Regulation of axon outgrowth by retrograde Ret signaling

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During development or following injury, axon terminals must navigate through a complex environment to form functional connections. Ret is a neurotrophin receptor which is present at the tips of growing axons. Ret activation and internalization induces retrograde transport of the activated receptor to the cell body triggering a transcriptional response, which in turn promotes axon outgrowth. Despite its essential role during nervous system development, it is not known how activated Ret receptor is trafficked in axons, the nature of the transcriptional response induced by the retrograde Ret signaling, and how these transcriptional targets ultimately promote axon growth. To address these questions, we are using the unique advantages of zebrafish, including live imaging and genetic approaches, to identify the molecular mechanisms that govern retrograde transport of a neurotrophin receptor Ret and the subsequent transcriptional response. In our preliminary studies, we discovered that retrograde transport of Ret depends on binding to the scaffold protein, Jip3, which links cargo to the retrograde motor for transport. Both ret and jip3 mutants display truncated sensory axons, and live imaging revealed abnormal growth cone dynamics and reduced axon terminal elaboration. Using RNA sequencing, we identified a number of factors that are transcriptionally induced in response to Ret-Jip3 retrograde signaling and are putative regulators of actin-based protrusive behavior in this context. Based on this data, we hypothesize that Jip3-dependent retrograde transport of Ret induces factors that promote growth cone dynamics required for sensory axon extension. We will test this hypothesis in the three specific aims. In Aim 1, we will define the molecular mechanisms of Ret retrograde transport and the role of Jip3 in this process. Experiments in the second aim will define the defects in growth cone dynamics that lead to the failure of axon extension in ret and jip3 mutants and determine the role of the Ret retrograde signaling in this process. The last aim will investigate the transcriptional response elicited by the retrograde Ret signaling and how factors that are regulated by this transcriptional program promote growth cone dynamics. Altogether, our study combines innovative assays in zebrafish with in vivo techniques to determine the specific role of retrograde neurotrophin signaling in axon outgrowth. Our work will further uncover the mechanisms by which long-range neurotrophin signals are transduced into cellular responses that regulate growth cone dynamics and axon extension.