[Show abstract][Hide abstract] ABSTRACT:
During development, neurons extend axons which follow highly stereotypic pathways to form a template for the development of a functional nervous system. In recent years, considerable progress has been made in elucidating the underlying molecular mechanisms which drive navigational decisions in the growth cone, primarily through detailed analysis of commissural axon guidance at the midline. Despite this, the molecular cues which contribute to the precise formation of the early axon scaffold in the embryonic vertebrate brain remain poorly defined. The Roundabout (Robo) family of axon guidance receptors and their ligand (Slit) have been demonstrated to play important roles in the regulation of midline crossing decisions and patterning of longitudinal axon pathways in the Drosophila central nervous system. In addition, three Robo receptors are known to be expressed in the vertebrate nervous system (Robo1, Robo2 and Robo3), where they have been implicated in the guidance of axons in the spinal cord and retinal pathway. In this thesis, the highly stereotypic and well-defined development of the zebrafish brain has been utilised to understand the role of the Robo family of axon guidance receptors in the development of the early scaffold of axon tracts in the embryonic vertebrate brain. The expression of each of the Robo receptors (Robo1, Robo2 and Robo3) and two candidate ligands (Slit1a and Slit2) were initially characterised with respect to the developing axon scaffold. All were found to be expressed in the brain during the period of axon outgrowth, in both unique and overlapping domains. The specific roles of each of these receptors and ligands were assessed using a morpholino knock down approach. These analyses revealed that both Robo and Slit have important roles in the development of the major longitudinal tract in the forebrain, the tract of the post-optic commissure (TPOC), as well as in some dorsoventral tracts in zebrafish forebrain, most notably, the supra-optic tract (SOT). In subsequent chapters (Chapters 4 and 5), a combinatorial knock down approach was utilised to investigate in greater detail the function of the Robo family of receptors in both the SOT and TPOC. Importantly, these studies identified two key mechanisms by which Robo-Slit signalling can influence the accurate guidance of axons navigating in the developing brain. Firstly, Robo-Slit signalling was found to have a prominent role in shaping the normal trajectory of axons navigating in the SOT. Simultaneous knock down of all, or unique combinations of Robo receptors had a profound impact on both the normal pathfinding and fasciculation of axons in this tract. The axon guidance defects observed in the SOT were indicative of a requirement for Slit1a and Slit2 to channel SOT axons into their appropriate linear pathway; consistent with the surround repulsion model previously described with reference to the guidance of axons in the peripheral nervous system. Secondly, a novel regulatory network comprising members of the Robo family of axon guidance receptors was identified and shown to be essential for the guidance of axons navigating in the TPOC. Using a combinatorial loss-of-function approach, a requirement for Slit1a mediated Robo2 activity to split the TPOC into discrete fascicles in the ventrocaudal diencephalon was established. Importantly, this process was negatively regulated by Robo1 and Robo3, which functioned redundantly in the TPOC to attenuate Robo2 activity through interactions involving both their intracellular and extracellular domains. Taken together, this thesis demonstrates a requirement for Robo-Slit signalling to accurately guide axons navigating in key trajectories in the embryonic vertebrate brain. Furthermore, this study highlights an emerging theme in the field, that is, the importance of context-specific interactions between axon guidance molecules to direct the navigation of growing axons along complex pathways.