The Non-canonical Role of Hox Genes in the Regulation and Maintenance of C. elegans Motor Neuron Terminal Identity

Hox transcription factors play fundamental roles during early patterning, but they are also expressed continuously, from embryonic stages through adulthood, in the nervous system. However, the functional significance of their sustained expression remains unclear. In C. elegans motor neurons, we identified a non-canonical role of Hox genes in the regulation and maintenance of ventral nerve cord motor neuron terminal identity. First, we are among the first groups to establish the MN expression profile of Hox genes with single cell resolution. Second, we demonstrated the functions of Hox proteins in regulating neurotransmitter pathway genes and a broad range of motor neuron terminal identity genes. LIN-39 (Scr/Dfd/Hox4-5) is continuously required during post-embryonic life to maintain neurotransmitter identity, a core element of neuronal function. LIN-39 acts directly to co-regulate genes that define cholinergic identity (e.g., unc-17/VAChT, cho-1/ChT). We further show that LIN-39, MAB-5 (Antp/Hox6-8) and the transcription factor UNC-3 (Collier/Ebf) operate in a positive feedforward loop to ensure continuous and robust expression of cholinergic identity genes. Third, we identified a two- component design principle for homeostatic control of Hox gene expression in adult MNs (Hox transcriptional autoregulation is counterbalanced by negative UNC-3 feedback). Last, we described a Hox transcriptional switch prevented by UNC-3 to safeguard cholinergic motor neuron identity throughout life. These findings not only highlighted the noncanonical role but also established a terminal selector role for Hox proteins during post-embryonic life, critically broadening their functional repertoire from early patterning to the control of motor neuron terminal identity.