Dissection of Genetic Pathways in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 142

Moreover, since the LIN-3 protein is normally required for VPCs to adopt vulval
fates and the LIN-1 protein inhibits the adoption of vulval fates of VPCs, the
interactive relationship between them can be inferred as LIN-3 negatively regulates
LIN-1 (see Fig. 1 ).
In cases where mutations involved in a common pathway display the same phe-
notype, epistatic analysis is not possible. However, some genes may have both gf and
lf alleles that display the opposite phenotypes. Such gain-of-function alleles have
proven to be very useful in deducing genetic hierarchies. The gf alleles can be either
mutagen-induced or artificially engineered. One example using an artificially engi-
neered transgene was a study examining the role of netrin signaling in axon out-
growth. A lf mutation in unc-40, encoding a netrin receptor, causes defects in axon
guidance. Several lf mutations in genes involved in actin cytoskeleton regulation also
display similar defective phenotypes. To order these genes into the unc-40 pathway,
Gitai et al. (2003) generated an artificial gf allele of unc-40 by overexpressing and
targeting the UNC-40 intracellular domain to the plasma membrane of the neuron.
The engineered transgenic strain displays excessive axon outgrowth. Through epi-
static analysis of the double mutants of this artificial unc-40 gf allele and the other
actin-regulating mutants, they found that these actin-regulating genes are epistatic to
unc-40 because the mutations in these actin-regulating genes suppress the excessive
axon outgrowth of the unc-40 gf allele. Further, these genes were found to form two
bifurcated pathways downstream of unc-40 as pairwise combinations of these muta-
tions showed synergistic suppression of the unc-40 gf phenotype.
2. Consideration Regarding Mutants Used for Epistatic Analysis and Limitations
of Epistatic Analysis
It is important to use null mutants or hypomorphic alleles with no activity in the
biological process being analyzed for epistatic analysis. An appropriate choice of
mutants (null or hypomorphic) based on their dosage effects is critical for success-
ful epistatic analysis. Using any mutants with residual activity may lead to a
misinterpretation of the results of epistatic analysis, particularly in cases where
one tests a gene with a hypomorphic allele that acts downstream of a gene with a
null allele. Because the phenotype of the double mutants of these two alleles will be
similar to that of the null allele, it will lead to an inaccurate conclusion that the gene
with the null allele is epistatic to (downstream of) the gene with the hypomorphic
allele ( Fig. 9 ).
An assumption of epistatic analysis is that the genes being ordered function in a
linear genetic pathway. However, many pathways regulating biological processes
have more complicated topological structures. They are nonlinear and often contain
feedback/feedforward loops or autoregulatory elements. Moreover, the genetic hier-
archy between the same components is sometimes context-dependent and can
change spatially or temporally. Such a complexity limits the applicable scope of
epistatic analysis. Fortunately, other tools are also available to C. elegans researchers
to complement epistatic analysis in constructing gene regulatory pathways.