TGF b -like and insulin--like signaling pathways regulate both dauer entry and recovery. Since the Daf-c (dauer formation constitutive) phenotype caused by
daf-2(lf) (insulin receptor-like) or
age-1(lf) (PI3 kinase) can be fully suppressed by
daf-16(lf) (fork-head-like transcription factor) at the non-permissive temperature of 25 deg C,
daf-16 has been considered to be the terminal dauer-promoting gene of the insulin-like pathway and the sole output of the
daf-2 /IR signal. Likewise,
daf-3 (smad transcription factor) is considered to be the terminal dauer-promoting gene of the
daf-7 /TGF b -like pathway. The two signaling pathways may converge to regulate dauer formation by affecting the
daf-12 (nuclear hormone receptor) activity, based on the genetic epistatic analysis. In addition, a third signaling pathway for dauer arrest has been implicated by the finding that
daf-16(0);
daf-3(0) animals can still form partial dauers in response to pheromone. The above working model is based on the hypothesis that the
daf-11 (guanyl cyclase) signaling cascade functions upstream of, instead of in parallel to TGF b -like and insulin--like signaling pathways. In order to identify the components in the putative third signaling pathway, we screened for Daf-c mutations in a
daf-16(0);
daf-3(0) background. No Daf-c mutations in either the TGF b or insulin-like pathways were expected to be isolated from this screen.
mg293, one of the two isolates from this screen, displayed a transient Daf-c phenotype in the original
daf-16(0);
daf-3(0) background and a non-recoverable Daf-c phenotype in the wild-type background. Unexpectedly,
mg293 turned out to be an allele of
daf-2, based on map position and a complementation test. We confirmed this result by constructing a
daf-16(0);
daf-2(0);
daf-3(0) strain, using a known
daf-2(0) allele. This triple mutant forms transient partial dauers at all temperatures tested. These results suggested that
daf-16(0) does not suppress the
daf-2 phenotype in the absence of the
daf-3 activity. This further suggests that there is a
daf-16 -independent
daf-2 output, which is normally masked by the
daf-3(+) function. It is noteworthy that this
daf-3(+) function is to repress dauer arrest. Different from the known
daf-16- dependent
daf-2 signal, which regulates both dauer entry and recovery, the
daf-16- independent
daf-2 signal specifically regulates dauer entry. Moreover, a
daf-16(0);
age-1(0);
daf-3(0) strain also showed the transient Daf-c phenotype, with lower penentrance compared to
daf-16(0);
daf-2(0);
daf-3(0), suggesting that
age-1 is involved in the
daf-16 -independent
daf-2 signal. Finally, introduction of a
daf-12(0) mutation into
daf-16(0);
daf-2(0);
daf-3(0) prevented the transient Daf-c phenotype, indicating that
daf-12 is a target of the
daf-16 -independent
daf-2 signal. Since the
daf-16 -independent
daf-2 signal regulates dauer entry but not recovery, some of its downstream effectors should also specifically regulate dauer entry. To identify such effectors, we performed a screen in the N2 background, aiming for mutants that mimic the transient Daf-c phenotype associated with
daf-16(0);
daf-2(0);
daf-3(0). We have obtained four candidate mutants from 12,000 mutagenized haploid genomes. Characterization of these mutants is underway.