The development of each of the 302 neurons in the C. elegans nervous system is tightly regulated, but mechanisms underlying specification of these neuronal cell fates are incompletely understood. For example, the tyraminergic RIM and octopaminergic RIC interneurons are involved in diverse sensorimotor behaviors, including reversals, head movements, and feeding, but the factors guiding the development of these neurons are largely unknown. To identify regulators of RIM/RIC neuronal development, we used genetic screens for genes that when mutated cause extra GFP-positive cells in
tdc-1::GFP transgenic worms, which express GFP specifically in the two RIM and two RIC neurons. We screened approximately 67,000 genomes and isolated 3 alleles of
eor-2 (
egl-1 suppressor/DiO uptake defective/raf enhancer).
eor-2 encodes a co-factor of the transcription factor EOR-1, a homolog of the promyelocytic leukemia zinc finger (PLZF). We found that
eor-1 and
eor-2 act in the same genetic pathway to prevent the generation of extra GFP-positive cells. A
ced-3(lf) mutation results in 8-9
tdc-1:GFP positive cells, and the addition of the
eor-2(
n5163) mutation, which generates a premature stop codon in the fifth exon of the 17 exons in
eor-2, increased this number to 10-11 cells. This observation suggests that EOR-1/EOR-2 act separately from CED-3 and regulate the fate of specific neurons rather than the deaths of the RIM and RIC sister cells. Next we used RIM- and RIC-specific reporters to ask whether the extra GFP-positive cells in
eor-1(
cs28) loss-of-function mutants were RIM-like or RIC-like. We found that the
eor-1(
cs28) mutants ectopically expressed both the RIM and the RIC reporter, and that the ectopic RIM and RIC reporters were expressed in different cells, suggesting that EOR-1 regulates the fate of at least two different cell types to prevent fate transformation to RIM-like or RIC-like cells. Additional experiments using other cell-specific markers suggested that the extra RIM-like cells normally differentiate to become GABAergic RME neurons. Our results demonstrate that the EOR-1 transcription factor and EOR-2 together regulate the cell-fate determination of specific neurons during development. We hope this study will provide novel insights concerning the functions of the evolutionarily conserved PLZF transcription factor in animal development and of the dysfunction of PLZF in human diseases, such as cancer.