Cellular and genetic analysis of pharyngeal morphogenesis in Caenorhabditis elegans

During development of the pharynx in the nematode Caenorhabiditis elegans, pharyngeal precursor cells cluster together to form a primordium that subsequently undergoes a program of differentiation and morphogenesis to form a linear digestive tube. I combined three experimental approaches to study how the pharyngeal primordium becomes a tube at the cellular and genetic levels. First, I studied the behaviors of pharyngeal and nonpharyngeal cells required for tube formation. Second, I screened a deletion library for genetic loci required for pharyngeal morphogenesis. Lastly, I performed a genetic screen for pharyngeal tubulogenesis mutants. I used time-lapse videomicroscopy and GFP reporter constructs to follow the behavior of the pharyngeal precursors during pharyngeal extension, the process whereby the pharyngeal primordium attaches to the buccal cavity, or mouth of the worm. Pharyngeal extension can be loosely divided into three stages (i) reorganization of cellular polarity within pharyngeal cells (specifically the pharyngeal epithelial precursors), (ii) formation of an epithelium that mechanically couples the pharyngeal cells to arcade cells in the nascent buccal cavity, and (iii) an apparent contraction that shifts the buccal cavity posteriorly and the pharynx anteriorly. Using Nomarski DIC microscopy and immunostaining, we have classified sixteen deficiencies into two groups that affect pharyngeal development. The first group of deficiencies gives rise to a pharynx primordium that fails to differentiate. The second group generates terminal embryos with specific defects in pharyngeal morphogenesis. One subset of this group produces small and/or unattached pharynges. A second subset of this group of deficiencies produces a pharyngeal lumen that branches at a reproducible location along the pharyngeal axis. I performed a genetic screen for mutants that arrest with a p&barbelow;harynx un attached (Pun) phenotype at the L1 larval stage. I recovered 13 mutants from an ENU mutagenesis of 2838 haploid genomes. One mutant that I found in the screen is an allele of the mitotic kinesin like protein 1 (MKLP1) ortholog in C. elegans, ZEN-4. MKLP1 orthologs have been implicated in cytokinesis. I have shown that the pharyngeal morphogenesis defect observed in our newly discovered mutant allele of zen-4 is not due to cytokinesis defects, but rather it appears to specifically affect epithelialization.