Date of Award
Doctor of Philosophy (PhD)
Genetics and Biochemistry
Robert R. H. Anholt
Trudy F. C. MacKay
Understanding the mechanisms by which genetic variation brings about phenotypic variation is essential for understanding variation in complex traits. Drosophila melanogaster is a powerful model organism for such studies. Flies are easy to raise in the laboratory under controlled genetic and environmental conditions and many genetic tools are widely available. To chart the genotype-phenotype map, we need to study how individual genetic variants contribute to phenotypic variation, as well as how environmental perturbations influence gene expression.
Regarding the former, I generated single nucleotide substitutions in Obp56h in a common genetic background. Obp56h, a member of the Odorant binding protein multigene family, is a small gene in a favorable genomic location for CRISPR-Cas9 mediated deletion. After deletion, I reinserted the gene at the endogenous locus with individual allelic variants chosen from those segregating in a wild-derived inbred population to produce five lines varying at single nucleotides in a common genetic background. Different alleles, both within and near the gene (potentially regulatory) and both common and rare, have different, large effects on organismal fitness traits as well as on genome-wide coregulated ensembles of transcripts. These effects are at the level of mean and microenvironmental variance in both fitness traits and the transcriptome. However, these alleles have only small effects on fitness traits in the wild-derived inbred population indicating that the effects of individual alleles can be context-specific and are perhaps suppressed in natural populations via epistatic interactions.
Next, I studied how acute cocaine consumption and developmental alcohol exposure affect the transcriptome at single-cell resolution. The Drosophila brain is small, allowing for comprehensive whole-brain studies. Further, previous studies have characterized effects of acute cocaine consumption and developmental alcohol exposure on flies, which resemble those in humans. Single-cell RNA sequencing revealed that the transcriptomes of cells in the fly brain are affected in a cell-type and sex-dependent manner after the flies consumed fixed amounts of cocaine or are exposed to developmental alcohol exposure. These effects are sexually dimorphic, with males showing a greater degree of differential expression and are particularly prominent in glial and mushroom body cells. Developmental alcohol exposure leads to a similar, but different, sexually dimorphic and cell-type dependent pattern of differential expression as cocaine consumption. Some mechanisms are shared between the experimental paradigms indicating common processes.
The strategies used in the studies described in this dissertation can be generally applied to explore genotype-phenotype relationships at high resolution.
Mokashi, Sneha Shreekant, "Of Single Nucleotides and Single Cells: Charting the Genotype-Phenotype Map at High Resolution Using Drosophila melanogaster" (2021). All Dissertations. 2904.