A systems-level study of signaling networks and differentiation

Statement of Work 1. We will use recombineering technology to modify interactions among the key network components of the Drosophila Yan gene network. We will mutate the miR7 binding sites in the Yan 3’UTR in various combinations, and will probe how disruption of the various feedback modules perturb network stability as measured by in vivo quantitation of photoreceptor determination. These results will provide insight into which components of the various regulatory loops are most critical for Yan network stability, and will directly inform subsequent rounds of model refinement and experimental testing. 2. We will measure Yan, Pnt, Propsero and miR-7 abundance in eye cells by fluorescence microscopy. Massive parallel measurement of fluorescence in thousands of cells will be done by automated computer segmentation. This will be used to model the basic robustness of developmental fate decisions in eye cells. 3. In particular, one interesting parameter we will examine is the number of mRNA transcripts per cell for Yan, Pnt, and miR-7. This parameter will be detected by single molecule fluorescecnce in situ hybridization.