The coordination of intracellular protein/organelle transport and microtubule dynamics plays an essential role in axon outgrowth and circuit formation during nervous system development and maintenance. Abnormalities in these processes are associated with a number of neurodevelopmental and neurodegenerative disorders, such as Charcot-Marie-Tooth disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis. Despite the pervasiveness of these disorders, there are no effective treatments. This deficiency can be partially attributed to the lack of an in vivo vertebrate system, which is needed to effectively analyze how alterations in axon transport or microtubule dynamics lead to failed axon extension or axonal degeneration. Our long-term goal is to define the genetic basis of the cellular and molecular mechanisms, which regulate axon transport and microtubule dynamics using the zebrafish as an in vivo model system. The overall objective of this proposal is to use forward genetic screening to find factors that regulate axon transport and microtubule dynamics during development of the vertebrate nervous system. Our preliminary studies confirm that this screen is feasible. We conducted a pilot screen that yielded 5 mutant strains exhibiting phenotypes consistent with defective axon transport and/or disrupted microtubule dynamics including: axon truncation, nerve degeneration and axonal swellings. We have also developed a number of assays to characterize microtubule-motor mediated transport and cytoskeletal dynamics in our mutant strains, including imaging approaches to visualize these processes in intact zebrafish embryos and larvae. Using these approaches, we have demonstrated that one of the mutants (llg1), which contains a mutation in an ortholog of Jnk-interacting protein 3 (Jip3) has specific defects in axon transport and simultaneously exhibits abnormal microtubule dynamics.
In Aim 1, we propose to continue our screening protocol to find additional mutants that exhibit dysfunctions in these processes.
In Aim 2, we propose to characterize the mechanisms by which these mutations alter axon development.
In Aim 3, we propose to map and identify molecular lesions that underlie mutant phenotypes indicative of defects in axon transport and/or microtubule dynamics. Our approach is innovative, because it combines the genetic advantages of the zebrafish system with new imaging approaches to define the roles of axon transport and microtubule dynamics during neural development. Public health relevance: Development of this vertebrate system, in which we can study the mechanisms of axon transport in an intact animal, will expedite the elucidation of causative factors of neurodevelopmental and neurodegenerative disorders whose etiologies lie in disruption of axon transport and/or microtubule dynamics.
Development of a zebrafish system, in which we can study the mechanisms of axon transport in an intact animal, will expedite the elucidation of causative factors of neurodevelopmental and neurodegenerative disorders whose etiologies lie in disruption of axon transport and/or microtubule dynamics.
