The Cxcl14 chemokine defines pioneer axon guidance and early circuit assembly in the inner ear.

The nervous system wiring requires the precise coordination of axon guidance, neuronal migration, and target cell recognition. Here, we show that inner ear circuit formation, relies on pioneer cells extending an axonal scaffold that selectively target nascent hair cells. High spatiotemporal imaging of pioneer axons reveal how they navigate through the cranial environment, establish dynamic cell-cell contacts with other axons to finally stabilize in target cells. These pioneer axons are required not only for follower axon growth but also for coordinated migration of follower neurons, revealing a cellular hierarchy underlying circuit assembly. We identify the chemokine Cxcl14 as a novel instructive guidance cue regulating pioneer axon extension, turning, and fasciculation at discrete cellular decision points. Loss of Cxcl14 disrupts axonal navigation, compromises synaptic organization in hair cells, and impairs mechanosensory-based behavior. Together, our findings establish a new chemokine-based mechanism linking pioneer axon guidance to early sensory circuit assembly necessary for building mechanosensory networks.