Project Summary The goals of my research program are to elucidate fundamental mechanisms underlying synapse and axon development and maintenance. We take multi-tiered approaches to investigate these processes, primarily using C. elegans because this animal model is well suited for in vivo imaging with high cellular resolution and because mechanistic dissection is within physiological relevant contexts and coupled with functional impacts. We examine the C. elegans locomotor circuit, because we can unambiguously observe the stereotyped patterns of synapses in all developmental stages, an essential readout to assess how synapses are dynamically regulated. We have consistently developed innovative technologies enabling visualization of organelles and subcellular structures in axons and synapses for in vivo analyses of dynamic cellular processes. We were the first to visualize synapse remodeling of juvenile motor circuit in living animals, which set two decades of vibrant research that have revealed multiple pathways coordinating temporal events and spatial organization of synaptic connections. We established C. elegans axon regeneration model, and have used large-scale genetic screens to discover genes that promote or repress axon regrowth in adults. Many pathways have been found to have conserved roles in axon regeneration in other animal models. In this R35 application, we will focus on mechanisms underlying both the developmental synapse plasticity in locomotor circuit maturation and maintenance, and also neuronal stress response and axon regeneration in adults. We will investigate how neuronal activity pattern coordinates with transcriptional regulation by combining in vivo imaging of synapse and calcium with single-cell RNA sequencing analysis over the entire period of circuit remodeling. We will gain a deep understanding of molecular dynamics associated with synapse and circuit plasticity. We have long-standing interest in the multifaceted roles of the conserved MAP3K DLK proteins, which have been shown to regulate diverse cellular processes from synapse formation and remodeling to axon regeneration and neurodegeneration. We will cross-examine mechanistic conservation between C. elegans and mice. Membraneless compartments generated through protein phase separation are now widely shown to play key roles in organizing cellular activities, including axon regeneration. We will take a holistic approach to investigate protein phase separation with physiological relevant expression and contexts, and also to model how genetic mutations in human homologs may alter protein phase separation properties. Overall, the findings will advance our knowledge on the cellular signaling network underlying normal brain function and will also provide insights into pathological processes associated with synapse dysfunction and axonal damage.