Axon Growth and Guidance in the Embryonic Vertebrate Forebrain

Wilson, Nicole Helen (2006). Axon Growth and Guidance in the Embryonic Vertebrate Forebrain PhD Thesis, School of Biomedical Sciences, University of Queensland.

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Author Wilson, Nicole Helen
Thesis Title Axon Growth and Guidance in the Embryonic Vertebrate Forebrain
School, Centre or Institute School of Biomedical Sciences
Institution University of Queensland
Publication date 2006
Thesis type PhD Thesis
Supervisor Associate Professor Brian Key
Abstract/Summary In the embryonic forebrain, pioneer axons establish a simple topography of dorsoventral and longitudinal tracts. In recent years, considerable progress has been made in elucidating the underlying molecular interactions that mediate pathfinding decisions in the growth cone, primarily through in vitro analyses, and examination of axon trajectories at the ventral midline or in the later-forming retinotectal projection. Despite this, the cues used by axons during the initial formation of the axon scaffold remain largely unknown. I have investigated the axon guidance role of Neogenin, a member of the immunoglobulin (Ig) superfamily with identical secondary structure to the established Netrin receptor, Deleted in Colorectal Cancer (DCC). Neogenin binds to the chemoattractive ligand Netrin-1, as well as to the chemorepulsive ligand Repulsive Guidance Molecule (RGMa). Despite this, an axon guidance role for Neogenin during early forebrain development is yet to be described. In this thesis, the relatively simple and well-characterised development of the Xenopus brain has been utilised to investigate the role of Neogenin in the development of the early scaffold of axon tracts in the vertebrate brain. A Xenopus orthologue of the Neogenin gene was cloned and characterised, and the expression of this gene in relation to the developing axon scaffold in the forebrain was examined. Neogenin was expressed in a principal nucleus of the developing forebrain, and in neuroepithelial cells underlying the main axon tracts. The specific role of Neogenin in the guidance of axons in the forebrain was assessed using a combination of gain-of-function and loss-of-function approaches. Misexpression of Neogenin led to aberrant development of axon tracts emanating from a specific forebrain nucleus, and ectopic differentiation of neurons in the dorsal brain. These results implicated Neogenin in both neuronal differentiation and axon guidance in embryonic vertebrate forebrain. Analysis of loss-of-function embryos generated by morpholino knock down and dominant-negative approaches confirmed that correct spatiotemporal activity of Neogenin was essential for the formation of a specific dorsoventral tract in the forebrain, the supraoptic tract (SOT). In the subsequent chapter, the axon guidance activity of Neogenin was examined in more detail by investigating ligands that could be interacting with Neogenin during axon guidance in the developing vertebrate forebrain. Two Neogenin ligands were examined: RGMa and Netrin-1. Both of these genes were expressed in the developing Xenopus forebrain in patterns that were consistent with their potential role as Neogenin ligands. Morpholino knock down of either Netrin-1 or RGMa produced similar mutant phenotypes in the formation of the SOT to those observed following loss of Neogenin. To examine genetic interactions amongst these genes, I utilised a simultaneous partial knock down approach, which revealed dosage-sensitive interactions and verified that these receptors and ligands were acting in the same pathway. The results confirmed that Neogenin acts as an axon guidance molecule in vivo, and is required for accurate pathfinding by axons navigating the initial axon trajectories. The findings support a model whereby Neogenin-expressing axons respond to a combination of attractive and repulsive cues as they navigate their ventral trajectory. Finally, this thesis describes the identification and initial characterisation of a Neogenin paralogue in Xenopus, termed Neogenin1b. Morpholino knock down analyses suggested that Neogenin1b may function distinctly from its sister gene, despite >90% amino acid identity between them. These results provide the framework for future investigations into the axon guidance role of the Neogenin proteins. Taken together, this thesis demonstrates that Neogenin, RGMa and Netrin-1 contribute to the complement of guidance cues and cellular interactions that are needed for the establishment of the earliest forming axon pathways in the embryonic vertebrate forebrain. Furthermore, this study highlights the remarkable integrative ability of a single axon guidance receptor, which appears to respond to a combination of opposing cues to direct growth along a specific route.

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