The nematode excretory canals provide a simple model of tubulogenesis in a single cell. Our laboratory studies the role of exc genes in regulating the lumenal diameter of these canals. The Exc phenotype causes the formation of fluid-filled cysts. Known exc genes include cytoskeletal proteins, a guanine exchange factor, and the RNA-binding protein ELAV. We have recently narrowed the position on LGX of three other exc genes preparatory to their cloning.
exc-1 mutants form large cysts predominantly at the tips of the canals.
exc-1 maps between
eat-20 and
lin-15 on LGX. Cosmid C02C6 appears to rescue the Exc-1 mutant phenotype. However, RNAi of the three predicted genes on this cosmid did not phenocopy the mutation. We are injecting fragments of C02C6 to narrow the
exc-1 position. Mutants in
exc-2 show very large cysts at the cell body. This gene maps between
dpy-8 and
mec-2. Microinjection eliminated most of the cosmids here as possible sites of
exc-2. RNAi of C03F11.1, a K channel, phenocopies the
exc-2 mutation. While microinjection of C03F11 into wild-type worms causes lethality, we found that blasting worms with C03F11.1 via the Biolistic PDS-1000, which may place fewer copies of a gene into animals than does microinjection, appears to rescue the mutant phenotype. Initial sequencing of the C03F11.1 coding region of the
rh90 allele shows a possible nonsense mutation in the fifth exon. Interestingly,
exc-4 mutants exhibit similar large cysts, and the gene also encodes an ion channel (O. Hobert, personal communication).
exc-3 mutants exhibit highly variable amounts of lumenal swelling in meandering canals. This gene maps between
him-4 and
unc-115. Microinjection of 12 of the 14 cosmids here failed to rescue the mutant phenotype. RNAi injection of the eight predicted genes on the two remaining cosmids was performed. Injection of dsRNA of either one of 2 genes, F42G10.1 and F19C6.4, resulted in phenotypes similar to those of
exc-3 mutants. RNAi of F42G10.1 generally resulted in more severe canal phenotypes. These genes appear to have arisen through gene duplication; both are predicted to encode peptidases. We are currently attempting mutant rescue through microinjection of these genes, and sequencing the predicted coding region of mutant alleles of these genes.