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

Abstract Motor neuron (MN) diversity is essential for producing the broad repertoire of animal movements, yet the molecular mechanisms that specify MN subtypes remain incompletely defined. Here, we investigate how Hox genes and their PBX cofactors shape cholinergic MN subtype identity along the anterior–posterior (A–P) axis of the C. elegans ventral nerve cord (VNC). In anterior MNs, we show that the anterior Hox genes ceh-13 (Lab/Hox1) and lin-39 (Scr/Dfd/Hox4–5) collaborate with the Hox cofactor ceh-20 (Exd/Pbx1–4) and the terminal selector unc-3 (Collier/Ebf1–4) to activate terminal identity genes. In posterior nerve cord MNs, the mid-body Hox gene mab-5 (Antp/Hox6–8) represses terminal identity gene expression by antagonizing unc-3 in a ceh-20 -dependent manner. Notably, 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, the posterior Hox gene egl-5 (Abd-A/Abd-B/Hox9–13) collaborates with unc-3 to activate lumbar-specific MN terminal identity genes in a ceh-20 -independent manner. We further find that ceh-20 is necessary for Hox gene expression ( ceh-13, lin-39, mab-5 ) in VNC MNs, supporting a model where Hox positive autoregulation requires PBX activity. Together, these findings reveal PBX-dependent and independent roles for Hox genes in establishing and maintaining MN identity, illustrating how combinatorial interactions between Hox factors and terminal selectors generate neuronal subtype diversity. AUTHOR SUMMARY Animals rely on many different types of motor neurons to generate precise and flexible movements, but how these neuron subtypes are specified remains an open question. In this study, we examine how a family of developmental genes called Hox genes, together with their cofactors, help define distinct motor neuron identities in the nervous system of the nematode Caenorhabditis elegans . We find that different Hox genes act in specific regions of the ventral nerve cord to either turn motor neuron identity genes on or keep them off. In anterior motor neurons, certain Hox genes work together with a cofactor called PBX and a neuron-specific regulator (UNC-3) to activate genes required for proper motor neuron function. In contrast, a mid-body Hox gene suppresses these genes in posterior neurons, while a more posterior Hox gene activates a unique set of genes in lumbar motor neurons through a different mechanism. Importantly, we show that some Hox genes and PBX are needed not only during early development but also later in life to maintain motor neuron identity. Together, our findings reveal how combinations of Hox genes and cofactors generate and preserve motor neuron diversity, providing insight into general principles of nervous system development.