Organs such as the kidney, lung and vasculature are systems of tubes providing an essential function by transporting gases or liquids. Tube formation is a critical developmental problem nature solves in three ways. The neural tube forms by wrapping, in which a tube branches off an epithelial sheet. The Drosphila proximal trachea forms by branching morphogenesis, whereby tubes extend from previously made epithelial tubes. Finally the proximal tubules of the kidney and C. elegans pharynx form by de novo epithelialization. Tubulogenesis by de novo epithelialization has been less well studied than the other two mechanisms. Surprisingly, many molecules important for epithelialization and/or polarity in other organisms (e.g. cadherin, catenins, discs large, spectrin, integrin,
par-1) are not required for tubulogenesis of the pharynx, as mutations in these genes do not generate a Pun (pharynx unattached) phenotype. To identify genes required for pharyngeal tube formation, we performed a genetic screen for arrested Pun mutants. From 3,000 haploid genomes we identified 13 Pun alleles that fall into two classes. First, during wildtype morphogenesis, pharyngeal epithelial cells reorient their apicobasal polarity to extend the pharyngeal lumen anteriorly. Five mutants disrupt this process. Second, in wildtype embryos, arcade cells form an epithelium de novo that links the buccal cavity to the pharynx. Eight mutants disrupt this process. We have characterized and cloned two loci, one from each class. The first locus,
px48, codes for a novel, conserved protein with sequence similarity to proteins that regulate polarity. In
px48 mutants, pharyngeal cells fail to reorganize apicobasal polarity during the first stage of pharyngeal morphogenesis. The second locus is defined by
px47, which is a weak allele of
zen-4, a mitotic-kinesin-like protein (MKLP1). Previous studies demonstrated that MKLP1 bundles midzone microtubules during mitosis. Our studies have revealed a new role for
zen-4/MKLP1, to form the apical surface during epithelialization of the arcade cells. Markers of the apical surface and adherens junctions are lost in
zen-4 (
px47) mutants and instead some of these markers accumulate in the cytoplasm. We speculate that
zen-4 is required for microtubule organization during epithelialization, similar to its role during mitosis, and that this function is critical to localize components to the apical surface and to adherens junctions. This model may bear on kidney tubule formation since targeting of apical components may play a key role in epithelialization of MDCK cells. Loss of apical targeting may lead to human diseases such as intestinal Microvillus Inclusion Disease (Ameen and Salas, 2000).