Disruption of Esrom and Ryk identifies the roof plate boundary as an intermediate target for commissure formation.

Growth cones are guided to their final destination by intermediate targets. Here, we identify intermediate targets and signaling components acting on zebrafish habenula commissural axons. Live imaging establishes that axons pause at the medial habenula before and after crossing the roof plate. esrom mutants axons fail to advance beyond the ipsilateral medial habenula. Tsc2 function is reduced in mutant axons, indicating cell autonomous defects in signaling. Consistent with signaling properties changing outside the roof plate, EphB is surface localized on axon segments within a zone demarcated by the medial habenula. wnt4a is expressed in the medial habenula and morpholino knockdown causes loss of the commissure. Electroporation of truncated Ryk causes axons to reenter the midline after reaching the contralateral habenula. These data identify Esrom as a mediator of growth cone navigation at an intermediate target and underscore the importance of midline boundaries as signaling centers for commissure formation.