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.     

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.     

Requirement of signalling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis

The majority of neurones and glia of the enteric nervous system (ENS) are derived from the vagal neural crest. Shortly after emigration from the neural tube, ENS progenitors invade the anterior foregut and, migrating in a rostrocaudal direction, colonise in an orderly fashion the rest of the foregut, the midgut and the hindgut. We provide evidence that activation of the receptor tyrosine kinase RET by glial cell line-derived neurotrophic factor (GDNF) is required for the directional migration of ENS progenitors towards and within the gut wall. We find that neural crest-derived cells present within foetal small intestine explants migrate towards an exogenous source of GDNF in a RET-dependent fashion. Consistent with an in vivo role of GDNF in the migration of ENS progenitors, we demonstrate that Gdnf is expressed at high levels in the gut of mouse embryos in a spatially and temporally regulated manner. Thus, during invasion of the foregut by vagal-derived neural crest cells, expression of Gdnf was restricted to the mesenchyme of the stomach, ahead of the invading NC cells. Twenty-four hours later and as the ENS progenitors were colonising the midgut, Gdnf expression was upregulated in a more posterior region - the caecum anlage. In further support of a role of endogenous GDNF in enteric neural crest cell migration, we find that in explant cultures GDNF produced by caecum is sufficient to attract NC cells residing in more anterior gut segments. In addition, two independently generated loss-of-function alleles of murine Ret, Ret.k- and miRet51, result in characteristic defects of neural crest cell migration within the developing gut. Finally, we identify phosphatidylinositol-3 kinase and the mitogen-activated protein kinase signalling pathways as playing crucial roles in the migratory response of enteric neural crest cells to GDNF.     

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.     

Pleiotropic effects of the bone morphogenetic proteins on development of the enteric nervous system

Formation of the enteric nervous system (ENS) from migratory neural crest-derived cells that colonize the primordial gut involves a complex interplay among different signaling molecules. The bone morphogenetic proteins (BMPs), specifically BMP2 and BMP4, play a particularly important role in virtually every stage of gut and ENS development. BMP signaling helps to pattern both the anterior-posterior axis and the radial axis of the gut prior to colonization by migratory crest progenitor cells. BMP signaling then helps regulate the migration of enteric neural crest-derived precursors as they colonize the fetal gut and form ganglia. BMP2 and -4 promote differentiation of enteric neurons in early fetal ENS development and glia at later stages. A major role for BMP signaling in the ENS is regulation of responses to other growth factors. Thus BMP signaling first regulates neurogenesis by modulating responses to GDNF and later gliogenesis through its effects on GGF-2 responses. Furthermore, BMPs promote growth factor dependency for survival of ENS neurons (on NT-3) and glia (on GGF-2) by inducing TrkC (neurons) and ErbB3 (glia). BMP signaling limits total neuron numbers, favoring the differentiation of later born neuronal phenotypes at the expense of earlier born ones thus influencing the neuronal composition of the ENS and the glia/neuron ratio. BMP2 and -4 also promote gangliogenesis via modification of neural cell adhesion molecules and promote differentiation of the circular and then longitudinal smooth muscles. Disruption of BMP signaling leads to defects in the gut and in ENS function commensurate with these complex developmental roles.     

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.     

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     

Granulocyte-colony stimulating factor: a new player for the enteric nervous system

The enteric nervous system (ENS) controls and modulates gut motility and responds to food intake and to internal and external stimuli such as toxins or inflammation. Its plasticity is maintained throughout life by neural progenitor cells within the enteric stem cell niche. Granulocyte-colony stimulating factor (G-CSF) is known to act not only on cells of the immune system but also on neurons and neural progenitors in the central nervous system (CNS). Here, we demonstrate, for the first time, that G-CSF receptor is present on enteric neurons and progenitors and that G-CSF plays a role in the expansion and differentiation of enteric neural progenitor cells. Cultured mouse ENS-neurospheres show increased expansion with increased G-CSF concentrations, in contrast to CNS-derived spheres. In cultures from differentiated ENS- and CNS-neurospheres, neurite outgrowth density is enhanced depending on the amount of G-CSF in the culture. G-CSF might be an important factor in the regeneration and differentiation of the ENS and might be a useful tool for the investigation and treatment of ENS disorders.     

Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system.

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.     

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.     

Enteric nervous system progenitors are coordinately controlled by the G protein-coupled receptor EDNRB and the receptor tyrosine kinase RET

The enteric nervous system (ENS) in vertebrates is derived mainly from vagal neural crest cells that enter the foregut and colonize the entire wall of the gastrointestinal tract. Failure to completely colonize the gut results in the absence of enteric ganglia (Hirschsprung's disease). Two signaling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We demonstrate that interaction between these signaling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhances the effect of RET signaling on the proliferation of uncommitted ENS progenitors. In addition, we reveal novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A is a key component of the molecular mechanisms that integrate signaling by the two receptors. Our data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals.     

BMP signaling regulates murine enteric nervous system precursor migration, neurite fasciculation, and patterning via altered Ncam1 polysialic acid addition

The enteric nervous system (ENS) forms from migrating neural crest-derived precursors that differentiate into neurons and glia, aggregate into ganglion cell clusters, and extend neuronal processes to form a complex interacting network that controls many aspects of intestinal function. Bone morphogenetic proteins (BMPs) have diverse roles in development and influence the differentiation, proliferation, and survival of ENS precursors. We hypothesized that BMP signaling might also be important for the ENS precursor migration, ganglion cell aggregation, and neurite fasciculation necessary to form the enteric nervous system. We now demonstrate that BMP signaling restricts murine ENS precursors to the outer bowel wall during migration. In addition, blocking BMP signaling causes faster colonization of the murine colon, reduces ganglion cell aggregation, and reduces neurite fasciculation. BMP signaling also influences patterns of neurite extension within the developing bowel wall. These effects on ENS precursor migration and neurite fasciculation appear to be mediated at least in part by increased polysialic acid addition to neural cell adhesion molecule (Ncam1) in response to BMP. Removing PSA enzymatically reverses the BMP effects on ENS precursor migration and neurite fasciculation. These studies demonstrate several novel roles for BMP signaling and highlight new functions for sialyltransferases in the developing ENS.     

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.     

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.     

Novel roles of the chemorepellent axon guidance molecule RGMa in cell migration and adhesion

The repulsive guidance molecule A (RGMa) is a contact-mediated axon guidance molecule that has significant roles in central nervous system (CNS) development. Here we have examined whether RGMa has novel roles in cell migration and cell adhesion outside the nervous system. RGMa was found to stimulate cell migration from Xenopus animal cap explants in a neogenin-dependent and BMP-independent manner. RGMa also stimulated the adhesion of Xenopus animal cap cells, and this adhesion was dependent on neogenin and independent of calcium. To begin to functionally characterize the role of specific domains in RGMa, we assessed the migratory and adhesive activities of deletion mutants. RGMa lacking the partial von Willebrand factor type D (vWF) domain preferentially perturbed cell adhesion, while mutants lacking the RGD motif affected cell migration. We also revealed that manipulating the levels of RGMa in vivo caused major migration defects during Xenopus gastrulation. We have revealed here novel roles of RGMa in cell migration and adhesion and demonstrated that perturbations to the homeostasis of RGMa expression can severely disrupt major morphogenetic events. These results have implications for understanding the role of RGMa in both health and disease.