Seamless single‐celled tubules form the excretory canals of the nematode Caenorhabditis elegans. These canals are narrow and extend over the whole length of the worm, and are an attractive model to understand how biological tubes maintain diameter and shape. In C. elegans, loss‐of‐function mutations in any of a set of exc genes cause the excretory canal lumen to swell into large fluid‐filled cysts. The exc‐2 mutant worm shows one the most severe phenotypes among the exc genes. In order to identify the exc‐2 gene, whole‐genome sequencing was performed on exc‐2 mutant allele rh90. The results suggested a frameshift mutation in the intermediate filament gene ifa-4. Knockdown of ifa-4 gene mimics the Exc-2 cystic-canal phenotype, and the strain RB1483 (∆ifa-4(ok1734)) showed a similar cystic phenotype. Unexpectedly, injection of the wild‐type ifa‐4 gene did not rescue the exc‐2 mutant; worms and mutants of the two genes genetically complemented each other, which surprisingly confirmed that ifa‐4 and exc‐2 are not the same gene. Next, whole‐genome sequencing was performed on three additional exc‐2 alleles (rh105, rh209, and rh247), which provided greater resolution and confidence in identifying the gene. Results showed that the non‐essential cytosolic intermediate filament gene ifc‐2 has mutations in all of the four alleles that were sequenced. The ifc‐2 gene has nonsense mutations in the alleles rh209 and rh247, while alleles rh90 and rh105 have deletions among coding regions of the gene. ifc‐2 is a relatively large gene (~13kb), with four isoforms: C and D take up the first and second halves of the gene, respectively, while isoforms A and B each cover almost all of the exons. Knocking down any combination of isoforms containing both A and B showed a phenotype similar to Exc‐2 cysts, while knockdown of isoform A by itself showed a much milder phenotype. We have rescued the exc‐2 gene by injecting PCR product contains full length of the ifc‐2 gene into exc‐2 mutant worms.
EXC‐2/IFC‐2 and IFA‐4 join the previously discovered role (Labouesse lab) of IFB‐1 in regulating the diameter of a model narrow seamless tube. Intermediate filaments could play important roles in other narrow seamless tubes as well. We are now investigating the time and position of expression of ifa-4 and ifc-2 isoforms to determine the interactions that form the distinctive shape of the excretory canals.