Dauer formation in C. elegans is controlled by the
daf-2 (insulin receptor-like) signal, in conjunction with the
daf-7 (TGF-beta-like) and the
daf-11 (guanylyl cyclase) signals. Although multiple components of the DAF-2/IR pathway have been identified in various Daf-c (dauer-formation constitutive) and Daf-c suppressor screens using
daf-2 and
age-1 (PI3 kinase) mutants, no loss-of-function (lf) mutation has been found in an insulin-like molecule, a presumed ligand for DAF-2/IR. Failure to isolate such a lf mutation may be attributed to the presence of 37 ins (insulin-like) genes in the C. elegans genome (Pierce et al., 2001), some of which may serve redundant functions in dauer formation. We have determined that the Daf-c gene
daf-28, defined by the dominant-negative mutation
sa191, encodes an insulin-like molecule. Given that
daf-28(
sa191) also shares additional phenotypic features with mutants of
daf-2 and other components in the insulin-like pathway, such as extended life span and interaction with
daf-16 (fork head transcription factor) (Malone and Thomas, 1993; Malone et al., 1996), we propose that DAF-28 exerts its function as a ligand of DAF-2/IR. We cloned
daf-28 by SNP mapping in conjunction with traditional 3-factor mapping. We have also found that high-copy transgenes of
daf-28 can effectively suppress the Daf-c phenotype associated with
daf-28(
sa191). This is consistent with previous genetic evidence showing that
daf-28 (
sa191) was a semi-dominant allele and that its product may poison the process in which wild-type DAF-28 functions (Malone and Thomas, 1993). Since increasing the dose of the wild-type DAF-28 reduces the Daf-c effect resulted from the mutant product, we predict that the wild-type function of DAF-28 is to inhibit dauer arrest. This hypothesis is further supported by evidence on how
daf-28 expression is regulated. A
daf-28::gfp transcriptional fusion is expressed in two pairs of sensory neurons and the hind gut from late L1 to adult. The sensory neurons are ASI and ASJ, which have previously been shown to be important for regulation of dauer formation.
daf-28::gfp expression in the sensory neurons is down-regulated in natural dauers, pheromone induced dauers and
daf-11(lf) dauers. These results suggest that in response to environmental cues and the
daf-11 signal,
daf-28 functions in sensory neurons to inhibit dauer arrest.
daf-28(
sa191)is an arginine to cysteine alteration at position 37. One possibility is that creation of an extra cysteine in DAF-28 results in the formation of inappropriate inter-molecular disulfide bonds that cause improper folding and association of DAF-28 with other insulins. Given the possible redundant function among the ins genes, it makes sense that it would take such a dominant-negative mutation to reveal functional involvement of insulins in dauer formation. Consistent with redundancy, transgenes encoding the wild-type INS-4 protein, a close relative of DAF-28, can also suppress the Daf-c phenotype associated with
daf-28(
sa191).