Worm Breeder's Gazette 1(2): 27
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The work on the genetics of dauer larva formation is aimed at identifying all the genes involved, and establishing the specific function of each of these genes. Two basic classes of mutants have been isolated: (1) mutants which enter the dauer larva state even in the presence of food (dauer-constitutive), and (2) mutants which fail to form dauer larvae when starved (dauer-defective). As reported in the previous issue of WBG, about half of the eleven dauer-defectives initially isolated seem to be sensory mutants. Therefore, it is expected that at least some mutants isolated as non-chemotactic, will also be found to be dauer-defective. Even before this work was begun, Jim Lewis and Jonathan Hodgkin observed that starved cultures of their non-chemotactic mutant, E1033, failed to accumulate dauers. Genetic mapping of the dauer-defective genes is in progress (fig. 1). So far, none of the dauer-defectives (daf 3, 5, 6, 10) has been found to be closely linked to a dauer-constitutive mutation; The 20 dauer-constitutive mutants characterized thus far fall into 6 complementation groups. Each of the 6 genes (daf 1, 2, 4, 7, 8, and 9) has been mapped (fig. 1). Fourteen of the 20 mutants fall into a single complementation group, daf 2. While working at Cambridge, I obtained many dauer-constitutive mutants from Drs. Sydney Brenner and Bob Edgar. Bob Edgar isolated several mutants using SDS resistance ( Cassada and Russell, Devel. Biol. 46, 326; 1975) as a positive selection. [See Figure 1] A surprisingly large fraction of dauer-constitutive mutants (12/20) are ts. This may reflect a bias toward selection of leaky mutants which is inherent in the SDS selection. However, 2 out of 5 dauer- constitutives isolated by visual inspection of F1 clones are ts. The remaining 3 are absolute lethals in the homozygous state because dauers are formed which cannot recover. These latter mutants (2 alleles of daf 2 and 1 allele of daf 9) must be maintained as heterozygotes. The most extreme ts mutants are ts lethals. At 25 C, 100% of the population forms dauer larvae which are unable to recover until the incubation temperature is lowered to 15 C. Growth at 15 C, on the other hand, is normal; no dauers are formed in the presence of food. The ts lethal dauer-constitutive mutants are quite suitable for reversion experiments. Selection for revertants (which grow at 25 C) provided the first indication of the relationship between constitutive and defective mutants. Revertants of daf 2, daf 4 and daf 7 alleles were selected, and nearly all revertants were found to be dauer- defective. That is, the revertants not only escape dauer formation at 25 C, but they fail to form dauers even when starved. Genetic analysis of the revertants revealed that they are, in fact, double mutants which not only carry the parental constitutive mutation, but are also homozygous for an epistatic dauer-defective mutation. Thus, some dauer-defectives are suppressors of dauer-constitutives. Suppression is generally recessive. Many revertants carried alleles of dauer-defective genes which had been already isolated and mapped. The spectrum of revertants quickly approached saturation and many alleles of a few dauer-defective genes were obtained. Thus it was clear that some, but not all, dauer-defective genes could suppress the constitutives. In order to determine which dauer-constitutive mutants could be suppressed by various defectives, a series of multiple mutants was constructed by genetic crosses. The data from these 'cross- suppression' tests is summarized in fig. 2. Suppressors fell into 4 classes based on their pattern of suppression of 4 dauer-constitutives tested. [See Figure 2] These data have been considered in the following way. The dauer- defective mutants are blocked in the natural pathway of dauer formation. The constitutives on the other hand, generate a false, internal signal which causes the mutant to form a dauer even in the absence of the natural signal (which accompanies starvation). If the pathway is blocked after the false signal, the double-mutant will be dauer-defective. However, if the false signal is generated after the block, the double-mutant is dauer-constitutive. Thus, the data in figure 2 can be organized in the form of a genetic pathway for dauer larva formation (fig. 3). The pathway in the figure is undoubtedly incomplete and may become more complex as more mutants are characterized. As of now, the order of the constitutive genes, daf 8, 7 and 4, in pathway I is unambiguous. The daf 2 gene presents a problem since it seems to be on the partially distinct pathway II. Pathway II shares at least one step in common with pathway I. However, pathway II must not be functional in wild-type animals since any block in pathway I produces the dauer-defective phenotype. In other words, pathway II must not respond to the natural signal for dauer larva formation, but only functions in daf 2 mutants. [See Figure 3] A block in pathway I is both necessary and sufficient to produce a dauer-defective phenotype. In contrast to blocks in pathway I, the M26 mutation (which blocks only pathway II) is not dauer-defective. The M26 mutation was selected as a suppressor of daf 2 and that is its only obvious phenotype. Over half (7/11) of the original collection of dauer-defectives are sensory mutants, at least some of which have defects in amphid structure. Such sensory mutants are not found among revertants of dauer constitutives. Furthermore, the sensory mutants which have been combined with dauer-constitutives, fail to suppress constitutive dauer formation at 25 C. Thus, it is concluded that these genes function early in the pathway, prior to daf 8. This is consistent with the hypothesis that the amphids mediate the primary sensory signal for dauer larva formation, while the dauer-constitutive mutations generate a false, internal signal.