The general objective of this proposal is the elucidation of the molecules and mechanisms of signaling specificity. Achieving the appropriate cellular response to a signal is vital to the well- being of an organism, as the wrong response can result in developmental defects or cancer. The MAP kinase signal cascade functions to promote differentiation in multiple tissues in C. elegans, including the vulva and the germline. A mechanism for determining signalling specificity in the vulva has already been determined, and involves activation of a tisssue-specific transcription factor, LIN-31. This proposal focuses on investigating the mechanism of signal specificity in the germline by identifying molecules acting downstream of MAP kinase in that tissue, and comparing them to those utilized in the vulva. Briefly, 1) a genetic screen design to isolate downstream targets of MAP kinase is being performed; 2) DNA microarrays will be used to pinpoint transcriptional changes that occur in the germline upon MAP kinase signaling; and 3) the targets identified in 1 and 2 will be molecularly cloned, and analyzed for expression pattern and a possible role as substrates of MAP kinase phosphorylation. The genetic screen is set up as follows: a mutation in map kinase, mpk-1(ga111), has a phenotype specific to the germline and is temperature-sensitive. At 20 degrees C, mpk-1(ga111) acts like wild type and the animals are fertile; at 25 degrees C, mpk- 1(ga111) acts like mutant, and the germ nuclei arrest in pachytene and the animal is sterile. By screening for suppressors of sterility at 25 degrees C, downstream effectors of MAP kinase will be isolated. The effectors will be analyzed using standard genetic procedures. DNA microarrays containing a portion of all the C. elegans cDNAs will be probed with fluorescently labelled cDNA extracted from the mpk-1(ga111) strain grown at 20 degrees C and at 25 degrees. This experiment identifies genes that are differentially regulated between the two strains. Controls will be included for temperature differences and germline formation, to aid in identification of the most relevant targets. Soon, microarrays containing every C. elegans cDNA will be made, and used in the above experiment. Genes which appear in both the suppressor screen and the microarray analysis as potential targets of MAP kinase will be given top priority in further studies. These genes will be cloned and their expression patterns determined by both RNA in situ analysis and antibody studies. These genes will be tested as possible MAP kinase substrates. Identification of the roles of these genes in MAP kinase signalling in the germline will determine the mechanism by which specificity is achieved in this tissue. The molecules and mechanisms used to generate the tissue-specific outcome of meiotic progression in the germline will be compared with those used in the vulva to increase the understanding of the generality of such mechanisms.
