An examination of behaviors in Drosophila Melanogaster for their utility in an Rnai screen of autism candidate genes

Open Access
Author:
Saxena, Ritu Shalini
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Scott Brian Selleck, Thesis Supervisor
  • David Scott Gilmour, Honors Advisor
Keywords:
  • Autism
  • Drosophila
  • Melanogaster
  • RNAi
  • screen
  • gene candidates
  • behavior
  • assays
  • circadian rhythm
  • geotaxis
  • social space
Abstract:
Autism awareness and research has become more prevalent and garnered much more attention over the last few decades due to increasing rates of incidence. With no known cure, scientists have been pressured to research the causes of autism and autism spectrum disorders with the hopes of discovering preventative measures. Recent research has indicated that autism is not caused by mutations in one gene, but many genes. The purpose of this project was to find a way to provide biological validation for genes identified by human genetic studies that may be related to autism spectrum disorders, using the model organism Drosophila melanogaster. This was attempted by selecting specific phenotypes, identified through literature review of Drosophila behavioral analysis, which could be indicative of autism related neurological changes. The well-established fly model of Fragile X syndrome, a known syndromic cause of autism, was selected as a positive control for these behavioral assays. In the future, if a scientist were to manipulate a gene candidate in a fruit fly and observe the same behavioral disturbances as the positive control, then that gene would be more attractive for further testing in higher organisms such as mice, where establishment of models is much more costly and time consuming. The behaviors tested in this project were social spacing, circadian rhythm, and negative geotaxis. UAS-Dicer II was used together with elav-GAL4 to increase the effectiveness of the RNA interference in neurons, decreasing the expression of the Fragile X mental retardation protein (FMR1). This, in effect, replicated Fragile X Syndrome. Similarly I chose to decrease Drosophila metabotropic glutamate receptor (DmGluRA) using RNAi and observe those behaviors, because mGluRA levels increase when FMR1 levels decrease. Decreasing mGluRA could potentially serve as a negative control. Circadian rhythm proved to be too sensitive to extraneous variables and was unable to produce a distinct phenotype, as well as with social spacing. The controls and experimental genotypes in both of these assays showed no difference in behavior, but seemed to be affected more by the p-element insertions and the presence of mini-white from the RNAi and Gal4 lines. I observed no gross morphological changes using immunohistochemistry and confocal imaging either. However, the negative geotaxis results showed that there is a significant difference in behavior when flies have decreased FMR1 expression. These flies were very inactive, with a 15% success rate compared to Canton-S (the control group), which had a success rate of 85%. This could indicate that negative geotactic ability is unaffected by the presence of p-elements and other variables that seemed to interfere with the other assays. Ways to improve upon this project in the future would be to backcross to potentially remove mini-white from the RNAi and Gal-4 lines, as well as homogenize the background of each line to be that of Canton S. If all variables are taken into account and the extraneous ones are eliminated, the implications of this project are invaluable.