PBX-dependent and -independent Hox programs establish and maintain motor neuron terminal identity.

Motor neuron (MN) diversity is essential for generating animal movement, yet the molecular mechanisms specifying MN subtypes remain poorly understood. We investigate how Hox genes and their PBX co-factors establish cholinergic MN identity along the anterior-posterior axis of the Caenorhabditis elegans ventral nerve cord. In anterior MNs, the Hox genes ceh-13 (Lab/Hox1) and lin-39 (Scr/Dfd/Hox4-5) collaborate with the co-factor ceh-20 (Exd/Pbx1-4) and terminal selector unc-3 (Collier/Ebf1-4) to activate terminal identity genes. In posterior MNs, the Hox gene mab-5 (Antp/Hox6-8) represses terminal identity genes by antagonizing unc-3 in a ceh-20-dependent manner. Both mab-5 and ceh-20 are required not only during early development but also in later life stages to maintain posterior MN identity. In lumbar MNs, egl-5 (Abd-A/Abd-B/Hox9-13) collaborates with unc-3 to activate lumbar-specific identity genes independently of ceh-20. We also find that ceh-20 is required for Hox gene expression in ventral nerve cord MNs, supporting a model in which Hox-positive autoregulation depends on PBX activity. Together, these findings provide a conceptual framework for understanding how spatial patterning information is integrated with terminal selectors to generate and maintain neuronal subtype diversity.