Netrin‐1 is required for efficient neural tube closure

During neural tube formation, neural plate cells migrate from the lateral aspects of the dorsal surface towards the midline. Elevation of the lateral regions of the neural plate produces the neural folds which then migrate to the midline where they fuse at their dorsal tips, generating a closed neural tube comprising an apicobasally polarized neuroepithelium. Our previous study identified a novel role for the axon guidance receptor neogenin in Xenopus neural tube formation. We demonstrated that loss of neogenin impeded neural fold apposition and neural tube closure. This study also revealed that neogenin, via its interaction with its ligand, RGMa, promoted cell–cell adhesion between neural plate cells as the neural folds elevated and between neuroepithelial cells within the neural tube. The second neogenin ligand, netrin‐1, has been implicated in cell migration and epithelial morphogenesis. Therefore, we hypothesized that netrin‐1 may also act as a ligand for neogenin during neurulation. Here we demonstrate that morpholino knockdown of Xenopus netrin‐1 results in delayed neural fold apposition and neural tube closure. We further show that netrin‐1 functions in the same pathway as neogenin and RGMa during neurulation. However, contrary to the role of neogenin‐RGMa interactions, neogenin‐netrin‐1 interactions are not required for neural fold elevation or adhesion between neuroepithelial cells. Instead, our data suggest that netrin‐1 contributes to the migration of the neural folds towards the midline. We conclude that both neogenin ligands work synergistically to ensure neural tube closure. © 2012 Wiley Periodicals, Inc., 2013     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

Repulsive guidance molecule A (RGMa): A molecule for all seasons

RGMa (repulsive guidance molecule a) was the first identified molecule that possessed the necessary functional activity to repulse and steer growth cones to their target in the brain. By binding to its neogenin receptor, RGMa caused the collapse of growth cones and encouraged axons to grow along specific trajectories in vitro. Although originally characterized in 1990, RGMa was not conclusively shown to mediate axon guidance in vivo for another 12 years. Loss-of-function analysis in mice revealed that RGMa may play a more important role in neural tube morphogenesis. RGMa has now emerged as a molecule with pleiotropic roles involving cell adhesion, cell migration, cell polarity and cell differentiation which together strongly influence early morphogenetic events as well as immune responses. RGMa can be regarded as a molecule for all seasons.     

Repulsive guidance molecule A (RGMa)

RGMa (repulsive guidance molecule a) was the first identified molecule that possessed the necessary functional activity to repulse and steer growth cones to their target in the brain. By binding to its neogenin receptor, RGMa caused the collapse of growth cones and encouraged axons to grow along specific trajectories in vitro. Although originally characterized in 1990, RGMa was not conclusively shown to mediate axon guidance in vivo for another 12 years. Loss-of-function analysis in mice revealed that RGMa may play a more important role in neural tube morphogenesis. RGMa has now emerged as a molecule with pleiotropic roles involving cell adhesion, cell migration, cell polarity and cell differentiation which together strongly influence early morphogenetic events as well as immune responses. RGMa can be regarded as a molecule for all seasons.     

Unc5B associates with LARG to mediate the action of repulsive guidance molecule

Neuronal axons are guided by attractive and repulsive cues in their local environment. Because the repulsive guidance molecule A (RGMa) was originally identified as an axon repellent in the visual system, diverse functions in the developing and adult central nervous system have been ascribed to it. RGMa binding to its receptor neogenin induces RhoA activation, leading to inhibitory/repulsive behavior and collapse of the neuronal growth cone. However, the precise mechanisms that regulate RhoA activation are poorly understood. In this study, we show that Unc5B, a member of the netrin receptor family, interacts with neogenin as a coreceptor for RGMa. Moreover, leukemia-associated guanine nucleotide exchange factor (LARG) associates with Unc5B to transduce the RhoA signal. Focal adhesion kinase (FAK) is involved in RGMa-induced tyrosine phosphorylation of LARG as well as RhoA activation. These findings uncover the molecular basis for diverse functions mediated by RGMa.     

Identification of the neogenin-binding site on the repulsive guidance molecule A

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the chick retinotectal system. RGMa, one of the 3 isoforms found in mammals, is involved in laminar patterning, cephalic neural tube closure, axon guidance, and inhibition of axonal regeneration. In addition to its roles in the nervous system, RGMa plays a role in enhancing helper T-cell activation. Binding of RGM to its receptor, neogenin, is considered necessary to transduce these signals; however, information on the binding of RGM to neogenin is limited. Using co-immunoprecipitation studies, we have identified that the RGMa region required for binding to neogenin contains amino acids (aa) 259-295. Synthesized peptide consisting of aa 284-293 directly binds to the extracellular domain (ECD) of recombinant neogenin, and addition of this peptide inhibits RGMa-induced growth cone collapse in mouse cortical neurons. Thus, we propose that this peptide is a promising lead in finding reagents capable of inhibiting RGMa signaling.     

Neogenin: A multi-functional receptor regulating diverse developmental processes

Neogenin, a close relative of the axon guidance receptor Deleted in Colorectal Cancer (DCC), has been shown to be a receptor for members of the Netrin and Repulsive Guidance Molecule (RGM) families. While Netrin-1-Neogenin interactions result in a chemoattractive axon guidance response, the interaction between Neogenin and RGMa induces a chemorepulsive response. Evidence is now accumulating that Neogenin is a multi-functional receptor regulating many diverse developmental processes, including neural tube and mammary gland formation, myogenesis and angiogenesis. Little is known of the function of Neogenin in the adult, however, a novel role in the regulation of iron homeostasis is now emerging. While the signal transduction pathways activated by Neogenin are poorly understood, it is clear that the functional outcome of Neogenin activation, at least in the embryo, depends on both the developmental context as well as the nature of the ligand.     

Neogenin: one receptor, many functions

Neogenin is a multifunctional transmembrane receptor belonging to the immunoglobulin superfamily. It displays identical secondary structure to deleted in colorectal cancer (DCC), a netrin receptor that is involved in axon guidance and cell survival. Like DCC, neogenin is able to transduce signals elicited by netrin. These neogenin-netrin interactions have been implicated in tissue morphogenesis, angiogenesis, myoblast differentiation and most recently in axon guidance. Neogenin is also a receptor for repulsive guidance molecule, a glycosylphosphatidylinositol-linked protein involved in neuronal differentiation, apoptosis and repulsive axon guidance. Numerous studies have been started to elucidate the in vivo functions of neogenin, and its role in multiple aspects of development and homeostasis.     

Spatiotemporal Expression of Repulsive Guidance Molecules (RGMs) and Their Receptor Neogenin in the Mouse Brain

Neogenin has been implicated in a variety of developmental processes such as neurogenesis, neuronal differentiation, apoptosis, migration and axon guidance. Binding of repulsive guidance molecules (RGMs) to Neogenin inhibits axon outgrowth of different neuronal populations. This effect requires Neogenin to interact with co-receptors of the uncoordinated locomotion-5 (Unc5) family to activate downstream Rho signaling. Although previous studies have reported RGM, Neogenin, and/or Unc5 expression, a systematic comparison of RGM and Neogenin expression in the developing nervous system is lacking, especially at later developmental stages. Furthermore, information on RGM and Neogenin expression at the protein level is limited. To fill this void and to gain further insight into the role of RGM-Neogenin signaling during mouse neural development, we studied the expression of RGMa, RGMb, Neogenin and Unc5A-D using in situ hybridization, immunohistochemistry and RGMa section binding. Expression patterns in the primary olfactory system, cortex, hippocampus, habenula, and cerebellum were studied in more detail. Characteristic cell layer-specific expression patterns were detected for RGMa, RGMb, Neogenin and Unc5A-D. Furthermore, strong expression of RGMa, RGMb and Neogenin protein was found on several major axon tracts such as the primary olfactory projections, anterior commissure and fasciculus retroflexus. These data not only hint at a role for RGM-Neogenin signaling during the development of different neuronal systems, but also suggest that Neogenin partners with different Unc5 family members in different systems. Overall, the results presented here will serve as a framework for further dissection of the role of RGM-Neogenin signaling during neural development.     

Overexpression of repulsive guidance molecule (RGM) a induces cell death through Neogenin in early vertebrate development

Repulsive guidance molecule (RGM) a is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein that has been implicated in chemorepulsive axon guidance. Although RGMa binds the transmembrane receptor Neogenin, the developmental events controlled by the RGMa-Neogenin interactions in vivo remain largely unknown. We have cloned full-length RGMa from Xenopus borealis for the first time and identified two homologous genes referred to as RGMa1 and RGMa2. Here we show RGMa1 overexpression at 2-cell-stage resulted in cell death, which lead to an early embryonic lethal phenotype of the embryos. Time-lapse photomicroscopy revealed that embryos began to show initial morphological defects from ∼5 h post-fertilization (hpf) which was then followed by extensive blastomere cell death at ∼11 hpf. This phenotype was rescued by simultaneous knock down of RGMa using translation blocking anti-sense morpholinos. Knock down of the RGMa1 receptor Neogenin in RGMa1 overexpressing embryos was also able to rescue the phenotype. Together these results indicated that RGMa1 was signalling through Neogenin to induce cell death in the early embryo. While previous studies have suggested that Neogenin is a dependence receptor that induces cell death in the absence of RGM, we have instead shown that Neogenin-RGM interactions induce cell death in the early embryo. The roles of RGMa1 and Neogenin appear to be context specific so that their co-ordinated and regulated expressions are essential for normal development of the vertebrate embryo.     

Repulsive guidance molecule/neogenin: a novel ligand-receptor system playing multiple roles in neural development

The repulsive guidance molecule (RGM) is a membrane-bound protein originally isolated as an axon guidance molecule in the visual system. Recently, the transmembrane protein, neogenin, has been identified as the RGM receptor. In vitro analysis with retinal explants showed that RGM repels temporal retinal axons and collapses their growth cones through neogenin-mediated signaling. However, RGM and neogenin are also broadly expressed at the early embryonic stage, suggesting that they do not only control the guidance of visual axons. Gene expression perturbation experiments in chick embryos showed that neogenin induces cell death, and its ligand, RGM, blocks the pro-apoptotic activity of neogenin. Thus, RGM/neogenin is a novel dependence ligand/receptor couple as well as an axon guidance molecular complex.     

Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury

Neuronal axons are guided by attractive and repulsive cues in their local environment. Since the identification of the repulsive guidance molecule (RGM) a (RGMa) as an axon repellent in the visual system, diverse functions, as part of the developing and adult central nervous system (CNS), have been ascribed to it. The binding of RGMa to its receptor neogenin has been shown to induce RhoA activation, leading to inhibitory/repulsive behavior and the collapse of the neuronal growth cone. In this paper, we provide evidence to suggest the involvement of RGMb, another member of the RGM family, in the rat CNS. RGMb inhibits neurite outgrowth in postnatal cerebellar granule neurons (CGNs) in vitro. RGMb is expressed by oligodendrocytes and neurons in the adult rat CNS, and the expression of this molecule is upregulated around the site of spinal cord injury. RGMb is present in myelin isolated from an adult rat brain. RGMb and neogenin are coexpressed in CGNs and entorhinal cortex neurons. These findings suggest that RGMb is a myelin-derived inhibitor of axon growth in the CNS. Inhibition of RGMb may provide an alternative approach for the treatment of spinal injuries.     

Ryk: a novel Wnt receptor regulating axon pathfinding

Members of the Wnt family and their receptors, the Frizzleds, are key regulators of pivotal developmental processes including embryonic patterning, specification of cell fate, and determination of cell polarity. The versatility and complexity of Wnt signaling has been further highlighted by the emergence of a novel family of Wnt receptors, the Ryk family. In mammals and flies, Ryk is a key chemorepulsive axon guidance receptor responsible for the establishment of important axon tracts during nervous system development. Although the function of Ryk is currently best understood with respect to this role, its widespread expression, both in developing tissues and in the adult, suggests that Ryk may regulate many essential biological processes. This hypothesis is supported by the multiple developmental phenotypes apparent in Ryk loss-of-function mice. These mice display a variety of embryonic abnormalities, including disruption of skeletal, craniofacial and cardiac development. Here we review Ryk structure and function focusing on its activity as an axon guidance receptor.     

Neogenin interacts with RGMa and netrin-1 to guide axons within the embryonic vertebrate forebrain

In the embryonic forebrain, pioneer axons establish a simple topography of dorsoventral and longitudinal tracts. The cues used by these axons during the initial formation of the axon scaffold remain largely unknown. We have investigated the axon guidance role of Neogenin, a member of the immunoglobulin (Ig) superfamily that binds to the chemoattractive ligand Netrin-1, as well as to the chemorepulsive ligand repulsive guidance molecule (RGMa). Here, we show strong expression of Neogenin and both of its putative ligands in the developing Xenopus forebrain. Neogenin loss-of-function mutants revealed that this receptor was essential for axon guidance in an early forming dorsoventral brain pathway. Similar mutant phenotypes were also observed following loss of either RGMa or Netrin-1. Simultaneous partial knock downs of these molecules revealed dosage-sensitive interactions and confirmed that these receptors and ligands were acting in the same pathway. The results provide the first evidence that Neogenin acts as an axon guidance molecule in vivo and support a model whereby Neogenin-expressing axons respond to a combination of attractive and repulsive cues as they navigate their ventral trajectory.     

Neogenin Regulates Skeletal Myofiber Size and Focal Adhesion Kinase and Extracellular Signal-regulated Kinase Activities In Vivo and In Vitro

A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.     

Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1

Molecular cues, such as netrin 1, guide axons by influencing growth cone motility. Rho GTPases are a family of intracellular proteins that regulate the cytoskeleton, substrate adhesion and vesicle trafficking. Activation of the RhoA subfamily of Rho GTPases is essential for chemorepellent axon guidance; however, their role during axonal chemoattraction is unclear. Here, we show that netrin 1, through its receptor DCC, inhibits RhoA in embryonic spinal commissural neurons. To determine whether netrin 1-mediated chemoattraction requires Rho function, we inhibited Rho signaling and assayed axon outgrowth and turning towards netrin 1. Additionally, we examined two important mechanisms that influence the guidance of axons to netrin 1: substrate adhesion and transport of the netrin receptor DCC to the plasma membrane. We found that inhibiting Rho signaling increased plasma membrane DCC and adhesion to substrate-bound netrin 1, and also enhanced netrin 1-mediated axon outgrowth and chemoattractive axon turning. Conversely, overexpression of RhoA or constitutively active RhoA inhibited axonal responses to netrin 1. These findings provide evidence that Rho signaling reduces axonal chemoattraction to netrin 1 by limiting the amount of plasma membrane DCC at the growth cone, and suggest that netrin 1-mediated inhibition of RhoA activates a positive-feedback mechanism that facilitates chemoattraction to netrin 1. Notably, these findings also have relevance for CNS regeneration research. Inhibiting RhoA promotes axon regeneration by disrupting inhibitory responses to myelin and the glial scar. By contrast, we demonstrate that axon chemoattraction to netrin 1 is not only maintained but enhanced, suggesting that this might facilitate directing regenerating axons to appropriate targets.     

Gene expression of the repulsive guidance molecules/neogenin in the developing and mature mouse visual system: C57BL/6J vs. the glaucoma model DBA/2J

We used in-situ hybridization to analyze the expression patterns of three known members (a, b and c) of the RGM ("repulsive guidance molecule") gene family and of the RGMa receptor neogenin in a glaucoma mouse model (DBA/2J strain) and the C57BL/6J strain, which served as a control. In order to understand the role of the RGMs and neogenin in glaucoma, we characterized their expression patterns in the developing and mature mouse retina and in the optic nerve. In all investigated stages from post-natal day (P) 0 to 15 months (M) RGMa, RGMb and neogenin expression was detected in the ganglion cell layer (GCL). From P10 to 15M, we found RGMa, RGMb and neogenin expression in the inner nuclear layer (INL) and the outer nuclear layer (ONL). In P10- and older mice, the expression patterns of RGMa and its receptor neogenin were similar, while that of RGMb differed from both. As expected, no specific retinal expression of RGMc was detected in any of the age groups investigated. C57BL/6J mice and DBA/2J mice displayed no differences in the expression pattern of RGMa, RGMb, RGMc and neogenin in the developing retina (gestational age 14.5 days (E14.5), P0 & P10). Interestingly, we found a higher expression of RGMa, RGMb and neogenin in the retinas of all glaucoma-affected mice than in the age-matched control strain. Furthermore, we detected a higher RGMa and RGMb expression in the optic nerves of glaucoma-affected DBA/2J-mice older than 11M than in C57BL/6J mice of the same age.     

Peptides derived from repulsive guidance molecule act as antagonists

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system [T. Yamashita, B.K. Mueller, K. Hata, Neogenin and RGM signaling in the central nervous system, Curr. Opin. Neurobiol. 17 (2007) 29-34]. Functional studies in Xenopus and chick embryos have revealed the roles of RGM in axon guidance and laminar patterning, while those in mouse embryos have demonstrated its function in regulating the cephalic neural tube closure. Importantly, RGM inhibition enhanced the growth of injured axons and promoted functional recovery after spinal cord injury in rats. Here, we identified two RGMa-derived peptides that functioned as antagonists against RGMa. The peptides studied in vitro dose-dependently suppressed the neurite growth inhibition and growth cone collapse induced by RGMa. Thus, these peptides are promising reagents to treat injuries of the central nervous system.     

Dual roles of the chemorepellent axon guidance molecule RGMa in establishing pioneering axon tracts and neural fate decisions in embryonic vertebrate forebrain

Repulsive guidance molecule A (RGMa) is a glycosylphosphatidylinositol‐anchored plasma membrane protein that was originally identified based on its chemorepulsive activity during axon navigation in the developing nervous system. Knock down of RGMa has previously shown to perturb axon navigation in the developing Xenopus forebrain (Wilson and Key, 2006). In order to further understand the in vivo role of RGMa in axon guidance, we have adopted an in vivo gain‐of‐function approach. RGMa was mosaically overexpressed in the developing Xenopus embryo by the injection of mRNA into single blastomeres. Ectopic expression of RGMa affected the morphology and the topography of developing axon tracts in vivo. Pioneer axons misrouted or aberrantly projected in response to ectopic RGMa in the developing Xenopus forebrain, confirming the in vivo chemorepulsive activity of this ligand. In addition, we show here for the first time that overexpression of RGMa acts cell‐autonomously to generate ectopic neurons in the developing embryonic brain. Taken together, the current study reveals a pleiotropic role of RGMa in early vertebrate embryonic brain in the spatial organization of axon tracts, pioneer axon guidance, and neural cell differentiation. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Repulsive guidance molecule A (RGM A) and its receptor neogenin during neural and neural crest cell development of Xenopus laevis

Background information. RGM A (repulsive guidance molecule A) is a GPI (glycosylphosphatidylinositol)‐anchored glycoprotein which has repulsive properties on axons due to the interaction with its receptor neogenin. In addition, RGM A has been demonstrated to function as a BMP (bone morphogenetic protein) co‐receptor. Results. In the present study, we provide the first analysis of early RGM A and neogenin expression and function in Xenopus laevis neural development. Tissue‐specific RGM A expression starts at stage 12.5 in the anterior neural plate. Loss‐of‐function analyses suggest a function of RGM A and neogenin in regulating anterior neural marker genes, as well as eye development and neural crest cell migration. Furthermore, overexpression of RGM A leads to ectopic expression of neural crest cell marker genes. Conclusions. These data indicate that RGM A and neogenin have important functions during early neural development, in addition to their role during axonal guidance and synapse formation.