The central nervous system (CNS) develops from the neural tube, a hollow structure filled with embryonic cerebrospinal fluid (eCSF) and surrounded by neuroepithelial cells. Several lines of evidence suggest that the eCSF contains diffusible factors regulating the survival, proliferation, and differentiation of the neuroepithelium, although these factors are only beginning to be uncovered. One possible candidate as eCSF morphogenetic molecule is SCO-spondin, a large glycoprotein whose secretion by the diencephalic roof plate starts at early developmental stages. In vitro, SCO-spondin promotes neuronal survival and differentiation, but its in vivo function still remains to be elucidated. Here we performed in vivo loss of function experiments for SCO-spondin during early brain development by injecting and electroporating a specific shRNA expression vector into the neural tube of chick embryos. We show that SCO-spondin knock down induces an increase in neuroepithelial cells proliferation concomitantly with a decrease in cellular differentiation toward neuronal lineages, leading to hyperplasia in both the diencephalon and the mesencephalon. In addition, SCO-spondin is required for the correct morphogenesis of the posterior commissure and pineal gland. Because SCO-spondin is secreted by the diencephalon, we sought to corroborate the long-range function of this protein in vitro by performing gain and loss of function experiments on mesencephalic explants. We find that culture medium enriched in SCO-spondin causes an increased neurodifferentiation of explanted mesencephalic region. Conversely, inhibitory antibodies against SCO-spondin cause a reduction in neurodifferentiation and an increase of mitosis when such explants are cultured in eCSF. Our results suggest that SCO-spondin is a crucial eCSF diffusible factor regulating the balance between proliferation and differentiation of the brain neuroepithelial cells.

Tanycytes are hypothalamic radial glia-like cells that form the basal wall of the third ventricle (3V) where they sense glucose and modulate neighboring neuronal activity to control feeding behavior. This role requires the coupling of hypothalamic cells since transient decreased hypothalamic Cx43 expression inhibits the increase of brain glucose-induced insulin secretion. Tanycytes have been postulated as possible hypothalamic neuronal precursors due to their privileged position in the hypothalamus that allows them to detect mitogenic signals and because they share the markers and characteristics of neuronal precursors located in other neurogenic niches, including the formation of coupled networks through connexins. Using wild-type (WT), Cx30^−/– and Cx30^−/–, Cx43^fl/fl:glial fibrillary acidic protein (GFAP)-Cre (double knockout, dKO) mouse lines, we demonstrated that tanycytes are highly coupled to each other and also give rise to a panglial network specifically through Cx43. Using the human GFAP (hGFAP)-enhanced green fluorescent protein (EGFP) transgenic mouse line, we provided evidence that the main parenchymal-coupled cells were astrocytes. In addition, electrophysiological parameters, such as membrane resistance, were altered when Cx43 was genetically absent or pharmacologically inhibited. Finally, in the dKO mouse line, we detected a significant decrease in the number of hypothalamic proliferative parenchymal cells. Our results demonstrate the importance of Cx43 in tanycyte homotypic and panglial coupling and show that Cx43 function influences the proliferative potential of hypothalamic cells.

Cerebrospinal fluid is a clear fluid that occupies the ventricular and subarachnoid spaces within and around the brain and spinal cord. Cerebrospinal fluid is a dynamic signaling milieu that transports nutrients, waste materials and neuroactive substances that are crucial for the development, homeostasis and functionality of the central nervous system. The mechanisms that enable cerebrospinal fluid to simultaneously exert these homeostatic/dynamic functions are not fully understood. SCO-spondin is a large glycoprotein secreted since the early stages of development into the cerebrospinal fluid. Its domain architecture resembles a combination of a matricellular protein and the ligand-binding region of LDL receptor family. The matricellular proteins are a group of extracellular proteins with the capacity to interact with different molecules, such as growth factors, cytokines and cellular receptors; enabling the integration of information to modulate various physiological and pathological processes. In the same way, the LDL receptor family interacts with many ligands, including β-amyloid peptide and different growth factors. The domains similarity suggests that SCO-spondin is a matricellular protein enabled to bind, modulate, and transport different cerebrospinal fluid molecules. SCO-spondin can be found soluble or polymerized into a dynamic threadlike structure called the Reissner fiber, which extends from the diencephalon to the caudal tip of the spinal cord. Reissner fiber continuously moves caudally as new SCO-spondin molecules are added at the cephalic end and are disaggregated at the caudal end. This movement, like a conveyor belt, allows the transport of the bound molecules, thereby increasing their lifespan and action radius. The binding of SCO-spondin to some relevant molecules has already been reported; however, in this review we suggest more than 30 possible binding partners, including peptide β-amyloid and several growth factors. This new perspective characterizes SCO-spondin as a regulator of cerebrospinal fluid activity, explaining its high evolutionary conservation, its apparent multifunctionality, and the lethality or severe malformations, such as hydrocephalus and curved body axis, of knockout embryos. Understanding the regulation and identifying binding partners of SCO-spondin are crucial for better comprehension of cerebrospinal fluid physiology.

During early stages of development, encephalic vesicles are composed by a layer of neuroepithelial cells surrounding a central cavity filled with embryonic cerebrospinal fluid (eCSF). This fluid contains several morphogens that regulate proliferation and differentiation of neuroepithelial cells. One of these neurogenic factors is SCO-spondin, a giant protein secreted to the eCSF from early stages of development. Inhibition of this protein in vivo or in vitro drastically decreases the neurodifferentiation process. Other important neurogenic factors of the eCSF are low density lipoproteins (LDL), the depletion of which generates a 60% decrease in mesencephalic explant neurodifferentiation. The presence of several LDL receptor class A (LDLrA) domains (responsible for LDL binding in other proteins) in the SCO-spondin sequence suggests a possible interaction between both molecules. This possibility was analyzed using three different experimental approaches: 1) Bioinformatics analyses of the SCO-spondin region, that contains eight LDLrA domains in tandem, and of comparisons with the LDL receptor consensus sequence; 2) Analysis of the physical interactions of both molecules through immunohistochemical colocalization in embryonic chick brains and through the immunoprecipitation of LDL with anti-SCO-spondin antibodies; and 3) Analysis of functional interactions during the neurodifferentiation process when these molecules were added to a culture medium of mesencephalic explants. The results revealed that LDL and SCO-spondin interact to form a complex that diminishes the neurogenic capacities that both molecules have separately. Our work suggests that the embryonic cerebrospinal fluid is an active signaling center with a complex regulation system that allows for correct brain development.

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. Developmental Dynamics 239:2584–2593, 2010.

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 β1. 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 β1. Our results suggest that SCO‐spondin has a role in the development of the PC through its interaction with integrin β1. Developmental Dynamics 238:2494–2504, 2009.