| dc.description.abstract | The formation of complex neuronal circuits is crucial for the proper development of the central nervous system. The wiring structure of the nervous system underlies its function in sensation and movement, and higher order functions such as learning, memory, and cognition. Disruption of this wiring leads to a number of neurodevelopmental disorders such as developmental disability syndromes, autism, and schizophrenia. Axon guidance is an important aspect in this process of development, as neurons are not born with axons but must actively extend these wires in the developing nervous system to reach their appropriate synaptic targets. The developing axons are led by growth cones, dynamic actin-based structures that sense and respond to extracellular guidance cues that drive the forward motion of the axon. Growth cones contain a dynamic lamellipodial body ringed by filopodial protrusions, both important in guiding the axon to its target destination. Motility and guidance behaviors of the growth cone are regulated by its actin and microtubule (MT) cytoskeleton through the modulatory influence of axon guidance cues, such as UNC-6/Netrin and its guidance receptors UNC-40/DCC and UNC-5 that are present at the leading edge of the growth cone. Netrin is a secreted guidance cue that acts as both an axon attractant and repellant in different receptor contexts. Though the role of Netrin and its receptors in axon pathfinding has been extensively studied, very little is known about how Netrin regulates growth cone behavior and morphology in vivo. Caenorhabditis elegans is a useful system to study axon pathfinding and growth cone development in vivo due to its simple, well-characterized nervous system, transparency and fully sequenced genome. The VD and DD motor neurons reside along the ventral nerve cord of the animal, and their axons normally extend straight dorsally to the dorsal nerve cord to form commissures. Though the DD axons develop during embryogenesis, the VD neurons develop post-embryonically in a well-described and stereotypical manner, making them great candidates to study in vivo growth cone development. After a brief introduction to axon guidance and growth cone morphology in chapter I, chapter II describes a novel role for the C. elegans flavin-containing monooxygenase (FMOs) genes in Netrin-mediated axon guidance and growth cone protrusion. We show that the FMO genes are required for VD/DD motor axon guidance and to restrict growth cone filopodial protrusions downstream of the Netrin receptors UNC-40 and UNC-5 and the Rac GTPases CED-10 and MIG-2 in Netrin-mediated growth cone repulsion. In chapter III we present new roles for UNC-6/Netrin in regulating the polarity and extent of growth cone protrusion through its receptors UNC-40 and UNC-5. We demonstrate that UNC-5 signaling regulates three aspects of growth cone morphology during growth away from UNC-6: (i) inhibition of growth cone protrusion (ii) dorsal leading-edge polarization of F-actin and (ii) restriction of MT entry into the growth cone, possibly via the cytoskeletal interacting protein UNC-33/CRMP. We also find that UNC-6 and UNC-40 can stimulate VD growth cone protrusion at the dorsal leading-edge, independent of dorsal F-actin polarity and growth cone MT plus-end accumulation. The characterization of the small GTPases RHO-1 and its guanine nucleotide exchange factor RHGF-1 in modulating growth cone protrusion and microtubule accumulation is detailed in chapter IV. We show that RHO-1 and RHGF-1 are required to prevent MT plus-ends from entering the growth cone as well as limiting excessive filopodial protrusions downstream of UNC-6/Netrin signaling. Chapter V provides new insights into the building blocks of MTs, the tubulins and their importance in MT organization and stability with respect to growth cone morphology. We show that certain missense mutations in the C. elegans alpha and beta-tubulin genes, tba-1 and tbb-1, have different effects on MT stability and in turn growth cone morphology. Mutations that lead to MT destabilitization inhibited growth cone protrusivity, while mutations that lead to the formation of hyperstable MTs caused excess protrusions. This reveals a causal relationship between MT organization and growth cone structure. To address whether other guidance systems cooperate with UNC-6/Netrin to regulate protrusion and directed growth cone migration, chapter VI demonstrates the role of the guidance receptor SAX-3/Robo in dorsal axon guidance and VD growth cone protrusion. We show that sax-3 is required for the guidance of dorsally-directed VD/DD axons and inhibits growth cone protrusion through the Rac GEF UNC-73/Trio. In summary, the results contained here provide new insights into the mechanisms by which Netrin signaling regulates several aspects of growth cone protrusion during the process of axon pathfinding. By studying established and novel genes in repulsion and growth cone inhibition, and by defining their effects on the growth cone cytoskeleton, we are starting to understand how these signaling pathways shape the morphological characteristics of the growth cone during directed migration in vivo. | |
