The HMX/NKS homeodomain transcription factor MLS-2 is required to initiate the expression of the AWC terminal selector
ceh-36, and as such the AWCs of
mls-2 loss-of-function mutant animals fail to express downstream AWC-specific terminal differentiation genes (Kim et al.,2010). Interestingly, loss of
mls-2 function was also shown to result in ectopic expression of ASH markers (
sra-6p::gfp and
osm-10p::gfp) in at least one neuron in a large percentage of
mls-2 mutant animals (Kimet al., 2010). The cells that ectopically express the ASH markers are adjacent to the native ASHs and, like native ASH neurons, dye-fill with lipophilic dyes such as DiD (Perkinset al.,1986; Kimet al.,2010). Since ASH expression of both
sra-6p::gfp (Baranet al., 1999; Wood and Ferkey, 2019) and
osm-10p::gfp (Wood and Ferkey, 2019), as well as
ceh-23p::gfp (Wood and Ferkey, 2019), depends upon the paired-like homeodomain transcription factor UNC-42, we assessed whether the ectopic expression of these ASH markers requires UNC-42 function as well. We first examined the expression pattern of stably integrated reporters for
sra-6p::gfp,
osm-10p::gfpand
ceh-23p::gfp in
mls-2(
cc615)loss-of-function mutant animals. In addition to being expressed in the native ASH neurons, for each transgene we confirmed ectopic marker expression unilaterally or bilaterally in the ectopic ASH-like cells of
mls-2(
cc615) mutant animals, which were identified by dye-filling (Figure 1). We note that there was no obvious directional bias as to which side of the bilateral ASH-like pair the unilateral ectopic expression arose (Figure 1B). We found that both native and ectopic expression of all three ASH markers was lost in the
unc-42(
gk598);
mls-2(
cc615)double mutants, although the native and ectopic cells retained dye-filling capacity (Figure 1A, C). Thus, the ectopic expression of these ASH markers in the absence of MLS-2 function depends upon UNC-42, as native ASH expression does (Baranet al.,1999; Wood and Ferkey, 2019).