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 (MNs), we find that 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. Finally, we identify 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. These findings uncover a noncanonical role for Hox proteins during post-embryonic life, critically broadening their functional repertoire from early patterning to the control of neurotransmitter identity.

Crossovers, the exchange of homolog arms, are required for accurate segregation during meiosis. Studies in yeast have established that the single end invasion intermediate is highly regulated to ensure crossover distribution. Single end invasions are thought to differentiate into double Holliday junctions that are resolved by MutLgamma (MLH1/3) into crossovers. Currently, we lack knowledge of early steps of mammalian crossover recombination or how intermediates are differentiated in any organism. Using comprehensive analysis of recombination and cytology, we infer that polymerized single-end invasion intermediates and nicked double Holliday junctions are crossover precursors in mouse spermatocytes. In marked contrast to yeast, MLH3 plays a structural role to differentiate single end invasions into double Holliday junctions with differentially polymerized 3' ends. Therefore, we show independent genetic requirements for precursor formation and asymmetry with regard to 3' end processing, providing mechanistic insight into crossover formation and patterning.

Hox transcription factors play fundamental roles during early patterning, but they are also expressed continuously, from embryo through adulthood, in the nervous system. The functional significance of their sustained expression remains unclear. In C. elegans motor neurons (MNs), we find that 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. Finally, we identify a two-component, design principle (Hox transcriptional autoregulation counterbalanced by negative UNC-3 feedback) for homeostatic control of Hox gene expression in adult MNs. These findings uncover a noncanonical role for Hox proteins during post-embryonic life, critically broadening their functional repertoire from early patterning to the control of neurotransmitter identity.