How are cells regulated to differentiate as one cell type rather than another? How are groups of cells coordinatedly regulated to adopt the same fate? We propose to approach these fundamental problems of metazoan development by analyzing the control of a major fate decision in the nematode Caenorhabditis elegans: determination of sexual fate. Over the past few years, we have laid the groundwork for experiments proposed. First, we have analyzed the tra-2 gene and its RNA products at the molecular level. This work suggests that tra-2 plays a pivotal role in worm sex determination by mediating cell communication and also by regulating transcriptional regulators of cell fate. Second, we have analyzed the fog-1 gene genetically and found that fog-1 is a terminal regulator of sexual fate in the germ line. During the next five years, we propose to investigate the mechanisms by which tra-2 promotes female development throughout the animal and by which fog-1 specifies spermatogenesis in the germ line. First, we will delineate the roles of the individual tra-2 products in sex determination. To this end, we will analyze the phenotypes of transgenic animals that express one of each of the three tra-2 RNAs. Second, we will raise antibodies to the tra-2 proteins, elucidate their cellular locations, and characterize them throughout development in each sex. Third, we will test the idea that the predicted membrane protein pTra2A may serve as a receptor. To this end, we will assay the autonomy of tra-2, either using tissue-specific promoters that drive tra-2 expression in particular cells of transgenic animals or by classic mosaic analysis. Fourth, we will explore the role of maternal tra-2 in early embryonic sex determination, using a combination of genetic maternal effect tests and RNA injection experiments. Fifth, we will identify domains in the tra-2 protein that mediate her-1 control and that promote female development by regulating one or more fem genes. Sixth, we will investigate translational regulation of tra- 2 RNA by its 3'UTR. Seventh, we will clone the fog-1 gene, sequence fog-1 cDNAs, and identify and characterize the fog-1 transcript. These studies will shed light on the mechanisms by which tra-2 and fog-1 are regulated and by which these two genes fulfill their key roles in determination of sexual fate. The health relatedness of this work derives from its contribution to an understanding of fundamental controls of cell fate, including cell communication and transcriptional and translational regulation. These controls are common to all metazoans, including man.
