The formation and regulation of tubular shape and diameter is important for the development and function of many tissues in organisms. The excretory canal cell of C. elegans is a single-cell tubular organ, and provides a simple model for understanding aspects of apical and basal epithelial cell proteins on regulation of tubule diameter. The EXC proteins regulate apical (lumenal) diameter; mutations in exc genes alter the diameter of the excretory canal lumen. The Exc phenotype results from mutations in genes including ion channels, a GDP-GTP exchange factor, and the cytoskeletal protein H-spectrin. Mutants of
exc-9 have wide, meandering excretory canals with some septate cysts, rather than maintain the normal tubular shape. We are continuing to narrow the position of the
exc-9 gene preparatory to cloning it.
exc-9 maps on LGIV near
unc-24. We are using SNP mapping to narrow the position of this gene prior to attempting rescue by microinjection. Using
bli-6 as a marker on the left, we have narrowed down the region to 18 cosmids (C06A6-D2096). We are further narrowing down the limits of this region through the use of
fem-3 as a marker. After that, we plan to microinject cosmids for rescue, and RNAi of likely genes. We have introduced a
vha-1-promoter-driven GFP (graciously supplied by T. Oka & M. Futai) into
exc-9 mutants via genetic cross.
vha-1 is a member of the Vacuolar H+-ATPase gene class. An unstable array of
vha-1::GFP is strongly expressed in the H-shaped excretory cell cytoplasm (Ota et al., JBC, '01). In addition to the excretory cell, the fusion protein was also detected in cells of the rectum and a pair of cells with parallel orientation posterior to the anus. We introduced this GFP construct into our worms via GeneGun, and TMP was used to incorporate the array into the chromosome. We have mapped this insertion to LGI, and are now using this construct to isolate new
exc-9 alleles, screen for new <i?exc mutants, and map
exc-9.