Description

We screened for mutants that affect expression of dopaminergic neuron identity, using a transcriptional reporter for expression of the dopamine transporter dat-1. We previously published and characterized a number of mutants that affect dat-1 expression in different neuron types (Doitsidou et al., 2008). Four alleles that we did not publish in our original screening paper are described here. While wild-type animals only display a single dat-1::gfp(+) neuron pair in the midbody region, the PDE neuron pair from the postdeirid lineage, all 4 mutant alleles display ectopic dat-1::gfp expression along the anterior/posterior axis of the animal (Fig.1A,B). Postdeirid lineage duplication defects were previously described in animals lacking the bHLH transcription factor lin-22/Hairy (Wrischnik and Kenyon, 1997). We find that the canonical lin-22 allele, n372, indeed displays dat-1::gfp expression defects similar to those observed in our mutants (Fig.1B). We sequenced the lin-22 locus in all of our four, independently isolated alleles. Two of them are premature stop codons, one is a missense mutation affecting a conserved leucine residue and all display a similar penetrance of defects (Fig.1B,C). The fourth and weakest allele, ot269, displayed no sequence alteration in the lin-22 coding sequence or in exon/intron boundaries. ot269 failed to complement ot267, ot268, ot287 and the canonical lin-22 allele n372. Furthermore, the ot269 phenotype was rescued by injection of the fosmid WRM0627dG07, which contains lin-22 and one additional complete gene. We found that ot269 harbors a single nucleotide change in the upstream intergenic region of lin-22, almost 5kb away from the start of the gene (sequence change shown in Fig.1B). Subsequent work has shown that this mutation affects a binding site for a GATA transcription factor (Katsanos et al. 2017).