Neuropilin asymmetry mediates a left-right difference in habenular connectivity

The medial habenular nuclei of the zebrafish diencephalon, which lie bilateral to the pineal complex, exhibit left-right differences in their neuroanatomy, gene expression profiles and axonal projections to the unpaired midbrain target - the interpeduncular nucleus (IPN). Efferents from the left habenula terminate along the entire dorsoventral extent of the IPN, whereas axons from the right habenula project only to the ventral IPN. How this left-right difference in connectivity is established and the factors involved in differential target recognition are unknown. Prior to IPN innervation, we find that only the left habenula expresses the zebrafish homologue of Neuropilin1a (Nrp1a), a receptor for class III Semaphorins (Sema3s). Directional asymmetry of nrp1a expression relies on Nodal signaling and the presence of the left-sided parapineal organ. Loss of Nrp1a, through parapineal ablation or depletion by antisense morpholinos, prevents left habenular neurons from projecting to the dorsal IPN. Selective depletion of Sema3D, but not of other Sema family members, similarly disrupts innervation of the dorsal IPN. Conversely, Sema3D overexpression results in left habenular projections that extend to the dorsal IPN, as well as beyond the target. The results indicate that Sema3D acts in concert with Nrp1a to guide neurons on the left side of the brain to innervate the target nucleus differently than those on the right side.