Very little is known of the means by which genes control the three- dimensional structure of the organs of an animal. In most screens for mutants affecting Drosophila melanogaster development, internal tissues have been ignored in favor of external tissues because they are not easily visualized. The objective of this proposal is to initiate a combined morphological, genetic, and biochemical study of the development of the Drosophila embryonic tracheal system, an elaborate but regular branched tubular structure which is the animal's respiratory organ. Formation of the tracheal system involves the invagination of a defined set of epithelial cells, the tracheal precursor cells, in each thoracic and abdominal segment to form blind-ended tubes; branching of the blind end and branch outgrowth; fusion of branches from adjacent segments to form a continuous tube; and further branching and tissue penetration by these secondary branches. The long-term goals of this research are to identify the gene products that control these morphogenetic processes, focusing on the biochemical mechanisms of branch formation and the outgrowth and fusion of the branches. The specific aims of this proposal are: 1. To characterize the normal growth and development of the embryonic tracheal system by fluorescence microscopy and high resolution confocal microscopy using a monoclonal antibody that stains trachea throughout embryogenesis. 2. To elucidate dynamic aspects of tracheal tube formation, branching, and branch outgrowth and fusion by real-time imaging of tracheal development after photo-activation of a caged fluorescein molecule in tracheal precursor cells. 3. To isolate genes involved in tracheal development, by screening for recessive mutations that alter tracheal anatomy and by screening lacZ enhancer-detector strains to identify genes expressed in or near the developing trachea. 4. To initiate the molecular cloning and molecular genetic characterization of one or more of the identified genes involved in tracheal development. 5. To purify tracheal precursor cells by a new approach called whole animal cell sorting to establish an in vitro system for tracheal development.