Robo2--slit and Dcc--netrin1 coordinate neuron axonal pathfinding within the embryonic axon tracts

In the embryonic vertebrate brain, early born neurons establish highly stereotyped embryonic axonal tracts along which the neuronal interconnections form. To understand the mechanism underlying neuron axonal pathfinding within the embryonic scaffold of axon tracts, we studied zebrafish anterior dorsal telencephalic (ADt) neuron development. While previous studies suggest the ADt neuronal axons extend along a commissural tract [anterior commissure (AC)] and a descending ipsilateral tract [supraoptic tract (SOT)], it is unclear whether individual ADt neuronal axons choose specific projection paths at the intersection between the AC and the SOT. We labeled individual ADt neurons using a forebrain-specific promoter to drive expression of fluorescent proteins. We found the ADt axonal projection patterns were heterogeneous and correlated with their soma positions. Our results suggest that cell intrinsic differences along the dorsal ventral axis of the telencephalon regulate the axonal projection choices. Next, we determined that the guidance receptors roundabout2 (Robo2) and deleted in colorectal cancer (Dcc) were differentially expressed in the ADt neurons. We showed that knocking down Robo2 function by injecting antisense morpholino oligonucleotides abolished the ipsilateral SOT originating from the ADt neurons. Knocking down Dcc function did not prevent formation of the AC and the SOT. In contrast, the AC was specifically reduced when Netrin1 function was knocked down. Further mechanistic studies suggested that Robo2 responded to the repellent Slit signals and suppressed the attractive Netrin signals. These findings demonstrate how Robo2-Slit and Dcc-Netrin coordinate the axonal projection choices of the developing neurons in the vertebrate forebrain.