Chondroitin sulfates modulate axon guidance in embryonic Xenopus brain

Chondroitin sulfate proteoglycans display both inhibitory and stimulatory effects on cell adhesion and neurite outgrowth in vitro. The functional activity of these proteoglycans appears to be context specific and dependent on the presence of different chondroitin sulfate-binding molecules. Little is known about the role of chondroitin sulfate proteoglycans in the growth and guidance of axons in vivo. To address this question, we examined the effects of exogenous soluble chondroitin sulfates on the growth and guidance of axons arising from a subpopulation of neurons in the vertebrate brain which express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM. Intact brains of stage 28 Xenopus embryos were unilaterally exposed to medium containing soluble exogenous chondroitin sulfates. When exposed to chondroitin sulfate, NOC-2(+) axons within the tract of the postoptic commissure failed to follow their normal trajectory across the ventral midline via the ventral commissure in the midbrain. Instead, these axons either stalled or grew into the dorsal midbrain or continued growing longitudinally within the ventral longitudinal tract. These findings suggest that chondroitin sulfate proteoglycans indirectly modulate the growth and guidance of a subpopulation of forebrain axons by regulating either matrix-bound or cell surface cues at specific choice points within the developing vertebrate brain. (C) 1998 Academic Press.

Multiple axon guidance cues establish the olfactory topographic map: how do these cues interact?

Each primary olfactory neuron stochastically expresses one of similar to1000 odorant receptors. The total population of these neurons therefore consists of similar to1,000 distinct subpopulations, each of which are mosaically dispersed throughout one of four semi-annular zones in the nasal cavity. The axons of these different subpopulations are initially intermingled within the olfactory nerve. However, upon reaching the olfactory bulb, they sort out and converge so that axons expressing the same odorant receptor typically target one or two glomeruli. The spatial location of each of these 1800 glomeruli are topographically-fixed in the olfactory bulb and are invariant from animal to animal. Thus, while odorant receptors are expressed mosaically by neurons throughout the olfactory neuroepithelium their axons sort out, converge and target the same glomerulus within the olfactory bulb. How is such precise and reproducible topographic targeting generated? While some of the mechanisms governing the growth cone guidance of olfactory sensory neurons are understood, the cues responsible for homing axons to their target site remain elusive.

Mapping of the binding domains of the axon guidance receptor Unc5B to Netrin-1 and FLRT3

Anàlisi de les interaccions, a nivell neuronal, que tenen lloc durant el desenvolupament embrionari entre el receptor Unc5B (receptor present a la membrana) i les proteïnes Netrin-1 i FLRT3 (fibronectin and leucine-rich transmembrane proteins). La interacció entre aquest receptor i Netrin-1 ha estat profundament estudiada fins al moment, de manera que es coneix que aquesta promou una repulsió en la guia d’axons durant el desenvolupament embrionari. A més, la interacció està implicada en la senyalització per a diferents processos com l’angiogènesi i la supervivència cel·lular. Per altra banda, la interacció entre neurones Unc5B positives i FLRT3, promou un retard en la migració de les neurones. Diversos estudis demostren que aquest retard en la migració està relacionat amb certes patologies mentals.

Missense mutations in TENM4, a regulator of axon guidance and central myelination, cause essential tremor.

Essential tremor (ET) is a common movement disorder with an estimated prevalence of 5% of the population aged over 65 years. In spite of intensive efforts, the genetic architecture of ET remains unknown. We used a combination of whole-exome sequencing and targeted resequencing in three ET families. In vitro and in vivo experiments in oligodendrocyte precursor cells and zebrafish were performed to test our findings. Whole-exome sequencing revealed a missense mutation in TENM4 segregating in an autosomal-dominant fashion in an ET family. Subsequent targeted resequencing of TENM4 led to the discovery of two novel missense mutations. Not only did these two mutations segregate with ET in two additional families, but we also observed significant over transmission of pathogenic TENM4 alleles across the three families. Consistent with a dominant mode of inheritance, in vitro analysis in oligodendrocyte precursor cells showed that mutant proteins mislocalize. Finally, expression of human mRNA harboring any of three patient mutations in zebrafish embryos induced defects in axon guidance, confirming a dominant-negative mode of action for these mutations. Our genetic and functional data, which is corroborated by the existence of a Tenm4 knockout mouse displaying an ET phenotype, implicates TENM4 in ET. Together with previous studies of TENM4 in model organisms, our studies intimate that processes regulating myelination in the central nervous system and axon guidance might be significant contributors to the genetic burden of this disorder.

Elucidation of the Retinoid Signalling Pathway Involved in Axon Guidance in Lymnaea stagnalis and Xenopus laevis

The vitamin A metabolite, retinoic acid (RA), is known to play a crucial role in several developmental processes including axial patterning and differentiation. More recently, RA has been implicated in the regenerative process acting through its classical signaling pathway, the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), to mediate gene transcription. Moreover, RA has been shown to act as a guidance molecule for growth cones of regenerating motorneurons of the pond snail, Lymnaea stagnalis. Our lab has recently shown that RA can induce this morphological response independent of nuclear transcription, however, the role of the retinoid receptors in RA-induced chemoattraction is still unknown. Here, I show that the retinoid receptors, RXR and RAR, may mediate the growth cones response to the metabolically active retinoic acid isomers, all-trans and 9-cis RA, in Lymnaea stagnalis. Data presented here show that both an RXR and RAR antagonist can block growth cone turning in response to application of both isomers. Because no prior investigations have shown growth cone turning of individual vertebrate neurons, I aimed to show that both retinoic acid isomers were capable of inducing growth cone turning of embryonic spinal cord neurons in the frog, Xenopus laevis. For the first time in Xenopus, I showed that both all-trans and 9-cis RA were able to induce significantly more neurite outgrowth from cultured embryonic spinal cord neurons and induce positive growth cone turning of individual growth cones. In addition, I showed that the presence of the RXR antagonist, HX531, blocked 9-cis RA-induced growth cone turning and the RARβ antagonist, LE135, blocked all-trans RA-induced growth cone turning in this species. Evidence provided here shows for the first time, conservation of retinoic acid-induced growth cone turning in a vertebrate model system. In addition, these data show that the receptors involved in this morphological response may be the same in vertebrates and invertebrates.

BOC, brother of CDO, is a dorsoventral axon-guidance molecule in the embryonic vertebrate brain

The early axon scaffolding in the embryonic vertebrate brain consists of a series of ventrally projecting axon tracts that grow into a single major longitudinal pathway connected across the midline by commissures. We have investigated the role of Brother of CDO (BOC), an immunoglobulin (Ig) superfamily member distantly related to the Roundabout (Robo) family of axon-guidance receptors, in the development of this embryonic template of axon tracts in the zebrafish brain. A zebrafish homologue of BOC was isolated and shown to be expressed predominantly in the developing neural plate and later in the neural tube and developing brain. Zebrafish boc was initially highly localized to discrete bands in the mid- and hindbrain, but, as the major brain subdivisions emerged, it became more evenly expressed along the rostrocaudal axis, particularly in dorsal regions. The function of zebrafish boc was examined by a loss-of-function approach. Analysis of embryos injected with antisense morpholinos designed against boc revealed highly selective defects in the development of dorsoventrally projecting axon tracts. Loss of boc caused ventrally projecting axons, particularly those arising from the presumptive telencephalon, to follow aberrant trajectories. These data indicate that boc is an axon-guidance molecule playing a fundamental role in pathfinding during the early patterning of the axon scaffold in the embryonic vertebrate brain. (c) 2005 Wiley-Liss, Inc.

Mechanisms of axon guidance in the developing nervous system

The human brain assembles an incredible network of over a billion neurons. Understanding how these connections form during development in order for the brain to function properly is a fundamental question in biology. Much of this wiring takes place during embryonic development. Neurons are generated in the ventricular zone, migrate out, and begin to differentiate. However, neurons are often born in locations some distance from the target cells with which they will ultimately form connections. To form connections, neurons project long axons tipped with a specialized sensing device called a growth cone. The growing axons interact directly with molecules within the environment through which they grow. In order to find their targets, axonal growth cones use guidance molecules that can either attract or repel them. Understanding what these guidance cues are, where they are expressed, and how the growth cone is able to transduce their signal in a directionally specific manner is essential to understanding how the functional brain is constructed. In this chapter, we review what is known about the mechanisms involved in axonal guidance. We discuss how the growth cone is able to sense and respond to its environment and how it is guided by pioneering cells and axons. As examples, we discuss current models for the development of the spinal cord, the cerebral cortex, and the visual and olfactory systems. (c) 2005, Elsevier Inc.

Expression and role of complex carbohydrates in axon guidance in the olfactory system

Primary sensory neurons in the vertebrate olfactory systems are characterised by the differential expression of distinct cell surface carbohydrates. We show here that the histo-blood groups Sda (or CT1 antigen) and H are expressed by primary sensory neurons in the olfactory system, while the blood group A carbohydrate is expressed by a subset of vomeronasal neurons only in the developing accessory olfactory system. We have used both loss-of-function and gain-of-function approaches to manipulate expression of these carbohydrates in the olfactory system. In null mutant mice lacking the alpha(1,2)fucosyltransferase FUT1, the blood group H and A carbohydrates were not expressed in the olfactory systems which caused delayed development of the nerve fibre and glomerular layers in the main olfactory bulb. In contrast, ubiquitous expression of blood group A on olfactory axons in gain-of-function transgenic mice perturbed the ability of vomeronasal axons to terminate in the accessory olfactory bulb and affected the selective targeting of axons in the main olfactory bulb. During regeneration following bulbectomy, vomeronasal axons were unable to effectively sort out from the main olfactory axons when blood group A was misexpressed. These results provide in vivo evidence for a role of specific cell surface carbohydrates during development and regeneration of the olfactory nerve pathways.

The Wnt receptor Ryk is required for Wnt5a-mediated axon guidance on the contralateral side of the corpus callosum

Ryk (receptor related to tyrosine kinase) has been shown to be a novel Wnt receptor in both Caenorhabditis elegans and Drosophila melanogaster. Recently, Ryk-Wnt interactions were shown to guide corticospinal axons down the embryonic mouse spinal cord. Here we show that, in Ryk-deficient mice, cortical axons project aberrantly across the major forebrain commissure, the corpus callosum. Many mouse mutants have been described in which loss-of-function mutations result in the inability of callosal axons to cross the midline, thereby forming Probst bundles on the ipsilateral side. In contrast, loss of Ryk does not interfere with the ability of callosal axons to cross the midline but impedes their escape from the midline into the contralateral side. Therefore, Ryk(-/-) mice display a novel callosal guidance phenotype. We also show that Wnt5a acts as a chemorepulsive ligand for Ryk, driving callosal axons toward the contralateral hemisphere after crossing the midline. In addition, whereas callosal axons do cross the midline in Ryk(-/-) embryos, they are defasciculated on the ipsilateral side, indicating that Ryk also promotes fasciculation of axons before midline crossing. In summary, this study expands the emerging role for Wnts in axon guidance and identifies Ryk as a key guidance receptor in the establishment of the corpus callosum. Our analysis of Ryk function further advances our understanding of the molecular mechanisms underlying the formation of this important commissure.

Expression and role of roundabout-1 in embryonic Xenopus forebrain

The receptor Roundabout-1 (Robo1) and its ligand Slit are known to influence axon guidance and central nervous system (CNS) patterning in both vertebrate and nonvertebrate systems. Although Robo-Slit interactions mediate axon guidance in the Drosophila CNS, their role in establishing the early axon scaffold in the embryonic vertebrate brain remains unclear. We report here the identification and expression of a Xenopus Robo1 orthologue that is highly homologous to mammalian Robo1. By using overexpression studies and immunohistochemical and in situ hybridization techniques, we have investigated the role of Robo1 in the development of a subset of neurons and axon tracts in the Xenopus forebrain. Robo1 is expressed in forebrain nuclei and in neuroepithelial cells underlying the main axon tracts. Misexpression of Robo1 led to aberrant development of axon tracts as well as the ectopic differentiation of forebrain neurons. These results implicate Robo1 in both neuronal differentiation and axon guidance in embryonic vertebrate forebrain. (C) 2002 Wiley-Liss, Inc.

Exogenous Slit2 does not affect ureteric branching or nephron formation during kidney development

In an attempt to elucidate the role of Slit2 invertebrate kidney development, the effect of adding exogenous human Slit2 protein (hSlit2) to developing murine metanephric kidney explants was examined. To confirm the activity of the recombinant Slit2 protein, neurons from 8 day old chick sympathetic nerve chain dorsal root ganglia were cultured with hSlit2 protein, which induced significant neurite branching and outgrowth. Using kidney explants as a model system, metanephric development in the presence of hSlit2 protein was examined. Addition of hSlit2 up to a final concentration of 1 mug/ml had no detectable effect on the formation of nephrons or on branching morphogenesis of the ureteric tree after 2 or 4 days in culture, as assessed via immunofluorescence for the markers WT1 and calbindin 28K respectively. Similarly, maturation of the nephrogenic mesenchyme occurred in a phenotypically normal fashion. In situ analysis of the Slit receptors, Robot and Robot, the vasculogenic markers VEGFA and Flk-1, and the stromal cell marker BF2 displayed no difference in comparison to controls.