FUNCTIONAL ANALYSIS OF REPULSIVE GUIDANCE MOLECULE A DURING EARLY VERTEBRATE DEVELOPMENT

The repulsive guidance molecule A (RGMa) is a contact mediated axon guidance molecule that has significant roles in CNS development. In thesis I have investigated the roles and possible genetic interactions between the Netrin-1 receptors, neogenin and UNC5B, in RGMa-mediated signalling in early vertebrate development using Xenopus frog as a model system. The functional roles of RGMa and some of its protein domains in cell migration and adhesion were initially examined using ex vivo animal cap explant assays. This was followed by a gain-of-RGMa-function approach to assess in vivo roles during gastrulation and neurulation. To further understand the role of RGMa in early brain development, gain-of-function embryos were generated that mosacially expressed RGMa. Finally, the genetic interaction of RGMa-neogenin signalling pathway with the UNC5B coreceptor was examined during neural tube closure in Xenopus embryos. In Chapter 2, the role of RGMa in cell migration and cell adhesion were examined using pluripotent Xenopus animal cap explants. RGMa stimulated cell migration from these explants in a neogenin-dependent manner. Although RGMa has been previously implicated as a BMP coreceptor, this RGMa-induced cell migration was independent of BMP activity. 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 characterise the role of specific domains in RGMa, the migratory and adhesive activities of domain deletion mutants were assessed. RGMa lacking the partial von Willebrand factor (vWF) type D domain preferentially perturbed cell adhesion, while mutants lacking the tri-amino-acid motif RGD affected cell migration. It was also revealed that manipulating the levels of RGMa in vivo caused major developmental defects during Xenopus gastrulation. RGMa delayed blastopore closure and caused aberrant blastocoel formation and epiboly, resulting in defects in neural tube closure and ultimately embryonic lethality. Taken together, I have revealed 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 recent reports proposing involvement of RGMa in certain tissue cancers. RGMa-neogenin was previously shown to mediate formation of pioneer axon tracts in the developing Xenopus forebrain (Wilson and Key, 2006). I hypothesised that ectopic expression of RGMa in axon tract pathways would inappropriately misguide axons and provide evidence for repulsive axon growth in vivo. In Chapter 3, I mosaically mis-expressed RGMa in Xenopus embryos and analysed the subsequent formation of the pioneer axon trajectories in the early developing forebrains. Ectopic expression of RGMa severely affected the topography of developing axon tracts in vivo. Pioneer axons misrouted or aberrantly projected in response to ectopic RGMa in the developing Xenopus forebrain. In addition to its previously reported role in axon guidance, I found that RGMa acted cell-autonomously to generate ectopic neurons in the embryonic brain. Taken together, RGMa has been shown here to be involved in the spatial organisation of axon tracts, pioneer axon guidance, and neural cell fate decisions in the early developing vertebrate embryonic brain. In Chapter 4, I have examined genetic interactions between the RGMa receptors neogenin and UNC5B during anterior neural tube closure. RGMa-neogenin was previously shown to mediate anterior neural tube closure in the neurulating Xenopus embryos (Kee et al., 2008). As UNC5B was shown as a coreceptor for RGMa-neogenin signalling during axon guidance in vitro (Hata et al.,2009), I hypothesised that UNC5B would genetically interact with RGMa-neogenin mediated anterior neural tube closure. Subthreshold amounts of morpholinos were used to simultaneously knock-down both receptors and the penetrance of the phenotype was examined. The neogenin/UNC5B co knock-down morphants showed synergistic increase in the penetrance of anterior neural tube closure, suggesting that neogenin and UNC5B genetically interact in a known RGMa-neogenin signalling pathway. Taken together, the current project revealed the pleiotropic role of RGMa in the embryonic development that has implication in the emerging roles of RGMa in pathogenesis. I have shown that RGMa signals via its functional domains, vWF and RGD, with immunoglobulin receptor neogenin in regulating cell adhesion and migration ex vivo and in vivo. Furthermore, RGMa was shown to be responsible for early brain development via neogenin-dependent and -independent mechanisms. Ectopic RGMa expression perturbed the spatial organization of axon tracts and pioneer axon guidance of subpopulation of axons expressing neogenin, whereas it affected neural cell fate decisions independent of neogenin expression. Finally, RGMa receptors neogenin and UNC5B showed genetic interaction in anterior neural tube closure during the embryonic vertebrate development. In conclusion, this thesis reports novel and multifaceted roles of RGMa in neural and non-neural development, and provides the foundation for future studies to further understand the context specific nature of the function of RGMa