found in eye tissue explants from 8-day-old embryos. However, when 5-bromo-deoxyuridine (25 microM) was introduced into the medium at the beginning of culturing (for 12 h), large eosinophilic cells containing alpha-, beta- and delta-crystallins were detected in retinal explants of the 8-day old embryos. Thus, retina and RPE of 3,5-5-day-old chick embryos are capable of lens differentiation after explantation in vitro without dissociation into individual cells. This capacity is lost during development.     

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.)     

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.     

Identification of HGF-like protein as a novel neurotrophic factor for avian dorsal root ganglion sensory neurons

HGF-like protein (HLP) is a member of the hepatocyte growth factor (HGF) family. Although HGF is shown to have neurotrophic activities on many of CNS and PNS neurons, the role of HLP in the nervous system is poorly understood despite the knowledge that Ron/HLP receptor is expressed in embryonic neurons. Here we show that HGF but not HLP promotes neurite extension and migration emanating from chick embryonic day 9 (E9) dorsal root ganglia (DRG) explants in the presence of low levels of NGF, however, HLP does promote neurite extension and cellular migration from E15 chick DRG explants with low levels of NGF. Ron-Fc, a chimeric molecule composed of the extracellular domain of Ron fused with immunoglobulin Fc, eliminated activities of HLP, such as cellular migration and long neurite extension emanating from E15 DRG explants in the presence of NGF, but did not eliminate short neurites. These results suggested that promotion of long neurite extension and migration depends on activities of HLP through its receptor/Ron. Taken together, we propose that HLP may play an important role in chick sensory ganglia at relatively late stages of development. This is the first evidence that HLP functions as a neurotrophic factor.     

Induction of alpha- and beta-crystallin synthesis in organ cultures of the adenohypophyseal anlage of chickens as affected by 5-iododeoxyuridine and 5-bromodeoxyuridine

The effects of 5-iododeoxyuridine and 5-bromodeoxyuridine on differentiation of the cells of adenohypophysis rudiment from 3, 4, and 5 day old chick embryos were studied in the in vitro organ culture. On the 7th day of cultivation most explants from 3 and 4 day old embryos formed lentoids and individual cells with the lens phenotype among the adenohypophysis tissue. Alpha-, beta- and delta-crystalline were immunochemically detected in them. When cultivating explants from 5 day old embryos, no lentoids formed. But the immunochemical study of serial sections made it possible to detect in individual explants single alpha-crystalline-containing cells. There is a period in the development of chick adenohypophysis, which lasts five days of incubation and during which the adenohypophysis rudiment retained its capacity for lens differentiation despite the fact that it is already determined in the adenohypophysis direction.     

Effects of alkaline phosphatase inhibitors on chick neural retinal cell differentiation in vitro: ultracytochemical studies

In the matured chick retina, alkaline phosphatase (ALPase) activity is specifically localized in the outer plexiform layer and in horizontal and Müller cells. In the developing chick retina, ALPase activity is first recognized in growing neurites from horizontal cells during the 13th day of incubation, when synaptogenesis begins in the outer plexiform layer. Intraocular administration of ALPase inhibitors to developing chick embryos resulted in developmental disturbances in differentiation of the outer plexiform layer and also of photoreceptor cells. We have now extended these studies to an in vitro system. ALPase activity was studied by ultracytochemistry in cultured retinal cells from chick embryos, and the effects of specific ALPase inhibitor on retinal development were also analyzed. Two cell types showed intense ALPase activity: 1) flat glial cell, which formed a multi-layered epithelial sheet and 2) neuronal cell found within cell aggregates. Some cellular processes forming a neuropil-like structure within these aggregates also showed ALPase activity. When the ALPase inhibitor bromotetramisole was present in the culture medium, there was delay in aggregate formation and the development of neuritic processes was also affected. Moreover, this treatment also caused a considerable reduction in the number of photoreceptor cells present in the culture. The present results indicate that ALPase activity plays a significant role in retinal cell differentiation.     

Ontogenetical development of the chick and duck subcommissural organ

Summary The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day of incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Reissner's fiber (RF) serum as the primary antibody was the principal method used.In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferentiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 i) blood vessels penetrated the SCO, ii) scarce hypendymal cells appeared, iii) the first signs of ventricular release of IRM were noticed, iv) appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct; day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 i) hypendymal cells proliferated, developed processes and migrated dorsally, ii) ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. The duck SCO appears to follow a similar pattern of development.Supported by Grant I/60 935 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile. M.H. was recipient of a personal grant from JNO (29-5-54), which is gratefully acknowledged     

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.     

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.     

The development of chick spinal cord in tissue culture. I. Fragment cultures from embryos of various developmental stages

Explants from neural tube and spinal cord of chick embryos at developmental stages 8 through 36 were cultured on collagen-coated cover glasses for 21 days. The cultures of neural tube at stages 10 to 14 contained many neuronal precursor cells which gave rise to mature neurons. This was verified by cumulative labeling of cultures with tritiated thymidine. Explants from spinal cords of stages 26 and 27 contained fewer precursor cells, and at stage 36, only 7% of mature neurons were labeled. Regardless of the stage of development at which explants were made (stages 8 through 36), all cultures had a similar appearance after 21 days, indicating that cells from explants taken from earlier developmental stages (before neurons were formed) "caught up" with the explants from later developmental stages, which already had formed neurons at the time of explantation.     

Antisera to basal lamina and glial endfeet disturb the normal extension of axons on retina and pigment epithelium basal laminae

In order to determine the role of the extracellular matrix in regulating the directed growth of embryonic neurites, antisera to retina (a-RBL I and II), to pigment epithelium (a-PBL) and to glomerular (a-GBL) basal lamina were probed for an effect on the ordered extension of neurites. In the assays, retina explants from chick and quail were cultured on basal lamina from embryonic chick retina and pigment epithelium either in the presence of anti-basal lamina antisera or in the presence of the corresponding preimmune sera. In the presence of all anti-basal lamina antisera, normal extension of axons was greatly inhibited both on retina and on pigment epithelium basal lamina. The antisera affected the growth pattern and the morphology of the individual axons in two ways: in the presence of a-RBL I the short axons were less directed, developed more and longer side branches, and the lamellipodia of the growth cones were reduced in size compared to axons from control cultures. In the presence of a-RBL II and a-GBL, axons grew slowly out from the explants as very thick bundles, strikingly different from axons in control cultures. The antiserum to pigment epithelium basal lamina induced both strong fasciculation and disorganization of the linear fiber extension, being intermediate between the two types of effects observed after antiserum addition. The results suggest that adhesive matrix molecules in basal laminae have important functions in elongation, fasciculation and in the morphology of growing axons.     

Formation of cartilage in organ-cultured leg buds of chick embryo

When the leg buds from chick embryos at stages 17-23 were cultured as organ cultures in F12 medium, which contained chick serum and an extract of chick embryos, leg cartilage structures developed that included pelvic girdle, femur, tibiofibula, and tarsometatarsus. The proportion of explants with definite distal elements increased when the leg buds from later embryos were used. Supplementation with chick serum was essential. These results suggest that the pattern of formation of limbs in chick embryos can be studied in organ culture.     

Accumulation of amylase by chick pancreas in organ culture : Effect of hydrocortisone

Accumulation of amylase by pancreas explants from chick embryos of 7 to 14 days of development was studied in organ culture. The explants produced amylase but there was no increase in their total DNA and little if any increase in their total protein. Most of the amylase in the cultures was released into the culture medium. The lower the developmental age at which the pancreas was taken, the greater was the relative increase of amylase activity in the culture. After several days of culture the accumulation of amylase slowed or stopped. Hydrocortisone markedly increased the amount of amylase produced by the explants. The hormone had little or no effect on the initial rate of accumulation of amylase by the explants. However, addition of hydrocortisone to the culture resulted in a continuing increase of amylase activity when accumulation of the enzyme had ceased in the controls without added hormone. The observations support the hypothesis, suggested earlier, that corticosteroid hormones are implicated in the later stages of differentiation of the embryonic chick pancreas.