The primary objective of the proposed study is to understand how master regulators, such as the homeotic genes, specify cell fate. Although homeotic genes carry out related functions in animals as diverse as flies and man, little is understood about how the homeotic genes control organ formation in any system. This work focuses on how the Drosophila homeotic gene Sex combs reduced (Scr) controls a relatively simple, well-defined event, the morphogenesis of the larval salivary gland. Scr is absolutely required for salivary gland formation and is the only known inducer of this tissue. In the absence of Scr function, there are no salivary glands, and when Scr protein is expressed in new places, additional salivary glands form in new places. Scr, like other homeotic genes, encodes a DNA-binding transcription factor and thus must control salivary gland formation through the transcriptional regulation of downstream genes. Identification of target genes directly regulated by Scr in the salivary gland: Salivary gland genes directly controlled by Scr should be expressed in the salivary gland primordia early when Scr is present, should require Scr for their expression in this tissue, and should directly associate with the Scr protein in vivo. Two distinct but convergent approaches have identified such candidate Scr target genes. Candidate genes were first identified by their early expression in the salivary gland and their dependence on Scr function for this expression. Secondly, the genes also may near binding sites for the Scr protein on the polytene chromosomes of the larval salivary gland. Using the in vivo chromosome binding assay in conjunction with in vitro binding studies, it will be determined whether the nearby binding sites on the chromosomes correspond to the salivary gland expressed genes and whether these binding sites mediate Scr regulation. Genes identified as direct Scr targets through these studies will be further characterized to determine their role in salivary gland formation. Analysis of target gene function in salivary gland morphogenesis: Scr and genes setting the dorsal-ventral limits on salivary gland formation probably directly determine the number of cells forming salivary glands. Genes downstream Scr may be involved in the early events of salivary gland differentiation such as nuclear movement, cell shape changes, invagination, initiation of polytenization and the transcriptional control of genes expressed later in salivary gland morphogenesis when Scr protein is no longer present. Genes identified as direct Scr targets will be mutagenized and their effects on each of these processes examined. Antibodies to the target gene products will be used to determine where in the cell the target gene product is active and to study genetic and cellular interactions among the salivary gland genes. It will be exciting to determine whether transcriptional hierarchies are related to functional pathways in the salivary gland.
