The diversity of neurons in the nervous system is specified by many genes, including those that encode transcription factors (TFs) and play crucial roles in coordinating gene transcription. To understand how the spatiotemporal expression of TF genes is regulated to generate neuronal diversity, we used one member of the LIM-Hox family,
lin-11, as a model that is necessary for the differentiation of amphid neurons in the nematode C. elegans and a related species C. briggsae. We characterized transcriptional regulation of
lin-11 and uncovered regulatory roles of two of the largest introns, intron 3 and intron 7. These introns promote
lin-11 expression in non-overlapping sets of neurons. Phenotypic rescue experiments in C. elegans revealed that intron 3 is capable of restoring
lin-11 function based on gene expression patterns and behavioral assays. Interestingly, intron 3-driven reporter expression showed differences in neuronal cell types between C. briggsae and C. elegans, indicating evolutionary changes in
lin-11 regulation between the two species. Reciprocal transformation experiments provided further evidence consistent with functional changes in both cis and trans regulation of
lin-11. To further investigate transcriptional regulation of
lin-11, we dissected the intronic regions in C. elegans and identified cell-specific enhancers. These enhancers possess multiple sequence blocks that are conserved among Caenorhabditis species and possess TF binding sites. We tested the role of a subset of predicted TFs and discovered that while three of them (SKN-1, CEH-6, and CRH-1) act via the intron 3 enhancer to negatively regulate
lin-11 expression in neurons, another TF (CES-1) acts positively via the intron 7 enhancer. Overall, our findings demonstrate that neuronal expression of
lin-11 involves multiple TF regulators and regulatory modules some of which have diverged in Caenorhabditis nematodes.