The excretory canal cell, which is required for osmoregulation, is a simple model to study tubulogenesis. It extends long processes first dorsally, then anterior and posteriorly, each with an intra-cellular lumen. Previous genetic analysis revealed that canal outgrowth requires some genes that also mediate neuronal outgrowth1-3; lumen formation is driven by regulation of fluid and ion transport 4,5,6; and lumen maintenance depends on the apical actin cytoskeleton7,8.
To identify new conserved genes involved in tubulogenesis, we used feeding RNAi in a sensitized background to test 243 conserved kinases9, identifying 9 that caused excretory canal phenotypes. We have initially focused on the kinase PIG-1/MELK. Loss of
pig-1 causes shortening of anterior and posterior canal arms, a mild cystic phenotype and regions with multiple lumens, similar to phenotypes seen in the uncloned mutant
exc-610. We cloned
exc-6, and found that it encodes an ortholog of the human formin INF2, a kidney disease gene11. INF2 promotes actin polymerization, de-polymerization and microtubule stability12. We have evidence that residues that regulate actin dynamics are required for EXC-6 function. Unexpectedly, EXC-6 may not predominantly co-localize with apical actin, instead accumulating with structures resembling microtubules. Thus, EXC-6 may coordinate actin and microtubule cytoskeletons in the excretory canal. Disease-causing INF2 mutations are dominant and have been proposed to result in constitutive activity. Using rescue of
exc-6(0), we have found functional evidence supporting this model. We are currently investigating the functional relationship between
pig-1 and
exc-6 in canal development. Given the conservation of INF2 function suggested by our rescue experiment, such a relationship might have potential relevance for disease.
1) Stringham et al., 2002. 2) Katidou et al., 2012. 3) Marcus-Gueret et al., 2012. 4) Khan et al., 2013. 5) Kolotuev et al., 2013. 6) Berry et al., 2003. 7) Tong and Buechner, 2008. 8)Buechner, 2002. 9) Shaye and Greenwald, 2011. 10) Buechner et al. 1999. 11) Brown et al. 2010. 12) Gaillard et al., 2011.