Robo1 and Robo2 have distinct roles in pioneer longitudinal axon guidance

► Pioneer longitudinal axons navigate using two Slit receptors, Robo1 and Robo2. ► We identified genetically separable guidance functions. ► Robo1 primarily functions in guiding ventral longitudinal axon populations. ► Robo2 is the main receptor for directing dorsal longitudinal axons. ► Robo2 is required to keep neuron cell bodies out of the floor plate.