Dissection of Genetic Pathways in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 144

of this approach is previous work with the TGF- b signaling pathway in C. elegans.
TGF- b signaling is involved in diverse developmental processes. Amutation in daf-4,
encoding a receptor for TGF- b superfamily ligands, results in small body size and
morphological defects in C. elegans male tails. These phenotypes are identical to
those of sma-2, sma-3,andsma-4 mutants, which initially suggested that these genes
were likely involved in the same biological process. To determine the hierarchical
relationship between daf-4 and sma-2, sma-3 and sma-4, Savage et al.(1996) cloned
and sequenced sma-2, sma-3,andsma-4, and found that they encode related proteins
homologous to a Drosophila TGF- b signaling component, Mad, suggesting that the
three genes might be also required for TGF- b signaling in C. elegans. This notion was
supported by the ensuing genetic analysis revealing that the sites of action of daf-4 and
sma-2 were in the same cell. The protein sequences of sma-2, sma-3,andsma-4 further
suggested a cytoplasmic or nuclear localization as no motifs were found to specify
extracellular or transmembrane localization. Two possibilities for function arose:
(1) sma-2, sma-3,andsma-4 might function as cytoplasmic targets downstream of
daf-4/TGF- b signaling, or (2) sma-2, sma-3,andsma-4 might act upstream of daf-4 by
regulating daf-4 expression. The latter possibility was ruled out by showing that
functional daf-4 driven by a heat-shock promoter failed to rescue the defects of
sma-2, sma-3,andsma-4 mutants. They then concluded that SMA-2, SMA-3, and
SMA-4 acted downstream of DAF-4, which was confirmed by later studies showing
that SMA-2, SMA-3, and SMA-4 form heterotrimers for propagation of TGF- b
signaling ( Savage-Dunn et al., 2000; Wu et al., 2001 ).
D. Localization Dependency of Gene Products for Gene Ordering
The function of a protein often relies on its precise cellular and subcellular
localization. In cases where this localization is tightly regulated, mutations in the
corresponding genes that genetically interact with these components may alter
protein localization. Therefore, examining localization patterns of a gene product
in different mutant backgrounds may provide useful information regarding the
relative positions of two genes in a pathway or interacting genetic network. This
strategy is termed as ''molecular epistasis'' in some studies. However, as it is so
distinct from the definition of ''epistasis''discussed above, it would be more precise
to term this strategy ''localization dependency'' to reflect its nature. Our lab
( Hagedorn et al., 2009 ) utilized this strategy to determine the hierarchical relation-
ship for two genes, ina-1 (encoding an a -integrin) and unc-40 (encoding a receptor
for the netrin ligand), during anchor cell invasion into the vulval epithelium. Both
ina-1 and unc-40 mutants are defective in AC invasion and both disrupt F-actin
localization at the invasive cell membrane. By tracking GFP fusion proteins, we
found that the normal localization pattern of UNC-40 in the AC was disturbed in
worms whose ina-1 activity is depleted by RNAi. In contrast, the localization pattern
of INA-1 remained normal in unc-40 mutants. Based on this result, we concluded
that INA-1 acts upstream of UNC-40 functionally in regulating F-actin formation in
the AC during AC invasion.