Joint actions of diverse transcription factor families ensure enhancer selectivity and robust neuron terminal differentiation

Identifying general principles for neuron-type specification programs remains a major challenge. Here we performed RNA interference (RNAi) screen against all 875 transcription factors (TFs) encoded in Caenorhabditis elegans genome to systematically investigate the complexity of neuron-specification regulatory networks. Using this library, we screened for defects in nine different neuron types of the monoaminergic (MA) superclass and two cholinergic motorneurons to search for common themes underlying neuron specification. We identified more than 90 TF candidates to be involved in neuron specification, of which 28 were also confirmed by mutant analysis. Correct specification of each individual neuron type requires at least nine different TFs. Individual neuron types do not usually share TFs involved in their specification but share a common pattern of involved TF families, composed mainly by five out of more than fifty in C. elegans : Homeodomain (HD), basic Helix Loop Helix (bHLH), Zinc Finger (ZF), Basic Leucine Zipper Domain (bZIP) and Nuclear Hormone Receptors (NHR). In addition, we studied terminal differentiation complexity focusing on the dopaminergic terminal regulatory program. We found three TFs (UNC-62, VAB-3 and MEF-2) that work together with the three known dopaminergic terminal selectors (AST-1, CEH-43, CEH-20). This complex combination of TFs provides genetic robustness and imposes a specific gene regulatory signature enriched in the regulatory regions of dopamine effector genes. Our results provide new insights on neuron-type regulatory programs in C. elegans that could help better understand neuron specification and the evolution of neuron types. ### Competing Interest Statement The authors have declared no competing interest.