In previous work, we showed that three different
srf-6 mutants carry mutations in gene
nsy-1(Van Sciver et al., 2019), and that a
srf-6mutant shows the 2AWCON phenotype characteristic of
nsy-1 mutants. We also showed that
srf-6(
yj13) fails to complement
nsy-1(
ok593)(Honzel et al., 2019). We concluded that
srf-6 and
nsy-1 are the same gene. C. elegans gene
nsy-1 encodes a MAP kinase kinase kinase (MAPKKK) that carries out the first step in a C. elegans MAP kinase pathway (Kim et al., 2002, Sagasti et al., 2001, Tanaka-Hino 2002). The pathway culminates in activation of a MAP kinase that is homologous to the human
p38 MAP kinase (Kim et al., 2002). This pathway is involved in at least three biological processes in C. elegans, i.e., immune responses to infection by pathogenic microbes (Kim et al., 2002), upregulation of serotonin biosynthesis in the ADF chemosensory neurons in response to pathogen exposure (Shivers et al., 2009, Zhang, Lu, and Bargmann 2005), and determination of asymmetric cell fate in the AWC chemosensory neuron pair (Sagasti et al., 2001). C. elegans
srf-6 mutants were originally identified by antibody staining at stages L2-L4 with mAb M37, which stains only the L1 stage in wild type C. elegans under normal growth conditions (Hemmer et al., 1991, Grenache et al., 1996). To determine whether loss of function in the C. elegans
p38 pathway also results in staining at stages L2-L4, we tested
nsy-1 (MAPKKK),
sek-1 (MAPKK) and
pmk-1 (MAPK) mutants for mAb M37 staining at these later larval stages. Results are shown in Figure 1. In these experiments,
nsy-1(
ok593)and
nsy-1(
ag3) also showed staining of L2-L4 stages (Figure 1). Staining of
sek-1(
km4) was also positive. The Toll-interleukin-receptor-like protein TIR-1 is required upstream of
nsy-1 for both immune responses and determination of AWC fate (Couillault et al., 2004, Chuang and Bargmann 2005). Positive antibody staining was also observed in
tir-1(
tm3036) mutants. It was surprising that no L2-L4 larvae of a mutant strain carrying
pmk-1(
km25) stained (out of a total of 105 worms in 4 biological replicates), because PMK-1 is required as the MAP kinase for immune responses (Kim et al., 2002). However, a requirement for PMK-1 has not been demonstrated for determination of AWC cell fate (Pagano, Kingston, and Kim 2015). It has been shown that function of the TIR-1 NSY-1 SEK-1 pathway is required for determination of AWC fate (Chuang and Bargmann 2005), and that PMK-1 and a second MAP kinase, PMK-2, function redundantly for determination of AWC cell fate (Pagano, Kingston, and Kim 2015). We therefore tested a
pmk-2 pmk-1 double mutant for M37 antibody staining. In contrast to
pmk-1, the
pmk-1 pmk-2 double mutant showed staining at a level similar to that of
sek-1(
km4). The
unc-43-encoded calcium dependent kinase CaMKII is required for upstream activation of the TIR-1 NSY-1 SEK-1 cascade in the AWC neuron, and the level of
unc-43 activity in AWC can determine whether the cell develops the AWCONor the AWCOFF fate (Troemel, Sagasti, and Bargmann 1999).We tested the mutant
unc-43(
n498gf), which shows a 2AWCOFF phenotype, and the 2AWCON mutant
unc-43(
n498n1186lf) for M37 antibody staining (Troemel, Sagasti and Bargmann 1999). Neither of these mutants stained with mAb M37.