The
let-60 ras/
mek-2/mpk-1 signalling cascade is used at multiple times and places during development in C. elegans. For instance, this cascade acts downstream of the receptor tyrosine kinase
let-23 in vulval precursor cells to promote vulval cell fate, in part through phosphorylation of a tissue-specific transcription factor, lin-31WH, by
mpk-1 (1). Another tissue that utilizes the ras/mek/mpk signalling cascade is the germline. A ras-mediated signal is required for progression of germ cells through pachytene into diakinesis of meiosis I. Specifically, mutations in
let-60 ras,
mek-2, or
mpk-1 cause germ cells to arrest during pachytene (2). The factors upstream and downstream of the ras/mek/mpk pathway in the germline differ from those in vulval development and remain unknown. To better understand how tissue-specific outcomes result from use of a common signalling pathway such as the ras/mek/mpk pathway, we intend to identify downstream targets of
mpk-1 in the germline of C. elegans using two approaches. First, we will use a traditional genetic method to identify downstream effectors of the ras/mek/mpk signalling cascade. A weak, loss-of-function allele of
mpk-1,
ga111, exhibits an apparent phenotype only in the germline, and not in other tissues that utilize
mpk-1. mpk- 1
(ga111) mutant animals display a temperature sensitive sterility. At 20?C, the animals are completely fertile; however, at 25?C, they are completely sterile, due to an arrest in pachytene by the germ cells. We will perform a genetic screen for suppressors of the 25?C sterility. A pilot screen has already been initiated, and multiple potential alleles have been indentified. Following complementation and mapping analysis, we will test each mutant for a phenotype in the germline and other tissues in which
mpk-1 acts, independently of its function as a suppressor in the germline. We are currently most interested in studying mutants that function primarily in the germline as potential tissue-specific targets of
mpk-1. Second, we will employ the new microarray technology to investigate changes in gene expression in the germline under various conditions. For instance, to understand how the ras/mek/mpk signalling cascade functions in the germline to alter gene expression and allow progression through pachytene, we will again utilize the
mpk-1(
ga111) allele described above. We will compare RNA isolated from young adult worms grown at 20?C with RNA from worms grown at 25?C for changes in gene expression that are related to map kinase signalling. This particular system allows for identification of both general and tissue- specific alterations that might occur. Several control experiments can be performed to help identify targets that are real and biologically significant. N2 worms grown at 20?C and 25?C will be used to control for temperature differences. Additionally,
glp-4(bn-2) worms, which exhibit a temperature sensitive defect in germline formation, can be utilized to identify targets that are specific to the germline. Certain mutations in
gld-1 that cause germ cells to remain in a mitotic state and never enter meiosis can also be utilized to identify meiosis- specific transcripts. Any genes whose expression patterns fulfill these criteria will be studied further. By combining a genetic screen with a method for intergration of gene expression patterns, we hope to better understand how tissue-specific outcomes emerge after use of a common signalling pathway. 1. Tan, P.B., Lackner, M. R., and Kim, S. K. Cell 93: 569-580. 1998. 2. Church, D. L., Guan, K.-L., and Lambie, E. J. Development 121: 2525- 2535. 1995.