In a genetic screen for genes affecting RMED/V neuron specification, we isolated two mutants,
ju190 and
ju198 (Huang et al., 2002; Huang et al., 2004; Huang and Jin, 2019). We mapped
ju190 to the X chromosome, a region covered by three cosmids (H01A20, C44H4 and F54E4), between
unc-9 and
unc-3, using the snip-SNP mapping strategy. The novel conserved nuclear transcription factor
eor-2 is contained in the cosmid C44H4, and
eor-2(
cs42) mutant animals exhibit similar behavior defects as
ju190 (Rocheleau et al., 2002). We introduced Punc-25GFP into
eor-2(
cs42), a null allele, and found no expression in RMED/V cell, as for
ju190 (Huang and Jin, 2019). DNA sequencing analysis of the
eor-2 genomic DNA from homozygous
ju190 animals identified a C to T nucleotide transition that results in an Opal stop at Arg721, in the conserved C-terminal domain (Figure 1A). Therefore, the RMED/V defects in
ju190 arise from a complete loss of EOR-2 function.
We mapped
ju198 to chromosome IV in a region near
eor-1. EOR-1 is a functional binding partner of EOR-2 (Howard and Sundaram, 2002; Howell et al., 2010). The phenotypic similarities between
eor-1 and
eor-2 and between
ju198 and
ju190 led us to suspect that
ju198 might be an allele of
eor-1. Indeed, we found that
eor-1(
cs28), a null allele, failed to complement
ju198 for the RMED/V phenotypes (Huang and Jin, 2019).
eor-1 encodes a C. elegans ortholog of mammalian promyelocytic leukemia zinc finger protein (PLZF) with a BTB domain and nine C2H2 zinc fingers (Figure 1B) (Rocheleau et al., 2002). We found that
ju198 is a missense mutation changing a conserved histidine to tyrosine in the last zinc finger (Figure 1B). Altogether, our data show that the complete loss of either
eor-1 or
eor-2 function results in identical differentiation defects in RMED/V neurons.