Cellular and molecular mechanisms promoting retinal ganglion cell axonal guidance during optic nerve regeneration

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PROJECT SUMMARY Visual information is transmitted from the retina in each eye to the brain through the optic nerve, which is composed of the axons of retinal ganglion cells (RGCs) and associated glia. Diseases that increase intraocular pressure and damage the RGC bodies and their axons, such as glaucoma, can ultimately result in irreversible blindness. After damage to RGC axons, regeneration of the mammalian optic nerve is largely deficient due to limited RGC axonal regrowth compounded by massive injury induced RGC death. Several RGC intrinsic signaling pathways are known to increase RGC survival and increase long range axonal growth after injury. However, enhancing RGC axonal growth often results in axonal misguidance during the initial stages of regeneration, as axons project inappropriately from the optic tract before and at the optic chiasm. Currently, the identity of extrinsic cues and mechanisms critical for guiding regenerating RGC axons are not well understood. In addition, the cellular responses and behaviors of glia, immune and other support cells that localize to the optic tract, which potentially provide guidance cues to regenerating RGC axons, have not yet been described. In contrast to most mammals, zebrafish exhibit a remarkable capacity for regeneration. In taking full advantage of the zebrafish system, the overall goal of the proposed research here is to use an optic nerve transection assay developed by the laboratory of Dr. Michael Granato in the optically transparent larval zebrafish, to identify and characterize mechanisms that promote optic nerve regeneration. Preliminary studies from a candidate genetic screen conducted using this assay identified mutations in three genes critical for guiding regenerating RGC axons: lh3, a glycosyltransferase critical for posttranslational collagen modifications, collagen18a1, a presumptive substrate of Lh3, and wntless, which is required for the secretion of Wnt ligands into the extracellular space. In all three mutants, regenerating RGC axons extend but fail to cross the optic chiasm, and instead project along aberrant trajectories, revealing that RGC axonal growth toward and across the optic chiasm requires critical extrinsic guidance cues during regeneration. To define the extrinsic cues and cell-cell interactions that guide RGC axons as they navigate towards the chiasm during regeneration, I propose to use live cell imaging to characterize RGC axonal and glial cell dynamics in vivo during the initial stages of optic nerve regeneration. I will also determine the cellular and molecular mechanisms by which Lh3, Collagen18a1, and Wntless promote correct axonal guidance during optic nerve regeneration and I will use an unbiased transcriptomics approach to identify additional genes required for this process. I will complete the mentored K99 phase of this proposal in the Granato lab at the University of Pennsylvania, a top research university with cutting-edge technologies and excellent mentoring resources. The results from this proposal will create a strong foundation to establish a focused independent research program and provide relevant insight to fundamental questions in the field of optic nerve regeneration.