Live imaging-based molecular dissection of a successful vertebrate RGC axonal regeneration program - While many interventions promote some degree of axonal regeneration in mice, we are still far from translating this knowledge into therapies, and this may be in part because we have very limited information as to how regeneration is accomplished in related vertebrate species that successfully regenerate their damaged axons. This project is focused on trying to systematically deconstruct how the frog Xenopus laevis recovers from axon injuries to retinal ganglion cells, managing to reconnect those damaged axons and regain vision. This proposal largely uses a new surgical optic nerve crush model and live imaging platform that we recently developed in very young tadpoles. With this system, we have already demonstrated that central players in conveying the injury signal back to the cell body, a pathway involving the gene Dlk, as well as how axons are removed from the optic nerve, a pathway involving the gene Sarm1, are highly similar in tadpoles as they are in mammals. However, in our studies we have some surprising findings that, if we further understand them, have potential to move forward the field. These findings include finding that the Dlk effect on regeneration does not go through the transcription factor Jun, a role for Sarm1 in axon regeneration as well as degeneration, and studies that implicate myeloid cells in the successful regeneration program, possibly acting downstream of Sarm1. The proposal focusses on the intrinsic pathways of Dlk and Sarm1 in the first Aim, mainly in tadpoles, though also validating results and searching for effector genes in adult frogs. In the second aim, in also largely live imaging-based experiments, we will use novel transgenes and varied genetic perturbations to parcel out the role of myeloid cells, including also searching for effector genes. In contrast to these two highly mechanistic aims, the third technique development aim moves to create a much more glaucoma-relevant axon injury model, while also optimizing genetic and pharmacological screening, so that hopefully in the near future we will be able to carry out very large unbiased screens in a glaucoma-relevant axon injury model which take full advantage of the unique properties of the frog as an experimental model organism.
