The excretory canal cell of C. elegans is a single-cell tubular organ. It provides a simple model for understanding the formation and regulation of tubular shape and diameter. The EXC proteins regulate apical (lumenal) diameter. Mutations in exc genes alter the diameter of the excretory canal lumen.
exc-9 mutants have wide, meandering excretory canals. While the normal canal is narrow and runs all the way to the tail, the posterior canal of
exc-9 mutants generally ends around the position of the vulva. In addition to canal defect,
exc-9 worms also show a non-penetrant tailspike defect. By SNP mapping, I narrowed the location of
exc-9 to a small region of 13 cosmids on LG IV. Injection of cosmid F20D12 rescued both canal and tail defects of the
exc-9(
n2669) mutant. RNAi of F20D12.5 showed a similar phenotype to that of allele
n2669. A 3 kb PCR fragment that includes the F20D12.5 coding sequence and 2 kb of upstream sequence rescued
exc-9, too. Finally, sequencing of the
n2669 allele revealed a G to A substitution that changes a coding codon to a stop codon. The truncated protein has 55 amino acids instead of 85, and disrupts a conserved domain.
exc-9 encodes a small protein containing a single LIM domain. Since there is no homoeodomain box in this protein, we do not believe that EXC-9 is a transcription factor, but rather functions to bind other proteins. EXC-9 shows closest homology to CRIP (Cysteine-Rich Intestinal Protein), first identified as a protein of the mammalian intestinal immune system. Others have found that CRIP is upregulated in regenerating leech neurons, and that overexpression of CRIP alters mouse cytokine expression. I have integrated F20D12.5 promoter into a GFP vector to see the expression pattern of this gene. It is expressed in several tissues including the excretory canals, the tailspike, the distal tip cells, several neurons, and it shows strong expression in the uterine seam cell. While low levels of expression of a translational GFP fusion partially rescue the cystic canal phenotype, high levels of expression appear to disrupt canal and tailspike shape and length, even when injected into N2 worms. Overexpression of a promoter fusion construct also disrupts canal shape in N2. We are currently testing antibodies to EXC-9 to use for co-immunoprecipitations in order to discover binding partners of this protein, and learn how this pathway functions.