Pioneer neurons are the first to extend axons to a particular region or target, acting as a guide and scaffold for “follower” axons. In most cases, pioneer neurons are essential in the developing central and peripheral nervous systems for the initial navigation to appropriate targets, proper follower axon pathfinding, and promoting follower axon outgrowth. Many studies have noted that pioneer neurons differ from followers in growth cone morphology and actin dynamics in multiple invertebrate and vertebrate model systems. These observations strongly argue that pioneer neurons possess a specific genetic program that controls distinct aspects of their growth cone morphology and behavior. Despite these critical roles and the unique axonal behavior of pioneer neurons, we still know little about 1) which genes are differentially expressed in pioneer versus follower neurons; and 2) how these transcriptional differences in turn promote specific pioneer neuron behaviors, such as enhanced axon outgrowth and protrusive activity in the growth cone. Our study will address this knowledge gap by identifying pioneer neuron-specific genes and testing their roles in axon growth. Our preliminary work has found that expression of a neurotrophin receptor Ret is highly elevated in a population of pioneering peripheral sensory neurons and is required for pioneer axon outgrowth. Furthermore, ret mutant pioneer axons display altered growth cone morphology, including reduced growth cone size and fewer filopodia. Thus, ret represents a unique marker of sensory pioneer axons. We will capitalize on this finding to isolate and interrogate a transcriptional profile of pioneer neurons. In Aim 1, we will use a single cell RNA-sequencing (scRNA-seq) to build a transcriptional profile of ret-positive pioneer neurons and identify genes that are enriched in the pioneer versus follower neuron subpopulations. In Aim 2, we will screen a list of genes from our scRNA-seq to identify candidates that play a role in growth cone dynamics and axon extension. In summary, our work will provide significant advancement by 1) generating a transcriptional profile of pioneer neurons during axon outgrowth to provide specific markers for studying pioneer neuron biology; and 2) testing the function of pioneer neuron-specific genes to identify new factors that promote pioneer axon growth in development.