Caroline Miller
Faculty Sponsor: Dr. Hales
Mitochondria undergo a variety of shape changes that are crucial for healthy cell function. Disruption in genes associated with this morphology results in aberrant mitochondria, the underlying cause of many human neurodegenerative disorders. Drosophila melanogaster spermatogenesis is used as a model system to observe distinct mitochondrial shape changes. The mitochondria first begin diffused throughout the cell before aggregating to the nucleus. Then mitochondria fuse into two derivatives, which wrap around each other to form the nebenkern during onion stage. The derivatives begin to elongate and eventually form the long sperm tails. By analyzing mutant fly strains defective in mitochondrial morphogenesis during these stages, we focus on identifying and characterizing the function of genes which play a role in shaping. Homozygous males in the Z2-2588 strain have a male sterile mutation resulting in abnormal mitochondrial shaping during onion stage and late elongation. Deficiency complementation mapping and RNA knockdown were used to identify CG5043 as the mutated gene. A related gene, CG5050, was knocked down using RNA interference to identify a mutant phenotype. A mild deformation in nebenkern formation was seen. CRISPR/Cas9 mutagenesis was used to induce mutations in the coding region of CG5050 in order to better visualize mitochondrial shaping dysfunction. Mutagenesis, however, failed and no phenotype was seen due to a single nucleotide polymorphism in the target region of the guide RNA. To further characterize the functions of CG5043 and CG5050, we performed RNA in situ hybridization using a DIG-labelled RNA probe to determine the stages of spermatogenesis at which the two genes are expressed. A negative control probe was also made using the sense strand sequence. RNA in situ hybridization staining patterns reveal wild type expression data of each gene. Mutant strain in situ is to be compared to wild type staining to determine the effects of mutation on expression of CG5050 and CG5043.
