Axon targeting of transcriptionally distinct pioneer neurons is regulated by retinoic acid signaling.

During nervous system development, pioneer neurons (pioneers) extend their axons toward distant targets, creating a scaffold for follower neurons and defining the initial structure of the nervous system. Despite years of study, whether pioneer neurons are transcriptionally distinct from followers is unknown. To address this question, we performed single-cell RNA sequencing (scRNA-seq) of zebrafish posterior lateral line (pLL) sensory neurons and found that pioneers and followers are transcriptionally distinct populations. Interestingly, expression profiling of differentiating pLL progenitors defines "follower" as the ground state and "pioneer" as a later developmental state, with retinoic acid (RA) signaling active in all pLL progenitors. Modulation of RA signaling within single pLL neurons demonstrated that its downregulation is necessary for expression of a neurotrophic factor receptor ret, which is required for correct targeting of pioneer axons. Our study reveals molecular heterogeneity between pioneers and followers and implicates RA signaling in fidelity of pioneer axonal targeting.