The C. elegans excretory system consists primarily of a large, tubular, H-shaped structure made from a single excretory canal cell. This cell, located near the terminal bulb of the pharynx, extends two hollow processes on either side of the animal to form two excretory canals that extend to the head and tail on both the left and right sides of the animal.
The EXC-5 protein is homologous to a human guanine nucleotide exchange factor (GEF) FGD1, which regulates formation of facial, genital, and limb structures. Null mutations in
exc-5 cause defects in lumen diameter in the form of large fluid-filled cysts, which in the worst cases can become as large as the entire body diameter of the worm. We believe these cysts form as a result of a mechanical failure of the actin cytoskeleton lining the apical surface of the canal. Canal extension during development generally ceases at the point where very large cysts form.
Recently, we performed an EMS non-complementation screen to identify new mutations in the
exc-5 gene. We have recovered 42 isolates. Two of these,
qp21 and
qp39, have been sequenced, and identified as having point mutations resulting in premature stop codons in the C-terminal Pleckstrin-Homology domain of the protein. The phenotype of these alleles is similar to the null-phenotype of the
rh232 deletion. This screen also produced several mutations that exhibit hypomorphic and temperature-sensitive defects in canal formation.
In addition to our allelic analysis, we are currently investigating the effects of
cdc-42 on canal formation and possible interactions between
cdc-42 and
exc-5, and between
exc-5 and
exc-9 (see abstract by X. Tong et al.). Preliminary data indicate that
exc-5 is necessary for both extension and maintenance of the canals.
Finally, in order to identify additional genetic interactions with
exc-5, we are developing a method to use RNAi solely in the excretory canals, in order to look at the effects of a canal-specific knockout of
cdc-42 and other genes.