Pioneer neurons establish the initial nervous system structure by extending axons toward distant targets, but their molecular distinction from follower neurons was previously unknown. Using single-cell RNA sequencing in zebrafish posterior lateral line neurons, we found that pioneers and followers are transcriptionally distinct. Further, we show that pioneers provide an indispensable scaffold for followers, which cannot extend in the absence of pioneers. Finally, retinoic acid signaling influences pioneer axon targeting by regulating the expression of the neurotrophic factor receptor ret.