Cloning and characterization of exc-9, a Caenorhabditis elegans CRIP homologue that regulates tubular structure

Abstract

Forming and maintaining tubular structure is fundamental to organismal development. The excretory canal cell of C.elegans forms a single-cell epithelial tubule, which provides a simple model for tubular structure study. The EXC proteins regulate maintenance of the apical (lumenal) cytoskeleton of the excretory canal. Loss of exc gene function allows formation of fluid-filled cysts in the excretory canal. exc-9 mutants exhibit short and cystic canals compared to wild-type worms. exc-9 mutants also exhibit tail defects in hermaphrodites and ray defects in male. By SNP mapping, cosmid rescue, and RNAi experiments, we proved that F20D12.5 encodes exc-9. EXC-9 is a homologue of the mammalian intestinal LIM-domain protein CRIP. exc-9 is highly expressed in the canal and tailspike, it is also expressed in some other cells, including UTSE, DTCs, and ALM neurons. Promoter regions important for exc-9 expression were studied. It was found that EXC-9 functions cell-autonomously and the free N-terminus of the protein is required for unextended canal phenotype. Overexpression of exc-9 constructs in an N2 background sometimes causes an "unextended canal" phenotype, in which the canal forms a large cell body filled with lumen with proper diameter, but has no canals along the length of the animal. Since canal extension is sensitive to expression levels of exc-9, injection of the construct also caused unextended canal phenotype in exc-9 mutants. Similar unextended canal phenotype was also found in animals showing high levels of exc-5 expression. I used the unextended canal phenotype to examine epistasis of EXC-9 function with that of other EXC proteins. EXC-9 appears to function upstream of EXC-5 to regulate cytoskeletal formation at the apical surface (possibly via CDC-42); and EXC-9 in turn may depend upon EXC-2 and EXC-4 function. A second well conserved CRIP homologue B0496.7 is highly expressed in multiple valves of C.elegans. It functions similarly to EXC-9 when ectopically expressed in the excretory canal. Expression of the mouse homologue CRIP in the canal failed to rescue exc-9 mutants, but with some modifications, mouse CRIP can function similar as EXC-9. Preliminary data of cloning of exc-2 is reported in this work too.