Meiosis is the specialized cell cycle that leads to the production of gametes (eggs or sperm) that carry a single copy of each chromosome. The most common cause of human birth defects is aneuploidy, which occurs when meiotic errors result in progeny with the wrong number of chromosomes, leading to conditions such as Down syndrome. Over 90% of human aneuploidies arise during the process of egg production, making the study of female meiosis a critical issue for human health. One step found in most species is congression, where the cell lines up its chromosomes in preparation for division. For example, the chemical Bisphenol A (BPA, a known endocrine disruptor) causes congression errors during female meiosis in mice, and those higher error rates are correlated with higher rates of aneuploidy. On the basis of this research, BPA is being phased out from consumer plastics. Therefore, increasing our understanding of the process of congression is a key component of the study of female meiosis and has applications to public health policy. This proposal will use the fruit fly Drosophila melanogaster as a model system to study congression during female meiosis.
The first Aim of this project is to identify genes that are required for normal congression. This will be done using a technique called RNA interference (RNAi), a technique that tricks cells into using a native antiviral pathway to knock down a targeted gene. This can be achieved by expressing a palindromic RNA molecule that matches the sequence of the target gene. Researchers have created large collections of fly stocks, each carrying an RNAi construct targeting a different gene. We will screen a collection of these constructs by driving expression of the RNAi construct in the female germline, and then examining eggs for visible defects using confocal fluorescent microscopy. If a gene is required for normal congression, then knocking it down should lead to defects. In a pilot project, our novel screening approach successfully identified eight gene `hits' as being required for female meiosis, half of which had never been recognized as being required for meiosis before. We will also incorporate this screen in the DePaul genetics class, allowing undergraduate students to participate in primary scientific research.
The second Aim of this project is to further characterize mustard, one of the hits identified by our pilot screen. This gene is a component of the innate immune system, which has not previously been recognized as required for female meiosis in Drosophila. We found that female germline RNAi of mustard caused high rates of meiotic errors, which we propose is due to mis-regulation of recombination. We will assay recombination rates in mustard-RNAi females and examine other members of the innate immunity pathway, in order to determine how this mutant causes meiotic errors. The third goal of this project is to further characterize nuf2, another of our screen hits. This gene is a component of the outer kinetochore, and in addition to causing semi-sterility and high rates of meiotic errors, we found that nuf2- RNAi also resulted in alteration of proteinaceous ooplasmic structures first identified by the PI, changing them from filaments to globules. The function of these filaments is unknown, but they appear to be conserved at least as far back as between flies and C. elegans. The filaments' structural components are unknown, but nuf2 is the first gene that has been found to alter their shape. We hypothesize that these structures act as surrogate kinetochores, to allow simultaneous signal generation throughout the oocyte. To test this, we will localize other kinetochore components, examine filament structures when other outer kinetochore proteins are knocked down and perform live imaging to determine if these proteins are used in their construction. This project will rely extensively on undergraduate research assistants, and is anticipated to provide salary and research material support for 6-12 undergraduates over the grant period, as well integrating primary research into the genetics class taken by hundreds of undergraduates. It is also anticipated to provide support for 1-2 M.S. students working with the PI for their thesis research projects. Students will be individually trained by the PI in doing Drosophila genetics, and if their data is used in published manuscripts they will be given coauthorship to credit their work. In addition, project funds will also be used to support these students to attend research conferences to present their work.
This project will use the fruit fly (Drosophila melanogaster) as a model system to study female meiosis, the specialized cell cycle that produces eggs. This is important for public health because chromosome segregation errors that occur during egg development are the single most common source of birth defects, and can cause diseases such as Down syndrome.
| Gilliland, William D; May, Dennis P; Colwell, Eileen M et al. (2016) A Simplified Strategy for Introducing Genetic Variants into Drosophila Compound Autosome Stocks. G3 (Bethesda) 6:3749-3755 |
| Gilliland, William D; Colwell, Eileen M; Lane, Fiona M et al. (2015) Behavior of aberrant chromosome configurations in Drosophila melanogaster female meiosis I. G3 (Bethesda) 5:175-82 |
| Gilliland, William D (2015) A Comment on Fine-Scale Heterogeneity in Crossover Rate in the garnet-scalloped Region of the Drosophila melanogaster X Chromosome. Genetics 201:1275-7 |
| Gilliland, William D; Colwell, Eileen M; Osiecki, David M et al. (2015) Normal segregation of a foreign-species chromosome during Drosophila female meiosis despite extensive heterochromatin divergence. Genetics 199:73-83 |
| Gillies, Shane C; Lane, Fiona M; Paik, Wonbeom et al. (2013) Nondisjunctional segregations in Drosophila female meiosis I are preceded by homolog malorientation at metaphase arrest. Genetics 193:443-51 |
