All-optical investigation reveals a hierarchical organization of vsx2+ reticulospinal neurons coordinating steering and forward locomotion

Locomotion requires the precise coordination of descending motor commands, but how distinct motor programs are organized and initiated at the brainstem level remains unclear. Reticulospinal neurons (RSNs) are known to transmit motor commands to the spinal cord. Yet, the nature of RSNs that initiate forward locomotion and coordinate with steering movement remains elusive. Here, we use a high-precision all-optical approach to map the spatial and functional organization of vsx2⁺ RSNs in larval zebrafish. From population imaging, we identify among them two clusters of putative ‘Start’ neurons that are consistently recruited ~300 ms before movement onset. Using patterned optogenetics and behavioral recordings, we demonstrate that RSNs triggering forward swimming are spatially segregated from those triggering steering. Caudal medullary vsx2⁺ RSNs in rhombomere (‘r’) 8 form a hub triggering forward locomotion and receive inputs from more rostral ‘Start’ and ‘Steering’ RSNs in retropontine (r6) and rostral medulla (r7). This hierarchical and modular circuit architecture across RSNs can allow rapid transitions between motor programs while maintaining stability — a hallmark of locomotion.