daf-28(
sal91) Defines a New Step in the Dauer Epistasis Pathway Elizabeth A. Malone and James H. Thomas Department of Genetics, University of Washington, Seattle, WA 98195 Dauer formation and dauer recovery are regulated by the same three external conditions: the concentration of dauer pheromone, temperature and the abundance of food. Previously all mutations that caused constitutive dauer formation (Daf- c) also inhibited dauer recovery. Together, these facts show that many genes are involved in regulating both dauer formation and dauer recovery. We recently described
sal91, a semi-dominant, temperature-sensitive Daf-c mutation that identifies a new gene,
daf-28 (Malone and Thomas, Genetics 136: 879-886). This mutation is unique because the dauers recover rapidly at all temperatures. We hypothesize that daf- 28
(sal91) perturbs only dauer formation, and not recovery. Because
sal91 is dominant, and because a deficiency of the locus does not cause a dominant Daf-c phenotype, we conclude that
sal91 is a gain-of-function mutation. As a first step in understanding the role of
daf-28 in dauer formation, we have constructed double mutants containing daf- 28
(sal91) and mutations in other genes that affect dauer formation. The mutations analyzed fall into two classes: mutations that prevent dauer formation (Daf-d) and other Daf- c mutations. Previous double mutant analysis suggests that these genes act in a complex, branched pathway (please see abstract by Schackwitz and Thomas for an abbreviated model). All but one of the
daf-28; daf-d double mutants are dauer constitutive, including those containing
daf-22,
daf-6, daf- 3,
daf-5, and mutations in several genes that affect the structure of amphid sensory cilia. These results suggest that
daf-28 functions downstream of these Daf-d genes. In contrast,
daf-12 completely suppresses the Daf-c phenotype of
daf-28, suggesting that
daf-12 acts downstream of
daf-28. Like
daf-28(
sal91), all of the Daf-c mutations that we tested are temperature sensitive. The phenotypes of the
daf-28;daf-c double mutants were examined at 15, the permissive temperature at which all of the single mutants form dauers at low frequency (0 to 77 %). Under these conditions, all but one of the
daf-28; daf-c double mutants formed 100% dauers, including those containing daf-ll,
daf-21, daf-l,
daf-4, daf- 7,
daf-8, and
daf-14. These synergistic interactions suggest that
daf-28 functions in parallel with these genes rather than in series. In contrast, the
daf-2;
daf-28 double mutant forms few dauers at 15. It is possible that
daf-28 functions in a sequential pathway with
daf-2. However, two results distinguish
daf-28 from
daf-2. First, a
daf-16 mutation suppresses
daf-2 but not
daf-28. Second,
daf-2;
daf-12 double mutants arrest in larval development, whereas the
daf-28;
daf-12 double mutant is viable and fertile. In summary, daf- 28
(sal91)is unique in its interactions with other mutations that affect dauer formation, and it may define a new branch of the network of genes that impinge on
daf-12. To further our understanding of the function of
daf-28, we reverted
daf-28(
sal91) with the aim of obtaining loss-of- function mutations and possible extragenic suppressors. Taking advantage of the fact that
unc-31;
daf-28 mutants recovery poorly from the dauer stage, we mutagenized these animals with EMS and screened for F2 progeny that did not form dauers. In a screen of 9,200 genomes, we identified 25 mutants, seven of which represent
daf-12 alleles. Of the remaining 18 revertants, we have determined that at least seven do not contain
daf-12 mutations. Since
daf-12 mutations are the only known Daf-d mutations that suppress daf- 28
(sal91), we expect that these revertants represent either loss-of-function mutations in
daf-28 or extragenic suppressors defining new genes.