The Subcommissural Organ and the Development of the Posterior Commissure

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The Subcommissural Organ and the Development of the Posterior Commissure Jesu´s M. Grondona,*,1 Carolina Hoyo-Becerra,*,† Rick Visser,*,‡ Pedro Ferna´ndez-Llebrez,*,2 and Marı´a Dolores Lo´pez-A´valos*,2 Contents 64 66

1. Introduction 2. Axon Guidance and the Development of Commissures 2.1. Importance of the midline as an intermediate target in axonal guidance 2.2. Glial structures in midline axonal guidance 2.3. Axon guidance molecules and its receptors 3. Posterior Commissure Development 3.1. Development of the tract of the posterior commissure 3.2. Nuclei that give rise to the posterior commissure 4. The Subcommissural Organ as a Specialized Ependyma 4.1. Structure and position in the brain 4.2. Secretory material of the subcommissural organ 4.3. Molecular features of the subcommissural organ secretory material 4.4. Sites of release of the secretory material 4.5. Ontogeny of the subcommissural organ 4.6. Classical functions of the subcommissural organ 5. SCO-Spondin as an Axonal Guidance Molecule 5.1. Similarities with other axon guidance molecules 5.2. In vitro activity of SCO-spondin on neurite outgrowth 5.3. Coculture experiments with SCO explants 5.4. SCO secretion in the floor plate

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* Departamento de Biologı´a Celular, Gene´tica y Fisiologı´a, Facultad de Ciencias, Universidad de Ma´laga, Ma´laga, Spain Department of Gastroenterology and Hepatology, University Hospital of Essen, Essen, Germany { Networking Research Center on Bioingeneering, Biomaterials and Nanomedicine (CIBER-BBN), Ma´laga, Spain 1 Corresponding author. E-mail: [email protected] 2 Both authors should be considered as last authors {

International Review of Cell and Molecular Biology, Volume 296 ISSN 1937-6448, DOI: 10.1016/B978-0-12-394307-1.00002-3

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2012 Elsevier Inc. All rights reserved.

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6. Expression of Axonal Guidance Molecules in the Subcommissural Organ 7. Subcommissural Organ-Posterior Commissure Alterations in Mutant Models 7.1. Pax6 mutant mice 7.2. Msx1 mutant mice 7.3. Other transgenic and mutant mice 8. Concluding Remarks Acknowledgments References

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Abstract 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 SCOspondin affect the neurite outgrowth or neuronal aggregation in vitro. Key Words: Subcommissural organ, Posterior commissure, Axonal guidance, SCO-spondin, CNS midline, Magnocellular nucleus, CNS development. ß 2012 Elsevier Inc.

1. Introduction Wiring of the developing nervous system occurs in a highly ordered way. This process depends on axonal pathfinding mechanisms that allow growing axons to find their final targets. Neuronal growth cones expose receptors that recognize environmental signals, the so-called axonal guidance cues that establish axonal pathways through which large axonal bundles develop. At early stages of development, the earliest axons that travel ahead and establish for the first time the route of a future tract are called pioneer axons and display peculiar characteristics (Easter et al., 1993). After the pioneer axons have formed the first scaffold, other axons follow such

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pathways and fasciculate with them to generate the tract (Van Vactor, 1998). In the long haul routes, axonal pathfinding progresses in sequential stages by means of regularly positioned sources of axon guidance molecules, referred to as intermediate targets. When axons have to cross to the opposite hemisphere, forming a commissure or a decussation, cell populations located at the midline of the brain represent important intermediate targets in the axonal trajectory (Kaprielian et al., 2001). Glial cells and, to a lesser extent, neuronal cells form part of such midline intermediate targets. Each commissure or decussation has an associated cell population and extracellular matrix (ECM) molecules produced by these cells to control the crossing of axons at the midline. Glial tunnels, the glial wedge, subcallosal sling, glial palisade, and floor plate (FP) are some of these structures (Chedotal and Richards, 2010). The subcommissural organ (SCO) is a specialized ependymal structure located in the roof plate of the prosomere 1 under the posterior commissure (PC). The SCO is an ancient and phylogenetically conserved structure present throughout vertebrate phyla (Oksche, 1961). SCO ependymal cells synthesize and secrete the SCO-spondin, a high-molecular-mass glycoprotein, which belongs to the thrombospondin superfamily due to the presence of numerous thrombospondin type 1 repeat (TSR) domains (Meiniel, 2001). The SCO-spondin also contains the TSR type 2 domain, which is shared by molecules involved in developmental processes such as R-spondin, F-spondin, and mindin. Although investigated since the early twentieth century, there is uncertainty about its functional role, albeit different functional hypotheses have been proposed. One of them concerns to the putative relationship of the SCO with the development of central nervous system (CNS) and, more recently, with the axonal guidance. Different evidences have suggested that the SCO is involved in the PC formation. First, there exists a close spatiotemporal relationship between the SCO and the PC during embryonic development. Second, the SCO is located at the dorsal midline, and the role of this region as intermediate target in axonal pathfinding is widely accepted. Third, data from different mutant mice indicate that animals lacking SCO or with SCO alterations fail to form a normal PC. Fourth, the SCO-spondin belongs to the thrombospondin superfamily, sharing type 2 TSR domains with molecules implicated in axonal pathfinding during the development of the nervous system, such as R-spondin, F-spondin, mindin, and semaphorins. Fifth, the SCO-spondin is expressed by two midline structures: the SCO and, transiently, the rostral FP, the latter having a well-known role in axonal guidance. Sixth, in vitro experiments using RF solubilized compounds or synthetic peptides derived from SCO-spondin in different cell culture systems have revealed a potential role of the SCOspondin in both neuronal aggregation and neurite outgrowth. Last, coculture experiments confronting SCO explants with ventral diencephalic

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explants, which give rise to axons forming the PC, have shown that SCO cells can exert attractive or repulsive effects on growing axons. Although none of these evidences is unequivocal, they altogether represent substantial information to support the role of the SCO in the development of the PC. Each of these evidences will be revised in detail in this review.

2. Axon Guidance and the Development of Commissures The establishment of the neuronal cytoarchitecture and neural circuits is essential for the brain functions. During embryonic development, wiring of the nervous system occurs in a highly patterned and ordered manner. This means that neurites from newborn neurons have to connect with their targets in a very precise way. To accomplish this, there are a number of axonal pathfinding mechanisms to guide the growing axons to their final target. The growing axon, and particularly its most distal portion, the growth cone, evaluates the extracellular milieu looking for environmental signals that accurately define the pathway to the target (Tessier-Lavigne and Goodman, 1996). These axon guidance mechanisms are mediated by evolutionarily conserved ligand–receptor systems. Such ligands are the so-called axonal guidance cues, which can be diffusible factors or be bound to the cell membrane molecules (Fig. 2.1). Growing axons contain receptors that recognize the axonal guidance cues and mediate chemotropic responses such as chemoattraction and chemorepulsion, meaning the attraction of the axons toward a target or its repulsion from a brain area, respectively. In general, the diffusible molecules have a long-range effect on axonal guidance, while those bound to cell membranes have a short-range, contact-dependent effect (Fig. 2.1). Therefore, axons and specifically growth cones have the ability to sense the extracellular environment and move toward or away from a particular brain region. It is thought that the growing axons behave this way because the axonal guidance molecules are distributed in concentration gradients, where the highest concentrations are found near the source and decrease with distance. Such molecular gradient induces a different response in different positions of the growth cone, triggering a cytoskeletal modification that results in the movement in this highly motile structure. When a large axonal tract or bundle is generated, not all the axons forming such bundle travel simultaneously. On the contrary, only relatively few axons establish the route for the first time, the so-called pioneering axons (Harris, 1986), which probably find their way using the axonal guidance mechanisms described above. These pioneers are necessary for the normal pathfinding of subsequently growing axons (Klose and Bentley, 1989). However, these latter axons may use an additional mechanism to reach their target as they travel through the route previously established by pioneering axons, and in fact,

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Gradient

Diffusible molecules Long-range effects

Chemo Chemo attraction repulsion

Chemo Chemo attraction repulsion

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Membrane-bound molecules Short-range effects or Contact-mediated effects

Figure 2.1 Schematic representation of the chemotropic actions of the two types of axonal guidance cues. These molecules can act as either diffusible or membrane-bound signals. Responses to axonal guidance molecules include chemoattraction and chemorepulsion. In general, the diffusible molecules have a long-range effect on axonal guidance, while the membrane-bound molecules have a short-range, contact-mediated effect.

they fasciculate with them (Tix et al., 1989). It is postulated that pioneer axons express on their surface molecules that serve to guide following axons and allow them to fasciculate with the pioneers (Van Vactor, 1998).

2.1. Importance of the midline as an intermediate target in axonal guidance As stated before, the pioneering axons from newborn neurons sometimes have to travel great distances along highly stereotyped pathways to reach their final target, which might even be located several millimeters away

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from the neuronal soma. Axonal pathfinding of such long-distance trajectories progresses in sequential stages, keeping axons in the appropriate direction by the use of regularly positioned sources of axon guidance molecules, referred to as intermediate targets (Ho and Goodman, 1982). In those animals with bilateral symmetry of the nervous system, the midline represents one of such important intermediate targets and a critical axonal choice point. In both vertebrates and invertebrates, axons that arrive at the ventral midline are exposed to certain axonal guidance cues to decide whether to stay on the same hemisphere (i.e., ipsilateral) or to cross to the opposite hemisphere (i.e., contralateral). In embryos of both vertebrates and invertebrates, midline axons are divided into two discrete longitudinal bundles that flank the midline bilaterally. Most axons cross the midline before joining one of these two longitudinal bundles. Commissures and decussations (see this section below for the exact definition) are the bundles of nerve fibers that cross from one hemisphere to the other. The selection of axons that will form part of a given commissure is a task that is tightly regulated by midline structures (Evans and Bashaw, 2010; Kaprielian et al., 2001). A paradigmatic and well-established example of this regulation occurs in the spinal cord, where the axons of commissural interneurons grow toward the ventral midline in response to attractant molecules. These axons do not respond to repellent molecules that are present at the midline. However, as soon as the axons cross the midline, they change their behavior and, as a result, they are not attracted anymore by the midline, being repelled instead. In this way, once the axons have crossed the midline, they avoid it and never cross back (Kidd et al., 1999; Long et al., 2004). An exquisite coordination of the axonal guidance molecules and their receptors is required to perform this switch from attraction to repulsion. A well-known example of such coordination is the attraction of precrossing axons by the midline molecule Netrin, which is mediated by receptors of the DCC (deleted in colorectal cancer) family (Dickson, 2002). Upon crossing, the axons upregulate Robo receptors that mediate the repulsion induced by the midline repulsive molecule Slit (Long et al., 2004). Precrossing axons do not express Robo and are not repelled from the midline (Kidd et al., 1998). In addition, Robo receptors interact with DCC receptors in postcrossing axons, which induces an inhibition of the DCC receptor activity preventing the recrossing of axons (Stein and Tessier-Lavigne, 2001). In the strictest sense, the commissures are formed by the fibers that interconnect corresponding structures in the two halves of the brain and their fibers neither ascend nor descend in the longitudinal axis of the neural tube. By contrast, decussations are a midline crossing of longitudinal projections that do not reciprocally connect equivalent structures but rather ascend or descend to project to different structures of the brain or spinal cord (Sarnat, 2008). In mammals, the main commissures are the corpus callosum, albeit only present in placental mammals (Sarnat and Netsky, 1985);

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the anterior commissure; the dorsal and ventral hippocampal commissures; the habenular commissure; the PC; the tectal commissures; and several commissures of the hindbrain, such as the inferior olivary commissure and numerous commissural fibers that cross the spinal cord midline, ventrally to the central canal. Other bundles that cross the midline are considered decussations, that is, the postoptic (or supraoptic) decussation, the optic decussation (optic chiasm), and several hindbrain and spinal cord commissures which, although resembling commissures and being called so, are in fact decussations: the trapezoid body of the auditory system, the decussation of the corticospinal tract at the caudal end of the medulla oblongata, and the axons that cross between the ventral funiculi of the spinal cord, ventral to the central canal (Sarnat, 2008). The PC is only partly commissural as some of its axons project toward hindbrain nuclei on the opposite side different from their nuclei of origin, so it can be as well considered a decussation. In the hindbrain and spinal cord, a high variety of commissural neurons cross the midline at all levels, rarely forming well-defined tracts (Sarnat, 2008). Despite the considerable number of commissures in the vertebrate nervous system, most studies concerning axonal guidance have been performed in one of two widely used models: (i) the development of the corpus callosum in the forebrain and (ii) the ventral midline crossing of commissural axons in the spinal cord.

2.2. Glial structures in midline axonal guidance In both insects and vertebrates, specialized populations of CNS midline cells define the plane of bilateral symmetry between the two halves of the neuroectoderm. In addition to their anatomical position, specialized midline cells in flies and vertebrates are also defined by the expression of specific molecular markers. In both metazoan groups, midline cells serve as inductive centers for the regional patterning of the adjacent neuroectoderm (Yamada et al., 1991). Concerning the axonal pathfinding, the midline of the CNS represents a boundary for neuronal growth cones, exhibiting both attractive and repulsive properties. Thus, cells that form the midline have special properties and functions (Kaprielian et al., 2001). Most, if not all, commissural and decussating projections of the brain are associated with a midline glial population (Silver et al., 1993). In addition to glial cells, transient neuronal populations participate as intermediate targets or as corridor cells in the axonal guidance process (Chedotal and Richards, 2010). In the subsequent sections, we present some glial structures that participate in axonal guidance at the midline. 2.2.1. Ventral midline structures In vertebrates, the ventral midline of the embryonic mesencephalon, rhombencephalon, and spinal cord is constituted by a layer of specialized neuroepithelial cells called the FP (Bronner-Fraser, 1994). The FP is a transient

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structure that is only present in the developing nervous system. In zebrafish, the ventral midline is made of a single column of cells, whereas in rats, it is about 15–20 cells wide (Tear, 1999). These cells are the first to be specified in the vertebrate nervous system, which speaks for its importance in the CNS development. The FP neuroepithelial cells produce signal molecules that control the cell differentiation pattern in the dorsoventral axis of the vertebrate neural tube (Yamada et al., 1991). It is well established that the FP cells secrete axonal guidance cues involved in the midline crossing of spinal commissural axons (Placzek and Briscoe, 2005). The FP of the zebrafish, chick, and mouse has been extensively studied as a paradigmatic model of axon pathfinding in vertebrates (Stoeckli et al., 1997). And so, a large amount of knowledge has been gained on axonal guidance mediated by the FP (Mastick et al., 2010). In the forebrain, the FP is missing, meaning that other glial cells should take over its function in midline guidance. A palisade of immature radial glia is involved in the formation of the optic decussation (optic chiasm) (Fig. 2.2). Retinal ganglion cell axons navigate over considerable distances from their site of origin in the eye to their targets in the diencephalon and midbrain. In mammals, retinal growth cones encounter specialized glia at the midline (Marcus et al., 1995). It is upon contact with these glial cells that ipsilateral and contralateral axons diverge from each other (Marcus et al., 1995). In zebrafish, a paired box (Pax) containing transcription factor called no-isthmus (Noi) seems to be involved in the axonal guidance (Macdonald et al., 1997). The noi gene shares considerable sequence homology and similar expression domains with the mouse pax-2 gene (Puschel et al., 1992). In zebrafish, the absence of functional Noi protein prevents glial cell differentiation in the optic nerve, as well as the fusion of the choroid fissure and axons in the optic chiasm shows significant pathfinding defects (Macdonald et al., 1997). These authors conclude that Noi is a key regulator of commissural axon pathway formation at the midline of the diencephalon, probably due to alterations in the expression of both Netrin and Sonic hedgehog also observed in noi mutant embryos (Macdonald et al., 1997). Specialized glial cells seem to be also involved in the development of the anterior commissure. Thus, GFAP and Vimentin-positive glial cells form a tunnel through which the anterior commissure fibers elongate. ECM molecules such as chondroitin sulfate proteoglycans, fibronectin, and laminin confine those crossing axons forming a true molecular tunnel (Fig. 2.3) (Lent et al., 2005; Pires-Neto et al., 1998). Similar glial tunnels have been described in the formation of the hippocampal commissure. In this case, the expression of chondroitin sulfate glycosaminoglycans is concomitant with the formation of the commissural tract and disappears when the commissure formation is completed. Partially overlapping with glycosaminoglycans expression, radial glial cells or their processes form a tunnel surrounding the hippocampal commissure, especially at its caudal portion (Fig. 2.4) (Braga-de-Souza and Lent, 2004).

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GP

OC Radial glia forming the glial palisade

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AC

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Tenascin

Fibronectin

Chondroitin sulfate

Laminin

GFAP astrocytes

Figure 2.2–2.3 (2.2) Scheme of a coronal section of a mouse embryo brain (E12) at the level of the developing optic chiasm (OC). Axons forming this commissure travel through the basal processes of radial glia cells, which form the so-called glial palisade (GP). (2.3) Scheme of a coronal section of the embryonic hamster brain (E14) at the level of the anterior commissure (AC). Glial cells expressing GFAP and several extracellular matrix molecules form a glial tube through which the axons forming the commissure will pass. Data for the scheme taken from Pires-Neto et al. (1998).

In invertebrates, CNS ventral midline cells have similar functions to those of the vertebrate FP as both structures are involved in the dorsoventral patterning of the neural tube and in axon guidance across the midline. The Drosophila CNS ventral midline contains about 20 individually identifiable cells per abdominal neuromere, four of them being glial cells (Klambt et al., 1991).

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CC VL

III v

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HC

Chondroitin sulfate glycosaminoglycans

GFAP astrocytes

CC IG

SCS GW

Indusium griseum 5

Subcallosal sling

Glial wedge

Figure 2.4–2.5 (2.4) Scheme of a horizontal section of the embryonic hamster brain (E15) at the level of the hippocampal commissure (HC). Chondroitin sulfate glycosaminoglycans and glial cells expressing GFAP form a glial tube that guides the passage of commissural axons. CC, corpus callosum; VL, lateral ventricle; III v, third ventricle. Data for the scheme taken from Braga-de-Souza and Lent (2004). (2.5) Scheme of a coronal section through the developing mouse cortex at E17. Three populations of cells are involved in the guidance of the pioneering axons of the corpus callosum (CC). Two of them are composed of glial cells: indusium griseum (IG) and glial wedge (GW). The third population, the subcallosal sling (SCS), consists mainly of neuronal cells, albeit they originally were identified as glial cells. Due to this reason, this structure was formerly named glial sling.

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Important functions have been assigned to these midline cells in Drosophila. Thus, it has been shown that (i) they are required for the development of neurons in the lateral nerve cord (Menne et al., 1997), (ii) they act as attractive cues for commissural growth cones growing toward the midline, and (iii) midline glial cells determine which axons cross the midline and which remain ipsilateral (Kidd et al., 1999). In Caenorhabditis elegans, the ventral nerve cord has an overall organization similar to the spinal ventral cord of vertebrates and insects. It consists of two axon bundles, separated by a midline structure (White et al., 1986). As a result, in early embryos the two axon bundles are separated by a dense and continuous row of motor neurons. Thus, in the C. elegans embryo, the ventral midline is, at least in part, established by the motor neurons, rather than by specialized glial-type cells as occurs in vertebrates or flies (Durbin, 1987). During larval development, hypodermal cells at both dorsal and ventral sides increase in volume, leading to a projection into the hypodermal tissue forming the so-called hypodermal ridge. These cellular extensions establish a distinctive anatomical midline structure in larval and adult animals (Podbilewicz and White, 1994). In spite of the unusual anatomical features of the C. elegans ventral midline, most, if not all, of the mechanisms of axon patterning and the molecules and receptors involved in axonal guidance at the ventral midline are strikingly conserved at the molecular level across phylogeny (Hobert and Bulow, 2003). 2.2.2. Dorsal midline structures Despite the significant number of dorsal commissures, only the corpus callosum has been frequently used as a model for the study of axonal guidance at the dorsal midline. Present only in placental mammals, the corpus callosum is the greatest commissure of the brain and contains approximately half of the commissural axons of the whole brain. Together with the anterior commissure, it directly interconnects corresponding regions of the cerebral neocortex on the two sides of the brain (Sarnat, 2008). Despite its importance, agenesis of the corpus callosum, one of the most common cerebral malformations, is neither lethal nor produces major neurological disabilities in most cases (Sarnat, 2008). In placental mammals, during the development of the corpus callosum, cortical axons from one cerebral hemisphere cross the midline to reach their targets in the opposite cortical hemisphere. These callosal axons cross through regions that are permissive for their growth, which are delineated by specific glial (indusium griseum and glial wedge) and neuronal (subcallosal sling) cell populations (Fig. 2.5). The subcallosal sling cells form a U-shaped cell layer contiguous with the subventricular zone, between the two cerebral hemispheres (Silver et al., 1982). The subcallosal sling cells migrate from the lateral ventricular zone to underlie the developing corpus callosum (Silver et al., 1982). Although this structure was originally named glial sling, thinking that they were glioblasts, later it was shown to be composed of neurons (Shu et al., 2003a). Two types of experiments suggest that

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subcallosal sling cells are required for the formation of the corpus callosum. First, either failure of the migration of subcallosal sling cells toward the midline (Schneider and Silver, 1990) or lesions of the subcallosal sling (Silver et al., 1982) prevent the formation of the corpus callosum. Second, in acallosal mice, the formation of the corpus callosum can be rescued by the insertion of a glia-covered scaffold in the midline (Silver and Ogawa, 1983). In addition to the subcallosal sling, two midline glial structures, known as the glial wedge and the indusium griseum glia, display a guidance activity for callosal axons (Shu and Richards, 2001). The glial wedge sends long radial-glial-like processes toward the midline, ventral to where the corpus callosum will form. The long radial processes of the glial wedge resemble those of radial glia (Rakic, 1972). Glia within the indusium griseum develop immediately dorsal to the developing corpus callosum. Axons of the corpus callosum avoid both these populations of glial cells. In organotypic slices, the reorientation of both glial structures causes callosal axons to turn away from the midline (Shu and Richards, 2001). Slits secreted by the glial wedge and by indusium griseum cells were shown to prevent callosal axons from entering ventral and dorsal domains (Shu et al., 2003b). Based on studies of null mutant mice, other molecules have been involved in the guidance of callosal axons: Slit-1, Slit-2, Robo-1, Robo-2, Wnt5a (Ryk), and Draxin (Andrews et al., 2006). Also in vertebrates, roof plate glia in the dorsal midline of the spinal cord prevent growing axons from crossing the midline (Snow et al., 1990) through the actions of bone morphogenetic proteins (Augsburger et al., 1999). The midline raphe glial structure (MRGS) is an extensive radial structure located in the midline raphe of the midbrain, hindbrain, and cervical spinal cord. Such glial structure exists both during development and in adulthood (Mori et al., 1990) and forms a continuous band of radial glial fibers separating the right and left brainstem but with some interruptions to allow for the passage of decussating fibers. Its structure suggests that the MRGS may be involved in sorting and organizing ipsilaterally and contralaterally projecting axons during development (Mori et al., 1990).

2.3. Axon guidance molecules and its receptors During the past 30 years, several families of axonal guidance molecules have been described in both vertebrates and invertebrates (Chedotal and Richards, 2010; Evans and Bashaw, 2010). The following sections describe the main families of axonal guidance cues discovered until present. 2.3.1. Slit–Robo Slit was first identified in the Drosophila embryo for being involved in the larval cuticle patterning (Nusslein-Volhard et al., 1984). However, the Slit family of secreted proteins was discovered in Drosophila as guidance cues involved in axonal midline crossing. In fly slit mutants, commissural axons

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from the two sides of the nerve cord exhibit defects in its behavior at the midline, and instead of crossing it, they fuse there (Rothberg et al., 1990). The Slit family comprises three members that are expressed in the ventral midline of the neural tube. While Slit-1 is mainly found in the CNS, Slit-2 and Slit-3 are also expressed outside the nervous system (Itoh et al., 1998). Slit proteins function as extracellular cues for axonal guidance (Kidd et al., 1999; Li et al., 1999) and as regulators of neuronal migration (Li et al., 1999) and axonal branching (Wang et al., 1999). The broad expression of the Slit family members during mouse and chick development suggests its participation in a diverse array of morphogenetic events (Piper and Little, 2003). Regarding the axonal guidance function, Slit is a chemorepulsive factor and a key regulator of midline crossing and axonal fasciculation (Rajagopalan et al., 2000a). The Slit protein contains an N-terminal signal peptide, four leucine-rich repeats, seven (in Drosophila Slit) or nine (in vertebrate Slits) EGF repeats, and a C-terminal cystine knot (Rothberg et al., 1990). The leucine-rich repeats are sufficient for the interaction of Slit with the Robo receptor (Chen et al., 2001). Posttranslational modifications of the Slit proteins can give rise to the different Slit isoforms. So, the processing of the human Slit-2 produces an N-terminal fragment (Slit-N) that contains all four leucine-rich repeats and five of the EGF repeats, and a corresponding C-terminal Slit fragment (Slit-C) that contains the rest of the protein (Wang et al., 1999). Both the full-length form and the fragments are secreted extracellularly (Rothberg et al., 1990; Wang et al., 1999). However, functional differences between the full-length and the fragments of Slit have been found (Nguyen Ba-Charvet et al., 2001). Heparan sulfate proteoglycans are involved in the binding of Slit to its receptors as removal of these extracellular molecules by the use of heparinase III prevents the repulsion induced by Slit (Hu, 2001). It will therefore be important to identify the molecules, such as co-receptors, that mediate such ligand specificity. Roundabout (Robo) proteins are Slit receptors (Kidd et al., 1999; Li et al., 1999). The first robo gene, robo1, was identified in Drosophila during a comprehensive screening for genes controlling CNS midline crossing; fly robo mutants display an increased number of axons crossing and recrossing the ventral midline (Kidd et al., 1998). Robo is an evolutionary conserved family of transmembrane receptors (Kidd et al., 1999; Rajagopalan et al., 2000b; Simpson et al., 2000; Zallen et al., 1998). Three robo genes have been identified in different organisms including C. elegans (Zallen et al., 1998), Drosophila (Rajagopalan et al., 2000b), and several species of vertebrates (Kidd et al., 1998; Lee et al., 2001). Throughout development, the expression pattern of Robo receptors is closely regulated in time and space. In Drosophila, the protein Midline, belonging to the T-box family of transcription factors (Stennard and Harvey, 2005), controls the transcription of Slit and Robo along the midline of the central and peripheral nervous systems (Liu et al., 2009).

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In Drosophila, commissural axons that grow toward the midline do not express Robo on their surface. This negative regulation is conducted by the transmembrane protein Commissureless (Comm), which redirects Robo to the endosomes for its degradation (Keleman et al., 2005). When growing axons reach the midline, an unknown mechanism diminishes the expression of Comm, resulting in an increase of the amount of Robo at the membrane, and consequently, the axon is repelled from the midline preventing it from recrossing (Keleman et al., 2005). The signaling pathways of Slit/Robo and Netrin-1/Dcc are intertwined and regulate one another at different levels. Thus, in the presence of Slit and Netrin-1, the intracellular domains of Dcc (P3) and Robo-1 (CC1) interact, suppressing Netrin-1 attraction (Stein and Tessier-Lavigne, 2001). As previously mentioned, the Slit/Robo pathway is not only involved in axonal guidance processes but also in a number of developmental processes, even outside the CNS. So, Slit/Robo interaction has an effect on tangential migration in several systems (Andrews et al., 2008) and on axonal targeting in the mouse olfactory system (Cho et al., 2009). 2.3.2. Netrins-DCC and UNC5 Netrins comprise a family of highly conserved secreted proteins that are structurally related to laminins (Tessier-Lavigne and Goodman, 1996). In the 1990s, UNC-6, the first member of the Netrin family, was identified and characterized in C. elegans (Ishii et al., 1992). Later on, two Netrins were described in Drosophila (Harris et al., 1996) and three Netrin proteins were identified in several vertebrates (Serafini et al., 1994). In the chick, Netrin proteins mimic the in vitro chemoattractant properties of the FP (Serafini et al., 1994). Gene expression studies showed that Netrin-1 was expressed in the FP and Netrin-2 in the ventral two-thirds of the spinal cord at a time when the first commissural axons are growing toward the ventral midline (Kennedy et al., 1994). Netrin mutants display severe pathfinding defects. In vertebrates, most commissural axons can no longer extend to reach the FP and thus axonal trajectories across the midline are severely altered (Serafini et al., 1996). It has been demonstrated that Netrins can act as short-range guidance cues as well as at long distances (Kennedy et al., 1994; Serafini et al., 1994). Two families of receptors mediate the axonal guidance function of Netrins: the UNC5 family (Leonardo et al., 1997) and the DCC and Neogenin family (Vielmetter et al., 1994). Netrins represent a paradigmatic case of doublefunction molecules in axonal guidance, as the same molecule can act as attractant (Keino-Masu et al., 1996) or as a repellent (Keleman and Dickson, 2001) depending on which receptor it binds. Recent data suggest that, in addition to its function in axonal guidance, Netrins are involved in developmental processes such as cell adhesion, cell motility, proliferation and differentiation in the nervous system (Schwarting et al., 2004), and nonneuronal tissues such as pancreas, lung, and mammary glands (Yebra et al., 2003). In this

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context, it is not surprising that two integrins (alpha6beta4 and alpha3beta1) have been shown to function as Netrin-1 receptors in epithelial cells and to mediate cell adhesion and migration on a Netrin-1 substrate (Yebra et al., 2003). 2.3.3. Semaphorins–plexins/neuropilins Semaphorins are members of a large, highly conserved family of molecular signals initially identified as repulsive axon guidance factors (Kolodkin et al., 1993; Luo et al., 1993) albeit some of them induce attractive responses (Dalpe et al., 2005). Semaphorins can be either secreted or appear associated to the membrane as transmembrane proteins or through glycosylphosphatidylinositol (GPI) linkage. This explains why they can mediate both longand short-range signaling. Transmembrane semaphorins can also release a signaling-competent extracellular domain (Wang et al., 2001) or function as receptors (Godenschwege et al., 2002). Semaphorins are characterized by a conserved extracellular amino-terminal “Sema” domain. More than 20 different semaphorins have been identified and classified into eight classes according to their phylogenetic relationship and the arrangement of additional domains. Two families of growth-cone receptors neuropilins and, more importantly, plexins are involved in semaphorin signaling (Fujisawa and Kitsukawa, 1998). Plexins can be divided into four classes and include two members from invertebrate species (PlexA and PlexB) and nine members from vertebrates (PlexinA1–PlexinA4, PlexinB1–PlexinB3, PlexinC1, and PlexinD1) (Tamagnone and Comoglio, 2000). Most semaphorins appear to bind and activate plexins directly, while the secreted (class 3) semaphorins require a complex of PlexinA1/2 and Neuropilin for binding and signaling (Rohm et al., 2000; Tamagnone et al., 1999). Therefore, vertebrate plexins and neuropilins form co-receptors that can distinguish between different classes of semaphorins. Neuropilins (NP1 and NP2) are found only in vertebrates (Tamagnone and Comoglio, 2000). The cytoplasmic domain of plexins is required for semaphorin signaling, whereas the small cytosolic tail of neuropilins is dispensable. In addition to binding secreted semaphorins, neuropilins are also vascular endothelial growth factor (VEGF) co-receptors (Gluzman-Poltorak et al., 2001) and are essential for vascular development (Takashima et al., 2002). Nonplexin receptors, such as CD72 (Kumanogoh et al., 2000) and Tim-2 (T cell immunoglobulin and mucindomain-containing 2) (Kumanogoh et al., 2002), provide further diversity to semaphorin function. As occurs in Slit/Robo interaction, several cell surface receptor proteins, such as heparan sulfate and chondroitin sulfate proteoglycans (Kantor et al., 2004), Nr-CAM (Falk et al., 2005), Off-track (Otk) (Winberg et al., 2001), L1 (Castellani et al., 2000), and Gyc76C (Ayoob et al., 2004), have been shown to be important for the formation of the

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semaphorin receptor complex. This fact suggests that semaphorin receptors are composed of multiple different subunits. Semaphorins (Sema3B and Sema3F), PlexinsA1, and Neuropilin-2 have been involved in the crossing of spinal cord commissural axons through the FP and their posterior rostral turn. Both pre- and postcrossing axons express Neuropilin-2 while PlexinA1 expression is higher in postcrossing axons. Only postcrossing axons respond to (i) Sema3B (expressed by the FP) which prevents axonal recrossing and (ii) Sema3F (expressed by the mantle zone of the spinal cord) which guides postcrossing axons to turn rostrally once they abandon the FP (Nawabi et al., 2010). In addition, Neuropilin-2 mutant mice display axonal midline defects in the spinal cord (Zou et al., 2000). Both results together suggest that Sema3B acts through a receptor complex formed by Neuropilin-2 and PlexinA1 (Derijck et al., 2010). In addition to its axonal guidance activity, semaphorins participate in other biological processes during the nervous system development, such as cell migration, cytokine release, cell death, and synapse formation. Besides, semaphorins, originally thought to be specific for axonal guidance in the nervous system, are now recognized to perform crucial functions in several cellular processes and systems. Hence, semaphorins have been involved in cardiogenesis (Toyofuku and Kikutani, 2007), angiogenesis (Serini et al., 2009), vasculogenesis (Gu et al., 2005), tumor metastasis (Capparuccia and Tamagnone, 2009), osteoclastogenesis (Takegahara et al., 2006), and immune regulation (Suzuki et al., 2008). In addition, human genetic analyses correlate semaphorins, their associated receptors, and cytosolic signaling molecules as causal and/or susceptibility genes in several diseases, such as neurodegenerative diseases, schizophrenia, and cancer (Mann et al., 2007). 2.3.4. Ephrins-Eph RTK Ephrins are membrane-bound guidance cues involved in a wide range of cellular responses in the developing nervous system, including axonal guidance, through contact-mediated attraction or repulsion, adhesion or de-adhesion, and migration. Ephrins are categorized into two classes: ephrin-As (EphrinA1– EphrinA5), which are GPI-anchored to the membrane, and ephrin-Bs (EphrinB1–EphrinB3), which have a transmembrane domain followed by a short cytoplasmic domain (Egea and Klein, 2007). Ephrin ligands bind to Eph receptors that represent the largest subfamily among receptor tyrosine kinases (RTKs). Eph receptors are divided into an A-subclass that contains eight members (EphA1–EphA8), and a B-subclass that contains five members (EphB1–EphB4, EphB6). Both EphAs and EphBs share a similar general structure but differ in amino acid sequences and binding affinities to different ligands. EphA receptors promiscuously interact with five A Ephrins, while EphB receptors bind preferentially to B-type Ephrins. An exception is found in EphA4 that binds both EphrinA-type and EphrinB-type ligands (Pasquale, 2005). A characteristic of the Ephrin-Eph signaling system is the capability to generate a bidirectional signaling, with Eph receptors eliciting a classical

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forward signaling via their intrinsic tyrosine kinase activity and transmembrane EphrinB ligands triggering a reverse signaling via their cytoplasmic domain (Mellitzer et al., 2000). The Eph/Ephrin system has been implicated in the formation of topographic maps in the visual system. The projection of retinal ganglion cells from the eye to their targets in the superior colliculus (or tectum in chicken) is one of the best-characterized axon pathways in the CNS. Axons of retinal ganglion cells are guided by the establishment of a precise spatial pattern: EphrinA2 and EphrinA5 are expressed in low anterior to high posterior gradients across the tectum/superior colliculus, while EphA2, EphA5, and EphA6 receptors are expressed in retinal ganglion cells in a corresponding high temporal to low nasal gradient in the retina. This expression pattern leads to the proper development of the visual system (Feldheim et al., 2000). Ephrins have also been involved in midline axonal guidance, acting in most cases as a midline repellent. Mutant mice with a disruption in EphrinB3mediated forward signaling display midline recrossing defects of the corticospinal tract (Yokoyama et al., 2001) and alterations in the decussation of ipsilateral axons in the spinal cord (Kullander et al., 2003). The same corticospinal tract defects are found in mice mutant for EphA4, a receptor that binds EphrinB2/B3 and ephrinAs (Kullander et al., 2001). EphrinB2 acts as a repulsive ligand at the optic chiasm midline where it determines that axons of retinal ganglion cells project ipsilaterally (Williams et al., 2003). The development of major forebrain commissures, the corpus callosum, and the anterior commissure is also regulated by the Eph-ephrin signaling system. Reverse signaling induced by EphrinB elicits the anterior commissure development (Henkemeyer et al., 1996), while both forward and reverse signaling through EphB and EphrinB interaction is necessary for corpus callosum development (Mendes et al., 2006). EphrinB3 is localized to the FP at the ventral midline of the embryonic vertebrate spinal cord (Kadison et al., 2006a). In mutant mice lacking EphrinB3 or multiple EphB receptors, numerous axons display aberrant trajectories at the ventral midline of the spinal cord (Kadison et al., 2006b). However, no defects were found in embryos with a disrupted EphrinB3 with the capacity of a forward but not a reverse signaling, which suggests that this reverse signaling is not necessary for midline axonal guidance in the spinal cord (Kadison et al., 2006b). As occurs in other groups of axonal guidance molecules, this significant family of Eph RTKs and its ephrin ligands exerts a pleiotropic activity, acting on a variety of biological processes in developing and adult organisms, which is a reflection of the complexity of its signaling. 2.3.5. Repulsive guidance molecule-Neogenin The repulsive guidance molecule (RGM) is a membrane-associated glycoprotein first discovered as a repulsive cue acting on growth cones of retinal axons (Monnier et al., 2002). The RGM family is formed by three members: RGMa, RGMb, and RGMc (Schmidtmer and Engelkamp, 2004).

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RGMa and RGMb (also called Dragon) are expressed in the developing and adult CNS, but with different expression patterns (Niederkofler et al., 2004; Samad et al., 2004). RGMc (also called HJV, HFE2, or DL-M) is expressed in the liver and the striated muscle but not in the nervous system and is involved in the regulation of iron metabolism (Kuninger et al., 2006). Transcripts of RGMa are present in several mouse embryonic brain regions: the hippocampus, the midbrain, the ventricular zone of the cortex, and parts of the brainstem and the spinal cord (Oldekamp et al., 2004). A similar expression pattern has been found in chick (Matsunaga et al., 2004) and zebrafish (Samad et al., 2004) embryos. Several lines of evidences point to Neogenin, a Netrin-binding protein, as a putative RGM receptor. So, anti-Neogenin antibodies and the soluble Neogenin ectodomain block the RGM repulsive activity on temporal retinal axons (Rajagopalan et al., 2004). Neogenin has been shown to belong to the dependence receptor family, a group of receptors that trigger apoptosis in the absence of the ligand (Mehlen and Thibert, 2004). In this case, RGMa downexpression in the developing chick neural tube induces apoptosis (Matsunaga et al., 2004). In a more recent article, the same authors demonstrated several additional roles of RGMa, being involved in neuronal proliferation, differentiation, and axon guidance (Matsunaga et al., 2006). Due to the spatial graded expression of RGM in the embryonic optic tectum and its repulsive effect on a subset of retinal axons, RGM was thought to be involved in the generation of the topographic map in the retinocollicular projections. However, RGMa null mutant mice display no defects in the projections and terminals of the retinal axons in the superior colliculus. This finding suggests that RGMa is not essential for the generation of the retinocollicular map in the mouse (Niederkofler et al., 2004). On the contrary, in chick embryos, overexpression of RGMa in the mesencephalon prevents the retinal axons from entering the affected areas in the tectum. Besides, in loss-of-function experiments by injecting siRNAs in the tectum, the retinal axons project ectopically, most of them posterior to the terminal zone in the optic tectum. These results strongly suggest RGMa to have a role in the formation of the retinotectal map in the chick (Matsunaga et al., 2006). By using these RGMa null mutant mice, an unexpected function of this axonal guidance molecule in the early embryonic development was found, as these mutant mice exhibited defects in the cephalic neural tube closure (Niederkofler et al., 2004). An important role of RGMa in the development of the projections from the entorhinal cortex to the hippocampal formation, that is, the perforant pathway, has been established. So, stripe and explant outgrowth assays demonstrated that RGMa produces an inhibition of entorhinal axons (Brinks et al., 2004). In animals where the RGMa function was altered, the entorhinal axons grew toward wrong areas of the hippocampus, with an alteration of the laminar termination pattern (Brinks et al., 2004).

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Besides its role in the nervous system, RGMa seems also to have a role in the immune system, particularly in the modulation of leukocyte recruitment and in the inflammatory process; several evidences support this: (i) leukocytes express RGMa, (ii) RGMa inhibits leukocyte migration through its receptor Neogenin, and (iii) RGMa suppresses the inflammatory response in vivo (Mirakaj et al., 2010). 2.3.6. Mindin-F-spondin and other extracellular matrix proteins The Mindin-F-spondin family includes rat F-spondin and Mindin, zebrafish Mindin1 and Mindin2, and Drosophila M-spondin (Feinstein et al., 1999; Higashijima et al., 1997). All members of this family are secreted ECM molecules that share two domains: (i) the spondin domain, a
200 amino acid module containing two conserved motifs, FS1 and FS2, which are exclusive to this family; and (ii) the TSRs, shared by a large group of proteins including thrombospondins, the Semaphorin 5 family, and the ADAM (a disintegrin and metalloproteinase) protein family (Feinstein et al., 1999). F-spondin is an ECM protein secreted by the FP (Burstyn-Cohen et al., 1999) and the caudal somites of birds (Debby-Brafman et al., 1999), which contains six TSRs (Bornstein et al., 1991; Lawler and Hynes, 1986) located at the carboxy-terminal half of the protein. The amino-terminal half contains (i) a domain that shares homology with the Reelin protein and (ii) the spondin domain (mentioned above). Cleavage of F-spondin by plasmin releases a diffusible protein containing four TSRs (Tzarfaty-Majar et al., 2001). F-spondin promotes the outgrowth of spinal cord commissural axons (Burstyn-Cohen et al., 1999) and inhibits the outgrowth of motor axons (Tzarfati-Majar et al., 2001), which suggest a role for F-spondin in axon guidance in the spinal cord. Mindin (also called Spondin 2) was originally identified in zebrafish and was found to selectively accumulate in the basal lamina (Higashijima et al., 1997). Rat Mindin is involved in the adhesion and outgrowth of hippocampal embryonic neurons in vitro (Feinstein et al., 1999). Mindin binds to bacteria and their components and acts as an opsonization agent that promotes macrophage phagocytosis of bacteria (He et al., 2004). Mindin null mutant mice display defects in the clearance of bacterial infections in vivo (He et al., 2004). Mindin has been shown to be a novel ligand for integrins and mindin– integrin interactions to be involved in inflammatory cell recruitment since (i) the adhesion of neutrophils to Mindin is blocked by different anti–integrin antibodies and (ii) the recruitment of macrophages and neutrophils is severely impaired in Mindin-deficient mice ( Jia et al., 2005). Other ECM proteins have been shown to have a role in neurite outgrowth in vitro and in vivo. ECM molecules can bind to and cooperate with classical axonal guidance molecules to modulate axonal outgrowth (Hynes, 2009; Myers et al., 2011). So, numerous experimental data point out that molecules such as collagen, laminin, tenascin, or fibronectin have a

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significant influence on axonal guidance (Myers et al., 2011). In addition, the existence of proteins that cleave some of these ECM molecules leads to an increased complexity in the regulation of axonal guidance (Rivera et al., 2010). Therefore, molecules such as matrix metalloproteases (MMPs), ADAMs, and plasminogens have been shown to act on both specific ligands present in the extracellular media and receptors on growth cones to modulate their motility (Chen et al., 2007). Integrin receptors represent one of the major physical linkages by which cells attach to the ECM molecules at distinct contact points. Integrins form heterodimeric receptors which are composed of an alpha and a beta subunit. In humans 18 alpha and 8 beta subunits have been identified, which can be combined into 24 different integrin heterodimers (Hynes, 2002). Ligand binding and receptor clustering are necessary for integrin activation. Ectopic expression experiments of specific integrins on nonresponding neurons elicit the axonal outgrowth on specific ECM substrata (Kwok et al., 2011). Besides, the axonal guidance activity of several molecules (Slits, Netrins, Semaphorins, and Ephrins) depends on their binding to integrin receptors (Nakamoto et al., 2004). Nogo-A, which is a myelin-associated protein, directly inhibits integrin receptors by an unknown mechanism, and this interaction provokes an inhibition of axonal growth (Hu and Strittmatter, 2008). Several other guidance cues such as Netrin (Yebra et al., 2003), Wnt5a (Kawasaki et al., 2007), and neurotrophins (Staniszewska et al., 2008) also bind to various integrin heterodimers. The TSR domains of SCO-spondin, the major SCO secreted protein (see Sections 4.3 and 5.2), induce neurite outgrowth in vitro, an effect that can be inhibited by functional-blocking antibodies against beta1 integrin, which indicates that the TRS motif acts through integrin receptors (Bamdad et al., 2004). Moreover, ECM proteins present within the basal lamina have the ability to bind Netrin molecules in vitro, providing a way of immobilizing Netrin in the extracellular space (Yebra et al., 2003). These and others results (Nakamoto et al., 2004) suggest a close relationship between some axonal guidance cues and integrin-mediated cell motility. Another important ECM receptor is Syndecan, a heparan sulfate proteoglycan. Some integrins and syndecans act as co-receptors and are critical for fibroblasts in the generation of focal adhesion on Laminin and Fibronectin (Morgan et al., 2007). Neuronal syndecan receptors are essential for a proper axonal response to a number of guidance cues (Kantor et al., 2004). So, disruption of the heparan chains or mutations in the syndecan genes result in an abnormal response to Slit in different animal models (Hu, 2001). Complex glycoconjugates such as proteoglycans, which consist of glycosaminoglycan chains linked to a core protein, form part of both the ECM and the cellular surface. There exist four classes of glycosaminoglycans: heparan sulfate/heparin, chondroitin sulfate/dermatan sulfate, keratan sulfate, and hyaluronic acid. Proteoglycans and glycosaminoglycans interact

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with a wide range of molecules, such as axon guidance molecules, morphogens, growth factors, and ECM proteins, influencing a variety of developmental processes like, for instance, proliferation, differentiation, neuronal migration, axonal pathfinding, and synaptogenesis (Maeda et al., 2010). Chondroitinase ABC, a bacterial enzyme that degrades chondroitin sulfate and hyaluronic acid, has been used to deplete chondroitin sulfate in tissue. Axonal pathfinding errors occur when the enzyme is injected into the embryonic nervous system, suggesting that chondroitin sulfate proteoglycans are involved in axonal guidance (Ichijo and Kawabata, 2001). An example of such relationship is the interaction of Sema5A with both chondroitin sulfate proteoglycans and heparan sulfate proteoglycans, which results either in attraction or repulsion on the fasciculus retroflexus development. Heparan sulfate proteoglycans located on the neuronal surface promote the attractive effect of Sema5A on habenular axons while forming the fasciculus retroflexus, suggesting that heparan sulfate proteoglycans might function as a co-receptor with Sema5A. However, Sema5A acts as an inhibitory guidance cue in the prosomere 2, which contains chondroitin sulfate proteoglycans. By use of chondroitinase in a stripe assay, it was shown that extracellular chondroitin sulfate proteoglycans convert the attractive effect of Sema5A into a repulsive one (Kantor et al., 2004).

3. Posterior Commissure Development At early stages of brain development, the arrangement of a series of longitudinal and commissural axon tracts, which act as an axonal scaffold, is a conserved feature in all vertebrates (Easter et al., 1993; Ware and Schubert, 2011). The presence of such axon tracts may serve as a framework to establish a much more complex wiring, characteristic of the later stages of development. The tract of the posterior commissure (TPC) is part of such early axonal scaffold, together with the ventral commissure and two basal longitudinal tracts: the medial longitudinal fascicle (MLF) and the tract of the postoptic commissure (TPOC) (Ware and Schubert, 2011). Therefore, the PC is the first dorsal commissure to be established in vertebrates (Ware and Schubert, 2011).

3.1. Development of the tract of the posterior commissure The TPC is a well-conserved transversal tract that travels parallel to the diencephalic–mesencephalic boundary in the alar plate. Its fibers subsequently take longitudinal ascending (rostral) or descending (caudal) courses within the basal plate (Diaz et al., 1999). In chick embryos, the first pioneer axons to cross the midline at the level of the PC could be detected by

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immunocytochemistry as early as stage HH18 (E3) (Hoyo-Becerra et al., 2010) (Figs. 2.6–2.9). In order to identify the neurons that generate this axonal tract, DiI crystals were placed onto the alar plate of the TPC of living chick embryos at HH23 (E4), which were allowed to survive until HH25 (E4.5-5.0). The DiI-labeled neurons were only found in the magnocellular

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Figure 2.6–2.9 (2.6) Lateral view of the head of an E5 chick embryo with a DiI crystal (arrow) placed into the posterior commissure. OT, optic tectum; Tel, telencephalon. Bar, 700 mm. (From Hoyo-Becerra et al., 2010). (2.7) Dorsal view of the head of the E5 embryo, shown in Fig. 2.6, 24 h after fixation. Diffusion of DiI toward the contralateral hemisphere is evident, although most of the DiI remains close to the DiI crystal (arrow). The dashed line indicates the brain midline. OT, optic tectum; Tel, telencephalon. Bar, 400 mm. (From Hoyo-Becerra et al., 2010). (2.8) Transverse section through the caudal diencephalon of the E5 chick embryo shown in Fig. 2.7. DiI diffuses toward the magnocellular nucleus of the posterior commissure (MNPC, star in Fig. 2.9) in both the ipsilateral and contralateral sides. The arrow indicates the location of the DiI crystal. Bar, 230 mm. (From Hoyo-Becerra et al., 2010). (2.9) Confocal image of the MNPC (dashed oval) at the contralateral side to the DiI crystal (boxed area from Fig. 2.8). Bar, 40 mm. (From Hoyo-Becerra et al., 2010).

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nucleus of the PC (MNPC) localized in the border region between the pretectum and the mesencephalon (Fig. 2.8). This result suggests that only axons coming from this nucleus contributed to the formation of the PC, at least at such early developmental stages (Hoyo-Becerra et al., 2010). Other experiments of TPC DiI labeling were performed in earlier chick embryos (HH19), obtaining similar results (Ware and Schubert, 2011). When DiI is applied in the alar plate of the caudal pretectum, ipsilateral TPC neurons located ventrally within the MLF axon tract are labeled. Such labeled neurons are intermingled with the dorsal and central populations of MLF neurons (Ware and Schubert, 2011). In human embryos, the close proximity between the TPC neurons and the MLF has also been reported (Keene and Hewer, 1933). Injection of DiI in the basal plate of the caudal pretectum causes the anterograde labeling of axons that project dorsally into the alar plate, while no retrograde labeled neurons were observed in the alar plate at this stage (Ware and Schubert, 2011). The same authors also reported the existence of neurons at the dorsal midline of the prosomere 1 at HH16, which project the pioneer axons of the PC toward the contralateral side. However, most of the axons forming the PC soon after come from neurons located at the ventral pretectum. These neurons most probably correspond to the ones labeled by Hoyo-Becerra and coworkers and identified as MNPC. Such neurons are located in the area commissuralis where they differentiate at HH16 (Puelles et al., 1987). At later stages, the TPC neurons seem to form two nuclei, the parvocellular and magnocellular interstitial nuclei of the PC (Kuhlenbeck, 1939). The magnocellular group is currently called MNPC (Ferran et al., 2009). At later stages in the chick development (HH21), the PC occupies the entire pretectal roof plate (Caprile et al., 2009; Ware and Schubert, 2011). In the mouse, a quite similar scenario with temporal variations has been reported (Mastick and Easter, 1996). The application of DiI onto the dorsal prosomere 1 in E10.5 dpc mouse embryos resulted in labeled axons coming from the contralateral side. Therefore, midline axons of the PC were originated from neurons located at two positions: (i) in the dorsal half of prosomere 1 and (ii) in a ventral cluster of neurons that extend into the mesencephalon. Such mesencephalic population was located ventrally to the axons of the mesencephalic tract of the trigeminal nerve. As occurs in the chick, the most ventral neurons of the TPC (the MNPC) are intermingled with dorsal neurons of the MLF in both prosomere 1 and mesencephalon (Mastick and Easter, 1996).

3.2. Nuclei that give rise to the posterior commissure The movements of the eyes are essential for visual object recognition (Sewards and Sewards, 2002). Eye movements are controlled by nerves III (oculomotor) and IV (trochlear) and their respective nuclei in the brainstem.

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Both nuclei receive positive and negative inputs from different parts of the brain. In a classical work, Keene (1938) described the organization and the putative anatomical origin of the fibers that cross the diencephalic roof, forming the vertebrate PC. In mammals, this author described fibers arising from the so-called nucleus of the PC (nucleus of Darkschewitsch for some authors) and indirectly from the interstitial nucleus (of Cajal) and the ipsilateral medial longitudinal bundle, the tegmentum and the capsule of the red nucleus, the thalamus, striatum and cortex, the habenula, the pineal gland, and the SCO. More recently, lesion studies have shown that the anatomical origin of the fibers crossing the PC are the rostral interstitial nucleus of the medial longitudinal fascicle (riMLF), located close to the nucleus of the III nerve, and the interstitial nucleus of Cajal (as an integrator). Through this system, vertical eye movements are controlled and so, when the system is damaged (either at the level of the nuclei or the PC), a palsy of the vertical eye movements occurs, including saccades and gaze (Alemdar et al., 2006). More precisely, upgaze paralysis is a consequence of a lesion affecting the PC (Pierrot-Deseilligny, 2011). Thus, it can be considered that the PC is at the service of the visual system.

4. The Subcommissural Organ as a Specialized Ependyma 4.1. Structure and position in the brain The SCO is an ependymal differentiation located in the midline roof plate of the caudalmost portion of the diencephalon (prosomere 1), under the PC (Figs. 2.11 and 2.24). Because of the secretory nature of the SCO, it has been regarded as a true brain gland. Concerning its histological structure, two layers of different cell types are present in the SCO: the ependymal and the hypendymal cell layers (Oksche, 1961). The ependymal layer is formed by a tall pseudostratified epithelium in contact with the cerebrospinal fluid of the third ventricle. It resembles the morphology of the FP epithelium, its cells being quite similar to radial glial cells (Fig. 2.10). On the other hand, the hypendymal cells are located under the ependymal layer of the SCO (Oksche, 1961). While the ependymal layer has not suffered substantial changes throughout phylogeny, the degree of development of the hypendymal layer differs considerably between lower vertebrates, where it is poorly developed, and mammals, in which the hypendyma forms a prominent subependymal layer (Oksche, 1961). The organization pattern of hypendymal cells varies among species. In some species, the hypendymal cells arrange in clusters preferentially situated in a fixed position of the SCO, while in other species like bovine, they form a continuous layer. Occasionally, the hypendymal cells form rosette-like aggregates with a central cavity (Olsson, 1958; Rodriguez

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Figure 2.10–2.12 (2.10) Schematic representation of an ependymal cell of the rat SCO. The secreted glycoproteins are shown at different steps of the maturation process: as coreglycosylated precursors stored in the RER (1), as complex-type precursor forms stored in immature secretory granules (2), as processed N-linked glycoproteins within mature secretory granules(3), and after their release in the ventricle, where they appear partially packed in a pre-RF state (4) and later densely packed forming the RF (5). The secretion that remains soluble in the cerebrospinal fluid is also depicted (6). BP, basal process ending on an expanded area of the perivascular space (PVS) partially filled with longspacing collagen (LSC). The double arrow points to extensions of the perivascular basal lamina. C, capillary. (Reprinted with kind permission from Springer Science þ Business Media: The Subcommissural Organ. Evidence for the release of CSF-soluble secretory material from the subcommissural organ, with particular reference to the situation in the human. Rodriguez et al., 1993, figure 1). (2.11) Sagittal section through the rat brain immunostained with an anti-Reissner’s fiber antibody. The subcommissural organ (SCO) is selectively stained. cc, cerebral cortex; c, cerebellum; p, pineal. Bar, 400 mm. (From Rodriguez et al., 1998). (2.12) Scanning electron micrograph of the Reissner’s fiber (RF) isolated from the bovine spinal cord. Bar, 12 mm. (From Rodriguez et al., 1998).

et al., 1984). In some mammalian species, the hypendymal cells are located within the PC sometimes close to the external limiting membrane (Rodriguez et al., 1984). Similarly, some hypendymal cells display long processes ending on the local blood vessels and the external limiting membrane and therefore crossing the PC.

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Ependymal SCO secretory cells are tall, cylindrical epithelial cells with a clear regionalization, allowing the recognition of several cytoplasmic regions: perinuclear, intermediate, subapical, and apical (Fig. 2.10). SCO ependymal cells display a basal process that project dorsally and contact with local blood vessels and with the external limiting membrane. In mammals, such processes are produced by both ependymal and hypendymal SCO cells, but in lower vertebrates, they are mainly present in the ependymal cells (Fernandez-Llebrez et al., 1987; Oksche, 1961; Rodriguez et al., 1984). In certain species of elasmobranchs and snakes studied, the endings of these basal processes contain numerous secretory related structures, such as RER, microtubules, and secretory granules, raising the possibility of a local release of the secretion into the leptomeningeal space (Grondona et al., 1994b; Peruzzo et al., 1990; Rodriguez et al., 1984). The subependymal space of the SCO is occupied by a dense capillary network whose vessels run closely to the hypendymal cells (Oksche, 1961). The presence of such vascular network in the basal region of the SCO and its apical contact with the ventricular cavity are both common characteristics of circumventricular organs; therefore, the SCO is regarded as one of them (Leonhardt, 1980). There exists a close relationship between the SCO cells and the PC (Figs. 2.13–2.17), which somehow reminds the relation between the FP with commissural axons of the spinal cord, or the one between the glial palisade and the axons of the optic chiasm (compare Figs. 2.2 and 2.17). The pioneer axons forming the PC cross the midline in close proximity to the basal region and the basal processes of the developing SCO (Caprile et al., 2009; Hoyo-Becerra et al., 2010; Meiniel et al., 1988; Schoebitz et al., 1986) (Figs. 2.15, 2.16, and 2.24). In accordance, SCO basal processes (both from ependymal and hypendymal cells) break through the nerve bundle of the PC, and even in some species, the hypendymal cells are located among axons of the PC bundles, proving the intimate association of the two structures (see earlier in this section). A detailed immunohistochemical study of the expression of the SCO secretion in the chick SCO and the relationships between the SCO cells and the axons of the PC allowed to define two regional domains within the SCO with functional implications. A lateral domain, where the SCO cells express its secretion and the PC axons associated to them are highly fasciculated, and a midline domain, where the SCO cells do not express immunoreactive secretory material and the associated axons, are defasciculated (see Fig. 2.24) (Stanic et al., 2010). It has been also proposed that the basal processes of the SCO-expressing lateral cells form tunnels that facilitate PC axon fasciculation and define their route toward the midline, where they start to defasciculate in the absence of secretion and face the options of crossing the midline or not (Stanic et al., 2010). A comparable, broadly studied situation occurs in the midline of the FP, where the organization

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Figure 2.13–2.17 (2.13) Sagittal section through the prosencephalon of an E3 chick embryo immunostained with an anti-Reissner’s fiber antibody (AFRU). Positive cells are found in the caudal portion of the diencephalon (prosomere 1) in contact with the cerebrospinal fluid of the third ventricle (V). Inset in Fig. 2.13 corresponds to a lower magnification image of a midline saggital section of an E3 chick embryo showing the location of the diencephalic roof plate (boxed area in the inset). SCO, subcommissural organ; OT, optic tectum; Tel, telencephalon; L, leptomeninge; V ventricle. Bars, 300 mm (inset) and 30 mm (Fig. 2.13). (From Hoyo-Becerra et al., 2010). (2.14) Sagittal section through the prosencephalon of an E3 chick embryo immunostained with antibeta-III tubulin. Pioneering axons forming the posterior commissure appear stained with the antibody. Inset in Fig. 2.14 shows the location of the diencephalic roof plate (boxed area in the inset) at a lower magnification. This section is similar to the one shown in the inset in Fig. 2.13. L, leptomeninge; SCO, subcommissural organ; OT,

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and routing of axons is a complex process involving the interplay of diverse axonal guidance cues.

4.2. Secretory material of the subcommissural organ The initial data regarding the molecular nature of the SCO secretion were compiled from histochemical techniques, which revealed that it consisted of a highly glycosylated material stained with periodic acid-Schiff and Gomori’s aldehyde fuchsin, demonstrating that it also has a high content of cysteine residues (Rodriguez et al., 1992). The precursor forms of this material accumulate in large RER cisternae and, according to its lectin-binding properties, present mannose as terminal residues of the sugar chains. Abundant apical secretory granules store the mature form of the secretion, which bears sialic acid as terminal residue of the sugar moieties (Fig. 2.10). The development of an array of antibodies allowed further characterization of the secretion (Fernandez-Llebrez et al., 2001a,b; Grondona et al., 1994b; Lopez-Avalos et al., 1996; Meiniel et al., 1988; Nualart et al., 1991; Rodriguez et al., 1986). Among these antibodies, the antisera against the purified RF (Rodriguez et al., 1984; Sterba et al., 1982) turned out to be a definitive tool to demonstrate the SCO origin of such thread-like structure (see Section 4.2.1. for more details). Although the bulk of the secretion is released into the ventricle and forms the RF (Rodriguez and Yulis, 2001), secretory granules have been optic tectum; Tel, telencephalon; V ventricle. Bars, 300 mm (inset), and 30 mm (Fig. 2.16). (From Hoyo-Becerra et al., 2010). (2.15) Confocal image of a sagittal section through the dorsal diencephalon of an E3 chick embryo, double-immunostained with a monoclonal antibody against the Reissner’s fiber (clone 4F7, red) and an antibody against Ng-CAM (Ng-CAM, green). The 4F7 antibody labels neuroepithelial cells that begin to differentiate into SCO cells at E3. The close relationship between the pioneering axons of the PC, labeled with anti-Ng-CAM (arrowheads), and the SCO cells can be observed. L, leptomeninge; SCO, subcommissural organ; V ventricle. Bar, 20 mm. (From Hoyo-Becerra et al., 2010). (2.16) Transversal section through the dorsocaudal diencephalon of an E4 embryo double-immunostained with AFRU (green) and anti-beta-III tubulin (red). Posterior commissure axons, labeled with anti-beta-III tubulin (arrowheads), cross the midline through the basal portion of the SCO cells (AFRU-positive, green). Bar, 60 mm. (From Hoyo-Becerra et al., 2010). (2.17) Scheme of a coronal section of the chick embryo brain (E6) at the level of the posterior commissure (PC). The basal processes of radial-like glial cells of the subcommissural organ (SCO) are in close relationship with the developing PC. At this stage, the SCO cells express SCO-spondin and some known axonal guidance cues.

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also described within basal processes of the SCO cells ending on the leptomeninge or on local blood vessels (Fernandez-Llebrez et al., 1987; Peruzzo et al., 1990; Oksche, 1961). The content of such granules shares properties with that stored in the granules of the apical pole of the cells, suggesting an additional basal route of release of the same type of glycoproteins (Peruzzo et al., 1990). The initial analysis of the SCO secretion was performed by immunohistochemistry and Western blot, employing both antibodies and lectins. These approaches revealed that the SCO secretion consists of quite large glycoproteins, synthesized as very high-molecular-mass precursors (540 and 320 kDa in the cow, 600 kDa in the dogfish, 540 kDa in chick embryos; Rodriguez et al., 1998), bearing mannose rich sugar moieties. These precursors undergo a maturation process that renders smaller products (450 and 190 kDa in the cow, 475, 400 and 145 kDa in the dogfish; Rodriguez et al., 1998) with sialic acid as terminal residue of the sugar chains. On the other hand, the use of several anti-RF or anti-SCO antibodies in different species throughout the vertebrate phylum demonstrated that the secretory compounds have some highly conserved epitopes, indicating phylogenetically conserved features of the SCO secretion, while other epitopes are preferentially class-specific (Grondona et al., 1994a; Nualart and Rodriguez, 1996). The large size and high degree of glycosylation of the SCO secreted compounds hindered its cloning and sequencing, but the efforts of two groups ended up with the full sequence of the so-called SCO-spondin (Gobron et al., 1996; Nualart et al., 1998), a huge protein of more than 5000 aminoacids with many TSRs, which make it a member of the thrombospondin family. 4.2.1. Reissner’s fiber Probably one of the most striking features of the SCO is the thread associated to its apical pole, which runs caudally all along the central canal of the spinal cord. The so-called Reissner’s fiber, named after the German anatomist Ernst Reissner, is, as mentioned before, the main destination of the SCO secretion. Upon release and once on the ventricular surface, the glycoproteins polymerize forming a cord (Fig. 2.12), which keeps growing anteriorly and depolymerizing caudally. Thus, the RF is a dynamic structure, with an estimated daily growth rate ranging from 10% of its length in the mouse to 1% in the carp (Ermisch, 1973). At the end of the spinal cord, the central canal enlarges forming the terminal ventricle, where the RF depolymerizes into a mass of fibrous material named massa caudalis. The unpacked glycoproteins seem to escape through openings in the walls of the terminal ventricle reaching the nearby blood vessels (Rodriguez et al., 1987a,b). But which is the physiological meaning of this peculiar structure? Although there is no definitive answer to this question, some authors suggest that the RF could control the concentration of biogenic amines in the CSF

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by reversibly binding them to its structure (see Section 4.6.3) (Caprile et al., 2003). Also, it has been speculated that it could participate in the detoxification of the CSF by binding waste molecules (see Section 4.6.3) (Olsson, 1958). Some experiments demonstrated abnormal CSF flow in the central canal after immunological destruction of the RF, pointing to a role of the fiber in CSF circulation (see Section 4.6.4) (Cifuentes et al., 1994). In large mammals such as the cow, the RF can be isolated by perfusing the central canal of the spinal cord, rendering an excellent purified source of secretion for analysis and antibody production (Fig. 2.12). Western blot analysis of the solubilized bovine RF reveals several high-molecular glycoproteins, ranging from 450 to 89 kDa (Nualart et al., 1991), all of them recognized by the antibodies raised against the RF. Similarly, very large compounds were detected by these antibodies in Western blots of SCO extracts from cow (Nualart et al., 1991), chick (Cifuentes et al., 1996; Didier et al., 1995), and dogfish (Grondona et al., 1994b; LopezAvalos et al., 1996). 4.2.2. CSF-soluble secretory material The RF is not the only destination of the SCO secretion. Some RF-related compounds remain soluble in the CSF and can be detected by the RF specific antibodies in embryos (Hoyo-Becerra et al., 2006) as well as in the adult (Rodriguez et al., 1993), or in a hydrocephalus model (Carmona-Calero et al., 2009; Irigoin et al., 1990). The release of CSF-soluble material has also been described in nonphysiological systems: (i) in SCO transplanted under the kidney capsule (Rodriguez et al., 1989), (ii) in SCO transplanted into the ventricular cavities of chick embryos (Hoyo-Becerra et al., 2005), and (iii) in the culture medium of bovine SCO explants maintained in vitro (Lehmann et al., 1993). More difficult to identify is the putative SCO secretion nonrelated to the RF glycoproteins described so far. Rodriguez et al. (1993) used an interesting approach, consisting of the production of antibody against soluble CSF glycoproteins from hydrocephalic children, and found that some of those antibodies labeled the SCO. These results represent evidence that the SCO releases other compounds to the CSF apart from the well-known RF glycoproteins.

4.3. Molecular features of the subcommissural organ secretory material As mentioned above, SCO-spondin is the main secretory product of the SCO that, upon secretion into the ventricle, is incorporated to the RF (Gobron et al., 1996; Nualart et al., 1998). Its large structure contains a series of very well-conserved domains (Fig. 2.18), resembling ECM proteins, which include several TSRs, low-density lipoprotein receptor (LDLr)

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Figure 2.18 Domain structure of the SCO-spondin and some proteins of the thrombospondin superfamily. The sizes and domain locations of the proteins are referred to the amino acid scale shown above. Thrombospondin-1, F-spondin, Semaphorin 5, and UNC-5 all bear the “thrombospondin type 1 repeats” (TSR), a characteristic domain of this superfamily along with other specific domains (sema, spondin domains FS1 and FS2). All of them are involved in axonal guidance in different processes. SCO-spondin shown below is a much larger protein belonging to the same superfamily. It is split into two halves for its schematic representation. The TSR domain is highly represented (26 times) and SCO-spondin repeats (SCOR, 16 times), which are specific to this protein, or the low-density lipoprotein receptor type A repeats (LDLrA, 10 times). At the bottom, the symbols for the different domains are depicted. vWC, von Willebrand C domain; vWD, von Willebrand D domain; emilin, emilin domain; FA5-8C, coagulation factor 5/8 type C domain; CTCK, C-terminal cystine knot. Data for the scheme taken from Meiniel and Meiniel (2007) and Tucker (2004).

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type A repeats, SCO-spondin repeats (SCORs), von Willebrand factor (vWF) domains, and single copies of the emilin (EMI) motif, the coagulation factor 5/8 type C (FA5-8C) motif, and a C-terminal cystine knot (CTCK) (Meiniel, 2001; Meiniel, 2007). In general, most of these domains are known to be involved in protein–protein interactions. The TSR domains are a common feature of the thrombospondin superfamily of proteins (Adams and Tucker, 2000) and allocate the SCO-spondin within this family. Also, the existence of several consensus sequences for N-glycosylation agrees with previous data of the high degree of glycosylation of this protein. The TSRs represent the identity feature of the thrombospondin superfamily (Tucker, 2004) and are involved in protein–protein interactions required for adhesion of the cells to substrates or cell aggregation processes. The TSRs are present in certain proteins of the complement cascade, in the malaria parasite, where they are significant for the infection process, and are as well important for blood coagulation (Lawler and Hynes, 1989). The FA5-8C motif, found once in SCO-spondin, is involved in coagulation as well. As much as 26 TSRs have been identified in SCO-spondin, which is a surprisingly high number compared to other proteins of the thrombospondin family, suggesting that the interaction with other proteins must be necessary for the function of this large protein. The TSRs are also present in other proteins sharing with SCO-spondin the property of the early expression in the embryonic CNS, that is, F-spondin (Spon1) and Mindin (Spon2), which have been shown to promote outgrowth of commissural, hippocampal, and sensory neurons (Burstyn-Cohen et al., 1999; Feinstein et al., 1999) (see Sections 2.3.6. and 5.1). The LDLr type A motif is present in numerous proteins with the common property of binding various ligands or forming large complexes, such as certain proteins of the membrane attack complex of the complement (Esser, 1994). SCO-spondin bears 10 copies of such domains, and the Drosophila protein nudel presents up to 11. The role of the Nudel protein might enlighten some putative unknown function of SCO-spondin. Nudel is an extracellular protease expressed in the Drosophila embryo, which serves two functions, the assembly and anchoring of a macromolecular complex of proteolytic enzymes, and the activation of the protease cascade, with the final aim of regulating the dorsoventral polarity of the embryo (Hong and Hashimoto, 1995). Furthermore, the LDL receptor-related protein (LRP), which also bears several type A repeats, has been shown to interact with soluble extracellular proteases and protease inhibitor complexes and, thus, regulating protease activity (Herz, 2001). The possible function of the SCO secretion in the development has been frequently raised, based on its early expression in embryos, the location of the SCO in the dorsal midline, and the transient expression of the secretion in some regions of the ventral midline (del Brio et al., 2000; Fernandez-Llebrez et al., 1996; Guin˜azu´

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et al., 2002; Lehmann and Naumann, 2005; Lopez-Avalos et al., 1997; Naumann, 1986; Richter et al., 2001; Rodriguez et al., 1996). The single EMI domain present at the NH2-end of the protein (Doliana et al., 2000) and the several vWF domains close to the same end are most probably involved in the multimerization of SCO-spondin. The CTCK motif found at the C-terminus of the protein serves likely for its dimerization, as has been described for numerous proteins presenting such domain, particularly growth factors (Isaacs, 1995). An initial dimerization mediated by the CTCK domain could be required for the posterior formation of oligomers of SCOspondin, a process that could involve the EMI and vWF domains. These would provide a molecular explanation for the formation of the RF and the general tendency of SCO-spondin to form aggregates (Meiniel, 2007). A thorough analysis of the SCO-spondin based on the alignment of the sequences of four mammalian species (mouse, rat, cow, and human) allowed the identification of a new consensus domain, the SCOR, which is characterized by a series of well-conserved cysteine residues. The SCOR module is repeated up to 16 times in SCO-spondin sequence (Fig. 2.18). It shows homologies with some protease inhibitors, particularly with serine-protease inhibitors and cysteine-rich protease inhibitors (Meiniel, 2007). The LDLr type A repeats, also present in SCO-spondin, have been related to the regulation of protease activity (Herz, 2001). Interestingly, SCOR domains could collaborate in this regulatory function, enlightening a putative role of SCO-spondin in protease activity regulation. The role of the SCO secretion has for long been elusive. The recent knowledge of SCO-spondin molecular structure has brought some light to its possible function. The well-characterized modular organization of the protein and the similarities found with other known proteins help to narrow the search for a role of SCO-spondin. Thus, it could participate in the regulation of extracellular protease activity, a process probably involving the formation of large complexes of proteins and particularly relevant in the context of the CNS development. The early expression of SCO-spondin during brain development and its structural similarities with other molecules known to act as CNS morphogens suggest its participation in axonal pathfinding. This particular issue will be discussed later.

4.4. Sites of release of the secretory material Several strong evidences support the ventricular release of at least part of the SCO secretory compounds into the CSF of the third ventricle. As described in Section 4.2., once released into the CSF, the SCO secretory material polymerizes to form the RF. Monoclonal and polyclonal antibodies raised against the RF specifically recognize the secretory material of the SCO (Rodriguez et al., 1984; Sterba et al., 1982). Likewise, antibodies against SCO extracts immunoreact with the RF (Fernandez-Llebrez et al., 2001a,b;

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Grondona et al., 1994a,b; Karoumi et al., 1990; Lopez-Avalos et al., 1996; Meiniel et al., 1988; Rodriguez et al., 1985). As previously mentioned, some of the SCO material released into the ventricular cavity remains soluble in the CSF (see Section 4.2.2). The existence of basal processes in both ependymal and hypendymal cells and the fact that the endfeet of such processes contact with local blood vessels and the external limiting membrane raise the possibility of a basal route of secretion, particularly when secretion-related structures and the secretion itself have been found in such distal endings (Fernandez-Llebrez et al., 1987; Oksche, 1961; Rodriguez et al., 1984). Numerous morphological studies have found a close relation between the SCO cells and the local blood capillaries (Fernandez-Llebrez et al., 1987; Oksche, 1961). In addition, perivascular endfeet contain immunoreactive material within secretory granules (Peruzzo et al., 1990). However, there are no definitive evidences for the release of SCO secretion to the blood vessels. A second target of the SCO terminal endfeet is the external limiting membrane of the brain. In the snake Natrix maura, these leptomeningeal endfeet contain granules which display the same immunocytochemical and lectin-binding properties than those found in the apical region of the SCO cells (Peruzzo et al., 1990). Due to their lectin-binding affinities, namely, WGA positive and ConA negative, it was concluded that those granules were post-Golgi structures. Because Golgi apparatus is absent of the terminal endfeet, the granules must be transported from the cell body to the endfeet. The transport of secretory granules and their accumulation in leptomeningeal endfeet strongly support the possibility of a release to the leptomeningeal space (Peruzzo et al., 1990). Apart from the three above-mentioned release sites of the SCO secretion considered classical fates, there exists a fourth one: the extracellular space situated at the basal portion of the ependymal and hypendymal layers. Immunocytochemical studies at the ultrastructural level have revealed the presence of anti-RF positive material in expanded areas of the extracellular space close to blood vessels (Rodriguez et al., 1987a,b). The only report that directly addresses this question is the one from Caprile et al. (2009). These authors showed that in chick embryos at HH29, SCO-spondin forms aggregates surrounding the basal processes. At this stage, the PC is well developed and its axons bear beta1 integrin. At this extracellular location, SCO-spondin could serve as a ligand for beta1 integrin, present at the surface of neurites of the developing PC (Caprile et al., 2009).

4.5. Ontogeny of the subcommissural organ Studies concerning SCO development have been carried out in several species (Rodriguez et al., 1992), but those performed in the chick provide a more detailed information of the process (Caprile et al., 2009; Cifuentes

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et al., 1996; Hoyo-Becerra, 2006; Hoyo-Becerra et al., 2005; HoyoBecerra et al., 2010; Karoumi et al., 1990; Meiniel et al., 1988; Naumann et al., 1987; Schoebitz et al., 1986; Stanic et al., 2010; Wingstrand, 1953). The SCO secretion has been detected in the roof plate of chick embryos as early as day 3 (Figs. 2.13 and 2.15) (Karoumi et al., 1990; Naumann et al., 1987; Schoebitz et al., 1986) (Figs. 2.13 and 2.15). This observation suggests that the SCO is one of the first structures in the brain to differentiate. AntiRF positive material was detected on the walls of the embryonic ventricles at day 7, indicating the release of a polymerizable secretion (Karoumi et al., 1990; Schoebitz et al., 1986; Wingstrand, 1953), albeit the RF proper was not detected until day 11 of incubation (Schoebitz et al., 1986). The absence of RF at stages when the secretion is already present in the ventricular cavity suggests that its polymerization to form the RF is not spontaneous but requires some extrinsic factor (Hoyo-Becerra et al., 2005). In addition, some data point out to an earlier ventricular release, that is, before day 7, although this early SCO secretion would remain soluble in the CSF (HoyoBecerra, 2006). Mouse SCO development has been investigated by the use of H3thymidine and autoradiography (Rakic and Sidman, 1968). These authors showed that in the mouse the SCO develops later than in the chick, being morphologically identifiable between 11 and 19 days post-coitum (dpc). The secretory activity was first detected at 14 dpc. Complete differentiation of the SCO cells takes place during the first postnatal month (CastaneyraPerdomo et al., 1983). The timing of the SCO development in the rat is quite similar. RF-immunopositive material is present at 15 dpc (Schoebitz et al., 1993) and a well-developed SCO is found at 17 dpc (Marcinkiewicz and Bouchaud, 1983). The RF is visible during the first postnatal week (Schoebitz et al., 1993). In the various species studied, the secretory activity of the SCO and the ventricular release of its secretion start very early in ontogeny (Naumann et al., 1993). In addition, the presence of soluble secretion within the ventricles seems to precede the RF polymerization (Hoyo-Becerra, 2006). These data would indicate that the SCO might play an important role during the development of the CNS. Most cordate species display a morphologically well-developed SCO, with an important secretory activity throughout the adult life (Oksche, 1961; Rodriguez et al., 1992). However, in anthropoid primates, including humans, the SCO is prominent during the fetal life but undergoes a regression after birth (Castaneyra-Perdomo et al., 1985; Oksche, 1961; Palkovits, 1965), and only remnants of SCO parenchyma can be distinguished in the adult (Oksche, 1961). An intriguing feature of the human SCO is that, despite of its secretory nature revealed by histochemical techniques (Oksche, 1961), the secretion is not recognized by the different antisera raised against the RF (Rodriguez et al., 1984; Rodriguez et al., 1990). However, an antibody raised

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against a human CSF-soluble compound was able to recognize the human fetal SCO and the adult rat SCO, suggesting that the human SCO has lost the ability to secrete RF-forming proteins while maintaining the CSF-soluble secretion (Rodriguez et al., 1993) (see Section 4.2.2).

4.6. Classical functions of the subcommissural organ Shortly after its discovery, various functional hypotheses were proposed for the SCO. Most of them were based on morphological evidences, which resulted in contradictory and unconnected hypotheses (Leonhardt, 1980; Rodriguez et al., 1992). The four most significant functional hypotheses are discussed in this section although, to date, none of them has been proven roundly nor are they mutually exclusive. In fact, most probably the SCO might have multiple roles due to the complexity of its secretion: several compounds, different release sites, andtherefore, various putative targets. 4.6.1. Morphogenetic function Some studies have shown that the presence of the RF in the regenerating tail and an active SCO is necessary for the normal regeneration of the amphibian’s tail (Hauser, 1972; Winkelmann, 1960). Furthermore, the destruction of the SCO in amphibians results in a deformation of the body axis (Murbach and Hauser, 1974; Ru¨hle, 1971). However, in experiments involving the SCO destruction, damage of the adjacent tissue might also occur; hence, caution must be taken raising conclusions. In postnatal rats, transection of the distal portion of the RF, named filum terminale, does not affect the normal growth of the tail (Sterba and Wolf, 1970). Therefore, with these evidences it is not possible to determine whether the RF plays a role in normal tail growth or not. In commercial cultures of the teleost Sparus aurata, a high incidence of axial deformities has been reported. Lordotic specimens showed important alterations in the RF and in the central canal of the spinal cord, and histochemical results suggested a hyperactivity of the SCO (Andrades et al., 1994). 4.6.2. Hydromineral balance regulation The SCO has been related to the water and electrolyte metabolism, being linked to volume reception, thirst, sodium excretion, diuresis, and aldosterone secretion. SCO ablation in rats produced a decrease in water intake (Gilbert, 1960). Gilbert proposed that the SCO could act as a volume receptor implicated in the regulation of fluid homeostasis in the body. In other experiments, the SCO ablation induced a decrease in urine output and sodium excretion, but water intake was unaffected (Upton et al., 1961). Further experiments of complete SCO ablation reported a reduction in water intake and urine production, but no changes were observed in sodium output (Brown and Afifi, 1965). Conversely, other authors did not find changes in any parameter

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related with the hydrosaline balance after SCO destruction (Bugnon et al., 1965). The different ablation methods used to remove the SCO might account for the variability of these results (Severs et al., 1987). Similar disparities were observed after the administration of SCO extracts. Some authors found no alteration in the hydrosaline balance (Wingstrand, 1953), while others reported an antidiuretic effect of the SCO extracts (Palkovits, 1965). Regarding the latter result, the presence of AVT (Rosenbloom and Fisher, 1975) or an AVT-related peptide (Dogterom et al., 1979) has been described in the SCO of several species. The presence in the SCO of hormonal receptors that control osmotic parameters suggests its participation in osmoregulation. Specific receptors for angiotensin II have been described in the SCO (Castaneyra-Martin et al., 2005; Nurnberger and Schoniger, 2001), and non-AT and non-AT2 binding site for Angiotensin II were recently described in the rat SCO (Karamyan and Speth, 2008). In the salamander Hydromantes genei, the pineal gland and the SCO showed a high concentration of atrial natriuretic factor (ANF) but did not contain ANF-binding sites (Mathieu et al., 2001). The rat SCO present scarce ANF-receptors (Mantyh et al., 1987) or is completely devoid of them (Bianchi et al., 1986). On the other hand, the SCO contains the glucocorticoid-inactivating enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a signature of aldosterone-sensitive tissues. Only a few brain sites contain aldosterone-sensitive neurons (Geerling et al., 2006). This result is in accordance with previous data obtained by intracerebroventricular infusion of aldosterone that suggest a relationship between the SCO and the adrenal gland (Dundore et al., 1987). The osmotic stimulation has been used as an alternative experimental approach for studying the participation of the SCO in water and electrolyte regulation. Parameters such as the amount of SCO material, ultrastructural changes, growth rate of the RF, turnover of the secretion, and incorporation of 35S-cysteine or 3H-leucine in the SCO have shown contradictory results (Rodriguez et al., 1992). Rodriguez and coworkers designed a set of experiments to unravel any relationship of the SCO with osmoregulation. No changes were found either in the amount of SCO secretion or in the ultrastructure of the SCO (Rodriguez et al., 1992). Only the immunological destruction of the RF resulted in some change in parameters related to water and electrolyte balance. Thus, rats immunologically deprived of RF showed alterations in the urine flow and in water intake (Rodrıguez, 1991). 4.6.3. CSF composition regulation Due to the biochemical nature of the RF, particularly its high sialic acid content, it has been proposed that the RF acts as a CSF detoxifier by binding waste molecules present in the CSF (Olsson, 1958). In vitro and in vivo studies have revealed that the RF specifically binds tyrosine and some

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biogenic amines such as adrenaline, noradrenaline, and serotonin (Sterba and Ermisch, 1969) More recently, by using different experimental approaches, Caprile et al. (2003) have shown that the RF regulates the CSF concentration of monoamines either by binding and transporting them away throughout the central canal of the spinal cord (L-DOPA, noradrenaline, adrenaline) or by transiently binding them and releasing them back to the CSF (serotonin). Furthermore, adrenaline and noradrenaline share the same binding site in the SCO-spondin, while serotonin has its own binding site in RF (Caprile et al., 2003; Rodriguez and Caprile, 2001). 4.6.4. CSF production and circulation More than 50 years ago, Overholser et al. (1954) first suggested that the fetal SCO secretion released to the CSF keeps the cerebral aqueduct open, allowing the circulation of the CSF between the ventricles. This hypothesis was supported by posterior data, as experimental alterations in the SCO development lead to hydrocephalus (Overholser et al., 1954; Takeuchi and Takeuchi, 1986). Moreover, in many cases of congenital hydrocephalus, the SCO epithelium is absent (Takeuchi et al., 1987) or reduced in size (Takeuchi et al., 1988). Immunological blockade of the SCO secretion with antibodies against RF can induce hydrocephalus (Rodriguez and Yulis, 2001) and rats devoid of RF display a decrease in the CSF flow (Cifuentes et al., 1994). Recently, null mutant mice for genes implicated in the development of the diencephalic roof plate and therefore, in SCO differentiation, were shown to develop congenital hydrocephalus (Galarza, 2002; Huh et al., 2009; Meiniel, 2007). Thus, mutant mice such as Wnt1Sw/Sw (Louvi and Wassef, 2000), PAX6Sey/Sey (Estivill-Torrus et al., 2001), and Msx1 (FernandezLlebrez et al., 2004) exhibit a congenital hydrocephalus associated with an abnormal or missing SCO. Also, mutant mice for transcription factors that regulate the expression of intraflagellar transport proteins exhibit communicating hydrocephalus with an absence or downregulation of the SCO secretion. This is the case of the RFX3/ mutant mouse (Baas et al., 2006) and the heterozygous RFX4_v3 mouse (Blackshear et al., 2003). Transgenic mice that overexpress the pituitary cyclase-activating polypeptide (PACAP) type I receptor exhibit hydrocephalus (Lang et al., 2006), along with a reduced SCO and abnormal cilia. A transgenic mouse line that expresses the G(i)-coupled RASSL (receptor activated solely by synthetic ligand) Ro1 in astrocytes develops hydrocephalus, partial denudation of the ependymal cell layer, altered SCO morphology, and obliteration of the cerebral aqueduct (Sweger et al., 2007). Conditional inactivation of the hdh gene (the mouse Huntington’s disease gene homolog) in Wnt1 cell lineages results in congenital hydrocephalus, which is associated with an increased CSF production by the choroid plexus and an abnormal SCO (Dietrich et al., 2009). On the other hand, p57 (Kip2), a cyclin-dependent kinase inhibitor, plays a key role in cell

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cycle arrest during development. In the mouse brain, p57 is predominantly expressed in the SCO and in cerebellar interneurons. Mice with brain-specific deletion of the p57 gene (Kip2) show a prominent nonobstructive hydrocephalus and cerebellar abnormalities (Matsumoto et al., 2011). Furthermore, the conditional inactivation of presenilin-1 in Wnt1 cell lineages results in congenital hydrocephalus and SCO abnormalities, which suggests a potential role of presenilin-1 in CSF homeostasis (Nakajima et al., 2011). All the above-related results represent multiple evidences of the possible implication of the SCO and its secretion in CSF production and/or flow, a process that, when altered, might lead to hydrocephalus.

5. SCO-Spondin as an Axonal Guidance Molecule The initial studies concerning the SCO–RF complex proposed functions related to the regulation of CSF composition and CSF flow, hydrosaline homeostasis, or some role in development (described in Section 4.6). The posterior description of the molecular features of the SCO secretion steered the attention to a potential participation in axonal guidance, based on the similarities found with other already known molecules displaying this function, and on the evidences obtained from in vitro experiments.

5.1. Similarities with other axon guidance molecules SCO-spondin belongs to the thrombospondin superfamily of ECM and membrane proteins, which are highly conserved and have a modular structure. Most members of this superfamily have at least one copy of the TSR domain, which frequently has functional significance. They are related to cell–cell and cell–matrix interactions, cell migration, and ECM organization in diverse tissues and body systems (Tucker, 2004). Thrombospondins (TSPs) form a subgroup within this superfamily, represented in vertebrates by TSP-1 through TSP-5. By interacting with a diversity of ECM components, growth factors, proteases, cell receptors, etc., TSPs contribute to complex processes such as angiogenesis, wound healing, and synaptogenesis (Adams and Lawler, 2011). TSP-1 and TSP-2 are structurally quite similar, each presenting three TSR domains (SCO-spondin has 26 TSRs). Their expression pattern in the developing brain and in vitro studies points to a role of TSPs in neuronal adhesion and neurite outgrowth (O’Shea et al., 1991). Specifically TSP-1 and TSP-2 are expressed by immature astrocytes and promote synaptogenesis (Christopherson et al., 2005). Moreover, TSP-1 supports neuroblast migration along the rostral migratory stream in the adult CNS (Blake et al., 2008). Another thrombospondin, TSP-4, has been also involved in neurite outgrowth in both the developing and the adult

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nervous system (Dunkle et al., 2007), although it must be noted that this thrombospondin is devoid of TSR domains. The TSP superfamily includes other nonthrombospondin members, such as those belonging to the F-spondin family, semaphorins, UNC-5, and the SCO-spondin (Adams and Tucker, 2000). The F-spondin family (see also Section 2.3.6) includes several members characterized by FS1 and FS2 domains as well as TSR motifs. Their most distinctive feature is the prominent expression at the FP during early stages of development (Higashijima et al., 1997). In vitro and in vivo experiments demonstrated that F-spondin promotes adhesion and neurite outgrowth of spinal cord, hippocampal, and sensory neurons, indicating a possible role in proper axon pathfinding both in the CNS and in peripheral nerves (Burstyn-Cohen et al., 1999; Feinstein et al., 1999). The contribution of F-spondin to this function is complex, as it has been shown that the cleavage of the F-spondin molecule renders two fragments with opposite functions, one acting as a repellent and the other as a permissive cue for commissural axons in the ventral midline (Zisman et al., 2007). Interestingly, in zebrafish F-spondin has been located associated to the Reissner’s fiber (Higashijima et al., 1997), suggesting a ventricular secretion of this protein similarly to the SCOspondin. On the other hand, it has been shown that F-spondin can also promote the differentiation of neural precursors, a function located outside the TSR domains (Schubert et al., 2006). Semaphorins (see Section 2.3.3) are phylogenetically conserved proteins that usually present up to seven TSR domains and a unique large semaphorin domain, some of which are secreted while others can be found bound to the cellular membrane (Adams and Tucker, 2000). They serve as axonal guidance molecules (Kolodkin et al., 1993). In Drosophila embryos, Semaphorin I acts as a repulsive cue and is required for guidance and defasciculation of motor axons, thus contributing to build neuromuscular connexions (Yu et al., 2000). Similar to the F-spondin mentioned above, Semaphorin 5A has been shown to be a bifunctional guidance cue. In mammals, it can promote either attraction or repulsion of developing axons, depending on the type of proteoglycan it is interacting with (Kantor et al., 2004). In zebrafish, the Semaphorin 5A bifunctionality has been further dissected: the axon growth stimulation resides in the TSR domain, whereas the repulsion effect that keeps axons from branching and defasciculation lies in the semaphorin domain (Hilario et al., 2009). UNC-5, which was first cloned in C. elegans, is a transmembrane protein with a distinctive cytoplasmic domain and an extracellular portion bearing two immunoglobulin and two TSR domains (see Section 2.3.2). The ligand of UNC-5 is the laminin-like molecule Netrin (or UNC-6), which has another receptor, DCC. UNC-5 homologues have been later found in mammals including humans. In C. elegans, UNC-5 is expressed by migrating cells and growth cones and guides dorsal movements, but not ventral or longitudinal

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migrations (Hamelin et al., 1993). The pattern of expression of its two vertebrate homologues in the mouse developing nervous system is consistent with a similar role in axon guidance and neuronal migration (Leonardo et al., 1997). Furthermore, Ackerman et al. (1997) described severe abnormalities in the cerebellum of a UNC-5 homologue mutant mouse, possibly as a result of a defective neuronal migration during development (Ackerman et al., 1997). Interestingly, Netrins are also bifunctional guidance molecules, depending on the type of receptor expressed by the migrating axons. Thus, DCC mediates an attractive response when expressed alone, but when it is co-expressed with UNC-5, both receptors form a complex upon Netrin binding that mediates a repulsion effect (Hong et al., 1999; Keleman and Dickson, 2001). Hong et al (1999) also presented evidence that the cytoplasmic domains of the DCC/ UNC-5 complex are sufficient to elicit the repulsive effect, but the extracellular domains (where the TSRs are located) are necessary for Netrin binding and triggering complex formation (Hong et al., 1999). The most significant structural feature that SCO-spondin shares with all the above-described molecules is the presence of TSRs. But the similarities with these other molecules further extend to their spatiotemporal expression pattern, frequently associated to early embryonic stages and particularly to midline structures of the CNS. In recent studies carried out in chicks, Caprile et al. (2009) demonstrate that SCO-spondin is present in the ECM at the basal portion of the SCO, where the basal processes of the ependymal cells intermingle with axons of the PC. They also demonstrate that, in this scenario, SCO-spondin colocalizes with beta1 integrin, which is expressed both by SCO cells and by axons of the PC (Caprile et al., 2009). This colocalization could denote the functional interaction between SCO-spondin and beta1 integrin, a possibility already suggested by other authors (Bamdad et al., 2004). The binding of SCO-spondin to beta1 integrin at the surface of the axons forming the PC points to a potential role of this ECM protein in the PC formation. Actually, integrin receptors have widely been studied as mediators of neuronal migration and axonal outgrowth and guidance (Clegg et al., 2003). Some authors suggest that the TSRs are the binding sites for beta1 integrin (Calzada et al., 2004). Furthermore, the inhibition of endothelial cell migration mediated by TSP-1 is dependent on its interaction with beta1 integrin through the TSR domain (Short et al., 2005).

5.2. In vitro activity of SCO-spondin on neurite outgrowth As there were raising evidences of a putative role of the SCO secretion on nervous system development, a series of in vitro studies were designed to address this question. The cultures employed consisted mainly of chick cortical or spinal cord neurons, or the rat neuroblastoma cell line B104. These cultures were treated either with the RF proper or with the solubilized RF, rendering slightly different results. Thus, while the RF proper induced neuronal

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aggregation in chick cortical and spinal cord neurons (Fig. 2.21) (Monnerie et al., 1997a,b), such effect was not observed when solubilized RF was applied to chick cortical neurons (Monnerie et al., 1996) or B104 cells cultured at low density (El-Bitar et al., 2001). The effects regarding enhancement of neurite outgrowth are more consistent, as both soluble and condensed RF material increased the number and length of neurites growing from chick cortical or spinal cord neurons (Monnerie et al., 1997a,b) and from B104 cells (El-Bitar et al., 2001). In a different experimental setting, the soluble RF was placed in a three-dimensional collagen gel, with explants obtained from the pretectal area of chick embryos immersed in it. In such situation, the RF material also produced an increase in the number and length of the axons sprouting from the explants, which was accompanied by an improved fasciculation of those axons (Stanic et al., 2010). Upon discovery of the molecular structure of SCO-spondin, a series of peptides were designed based on the unveiled sequence to be used in similar in vitro studies (Monnerie et al., 1998). Two peptides included in two different TSRs of the protein induced cell adhesion and aggregation and promoted neuritic outgrowth in chick embryonic cortical neurons (Monnerie et al., 1998), particularly the peptide WSGWSSCSRSCG, which presented a more potent activity (Fig. 2.20). In subsequent studies, this peptide has been shown to favor aggregation and neuritic outgrowth also in B104 cells (Bamdad et al., 2004; El-Bitar et al., 2001). Interestingly, in chick spinal cord neurons, an antiaggregative effect of this peptide was reported (Monnerie et al., 1998), despite it maintained a proneuritic effect. This apparent discrepancy is in accordance with the complexity of the developing systems, where the same cue can render opposite effects depending on the target cell or the specific environment. The enhanced aggregation and neuritic outgrowth promoted by the WSGWSSCSRSCG peptide on B104 cells were blocked by pretreatment of the cells with beta1 integrin function-blocking antibodies (Bamdad et al., 2004). The participation of the beta1 integrin signaling pathway in axon outgrowth and/or guidance mediated by SCO-spondin is further supported by recent studies showing the expression of beta1 integrin at the basal domain of the SCO cells and in the axons of the PC (Caprile et al., 2009). All these in vitro results demonstrate a role of the SCO secretion on neuronal development, particularly in the process of neurite outgrowth. To uncover the precise function of SCO-spondin in each specific stage of development, a more detailed analysis involving in vivo models will be required.

5.3. Coculture experiments with SCO explants The culture in three-dimensional matrices has emerged as a powerful approach for axonal guidance studies, as it mimics the in vivo environment and provides a semisolid medium where growth and auto- and paracrine

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Figure 2.19–2.21 (2.19) Effects of peptides derived from SCO-spondin on cortical neurons in primary cell cultures. In control cultures, after 5 days, cortical neurons adhered poorly on plastic wells and formed aggregates from which very short or no neurites extended. Bar, 50 mm. (From Meiniel, 2001). (2.20) Effects of peptides derived from SCO-spondin on cortical neurons in primary cell cultures. In experimental cultures, the presence of the TSR-derived peptide WSGWSSCSRSCG induces the extension of prominent neuritic processes after the same period of time in culture. Bar, 50 mm. (From Meiniel, 2001). (2.21) In experiments similar to those shown in Figs. 2.19 and 2.20, Reissner’s fiber (RF) pasted onto the well surface induced neuronal aggregation and enhanced neurite outgrowth. Photographs were taken in phase contrast to show the impressive extension of neurites in the vicinity of the RF. Bar, 110 mm. (From Meiniel, 2001).

interactions are allowed (Lumsden and Davies, 1983). The collagen gel is the most widely used matrix of this kind and has been employed also to analyze the role of the SCO and its secretion on axonal guidance. In the chick embryo model, the source of the PC pioneering axons that start crossing the PC at embryonic day (E) 3 was located by application in ovo of the lipophilic tracer DiI in the PC (see Section 3.1). The MNPC (Ferran et al., 2009; Puelles et al., 1996) was identified as the origin of the pioneering axons (Hoyo-Becerra, 2006; Hoyo-Becerra et al., 2010) and was subsequently used to prepare explants for coculture experiments. The MNPC

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explants were used as targets for the SCO explants, which were considered as the potential source of axonal guidance cues. The faced explants were immobilized and kept at a defined distance, immersed in a rat-tail collagen matrix. MNPC explants from E4 embryos were confronted to SCO explants from early (E4) and late (E13) stages of development. The number and length of the neurites emerging from the MNPC explant at the side facing the SCO explants was evaluated (proximal quadrant, PQ) and compared with the side opposite to the SCO explant (distal quadrant, DQ), which served as an internal control. Interestingly, the E4 SCO promoted neurite outgrowth in the PQ of the MNPC explants (Fig. 2.22), while the E13 SCO inhibited such effect (Fig. 2.23) (Hoyo-Becerra, 2006; HoyoBecerra et al., 2010). These results suggest that, at early developmental stages, the SCO exerts a stimulatory effect on neurite outgrowth from MNPC cells, whereas at later stages, it induces inhibition of such outgrowth. Hence, the SCO should produce guidance molecules able to diffuse through the collagen matrix, acting as an attractive and/or repellent signal for the neurites growing from the MNPC. SCO-spondin, whose expression by SCO cells is concomitant with the onset of PC formation (Hoyo-Becerra, 2006; Hoyo-Becerra et al., 2010), could be responsible for these in vitro effects. However, against this hypothesis is the fact that the high-molecular weight of SCO-spondin could prevent its diffusion through the collagen matrix toward the target cells. Therefore, the possibility that other guidance cues might account for the described effects of the SCO explants cannot be ruled out. In fact, the expression of several axonal guidance cues by the SCO has been previously reported (see Section 6). In other in vitro studies, SCO-spondin was dissolved within the collagen gel matrix, avoiding any possible diffusion drawback (Stanic et al., 2010). In this situation, the target explants were exposed to SCO-spondin from every side. An increase in the number and length of neurites, along with an improved fasciculation of the axons, was observed all around the explants exposed to SCO-spondin (compare Figs. 2.25 and 2.26) (Stanic et al., 2010). These bioassays may be considered a first approach to study the in vivo function of the SCO, but the specific role of SCO-spondin (or any other molecule released by the SCO) in the formation and/or maintenance of the PC remains to be determined.

5.4. SCO secretion in the floor plate The functional role of the FP as a ventral midline structure implicated in both the patterning of the ventral nervous system (Yamada et al., 1991) and the guidance of commissural axons (Feinstein and Klar, 2004; Imondi and Kaprielian, 2001; Zou et al., 2000) is quite well established. Molecules expressed in this region have a good chance of being involved either in morphogenetic events or in axonal guidance processes. Olsson (1956) was

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Figure 2.22–2.26 (2.22) Coculture of explants from E4 chick embryos. Explants of the magnocellular nucleus of the posterior commissure (MNPC) were confronted to subcommissural organ (SCO) explants. After 24 h in culture, neurites growing from the MNPC toward the SCO (PQ, proximal quadrant facing the SCO) are more abundant than those growing in the opposite side (DQ, distal quadrant opposite to the SCO). SCO at early stages of development should synthesize and release attractive axonal cues to induce such response in neurite outgrowth. Bar, 250 mm. (From Hoyo-Becerra et al., 2010). (2.23) Coculture of explants from chick embryos. Explants of the magnocellular nucleus of the posterior commissure region (MNPC) at E4 were confronted to subcommissural organ (SCO) explants obtained from later embryos (E13). Conversely to the results shown in Fig. 2.22, after 24 h of culture, a lower number of neurites are growing in the proximal quadrant (PQ, quadrant facing the SCO) compared to those growing in the distal quadrant (DQ, quadrant opposite to the SCO). At contrary, the SCO at later stages of development would synthesize and release some repulsive signal. Bar, 500 mm. (From Hoyo-Becerra et al., 2010). (2.24) Transversal section through the

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the first to report a hindbrain FP region exhibiting secretory features. This FP region was called flexural organ and seems to be the source of RF-like compounds prior to the differentiation of the SCO (Olsson, 1956; Olsson, 1958). Several decades later, numerous studies by using antibodies against the RF have shown that the hindbrain FP synthesizes compounds related to the SCO–RF secretion (del Brio et al., 2000; del Brio et al., 2001; Fernandez-Llebrez et al., 1996; Fernandez-Llebrez et al., 2001a,b; LopezAvalos et al., 1997; Naumann, 1986; Naumann et al., 1993; Rodriguez et al., 1996). By using a similar but different anti-RF antibody, another group has reported that this RF-like compound is not only present in the anterior portion of the FP, but also along its entire length during early developmental stages in different vertebrate species. Later in development, the RF-like substance expression disappears, first in the most rostral areas and later in the spinal cord (Lichtenfeld et al., 1999). A more definite result was reported by Richter et al. (2001), who demonstrated by RT-PCR that the bovine FP expresses sco-spondin gene. In addition, bovine FP explants in culture synthesize and release SCO-spondin to the medium where it remains soluble (Guin˜azu´ et al., 2002). Zebrafish mutants cyclops (cyc) and one-eyed pinhead (oep), both lacking a FP, exhibit an aberrant axonal growth of the MLF. These defects are probably related to the absence of axonal guidance cues associated with or released by the FP. Some authors suggest that the RF-like compound could be one of such missing cues (Lehmann and Naumann, 2005).

6. Expression of Axonal Guidance Molecules in the Subcommissural Organ SCO-spondin represents the major compound synthesized by the SCO cells. As discussed previously in this review, it is presumably involved in the formation of the PC. As occurs in the FP, midline structures make use of a wide variety of axonal guidance cues to regulate the crossing of axons. SCO of a chick embryo at HH39 (E13) immunostained with an anti-RF antibody (AFRU). SCO-spondin expression is restricted to the lateral diencephalic roof plate, where it is located within the cellular bodies as well as in the extracellular matrix that contacts with the axons of the PC. Right at the midline, a small domain of the SCO is devoid of SCO-spondin. Bar, 50 mm. (2.25) Chick embryo pretectal explant from HH34 (E8) chick embryos, cultured in a three-dimensional collagen gel matrix for 48 h. It was cultured without SCO-spondin. Bar, 200 mm. (From Stanic et al., 2010). (2.26) Chick embryo pretectal explant (similar to that showed in Fig. 2.25) grown in presence of 18 ng of SCO-spondin/ml collagen matrix. The presence of SCO-spondin results in an increase in the number and length of neurites. Bar, 200 mm. (From Stanic et al., 2010).

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Interestingly, some well-characterized axonal guidance molecules are also expressed by the SCO cells at the time of PC development, so they should be considered as potential regulators of the formation of that commissure. In Xenopus, Slit-2 is expressed in the FP and in the midline diencephalic roof plate (Fig. 2.27) (Hocking et al., 2010). Similarly, in the chick embryo, it is present in the dorsal and ventral midlines of the brain (including the diencephalic roof plate and the SCO) (Holmes and Niswander, 2001), and in mouse embryos (from E11.5 through E14.5), in the roof plate of the diencephalon and the midbrain (Fig. 2.28) (Bagri et al., 2002; Holmes et al., 1998). On the other hand, the semaphorin Sema3D is expressed in the dorsal diencephalon of chick embryos between HH12 and HH22 (E3,5) (Fig. 2.29) (Bao and Jin, 2006), when the PC is already formed. In zebrafish, the Sema3D knockdown by morpholinos demonstrated its requirement for the correct formation of two early axon pathways: the medial longitudinal fasciculus and the anterior commissure. However, the PC was not affected (Wolman et al., 2004). Regarding the ephrin/Eph signaling system, the expression of the receptor EphA7 has been reported in the developing chick diencephalon from very early stages (HH11, Baker and Antin, 2003; Garcia-Calero et al., 2006), becoming stronger in the roof plate at later (HH19 to HH35/E9) stages (Fig. 2.30) (Garcia-Calero et al., 2006) and later in development (HH35, E9). Another member of the Eph family, EphA4, is also present in the chick SCO at E6 (Fig. 2.31) (Hoyo-Becerra, 2006) and at E9 (Marin et al., 2001), while the ligand EphrinB1 was found to be present in the alar portion of the prosomere 1 (Fig. 2.32) (Hoyo-Becerra, 2006). To unravel the mechanism involved in the guidance of the axons forming the PC, receptors expressed by the growing axons and the guidance cues produced by the SCO are equally important, and there are a number of reports dealing with this subject. In mice, the rostral portion of the prosomere 1 expresses Sema3F, and both the fasciculus retroflexus and the PC express Neuropilin-2 (Funato et al., 2000). Sema3F/Neuropilin-2 interactions seem to participate in the formation of the fasciculus retroflexus (Funato et al., 2000). Both Sema3F and Neuropilin-2 null mutant mice display abnormalities in the anterior commissure and in the fasciculus retroflexus, among other brain defects (Giger et al., 2000; Sahay et al., 2003), but the PC is unaffected. Several cell adhesion molecules are expressed by the axons of the PC: Axonin1 (Redies et al., 1997) and Ng-CAM in chick (Hoyo-Becerra, 2006; Redies et al., 1997), and Nr-CAM in both chick and mouse (Hoyo-Becerra, 2006; Lustig et al., 2001). Two members of the cadherin superfamily, Cadherin6B and Cadherin7, are also present in the chick PC (Yoon et al., 2000). Caprile and coworkers demonstrated the presence of alpha6 integrin and beta1 integrin in the diencephalic roof plate of chick embryos (HH24 and HH43) by using RT-PCR and Western blot

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Figure 2.27–2.32 (2.27) Transverse sections through the diencephalon of Xenopus embryos (stage 37/38) processed for Slit-2 wholemount in situ hybridization. This axonal guidance molecule is expressed in the floor plate and the roof plate. Bars, 50 mm. (From Hocking et al., 2010). (2.28) Expression pattern of Slit-2 mRNA in the E11.5 embryonic mouse brain. Slit-2 is expressed in the floor plate (fp), and in the floor (long arrow) and roof (small arrows) of the diencephalon (di). A patch of expression is also seen rostral to the optic stalk (os); te: telencephalon. (From Holmes et al., 1998). (2.29) Expression of Sema3D in E3.5 chick embryos. The diencephalic roof plate is positive for this probe. Dien, diencephalon; oft, outflow tract; sec, surface ectoderm. (From Bao and Jin, 2006). (2.30) Chick embryo wholemount hybridized with an EphA7 probe. EphA7 is strongly expressed in the diencephalic roof plate at the embryonic stage HH24. Bars, 1 mm. (From Garcia-Calero et al., 2006). (2.31) Brain coronal section of an E6 chick embryo through the developing subcommissural organ (SCO). Section was immunostained with an antibody against EphA4. The apical portion of the

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(Caprile et al., 2009). In addition, by using immunocytochemical methods, they showed that at earlier stages (HH18 and HH23) both integrins are missing in axons of the developing PC while they are present in the SCO cells from HH18 onward. At HH29 and HH37, axons of the PC express beta1 integrin but not alpha6 integrin (Caprile et al., 2009). All these data strengthen the proposed function of the SCO in the organization of the axons that will form the PC, a complex process that probably would require the collaboration of SCO-spondin with a variety of other molecules.

7. Subcommissural Organ-Posterior Commissure Alterations in Mutant Models During the development of vertebrates, neural cells are produced in the ventricular zone of morphologically defined territories that divide the embryonic neural tube in a series of dorsoventral and caudorostral fields (Puelles and Rubenstein, 1993). Each of these regions is characterized by the expression of specific regulatory genes that control the expression of other regulatory genes, growth factors, cell surface receptors, ECM proteins, cell adhesion molecules, guidance cues, or neural cell type specific proteins. The expression of all these genes occurs according to an ontogenetically established program that is only partly known. The SCO and the underlying PC are derivatives from the roof plate of the prosomere 1 (Puelles and Rubenstein, 2003), located at the caudalmost portion of the embryonic diencephalon and just at the border of the primitive mesencephalon. The correct development of both structures depends on the spatiotemporal expression of a number of regulatory genes. In this section, we review the phenotypes of mice with an altered expression of some of the genes involved in the development of prosomere 1. These null mutant or transgenic animals display alterations of the SCO development and, most of them, of the formation of the PC. SCO cells is positive to EphA4. Bar, 40 mm. (2.32) Brain coronal sections of an E6 chick embryo through the developing subcommissural organ (SCO). Section was immunostained with an antibody against EphrinB1. Immunoreactivity is present in the alar plate of the diencephalon, while the middle portion of the SCO is negative. Bar, 40 mm.

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7.1. Pax6 mutant mice Pax6 is a developmentally regulated transcription factor that is widely expressed in the developing and adult CNS and in peripheral tissues. In the mammalian CNS, Pax6 expression begins at early embryonic stages in the dorsal forebrain, and its area of expression terminates abruptly in the border of the prospective diencephalon with the mesencephalon (Walther and Gruss, 1991), just in the area where the SCO and the PC will develop. A characteristic feature of the mesencephalon is the absence of Pax6 expression. The small eye (Sey) mouse mutant does not express Pax6 expression (Hill et al., 1991). Sey/Sey mice show severe developmental defects and die at birth with numerous anomalies in the peripheral tissues, the eyes, and the CNS (Schmahl et al., 1993). Many of these anomalies derive from acute alterations in neurogenesis and radial glia formation (Estivill-Torrus et al., 2002). In the Sey/Sey diencephalon, the identity of prosomere 1 is altered, and hence, severe defects in the development of roof structures including the PC and the SCO occur (Figs. 2.33–2.36) (Estivill-Torrus et al., 2001; Mastick et al., 1997; Schwarz et al., 1999). Axons originated from neurons located at the dorsal prosomere 1 are lost, and only a few ventral neurons probably send the scarce axons that will form a reduced PC. Thus, the Sey mutation results in a deficit of dorsal prosomere 1 neurons and axons, which could be due to a specification error or to a defect in the formation of such neurons. We suggest that a putative alteration in the specification of the correct directionality of the axons might be related to defects in the formation of the underlining SCO. Our group reported that the homozygous mutant Sey/Sey lacks any structure that could be regarded as a true SCO (Figs. 2.35–2.36) (EstivillTorrus et al., 2001), based on (i) the absence of a tall pseudostratified ependyma and (ii) no immunoreactivity to the anti-RF antibodies (Fig. 2.35) (AFRU, see Section 4.2.1). Besides, the expression of certain genes, such as lim1 or gsh1, in the caudal prosencephalon is lost in Sey mutants (Mastick et al., 1997). In addition, the mesencephalic marker gene dbx extends rostrally toward the prosencephalon, and the prosomere 1 disappears. In this scenario, a deficit in the axons that otherwise will form the PC occurs (Mastick et al., 1997), and both the PC and the SCO are absent. In contrast to pax6, pax2 and pax5 genes are expressed in the mesencephalon/metencephalon, and their expression terminates in the rostral roof at the borderline with the diencephalon, just where pax6 appears (Schwarz et al., 1999). Double mutant mice lacking both Pax2 and Pax5 show an enlarged PC which laid over a columnar epithelium resembling the SCO (Schwarz et al., 1999). Moreover, Pax6 null mutant mice with Pax6 expression under the Pax2 promoter lack a pretectum but develop an ectopic PC in the rostral-most mesencephalon, thus reinforcing the idea that Pax6 induces the formation of the PC, and probably also the underlying SCO (Schwarz et al., 1999).

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Figure 2.33–2.36 (2.33) Transverse section at the level of the dorsal prosomere 1 (pretectum) of a wild-type E16.5 mouse immunostained with an antibody against the neuronal protein GAP43. A prominent posterior commissure is present compared to that of Pax6 null mutants (see Fig. 2.34). This figure is a gift of Dr. G. Estivill-Torru´s. (2.34) Transverse section at the level of the dorsal prosomere 1 (pretectum) of a Pax6 null mutant mouse of E16.5 dpc immunostained with an antibody against the neuronal protein GAP43. Only minor GAP43 positive fibers can be observed at the posterior commissure. This figure is a gift of Dr. G. Estivill-Torru´s. (2.35) Sagittal section through the prosomere 1 (pretectum) roof plate of a wild-type E16.5 mouse embryo immunostained with anti-RF antibody (AFRU) and counterstained with hematoxylin. Wild-type embryos present a well-developed AFRU-positive SCO. This figure is a gift of Dr. G. Estivill-Torru´s. (2.36) Sagittal sections through the prosomere 1 (pretectum) roof plate of a Pax6 null mutant mouse of E16.5 immunostained with anti-RF antibody (AFRU) and counterstained with hematoxylin. In the Pax6 null mutants, no signs of AFRU immunoreactivity or SCO cells are found in an equivalent area, where the wildtype SCO is present. This figure is a gift of Dr. G. Estivill-Torru´s.

But, why does the lack of Pax6 lead to the absence of the prosomere 1 derivatives SCO and PC? It is known that Pax6 controls the expression of cadherins (Stoykova et al., 1997), membrane-bound proteins involved in cell recognition and adhesion and in axonal and dendritic guidance (BergerMuller and Suzuki, 2011). In the diencephalon, the expression pattern of the different cadherins defines boundaries (Redies et al., 2000). The absence of Pax6 could be also related with cadherin expression abnormalities. Cadherins are highly expressed in the neuroepithelium during embryonic development, as well as in the SCO cells, including their basal processes. Specifically, R-cadherin and OB-cadherin are highly expressed in mouse

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SCO cells. This expression decreases in the SCO basal processes of heterozygous Sey/þ mutants and virtually disappears in homozygous Sey/Sey mutants (Estivill-Torrus et al., 2001). If cadherins of the SCO basal processes were involved in the formation of the PC, their absence in Sey mutants would contribute to the paucity of axons in the mutant PC. Another neuronal cell adhesion molecule that could be related to PC malformations is L1, a molecule involved in axonal guidance and fasciculation during embryonic development whose expression is Pax6 dependent (Michelson et al., 2002). Indeed, Sey/Sey mutants with severe defects in the diencephalic roof plate also display an altered expression of L1 (Caric et al., 1997). L1 is present in the axonal growth cone but not in the neuroepithelium, so it would be the putative expression in the axons forming the PC, which could influence the formation of this structure. On the other hand, L1 has high affinity to sialic acid (Varki, 2007), a sugar quite abundant in the moieties of SCO-spondin (Grondona et al., 1998). Thus, the sialic acidmediated interaction between SCO-spondin and the L1 present in the PC growing axons might be necessary for the correct formation of the PC.

7.2. Msx1 mutant mice Msx genes encode homeodomain transcription factors expressed along the vertebrate neural tube (Sharman et al., 1999). In the mouse, all three members of the family are expressed in the dorsal midline of the brain from early embryonic stages (Shimeld et al., 1996). Msx1 homozygous mutants die at birth with severe defects in the cleft palate and other parts of the craniofacial skeleton (Satokata and Maas, 1994). The main features of the null mutant brains are the defects in the patterning of the dorsal diencephalon (Bach et al., 2003; Fernandez-Llebrez et al., 2004). In most of these mice, the SCO is completely absent, and only few axons are visible in the PC. Only some mutants show a moderate number of PC axons, albeit with frequent directional errors. Surprisingly, a few individuals develop an apparently normal PC and a SCO, although devoid of anti-RF immunoreactivity (Fernandez-Llebrez et al., 2004). This fact suggests that the PC can develop in the absence of SCO-spondin and contradicts the evidences reported in other models about the involvement of SCO-spondin in the formation of the PC (Hoyo-Becerra et al., 2010; Stanic et al., 2010). The diversity of diencephalic roof phenotypes found in Msx1 mutants might indicate a redundancy of msx1 and msx2 genes, whose importance may depend on the individual genetic background. The mechanism by which msx1 gene regulates the development of the diencephalic roof is unknown. Misexpression of msx1 could influence the expression of other genes involved in the formation of the dorsal CNS, such as BMPs or Wnt (Bach et al., 2003 and references therein). Indeed, diffusible molecules such as BMPs have been shown to participate in establishing

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the expression boundaries of other genes such as pax6 and msx1 (Timmer et al., 2002). On the other hand, msx1 and msx2 genes regulate cadherinmediated cell adhesion and cell sorting (Lincecum et al., 1998), and it is known that cadherin expression defines developing CNS territories including the prosomeres in the diencephalon (Redies et al., 2000). Thus, changes in the pattern of expression of cadherins could account for the defects observed in Msx1 mutants.

7.3. Other transgenic and mutant mice In addition to pax6 and msx1, other genes are involved in the patterning of the diencephalic roof plate. The wnt1 gene codifies for a secreted glycoprotein present in the dorsal midline of the diencephalon and mesencephalon of the embryonic CNS (Parr et al., 1993). Wnt1 null mutants do not develop a normal SCO, although they have a slightly altered PC (Louvi and Wassef, 2000). The development of a PC (though somehow altered) in the absence of SCO, as also occurs in rare Msx1 mutants (see Section 7.2), speaks in favor of alternative mechanisms to drive its development. Some authors suggest that Wnt1 signaling might be involved in regulating the expression of E-cadherin and alpha-N-catenin in restricted regions of the embryonic brain (Shimamura et al., 1994), providing a mechanism that would relate Wnt1 with the correct development of the SCO. On the other hand, Wnt1 expression has been proposed to be Msx1 dependent (Bach et al., 2003). Thus, the defects in the SCO development observed in Msx1-null mutants could be attributed to other downstream genes such as wnt1. Engrailed-1 (En1) is a gene expressed in the dorsal neuroepithelium of the mouse embryo at the mid-hindbrain junction. The En1 protein expression pattern includes a large portion of the mesencephalon and most of rhombomere 1 (Davis and Joyner, 1988). Ectopic expression of En1 in the territory of Wnt1, that includes the dorsal midline of P1, leads to agenesis of the SCO and severe errors in axonal pathfinding in the PC (Louvi and Wassef, 2000). The expression of En1 (and also Pax2) seems to be inhibited by Pax6 (Matsunaga et al., 2000). Thus, while certain genes induce the differentiation of SCO/PC, others such as en1 seem to repress the formation of both structures. This further proves that the development of both structures is somehow linked. RFX4_v3 is a member of the regulatory factor X family of winged helix transcription factors (Blackshear et al., 2003). Fetal mice completely lacking RFX4_v3 expression showed acute defects in the formation of the dorsal midline of the brain and died perinatally. The SCO was totally absent in these mice, but according to the authors, the PC was present (although a detailed study of this structure was missing). RFX4_v3 may be necessary for the expression of genes that are crucial for brain morphogenesis, such as Wnt, BMPs, and those involved in retinoic acid pathways (Zhang et al., 2006). This

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result reinforces the possibility that the SCO participates in the proper organization of the PC but is not essential for its development. Sonic hedgehog (Shh) is a protein secreted by the spinal FP involved in the specification of the ventral spinal cord. In addition to the FP, Shh (and axial) is also expressed in some regions of the dorsal diencephalon (Strahle et al., 1996). In cyclops zebrafish mutants, Shh expression is absent along the entire midline of the neuroectoderm (Strahle et al., 1996). Cyclops mutants lack a FP and show severe defects in the ventral midline, and 60% of the mutants lack a PC (Hatta et al., 1994). However, the SCO is present and is immunoreactive to the anti-SCO-spondin antibody (Fernandez-Llebrez et al., 2001a,b). It was reported that Shh specifically inhibits the expression of Pax6 and hence could determine the fate of different cell types (Ericson et al., 1997). It was also shown that the ectopic expression of Shh in the tectum of the chick embryo inhibits the expression of other genes related to the tectum development, such as Pax6, En2, Pax7, Pax2, Pax5, Wnt1, and Msx1 (Watanabe and Nakamura, 2000). Thus, the mechanism of action of Shh in regulating the formation of the PC could involve the Pax6 pathway. In this sense, it was recently reported that the growth of diencephalic and mesencephalic primordia is regulated by a mechanism that depended on the expression of Shh in the early mouse embryo, probably by interfering FGF15 and Wnt1 signaling (Ishibashi and McMahon, 2002). Musashi1 (Msi1) is an RNA-binding protein that is mainly expressed in embryonic proliferating pluripotent neural precursors. Msi1 null mutant mice develop obstructive hydrocephalus and an abnormal proliferation (and polyposis) of ependymal cells surrounding the Sylvius aqueduct as well as of the SCO cells. In the Msi1 mutants, a PC was not detectable (Sakakibara et al., 2002). Gdf7 (growth differentiation factor 7) is a member of the BMP family (BMP12) that is expressed in the dorsal rostral roof of the developing CNS and has been reported to be essential for the formation of the choroid plexus (Currle et al., 2005). Gdf7 is important for the specification of the neuronal identity of the dorsal interneurons in the spinal cord (Lee et al., 1998), and its expression seems to be also essential for the formation of the SCO (Louvi and Wassef, 2000). However, the potential role of this gene in the formation of the SCO/PC is unknown. Pcp4l1 (Purkinje cell protein 4 like 1) is a gene expressed in the floor/ basal plate of the spinal cord in mice during their early development, as well as later in the cerebral cortex, diencephalon, and mesencephalon. By E12.5, Pcp4l1 expression is restricted to the roof plate of the mesencephalon, and to the structures that will give rise to the circumventricular organs including the SCO (Bulfone et al., 2004), but unfortunately there are no reports on the influence of this gene on the SCO/PC development. Otx genes are involved in specification, regionalization, and terminal differentiation of the rostral part of the developing CNS (Boyl et al., 2001).

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Larsen and coworkers (2010) showed that otx2 was expressed in the developing human SCO and PC, but studies dealing with its involvement in the development of these structures are missing. A broad array of compounds, including regulatory factors, ECM proteins and enzymes, have been reported to participate in the formation of the SCO/PC. Some of them are described elsewhere in this review (see Section 4.6.4).

8. Concluding Remarks The formation of commissures and decussations in the CNS is a complex process controlled by a plethora of axonal guidance cues. A set of glial and, to a lesser extent, neuronal cell populations located at the midline control their formation. Among them, the SCO, located at the midline of the diencephalic roof, has been associated with the development of the PC. The most remarkable feature of the SCO is its secretion: a giant glycoprotein, the SCO-spondin, belonging to the thrombospondin superfamily. Unlike other groups of midline glial cells, SCO cells have received little attention regarding their role in the formation of the PC. Despite being one of the components of the first axonal scaffold in the vertebrate embryonic brain, the development of this commissure has been poorly investigated, and only a few studies have been devoted to the axonal guidance process during its formation (Caprile et al., 2009; Hoyo-Becerra et al., 2010; Stanic et al., 2010). The chick embryo provides a great opportunity for the study of such axonal pathfinding events in this early and clearly identifiable axonal tract, and its relationship with SCO cells. Here, we have reviewed the interrelation between the SCO and the PC at different levels. The SCO is located at the midline underneath the PC, and its basal processes come into close contact with pioneer axons of the PC. Both the SCO and the PC develop simultaneously, albeit not in all cases. Mutant mice for genes involved in the SCO development display abnormalities in the SCO and the PC, which suggests that the development of both structures may be tightly linked. SCO-spondin shares domains with axonal guidance molecules and is transiently expressed by another midline structure, the FP, suggesting a putative role for SCO-spondin as an axonal guidance cue. Different forms of SCO-spondin (soluble, aggregated, or certain synthetic peptides) display an in vitro activity on neuronal aggregation and/or neuritogenesis. In coculture experiments, SCO explants modify the pattern of neuritogenesis of diencephalic explants in three-dimensional collagen matrixes. All these evidences suggest a participation of the SCO in the development of the PC. The best-known secretion of the SCO is the large protein SCO-spondin, which, according to its domain composition, might possibly exhibit axonal

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guidance properties. In addition, the SCO releases SCO-spondin toward the extracellular spaces at their basal processes and hence close to the axons of the PC. However, two evidences indicate that SCO-spondin may not be the unique factor produced by the SCO that determines the formation of the PC. First, in some instances, the PC develops in the absence of a differentiated SCO. And second, a few mutants display a normal PC in the absence of SCO-spondin. Therefore, other attractive and/or repulsive signals might be produced by the early SCO or even by the undifferentiated neuroepithelium. In agreement to this, it was reported that the SCO and/or the neuroepithelium in the zone where the PC will develop also produces axonal guidance molecules such as Slit-2, Sema3D, EphA7, and EphA4. Consequently, we should ask what exactly the role of SCO-spondin is. In a recent report, Stanic et al. (2010) suggested that SCO-spondin may act on the fasciculation of PC axons by interacting with the beta1-integrin present in the axonal membrane. Thus, the extracellular SCO-spondin would bind to both the membrane of SCO cells through the alpha6integrin and the membrane of PC axons through beta1-integrin. Therefore, the embryonic SCO may have multiple roles in the formation of the PC: at early stages, controlling the crossing of pioneer axons by synthesizing axonal guidance molecules, similar to those used by other midline glial cells, and later in development, regulating the process of axonal fasciculation of the PC. Obviously, further studies are required to clarify the role of the SCO in the PC development. In this sense, knocking down the expression of SCOspondin in vivo would be necessary to assess to what extent the formation of the PC is dependent on this particular protein. On the other hand, the search for the molecular targets of SCO-spondin is also of major interest. The recent knowledge of the modular domain organization of this protein will be of crucial value for this task. In vitro bioassays using fragments of the protein as well as function-blocking antibodies will help to identify those targets. In addition, a comprehensive analysis of the expression of axonal guidance cues in the roof plate neuroepithelium at early developmental stages, particularly in relation to the routing of the PC pioneer axons, is required.

ACKNOWLEDGMENTS The authors are grateful to Dr. Guillermo Estivill-Torrus (IMABIS, Ma´laga, Spain) for the generous gift of images of Pax6 null mutant mice, to Dr. Teresa Caprile (Universidad de Concepcio´n, Chile) for her kindness in sending some original images and for her critical reading of some portions of the chapter, and to Dr. Harvey B. Sarnat (Faculty of Medicine and Alberta Children’s Hospital, Calgary, Canada) for helpful information about commissures and decussations. Rick Visser is a member of CIBER-BBN. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, and CIBER Actions, and financed by the Instituto de Salud Carlos III

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with assistance from the European Regional Development Fund. This work was supported by grants from Junta de Andalucı´a, P07-CVI-03079; Junta de Andalucı´a, SAS 08-0029; Ministerio de Ciencia e Innovacio´n, SAF2010-19087; Junta de Andalucı´a, SAS PI-05412010, SAS 2010-111224; Ministerio de Sanidad y Consumo, PNSD 2010/143.

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