Many important advances in TSC research have been realized over the past few years. We now have a very solid framework of the genes, proteins, and signaling pathways that are involved in TSC. Despite these exciting advances, many important questions remain unanswered, and more effective and safe therapies have not yet been realized. This is due in part to the high complexity of human genetic diseases such as TSC but also due to a relative lack of translational research aimed at bridging basic science discoveries to impact the care of people with TSC. Mouse models have proved beneficial to advance this field but have several disadvantages including cost and unsuitability for high-throughput discovery programs. While we are continuing to develop and use novel mouse models of TSC, we have embraced zebrafish as a new model system for TSC. This organism has become extremely popular for many avenues of research. This is due to the ease and low cost of breeding fish, large numbers of embryos (hundreds from each zebrafish cross versus 6-10 for mice), ease of studying development as the fish are transparent, and simplicity of introducing other genetic modifications to study gene interactions. Our tsc2-deficient zebrafish die prematurely but, due to the accelerated development of zebrafish, attain later developmental stages than comparable Tsc2-deficient mice or rats. While multi-organ pathology is seen, brain malformations are prominent and consistent with tuber formation. Strikingly, greatly increased mTOR activity as well as response to rapamycin are seen in this new model, indicating evolutionary conservation of this critical pathway. These conserved features strongly support the use of our zebrafish model to advance TSC research given the many advantages of this model system that are simply not feasible in mice or rats.

The goals of this proposal are to (1) complete characterization of brain abnormalities in this zebrafish model of TSC, (2) study hamartoma formation in the zebrafish brain and liver using mosaic embryos transplanted with fluorescent tsc2-deficient cells, and (3) investigate genetic interactions affecting the mTOR signaling pathway. These studies should greatly increase our understanding of the biological function of the tsc2 gene. Future experiments using this validated zebrafish TSC model will focus on high-throughput screening. Overall, our proposal should lead to the identification of new approaches and drugs that can reverse signaling pathways that are abnormal in TSC and hopefully lead to the development of much more effective therapies.