ras/map kinase signal transduction pathway in meiotic prophase progression

Min-Ho Lee1, Eric Lambie2, Mitsue Ohmachi1, Ross Francis1, Tim Schedl1
1
Dept. of Genetics, Washington Univ. School of Medicine
2
Dept of Biology, Dartmouth College

A RAS/MAP kinase signal transduction pathway in male and female germ cells is essential for meiotic prophase progression. Strong loss-of-function (lf) mutations in let-60 RAS, lin-45 RAF, mek-2 MAPKK, and mpk-1 MAPK result in germ cells arrested in pachytene. This suggests that the RAS/MAP kinase cascade transduces a signal necessary for the progression through pachytene and/or for the transition from pachytene to diplotene/diakinesis (Church et al., 1994). To further understand germline RAS/MAP kinase signaling, we used an antibody that specifically recognizes only the doubly phosphorylated active form of MPK-1 in western analysis as well as in germline staining of wild type, lf and gain-of-function (gf) mutants in this pathway.

MPK-1 has two isoforms, 55 KD and 45 KD; 55 KD isoform has 68 amino acids more at the N-terminus (Wu and Han, 1994; Lackner and Kim, 1998) and is expressed only in the germline whereas 45 KD isoform is expressed in the germline and in the soma. In strong lf mutants of let-60 RAS and mek-2 MAPKK, the doubly phosphorylated active MPK-1 is not detected or severely reduced. In wild type hermaphrodite germlines, MPK-1 is doubly phosphorylated and becomes active in two regions: in pachytene stage germ cells and in a few most proximal oocytes. The activation of MPK-1 first appears in the middle of pachytene region and continues till the end of pachytene region, which strongly supports the genetic data. Phosphorylated MPK-1 then decreases rapidly as germ cells progress from pachytene to diplotene/diakinesis. The reactivation of MPK-1 in the few most proximal oocytes suggests this pathway may also function in a late stage of oogenesis. In fact, reactivation of MPK-1 in oocytes is abolished in feminized germlines where oocytes are arrested in diakinesis. The sperm dependent signal for maturation/ovulation (McCarter et al., 1999) may thus induce MPK-1 activation.

To distinguish whether activation of MPK-1 is required for progression through pachytene or for the transition from pachytene to diplotene/diakinesis, we employed let-60 RAS gf mutants, which display earlier activation of MPK-1 in pachytene stage germ cells. The early activation of MPK-1 in let-60 RAS gf mutants is not sufficient to drive germ cells into diplotene earlier than in wild type. This suggests that RAS/MAP kinase signaling may be necessary for progression of germ cells from an early to a late stage of pachytene while the transition from pachytene to diplotene is controlled by a separate process. Alternatively, the transition from pachytene to diplotene may require not only RAS/MAP kinase signaling but the execution of additional events.

2000 Midwest Worm Meeting abstract 85

let-21, a putative RhoGEF gene, is required for germline development

M. Ohmachi1, P. Kuwabara2, E. Lambie3, A. Godbey1, D. Jackson1, R. Francis1, T. Schedl1
1
Genetics, Washington Univ. School of Medicine, St. Louis, MO 63110
2
MRC Laboratory of Molecular Biology
3
Biology, Dartmouth College

The progression of germ cells through pachytene and the transition to diplotene/diakinesis are important step in meiotic development. We have identified a set of mutants that have pacytene arrested germ cells with clumped nuclei, similar to what is observed for mutants in the RAS/MAP Kinase pathway. Two mutants, with no obvious somatic defects, were found to be allelic to let-21(e1778), originally identified by E. Hedgecock. e1778 mutants also display a weak Unc and incompletely penetrant P-Vul phenotype. Two probable null deletion alleles, dx85 and dx86, show a strong Sterile-Unc phenotype with many animals arrested as larvae.

Sequence analysis shows that let-21 is the worm ortholog of mammalian proto-oncogene ect2 and Drosophila pebble, which both function in cytokinesis. These gene products have two N-terminal BRCT domains, which are thought to be protein-protein interaction modules. They have a centrally located DH (Dbl Homology) domain and its associate PH (Pleckstrin Homology) domain that function as guanine nucleotide exchange factors for Rho-family GTPases. e1778 has in-frame deletion that delete BRCT1 domain completely. oz204 has two missense mutations. One is in the BRCT2 domain and the other is in the DH domain. oz93 has splice site mutation of exon7 that contains C-terminal part of BRCT2 and N-terminal part of DH domain. These suggest that the BRCT domains as well as the DH domain are important for let-21 function.

Phenotypic analysis of mutants and RNAi embryos indicate a cytokinesis defect, consistent with studies of ect2 and pebble. Current work is directed at understanding the germline phenotype and whether the pachytene arrest is a direct or indirect effect of loss of let-21 function.

2000 Midwest Worm Meeting abstract 40

syt-1, but not teg-1, is in the GLD-1 pathway for entry into meiosis.

Dave Hansen, Laura Wilson-Berry, David Schneider, Diana Jackson, Tim Schedl

Dept. of Genetics, Washington University School of Med., St. Louis, MO, 63110

The germline is a polar tissue with cells in the distal end proliferating in a mitotic cell cycle, while more proximal cells enter meiosis eventually differentiating as sperm or oocytes. The distal mitotic stem cell population is maintained by a signal from the somatic distal tip cell (DTC) that activates the GLP-1/Notch signaling pathway. When this signal is removed, germs cells enter meiosis prematurely and the stem cell population is lost. DTC/GLP-1 signaling serves to down regulate two genetic pathways, one containing GLD-1, and the other containing GLD-2 (Kadyk and Kimble, 1998). Each of these pathways is able to promote meiotic entry as single mutants in gld-1 or gld-2 are able to enter meiosis normally (Francis et al., 1995, Kadyk and Kimble, 1998). It is only when both pathways are not functioning that germ cells fail to enter meiosis. The result is that gld-2 gld-1 double mutants form a germline tumor of mitotic cells, similar the tumor caused by a constitutive activate form of GLP-1 (Kadyk and Kimble, 1998, Berry et al., 1997).

We have undertaken two screens to identify genes that are involved in regulating entry into meiotic prophase. In the first we screened for mutations that enhance a weak gain-of-function allele of glp-1. These mutations correspond to three genes, teg-1, teg-2 and teg-4. We are currently attempting to clone teg-1 and have narrowed down its location to a ~30 kbp region to the left of dpy-18 on chromosome III.

The second screen was designed to identify genes that specifically function in the GLD-1 pathway for entry into meiosis. Since the GLD-1 and GLD-2 pathways are each sufficient for entry into meiosis, we screened for mutations that are synthetic tumorous with a loss-of-function allele of gld-2. To date, we have identified mutations corresponding to at least five genes. As expected, one of these is gld-1. We also isolated another allele of teg-1. The remaining genes are termed syt as they are synthetic tumorous with gld-2. Since this second screen was designed to identify genes that function in the GLD-1 pathway, we performed genetic tests to confirm their position. Our criteria for determining if these genes function in the GLD-1 pathway are the following: 1) The mutation must be synthetic tumorous with gld-2(lf). 2) The mutation is not tumorous on its own. 3) The mutation is not synthetic tumorous with gld-1(lf). 4) The synthetic tumorous phenotype with gld-2(lf) must be epistatic to glp-1(lf). While teg-1 meets the first three criteria, gld-2; glp-1 teg-1 animals do not have tumorous germlines but rather show a Glp phenotype. This suggests that teg-1 is not in the GLD-1 pathway, but rather may function further upstream.

Another gene that was identified in the second screen, syt-1, does meet the four criteria for functioning in the GLD-1 pathway for entry into meiosis. In particular, gld-2; syt-1; glp-1 animals have tumorous germlines. We have mapped syt-1 to linkage group II near unc-4. We are also in the process of mapping the other mutations that were identified in the second screen.