Zic2-dependent axon midline avoidance controls the formation of major ipsilateral tracts in the CNS.

In bilaterally symmetric organisms, interhemispheric communication is essential for sensory processing and motor coordination. The mechanisms that govern axon midline crossing during development have been well studied, particularly at the spinal cord. However, the molecular program that determines axonal ipsilaterality remains poorly understood. Here, we demonstrate that ipsilateral neurons whose axons grow in close proximity to the midline, such as the ascending dorsospinal tracts and the rostromedial thalamocortical projection, avoid midline crossing because they transiently activate the transcription factor Zic2. In contrast, uncrossed neurons whose axons never approach the midline control axonal laterality by Zic2-independent mechanisms. Zic2 induces EphA4 expression in dorsospinal neurons to prevent midline crossing while Robo3 is downregulated to ensure that axons enter the dorsal tracts instead of growing ventrally. Together with previous reports, our data reveal a critical role for Zic2 as a determinant of axon midline avoidance in the CNS across species and pathways.