Organs such as the kidney, lung and vasculature are systems of tubes that provide an essential function by transporting gases or liquids. Epithelial tubes can arise by remodeling of pre-existing epithelia, as occurs during branching morphogenesis of the lung. Epithelial tubes are also constructed by groups of cells induced to form epithelia de novo. We are studying attachment of the C. elegans pharynx to the buccal cavity as a model for tube formation by de novo epithelialization. To identify genes required for pharyngeal tube formation, we performed a genetic screen for arrested Pun (pharynx unattached) mutants. From 3,000 haploid genomes we identified 13 Pun alleles in two classes. First, during wildtype morphogenesis, pharyngeal epithelial cells reorient their apicobasal polarity to extend the pharyngeal lumen towards the anterior. Five mutants disrupt this process. Second, in wildtype embryos, the arcade cells form an epithelium de novo linking the buccal cavity to the pharynx. Eight mutants disrupt this process. We have characterized a mutant from the second class,
px47 , and demonstrated it was a weak allele of the mitotic-kinesin-like protein
zen-4 1 . Previous studies demonstrated that ZEN-4 and its orthologues bundle midzone microtubules during mitosis and are required to complete cytokinesis 2,3 . Our studies have revealed a new, post-mitotic role for ZEN-4, to epithelialize the arcade cells. Markers of the apical domain and adherens junctions are lost from the cell surface in
zen-4(
px47) mutants and at least some of these proteins accumulate in the cytoplasm (e.g.
cdh-3::GFP and
ajm-1::GFP). Basolateral markers such as LET-413::GFP/Scribble and
unc-70 /spectrin are uniformly distributed around the cell cortex of
zen-4(
px47) mutants. Normally, ZEN-4 associates with the RhoGAP CYK-4 during cytokinesis 4,5 and our data demonstrate CYK-4 is required during pharyngeal attachment as well. These findings suggest that ZEN-4 and CYK-4 are required to organize the microtubule and actin cytoskeleton and this function is critical to polarize the arcade cells during epithelialization. This model may bear on other examples of tube formation by epithelialization such as kidney tubule formation. Our current goal is to elucidate the role of ZEN-4 and CYK-4 during epithelium formation and identify additional components in the pathway for pharyngeal attachment. 1. Portereiko MF, Saam J, Mango SE. ZEN-4/MKLP1 Is Required to Polarize the Foregut Epithelium. Curr Biol. 2004 Jun 8;14(11):932-41 2. Severson AF, Hamill DR, Carter JC, Schumacher J, Bowerman B. The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis. Curr Biol. 2000 Oct 5;10(19):1162-71. 3. Raich WB, Moran AN, Rothman JH, Hardin J. Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesin-like protein ZEN-4. Mol Biol Cell. 1998 Aug;9(8): 2037-49. 4. Mishima M, Kaitna S, Glotzer M.Central spindle assembly and cytokinesis require a kinesin-like protein/RhoGAP complex with microtubule bundling activity. Dev Cell. 2002 Jan;2(1): 41-54. 5. Jantsch-Plunger V, Gonczy P, Romano A, Schnabel H, Hamill D, Schnabel R, Hyman AA, Glotzer M. CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis. J Cell Biol. 2000 Jun 26;149(7): 1391-404.