Tubes are required in organisms for a variety of functions, including ingestion and excretion, gas exchange, and transportation of nutrients. Many tubes are multicellular, but the simplest are unicellular. For a single cell to form a tube requires a few achievements: adopting the correct fate, correct positioning and overall morphology of the cell, formation and elongation of a hollow interior lumen, and connection of the ends of the tube to other cells. To address the most basic questions regarding the formation and maintenance of tubes, we study the C. elegans excretory system. This ''primitive renal system'' includes three tandem unicellular tubes: the excretory canal cell, the duct cell, and the pore cell. Fluid is passed from the canal cell to the duct cell and then pore cell through a continuous lumen. To better understand the development of the excretory system, we are investigating the role of let-4, which causes an excretory system defect. The previously uncloned mutant let-4(mn105) is a recessive zygotic-effect mutation with a >99% penetrant lethal excretory system defect. let-4 mutants begin to accumulate fluid in the canal cell just before hatching, and die as fluid-filled L1s. We have cloned let-4, and found that it encodes a predicted transmembrane protein with an extracellular leucine-rich repeat(LRR) domain and a conserved intracellular domain. We generated transcriptional and translational reporters to determine where let-4 is expressed. let-4 is expressed in the canal cell, duct cell, and pore cell, as well as in epithelial cells of the vulva, rectum, and hypodermis. A functional LET-4::GFP fusion protein localizes to the apical side of the hypodermal, vulval and rectal cells. The protein is present in the canal cell, duct cell, and pore cell, but apical localization hasn''t yet been confirmed in these cells. We hypothesize that let-4 mutants have a defect in excretory system lumen connectivity. Our phenotypic characterization so far suggests that all three unicellular tubes of the excretory system are present and properly positioned; however, fluid does not appear to pass from the canal cell through the duct and pore. This phenotype is similar to that seen in lpr-1 mutants (see abstract from Stone, Hall and Sundaram). To visualize the lumen, we are performing transmission electron microscopy analysis in collaboration with David Hall. We are also performing immunohistochemistry to label the lumen of the duct cell and pore cell in let-4 mutants, and to test for possible mislocalization of apical cytoskeletal factors.