Chapter 10, February 2009

Andersen, E. C., A.M. Saffer, and H.R. Horvitz. (2008). Multiple levels of redundant processes inhibit Caenorhabditis elegans vulval cell fates. Genetics 179, 2001-2012 [DOI: 10.1534/genetics.108.092197]

Synthetic enhancement was used to elucidate the genetic pathway leading to vulval cell fates in C. elegans, as described on pages 423-426. As a brief reminder, two principal and functionally redundant pathways were identified, as defined by two sets of genes, the class A genes and the class B genes. A mutant worm that is knocked out for a gene in one class has a wild-type phenotype at the normal growth temperature of 20 ° C. (All mutations are recessive, so the mutants are homozygous for all of the mutations under discussion.) By contrast, a double mutant worm with genes from both class A and class B knocked out has a multivulva (Muv) mutant phenotype. For example, the original genes were lin-8 and lin-9. A worm homozygous mutant for lin-8 is wild-type, as is a worm that is homozygous mutant for lin-9. However, a double mutant that is homozygous for both lin-8 and lin-9, that is, of genotype, lin-8/lin-8; lin-9/lin-9, has a Muv mutant phenotype. This synthetic enhancement is the property that defined the two classes of genes; lin-8 is a member of class A, lin-9 is member of class B. For clarity, we will symbolize these as A; B double mutants. Four different class A genes have been identified, while more than 20 different class B genes have been found so far.

It was reported previously that mutations within the same class - that is, in two class A genes - do not enhance one another. Using our previous notation, A1; A2 or B1; B2 double mutants have a wild-type phenotype. This led to the notion that there are two parallel pathways, one defined by class A genes and one defined by class B genes, as shown in Figure 10-13 on page 425. A re-investigation of these genes from the same lab, described in the Andersen et al. paper, has re-enforced the original distinction between class A and class B genes, but has also found that more subtle distinctions can be found among genes within each class. Interestingly, these intra-class distinctions may provide another genetic method to identify genes whose products are not only in the same process or pathway but are actually in the same macromolecular complex.

The new investigation used two different methods to look for these more subtle intra-class distinctions among the genes. All of the original research had been done at the normal growth temperature of 20 ° C, at which the single mutant strains are wild-type. However, at the elevated growth temperature of 25 ° C, some of the single mutants from class A have a mutant Muv phenotype at low penetrance; none of the class B mutant shows this phenotype in single mutant strains. This low penetrance mutant phenotype is observed even with apparent null alleles of the class A genes, indicating that the temperature dependence is not a property of the allele itself but of the process that the gene affects. (This type of reasoning is explained in Chapter 9 on page 362 and in Case Study 9-1.)

The temperature dependence provided one approach to sensitize the double mutant strains in order to find subtle effects. Surprisingly, many of the class A mutants do now show synthetic enhancement among themselves under these more challenging growth conditions - that is, A1;A2 double mutant worms at high temperature have a mutant phenotype that is not observed for the same strains at the normal growth temperature. Since synthetic enhancement is interpreted to mean that the genes affect different pathways or processes, this result suggests that the class A mutants do not lie in a single pathway but rather in several different, but related, pathways.

A different method was used to make a sensitized background to observe intra-class effects among the other class A and the class B mutants. For these, a triple mutant strain was constructed, in which two of the mutants were from one class and the third was a weak hypomorphic allele of a gene in the other class. (In our notation, these strains would be A1;A2; B (weak) or A (weak); B1; B2.) In nearly all cases, the triple mutant had a higher penetrance mutant phenotype than did any of the double mutants.

For example, both lin-35 and lin-37 are class B genes, and a lin-35; lin-37 double mutant has a wild-type phenotype (shown in their Table 4). However, in a triple mutant with lin-35, lin-37, and the weak class A mutant lin-15 (n767), all of the worms have a Muv mutant phenotype at 20 ° C (their Table 5). Thus, in a sensitized genetic background in which the class A pathway is impaired but not eliminated, synthetic enhancement is also seen among class B mutants. This result indicates that the class B genes, like the class A genes, define several different but related pathways that affect vulval cell fate. The re-interpreted pathways are shown in Figure 1, with the class A genes in red and the class B genes in blue.

The most interesting result may be those few cases for which no synthetic enhancement was found. For example, both lin-35 and dpl-1 are class B mutants, and both are enhanced by weak class A mutations such as lin-15 (n767). Furthermore, in the sensitized genetic background, both lin-35 and dpl-1 show synthetic enhancement with many other class B mutations. However, even in the sensitized genetic background, lin-35 and dpl-1 do not enhance each other (see their Table 4 at 15 ° C and 17.5 ° C).

The products of these two genes provide the explanation for this result. Both genes have orthologs in many other organisms, including mammals. lin-35 encodes the C. elegans ortholog of the RB protein, whereas dpl-1 encodes the worm ortholog of the DP protein. In other organisms, these proteins are known to be part of a multimeric protein complex that also includes the E2F protein (encoded by the efl-1 gene in worms). The most likely interpretation of this result is that these two genes do not show synthetic enhancement because they affect not only the same overall process but, in fact, the same protein complex. Knocking out one of two proteins disables the complex, so knocking out the second has no additional effect. In support of this interpretation, no synthetic enhancement is observed in interactions between dpl-1 or lin-35 and the third gene of this complex, efl-1.

The re-investigation described in this paper demonstrates that class A and class B mutants do form two distinct groups affecting two different but functionally redundant processes. As before, a double mutant worm that is A; B has a mutant phenotype even at normal growth temperatures. However, by using sensitized growth conditions and genetic backgrounds, distinctions are seen within each class. Thus, functional redundancy also exists within each class. Finally, those few genes that do not show synthetic enhancement even under the most sensitized conditions appear to encode proteins that are part of the same multimeric complex. Thus, a failure to observe synthetic enhancement might be just as informative, and perhaps even more informative, as finding synthetic enhancement.

Figure 1. A revised model for vulval cell fates in C. elegans.
Two functional redundant pathways block hypodermal cells from adopting vulval cells, as illustrated by the shaded bars. The class A genes are in red, the class B genes in blue; a worm has a mutant phenotype when a gene in each class is knocked out. Andersen et al. have used sensitizing conditions to show that there is also functional redundancy within each class, as shown by the genes on the different lines. For example, lin-38 and lin-8 are class A genes that enhance each other under sensitizing conditions. However, not all of the genes show synthetic enhancement, as illustrated by genes on the same line. For the case of the class B genes dpl-1, efl-1, and lin-35, the genes encode proteins that are known to interact in the same macromolecular complex. Redrawn from Andersen et al., 2008.