During C. elegans development the NSMs differentiate into pharyngeal neurosecretory motoneurons, their sisters, however, die shortly after they are born as a result of programmed cell death. Our results indicate that the death of the NSM sister cells is specified by the asymmetric expression of the cell-death activator gene
egl-1 (egl, egg laying defective), which codes for a BH3-only protein.
egl-1 is transcriptionally active in the NSM sister cells, which are destined to die, but not in the NSMs, which are destined to survive. We show that a weak loss-of-function mutation of the only C. elegans Daughterless-like bHLH-gene
hlh-2 (hlh, helix-loop-helix),
hlh-2(
bx108), causes specifically the NSM sister cells to survive with a low frequency. A similar phenotype can be observed in animals treated with dsRNA of the achaete-scute-like bHLH-gene
hlh-3 (
hlh-3(RNAi)). In addition,
hlh-3(RNAi) strongly enhances the NSM sister cell survival phenotype of
hlh-2(
bx108) mutants, suggesting that
hlh-2 and
hlh-3 act together to kill the NSM sister cells. A heterodimer composed of HLH-2 and HLH-3, HLH-2/HLH-3, can bind to four E-box motifs (the DNA binding sites for bHLH proteins) in the
egl-1 promoter in vitro, which are required for
egl-1 expression in the NSM sister cells in vivo. We therefore propose that HLH-2/HLH-3 acts as a direct activator of
egl-1 transcription. The death of the NSM sister cells can also be prevented by a gain-of-function mutation (gf) in
ces-1 (ces, cell-death specification), which is thought to cause overexpression of CES-1, a Snail-like transcription factor, in the NSM sister cells. We found that the
ces-1(gf) mutation blocks this cell-death event by repressing
egl-1 expression in the NSM sister cells. Furthermore, in vitro, CES-1 binds to four Snail-binding sites in the
egl-1 promoter, which overlap with the four E-box motifs, and the capability of CES-1 to repress
egl-1 expression in vivo is dependent on its ability to bind to these sites. We therefore suggest that in
ces-1(gf) animals the NSM sister cells survive because CES-1 prevents an activator of
egl-1 expression, most likely HLH-2/HLH-3, from binding to the
egl-1promoter.Finally, we have identified additional enhancers of the weak NSM sister cell survival phenotype in
hlh-2(
bx108) mutants. Their characterisation may elucidate new factors required for the specification of the NSM sister cell death and reveal the mechanisms involved in this process.