Animal and plant development are complex biological processes where regulated cell division, signalling, differentiation and apoptosis lead from a single cell to a complete multicellular organism. Regulated gene expression drives development: genes need to be expressed in precise locations, times and at proper levels. Mis-regulation of this process often leads to major negative developmental consequences including disease and developmental failure. Despite the intrinsic stochasticity of gene expression, development is usually a stereotyped process, with little to no variation between different individuals. Thus, gene expression has to be extremely tightly controlled and modulated at several levels. One class of factors that have been found to modulate and buffer gene expression at a post-transcriptional level are microRNAs: tiny RNA molecules involved in inhibiting translation of target mRNAs. Many studies have hinted that microRNAs act as buffering factors, but puzzling and sometimes controversial results from the analysis of microRNA KO animalsâ phenotypes, have left many questions unanswered. This work aims to investigate microRNAsâ functions in regulating target genes expression at the single-cell level by analysing and quantifying single expressing cells in fixed Drosophila melanogaster embryos and larval tissues. Specifically, I have investigated how miR-9a regulates the transcription factor senseless, a protein required for peripheral nervous system (PNS) development, and the protease rhomboid, required for epidermal growth factor signalling and developmental patterning. Using such a single-cell quantitative approach I show that during PNS development miR-9a and senseless exhibit reciprocal dynamics that differ between embryonic and larval developmental stages. I then introduce how it is possible to extract single-cell data from high resolution microscopy relying on a âsegmentableâ membrane. I apply this method to study rhomboid expression and show that rhomboid mRNA abundance per cell is higher in miR-9a KO flies. I then investigate how this affects rhomboid protein levels, finding that rhomboid protein is detectable much earlier in development in miR-9a KO flies. This work not only brings new understanding of senseless and miR-9a reciprocal dynamics and rhomboid regulation by miR-9a, but it also introduces methods and tools that can be applied to many other microRNA-target gene networks.
- Peripheral Nervous System
- Drosophila melanogaster