Our studies began with the C. elegans orthologs of the cdc25 and cdc2 genes from Schizosaccharomyces pombe, two genes that drive cells from G2 to M-phase of the cell cycle. In all eukaryotes, the CDC2 (a.k.a. CDK1) protein is a serine/threonine kinase that universally regulates G2 to M-phase progression. Such progression is prevented by the phosphorylation of a threonine and tyrosine residue in the amino-terminus of CDC2, which renders it inactive. CDC25 functions as a dual specificity phosphatase that removes the phosphate groups from CDC2 and thus activates CDC2 during G2, driving cells into M-phase.
To assess the consequences on development when the expression of CDC2 or CDC25 is perturbed, we used RNA-mediated interference (RNAi). RNAi functionally "knocks out" the expression of C. elegans genes when animals are injected with double stranded RNA (dsRNA) specific to those genes. Progeny of injected animals are then analyzed for any defects in development caused by the deficiency of the targeted genes. Using this approach, we determined that the C. elegans versions of CDC2 and CDC25 are involved in the completion of oocyte meiosis.
C. elegans oocytes remain in prophase of meiosis I as they pass through the spermatheca where they are fertilized by sperm. After fertilization, the fertilized oocyte completes both meiotic divisions, extruding two polar bodies. The haploid oocyte pronucleus then migrates to join the haploid sperm pronucleus. The 1-cell embryo then undergoes its first mitosis. RNAi with a combination of three of the four known C. elegans cdc25 orthologs generates dead embryos. These embryos arrest at the 1-cell stage, during metaphase of meiosis I: the oocyte chromosomes organize on a metaphase plate, set up a meiotic spindle, and the sperm chromosomes remain condensed at the posterior of the embryo.
In collaboration with Dr. Diane Shakes and her students at the College of William and Mary, we have observed the same 1-cell arrest phenotype using RNAi with the C. elegans orthologs of a number of Anaphase Promoting Complex or cyclosome (APC/C) subunit genes (4; see Table below). The APC/C is a multi-subunit complex with E3 ubiquitin ligase activity that targets proteins for destruction during the metaphase to anaphase transition of the mitotic cell cycle. One target of the APC/C is securin, a protein that complexes with and inhibits separase, a protease that cleaves the cohesin complex that holds sister chromatids together at metaphase. When securin is ubiquitinated and subsequently destroyed by the 26S proteasome, separase is free to cleave the cohesin complex. Destruction of this complex allows the chromatids to segregate to opposite poles of the cell during anaphase and telophase. To avoid chromosome segregation defects, a cell cycle checkpoint exists at the metaphase to anaphase transition that negatively regulates the APC/C and thus prevents cell cycle progression until chromosomes properly align at the metaphase plate. The metaphase to anaphase transition has been shown to be tightly regulated during the mitotic cell cycle in other organisms, and our findings suggest that this phase of the cell cycle is also regulated during the meiotic cell cycle in C. elegans.
The mat mutants:
While RNAi is an excellent technique for determining the earliest function of a given gene, it does not allow for the study of such genes throughout development. For this reason, genetic mutants are more desirable for determining the multiple functions a gene may perform. Our RNAi observations led us to look for other genes involved in meiotic progression. In collaboration with Matt Wallenfang and Geraldine Seydoux (Johns Hopkins University School of Medicine), Danielle Hamill and Bruce Bowerman (University of Oregon), and Diane Shakes, we isolated temperature-sensitive (ts) mutants that arrested as 1-cell embryos when mothers were shifted to the non-permissive temperature of 25°C (6). Almost 40 such mutants were recovered in this screen; all but five were arrested in metaphase of meiosis I. The remaining five arrested as 1-cell embryos because of cytokinesis defects.
The meiotic arrest mutants break down into five complementation groups. Members from two groups were found to be allelic with emb-27 and emb-30 (emb= embryonic lethal), two genes that were previously identified in screens from 20 years ago. The remaining alleles defined three new complementation groups, which we have named mat for m etaphase to a naphase t ransition defective (6). The mat-1, mat-2, and mat-3 genes are each represented by multiple ts alleles. In addition to emb-27, emb-30, and the three mat genes, Diane Shakes has shown that a sixth gene, emb-1, belongs in this class of mutants. Similar to emb-27 and emb-30, this gene was previously identified by others in a screen for embryonic lethality. Though our screen did not yield any new emb-1 alleles, we have been characterizing the two existing ts alleles because of the similarity in phenotype to the mat mutants.
One-cell embryos from our mat mutants all share the same arrest phenotype when adults are shifted to 25°C: (a) the oocyte chromosomes are arranged on a metaphase plate or "pentagonal array", (b) the oocyte chromosomes organize a morphologically normal meiotic I spindle, and (c) the sperm chromosomes remain highly condensed at the other end of the embryos. The embryo does not progress further. No anaphase figures are observed and no polar bodies are extruded. The chromosomes do not decondense or undergo DNA synthesis. Maternal and paternal pronuclei do not form. The sperm centrosomes remain quiescent and do not nucleate microtubule asters. Staining with a number of M-phase marker antibodies further suggests that these meiotic arrest mutants persist in an M-phase-like state. The meiotic metaphase arrest observed in all these mutants suggests that key cell cycle regulator genes are functionally defective at the non-permissive temperature.
To address whether these genes also have roles in spermatocyte meiosis, Don Fox and Penny Sadler (College of William and Mary) analyzed ts mutant males shifted to the non-permissive temperature prior to the onset of spermatogenesis. For most of the alleles, defects in spermatocyte meiosis were observed. Specifically, primary spermatocytes failed to progress from metaphase to anaphase of meiosis I. The spermatocyte chromosomes congressed on a metaphase plate, established a meiotic I spindle, but failed to separate their chromosomes.
To address whether the mat genes also have roles in mitotic cell cycles, we analyzed mutants that were shifted to the non-permissive temperature as L1 larvae. For the most part, the somatic cells of these larvae continued to develop normally. For the majority of alleles however, the resulting adults were sterile. For some alleles, the adults were fertile, but produced dead 1-cell embryos. For a few alleles, somatic defects are apparent, such as protruding vulvae, abnormal male tails, or uncoordinated movement. These observed abnormalities are indicative of cell cycle defects. We did not, however, detect any developmental abnormalities during embryogenesis. When 2-cell embryos are shifted to the non-permissive temperature, the majority hatch and develop into adults. For some alleles, these adults then produce 1-cell embryos; for other alleles, the adults are sterile.
Each of the mat and emb genes has been mapped and cloned. Furuta et al. (2000) cloned emb-30, which was shown to be an ortholog of APC4, a component of the APC/C. The emb-27 gene encodes a CDC16 ortholog, mat-1 encodes a CDC27 ortholog (in collaboration with Jill Schumacher at the University of Texas), mat-2 encodes an APC1 ortholog, and mat-3 encodes a CDC23 ortholog. Thus, all of our mutants correspond to APC/C subunit genes (2, 4, 6).
| APC/C |
|
Genetic |
F1 |
| Component |
ORF (Chromosome) |
Mutant |
RNAi phenotype |
| APC-1 |
W10C6.1 (II) |
mat-2 |
Mei. 1-cell |
| APC-2 |
K06H7.6 (III) |
|
Mei. 1-cell |
| CDC-27 |
Y110A7A.17 (I) |
mat-1 |
Mei. 1-cell |
| APC-4 |
F54C8.3 (III) |
emb-30 |
Mei. 1-cell |
| APC-5 |
M163.4 (X) |
|
MC embryos |
| CDC-16 |
F10B5.6 (II) |
emb-27 |
Mei. 1-cell |
| CDC-23 |
F10C5.1 (III) |
mat-3 |
Mei. 1-cell |
| APC-10 |
F15H10.3 (V) |
|
MC embryos |
| APC-11 |
F35G12.9 (III) |
|
Mei. 1-cell |
Each of the genes in this Table is represented by at least one Expressed Sequence Tag (EST), and most are represented by many ESTs.
"Mei. 1-cell" refers to meiotic 1-cell embryos arrested at metaphase of the first meiotic division.
"MC embryos" refers to a mixed population of dead and hatching multicellular embryos.
Suppressor screen for regulators and substrates of the APC/C:
Given the fact that all of the genes discussed above encode APC/C components, we are eager to identify other proteins that functionally interact with or regulate the APC/C. In addition, we would like to identify substrates of the APC/C that might be meiosis-specific. One genetic approach we have undertaken in order to identify such proteins is a suppressor screen in which we mutagenized mat-3 mutants and looked for animals that could then survive at the non-permissive temperature. We have identified 29 such suppressors, the majority of which are extragenic and semi-dominant. Only three alleles were recessive. By snip-SNP mapping, these suppressors define at least five complementation groups. A few suppressor alleles are linked to mat-3 and we are currently determining whether these are intragenic suppressors. The fine mapping of all the suppressors is underway and the molecular identification of these genes should reveal regulators and/or substrates of the APC/C. In addition, we are determining whether these suppressor mutants have phenotypes on their own and whether they suppress mutations in the other mat genes (mat-1, mat-2, emb-27, and emb-30).
We have also taken a similar approach to identify factors that interact with or regulate separase, the protease that cleaves cohesin to allow sister chromatid separation at anaphase. We have mutagenized a mutant strain carrying a ts allele of sep-1, the C. elegans separase gene, and are currently characterizing three extragenic semi-dominant suppressors.
Future studies for the APC/C:
In the future, we hope that our genetic studies identify novel regulators and substrates of the APC/C that have not been found in other model systems. We are also undertaking some biochemical approaches to identify the substrates of the APC/C that apparently are not being ubiquitinated and degraded in the 1-cell arrested mat embryos.
We know from our RNAi studies that seven of the nine APC/C orthologs that have already been identified in the C. elegans genome play a role in meiosis I. However, RNAi of the orthologs of APC5 or APC10 does not result in a 1-cell arrest (see Table above), suggesting that perhaps the complex varies during meiosis and mitosis. To address this observation, we plan to use proteomic approaches to identify the components of the C. elegans APC/C and to determine whether this complex varies in its composition during development.
A Myt1 ortholog is essential for normal oocyte development and maturation
Myt1, a member of the WEE1 kinase family, is an essential negative regulator of the CDK/cyclin complex that acts during the G2/M transition of the meiotic cell cycle in Xenopus oocytes. A former post-doctoral fellow in the lab, Penny Sadler, has examined the developmental consequences of depleting animals of the C. elegans Myt1 ortholog. Using RNA interference (RNAi), she has shown that C. elegans oocytes depleted of the Myt1 homolog, WEE-1.3, fail to develop and mature normally within the oviduct of the hermaphrodite. Despite the precocious G2/M phase transition at the earliest stages of WEE-1.3 depletion, oocytes are ovulated normally. However, such WEE-1.3-depleted oocytes are fertilization-incompetent in the presence of wild-type sperm and appear to remain in an extended M-phase state within the uterus. With longer exposure to wee-1.3 RNAi, the ability of the hermaphrodite germline to produce normal oocytes becomes greatly compromised. Our explanation is that CDK-1, the presumed WEE-1.3 target, is an abundant maternal protein that needs to be kept inactive during critical phases of oocyte development. This model is strengthened by our observation that CDK-1 depletion suppresses the wee-1.3 RNAi maturation/fertilization defects. Our experimental results confirm that WEE-1.3 acts through CDK-1 in the proper regulation of the G2/M transition during C. elegans oocyte development and maturation.
In the future, we plan to examine other proteins that are known in other systems to function with, and regulate, Myt1. We will use RNAi to examine the developmental consequences when these proteins are depleted. We expect to identify genes that mimic the wee-1.3 RNAi phenotype and potentially other genes that suppress the wee-1.3 RNAi sterile phenotype. In addition, we are curious to determine the other functions of the wee-1.3 gene, as it is a widely expressed gene in C. elegans (Lamitina and L´Hernault 2002).
Staff Members
Chris D Richie, Ph.D., Post-doctoral fellow chrisr@intra.niddk.nih.gov 301-594-4369
Chris joined the lab in the spring of 2003 after acquiring his Ph.D. from The University of Texas in Houston. Chris is currently epitope-tagging various APC/C subunits for expression in transgenic animals for the purposes of immuno-purifying the APC/C complex from C. elegans extracts. He is also working on biochemical strategies to identify the proteins that persist in the 1-cell mat mutants because they are not ubiquitinated. Chris is also working on the sep-1 suppressors.
Kathryn Kreimborg Stein, Ph.D., Post-doctoral fellow steink@niddk.nih.gov 301-594-4368
Katie received her Ph.D. In Cell and Developmental Biology from the University of California, Davis in 2005. She is currently working on identifying and characterizing paternal effect mutants in C. elegans in order to better understand the contribution of the sperm to embryonic development. She is conducting a suppressor screen of an existing paternal effect gene, spe-11, to identify other genes that may play a role in this process. In addition, Katie is working on characterizing and mapping the mat-3 suppressor alleles.
Akila Moore, Graduate Student mooreak@mail.nih.gov 301-594-4505
Akila received her B.A. from University of California at Berkeley in 1997 and her MA of Science in 2000 from Cal State L.A. She is currently a pre-doctoral candidate from Johns Hopkins University School of Public Health in the Department of Molecular Microbiology and Immunology. She is currently characterizing the role of mitochondrial proteins during meiosis and mitosis and their subsequent roles in embryogenesis. In addition, Akila is looking at the roles of these proteins during spermatogenesis and oogenesis.
Ben Ross, Research Technician
Ben graduated from Lewis and Clark College in Portland, Oregon in the Spring of 2005 with a B.A. in Biochemistry and Molecular Biology. He joined the lab this fall and is currently working on the molecular identification of some our APC suppressors. In addition, he is carrying out immunoprecipitation assays, western blot analysis, and immunocytochemistry to examine phospho-CDK-1 expression.
Research Technician:
Tom Hays hayst@niddk.nih.gov 301-594-4505
Tom graduated from Penn State with a B.S. in Environmental Resource Management in 2004. While there he worked in Jeff Peters' lab and completed a thesis in liver carcinogenesis. Tom's currently working to map and identify mat-3 suppressors.
Our favorite links:
C. elegans WWW Server: http://elegans.swmed.edu/
WormBase:http://www.wormbase.org/
C. elegans ORFeome project: http://worfdb.dfci.harvard.edu/
Trans-NIH C. elegans Initiative: http://www.nih.gov/science/models/c_elegans/
Caenorhabditis Genetics Center: http://biosci.umn.edu/CGC/CGChomepage.htm
C. elegans Movies: http://www.bio.unc.edu/faculty/goldstein/lab/movies.html
