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.     

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.     

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.     

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     

Inhibition of repulsive guidance molecule,RGMa, increases afferent synapse formation with auditory hair cells

The peripheral fibers that extend from auditory neurons to hair cells are sensitive to damage, and replacement of the fibers and their afferent synapse with hair cells would be of therapeutic interest. Here, we show that RGMa, a repulsive guidance molecule previously shown to play a role in the development of the chick visual system, is expressed in the developing, newborn, and mature mouse inner ear. The effect of RGMa on synaptogenesis between afferent neurons and hair cells, from which afferent connections had been removed, was assessed. Contact of neural processes with hair cells and elaboration of postsynaptic densities at sites of the ribbon synapse were increased by treatment with a blocking antibody to RGMa, and pruning of auditory fibers to achieve the mature branching pattern of afferent neurons was accelerated. Inhibition by RGMa could thus explain why auditory neurons have a low capacity to regenerate peripheral processes: postnatal spiral ganglion neurons retain the capacity to send out processes that respond to signals for synapse formation, but expression of RGMa postnatally appears to be detrimental to regeneration of afferent hair cell innervation and antagonizes synaptogenesis. Increased synaptogenesis after inhibition of RGMa suggests that manipulation of guidance or inhibitory factors may provide a route to increase formation of new synapses at deafferented hair cells. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 457–466, 2014     

Repulsive guidance molecule (RGMa), a DRAGON homologue, is a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta (TGF-beta) superfamily of ligands, which regulate many mammalian physiologic and pathophysiologic processes. BMPs exert their effects through type I and type II serine/threonine kinase receptors and the Smad intracellular signaling pathway. Recently, the glycosylphosphatidylinositol (GPI)-anchored protein DRAGON was identified as a co-receptor for BMP signaling. Here, we investigate whether a homologue of DRAGON, repulsive guidance molecule (RGMa), is similarly involved in the BMP signaling pathway. We show that RGMa enhances BMP, but not TGF-beta, signals in a ligand-dependent manner in cell culture. The soluble extracellular domain of RGMa fused to human Fc (RGMa.Fc) forms a complex with BMP type I receptors and binds directly and selectively to radiolabeled BMP-2 and BMP-4. RGMa mediates BMP signaling through the classical BMP signaling pathway involving Smad1, 5, and 8, and it up-regulates endogenous inhibitor of differentiation (Id1) protein, an important downstream target of BMP signals. Finally, we demonstrate that BMP signaling occurs in neurons that express RGMa in vivo. These data are consistent with a role for RGMa as a BMP co-receptor.     

Repulsive guidance molecule RGMa alters utilization of bone morphogenetic protein (BMP) type II receptors by BMP2 and BMP4

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of multifunctional ligands that transduce their signals through type I and II serine/threonine kinase receptors and intracellular Smad proteins. Recently, we identified the glycosylphosphatidylinositol-anchored repulsive guidance molecules RGMa, DRAGON (RGMb), and hemojuvelin (RGMc) as coreceptors for BMP signaling (Babbit, J. L., Huang, F. W., Wrighting, D. W., Xia, Y., Sidis, Y., Samad, T. A., Campagna, J. A., Chung, R., Schneyer, A., Woolf, C. J., Andrews, N. C., and Lin, H. Y. (2006) Nat. Genet. 38, 531-539; Babbit, J. L., Zhang, Y., Samad, T. A., Xia, Y., Tang, J., Schneyer, A., Woolf, C. J., and Lin, H. Y. (2005) J. Biol. Chem. 280, 29820-29827; Samad, T. A., Rebbapragada, A., Bell, E., Zhang, Y., Sidis, Y., Jeong, S. J., Campagna, J. A., Perusini, S., Fabrizio, D. A., Schneyer, A. L., Lin, H. Y., Brivanlou, A. H., Attisano, L., and Woolf, C. J. (2005) J. Biol. Chem. 280, 14122-14129). However, the mechanism by which RGM family members enhance BMP signaling remains unknown. Here, we report that RGMa bound to radiolabeled BMP2 and BMP4 with Kd values of 2.4+/-0.2 and 1.4+/-0.1 nm, respectively. In KGN human ovarian granulosa cells and mouse pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling required BMP receptor type II (BMPRII), but not activin receptor type IIA (ActRIIA) or ActRIIB, based on changes in BMP signaling by small interfering RNA inhibition of receptor expression. In contrast, cells transfected with RGMa utilized both BMPRII and ActRIIA for BMP2 or BMP4 signaling. Furthermore, in BmpRII-null pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling was reduced by inhibition of endogenous RGMa expression, and RGMa-mediated BMP signaling required ActRIIA expression. These findings suggest that RGMa facilitates the use of ActRIIA by endogenous BMP2 and BMP4 ligands that otherwise prefer signaling via BMPRII and that increased utilization of ActRIIA leads to generation of an enhanced BMP signal.     

Emerging evidence for cell-autonomous axon guidance

Current models of axon guidance within the central nervous system (CNS) involve the presentation of environmental cues to navigating growth cones. The surrounding and target tissues present a variety of ligands that either restrict or promote growth, thus providing pathfinding instructions to developing axons. Recent findings show that RGMb, a GPI anchored extracellular protein present on retinal ganglion cells, down-regulates Wnt3a signaling by lowering LRP5 levels at the membrane surface. When RGMb is phosphorylated by the extracellular tyrosine kinase VLK, phosphorylated RGMb (p-RGMb) is internalized and carries LRP5 towards intracellular compartments. In the eye, a dorsal-high ventral-low gradient of VLK generates a dorsal-low ventral-high gradient of LRP5 that modulates Wnt3a signaling. These molecules, which are all expressed by individual RGCs, generate Wnt-signal gradients along the dorso-ventral axis of the retina, resulting in differential axon growth which in turn regulates proper retino-tectal/collicular map formation. This pathway represents a regulatory mechanism whereby extracellular phosphorylation generates what may be the first example of a unique self-guiding mechanism that affects neuronal-target connections independent of paracrine signals from the surrounding target tissue.     

Repulsive guidance of axons of spinal sensory neurons in Xenopus laevis embryos: roles of Contactin and notochord-derived chondroitin sulfate proteoglycans

An immunoglobulin superfamily neuronal adhesion molecule, Contactin, has been implicated in axon guidance of spinal sensory neurons in Xenopus embryos. To identify the guidance signaling molecules that Contactin recognizes in tailbud embryos, an in situ binding assay was performed using recombinant Contactin-alkaline phosphatase fusion protein (Contactin-AP) as a probe. In the assay of whole-mount or sectioned embryos, Contactin-AP specifically bound to the notochord and its proximal regions. This binding was completely blocked by either digestion of embryo sections with chondroitinase ABC or pretreatment of Contactin-AP with chondroitin sulfate A. When the spinal cord and the notochord explants were co-cultured in collagen gel, growing Contactin-positive spinal axons were repelled by notochord-derived repulsive activity. This repulsive activity was abolished by the addition of either a monoclonal anti-Contactin antibody, chondroitin sulfate A or chondroitinase ABC to the culture medium. An antibody that recognizes chondroitin sulfate A and C labeled immunohistochemically the notochord in embryo sections and the collagen gel matrix around the cultured notochord explant. Addition of chondroitinase ABC into the culture eliminated the immunoreactivity in the gel matrix. These results suggest that the notochord-derived chondroitin sulfate proteoglycan acts as a repulsive signaling molecule that is recognized by Contactin on spinal sensory axons.     

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.     

CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro

Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.     

A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.     

Emerging roles for neogenin and its ligands in CNS development

It is now well established that the netrin guidance cues and their receptors comprise a major molecular guidance system driving axon pathfinding during nervous system development. One netrin receptor, neogenin, is now emerging as a key regulator of many developmental processes throughout the embryo. Unexpectedly, a new family of neogenin ligands, the repulsive guidance molecule (RGM) family, has recently been identified. The functional outcome of neogenin activation is dictated by both the nature of the ligand as well as the developmental context. Netrin-1–neogenin interactions mediate chemoattractive axon guidance, while RGMa–neogenin interactions repel axons. Neogenin is required for the establishment of the pseudostratified epithelium of the neural tube, probably by promoting cell adhesion. In addition, a role for RGMa and neogenin in neuronal differentiation has been demonstrated. While neogenin signaling cascades are poorly understood, the opposing responses of neogenin to RGMa and netrin-1 in the context of axon guidance indicates that neogenin signaling is complex and subject to tight spatiotemporal regulation. In summary, neogenin is a multifunctional receptor regulating diverse developmental processes. Thus, its contribution to neural development is proving to be considerably more extensive than originally predicted.     

Myosin IIA is required for neurite outgrowth inhibition produced by repulsive guidance molecule

Although myelin-associated neurite outgrowth inhibitors express their effects through RhoA/Rho-kinase, the downstream targets of Rho-kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho-kinase, by using blebbistatin – a specific myosin II inhibitor – and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho-kinase-dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up-regulated, and this effect depends on Rho-kinase activity. Further, RGMa-induced F-actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho-kinase-dependent activation of MIIA via MLC phosphorylation induces F-actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.     

Noggin is required for correct guidance of dorsal root ganglion axons

Members of the bone morphogenetic protein family of secreted protein signals have been implicated as axon guidance cues for specific neurons in Caenorhabditis elegans and in mammals. We have examined axonal pathfinding in mice lacking the secreted bone morphogenetic protein antagonist Noggin. We have found defects in projection of several groups of neurons, including the initial ascending projections from the dorsal root ganglia, motor axons innervating the distal forelimb, and cranial nerve VII. The case of the dorsal root ganglion defect is especially interesting: initial projections from the dorsal root ganglion enter the dorsal root entry zone, as normal, but then project directly into the gray matter of the spinal cord, rather than turning rostrally and caudally. Explant experiments suggest that the defect lies within the spinal cord and not the dorsal root ganglion itself. However, exogenous bone morphogenetic proteins are unable to attract or repel these axons, and the spinal cord shows only very subtle alterations in dorsal-ventral pattern in Noggin mutants. We suggest that the defect in projection into the spinal cord is likely the result of bone morphogenetic proteins disrupting the transduction of some unidentified repulsive signal from the spinal cord gray matter.     

Specific projections of sympathetic preganglionic neurons are not intrinsically determined by segmental origins of their cell bodies

Sympathetic preganglionic projections of the chick are segmentally specific. Neurons from the 16th cervical (C16) and the first thoracic (T1) spinal cord segments project almost exclusively in the rostral direction, while those from the fifth thoracic (T5) to the first lumbar (L1) spinal segments project almost exclusively in the caudal direction. Neurons from the intervening spinal cord segments (T2–4) project in rostral and caudal directions. There is also a tendency for rostrally located neurons in each segment to project rostrally and caudally located neurons to project caudally. To investigate whether specific projections of preganglionic neurons are intrinsically determined by segmental origins of their cell bodies, neural tube segments were transplanted or rotated in embryos at stages 19–26; these stages include times during and after preganglionic cell birth and just prior to axon outgrowth. When the T1 neural tube segment was replaced with the T5 or T7 neural tube segment, the transplanted T5 or T7 preganglionic neurons, now in the T1 position, projected rostrally. Conversely, when the T5 or T7 neural tube segment was replaced with the T1 neural tube, the transplanted T1 preganglionic neurons projected caudally. In addition, when individual T3 spinal cord segments were rotated 180° along the A-P axis, neurons which were originally in the caudal part of the segment projected rostrally, whereas neurons originally from the rostral part of the segment projected caudally. These results show that specific projections of preganglionic neurons are not intrinsically determined by segmental origins of their cell bodies. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 371–378, 1998     

Repulsive guidance molecule B (RGMB) plays negative roles in breast cancer by coordinating BMP signaling

Repulsive guidance molecules (RGMs) coordinate axon formation and iron homestasis. These molecules are also known as co-receptors of bone morphogenetic proteins (BMPs). However, the role played by RGMs in breast cancer remains unclear. The present study investigated the impact of RGMB on functions of breast cancer cells and corresponding mechanisms. RGMB was knocked down in breast cancer cells by way of an anti-RGMB ribozyme transgene. Knockdown of RGMB resulted in enhanced capacities of proliferation, adhesion, and migration in breast cancer cells. Further investigations demonstrated RGMB knockdown resulted in a reduced expression and activity of Caspase-3, accompanied with better survival in RGMB knockdown cells under serum starvation, which might be induced by its repression on MAPK JNK pathway. Up-regulations of Snai1, Twist, FAK, and Paxillin via enhanced Smad dependent sigaling led to increased capacities of adhesion and migration. Our current data firstly revealed that RGMB may act as a negative regulator in breast cancer through BMP signaling.