Chromosome Congression in Drosophila Female Meiosis - 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.
