The genetic basis of sex determination has been analyzed extensively in the nematode, which has two naturally occurring sexes, an XX hermaphrodite and an XO male. Most of the sexual fate decision genes have been identified and have been shown to function in a cascade of negative regulation. Sex determination requires post-transcriptional regulation of a key gene,
tra-2. Genetic studies have shown that
tra-2 promotes female development since loss-of-function mutations of
tra-2 transform XX animals into pseudomales that do not produce oocytes. TRA-2 is thought to be a large transmembrane protein, and is controlled by its ligand HER-1, by proteolytic cleavage of intracellular domain (ICD), by alternative splicing of mRNA, by nuclear export of mRNA and by translational regulation through 3'UTR-binding proteins. However, the cellular behavior of endogenous TRA-2 protein is still enigmatic as a high titer antibody had not been available.
To detect the endogenous TRA-2 protein directly, we recently established an antiserum to the 7th intracellular domain of TRA-2 (TRA-2 ICD). With this antibody, we found the nuclei of hermaphrodite intestine, a female somatic tissue, were clearly immunostained. We believe that the staining is specific to endogenous TRA-2 protein, as our
tra-2(RNAi) on
fem-3 (
e1996lf) greatly reduced the intensity of nuclear staining. Furthermore, we confirmed that nuclei of
tra-2(
e2020gf) were stained much stronger than that of wild-type, while there are essentially no nuclear signals in
tra-2(
e1095lf) or in wild-type males. Lum et al. (2000) reported that GFP-TRA-2c, overproduced from the heat shock promoter, localized predominantly to nuclei in some somatic cells, and caused completely somatic feminization of XO animals. Our results supporting the possibility that endogenous TRA-2 (ICD) and nuclear transcriptional regulator TRA-1 have the opportunity to interact in vivo. With this antibody, we are now trying to examine the TRA-2 distribution in C. elegans germline during the course of development. Our results will be discussed in the meeting.
We thank Dr. S. Mitani, Dr. K. Gengyo-Ando, Dr. T. Schedl, Dr. B.Grant and the Caenorhabditis Genetics Center (CGC) for providing the mutant strains. We also thank Dr. H. Sorimachi,Dr. I. Kawasaki, Drs. Ken and Miyuki Sato and many other people in C. elegans society for helpful suggestions and discussions.