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 spinalcord. 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 therostromedial 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.
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We thank A. Barco, CA. Mason, and P. Bovolenta for critical reading of the manuscript. We are also grateful to S. Ferreiro for the Zic2 EGFP retina picture, C. Vegar for genotyping and maintenance of the mice, Y. Coca for in utero electroporations, and J. López-Atalaya for help with the ChIP assays. We are indebted to T. Jessell, C. Birchmeier, L. Cheng, G. L-Bendito, K. Brose, and S. Martinez for generously providing reagents. We also thank the RIKEN BioResource (Japan) for providing the Zic2 kd mice and the MMRRC Repository for the Zic2 EGFP mice. A.E. holds an FPU fellowship from the Spanish Government. Research in the laboratory of E.H. is funded by grants from the Regional Government (Prometeo2012-005), the Spanish Government (BFU2010-16563, CONSOLIDER-Ingenio Program-CDS2007-023), and the European Research Council (ERC-2011-StG20101109).