| Abstract |
Spatio-temporal regulation of signalling pathways plays an important role in generating diverse responses during the development of multicellular organisms. While increasing number studies are uncovering the importance of signalling dynamics in controlling tissue patterning and morphogenesis, the precise role of signal dynamics in transferring information in vivo is incompletely understood owing to the lack of methods to manipulate protein activity at the relevant spatio-temporal scales. In this PhD thesis, I employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), at its endogenous locus. Light mediated activation of opto-Delta disrupts Notch signalling during different developmental stages. Using clonal analysis, I show that optogenetic activation blocks Notch activation through cis-inhibition in signal-receiving cells. To investigate how a Notch input is dynamically translated into a differentiation output, I focused on mesectoderm specification during early Drosophila embryogenesis. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals different modes of regulation at the tissue and cellular level. While at the tissue-level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation needs to reach a minimum threshold to generate a response. Taken together these results provide novel insights into the dynamic input-output regulation of Notch signalling, supporting a model in which the Notch receptor is an integrator of (noisy) analog signals that generates a digital switch-like behaviour at the level of target gene expression during tissue differentiation.
In order to further test this model, I attempted to develop an optogenetic system to activate Notch in vivo (opto-Notch). Despite showing light-responsive changes in localization, a certain level of Notch is activated even prior to photo-activation, thus necessitating further optimization. Finally, I describe efforts for further characterization of opto-Delta as a tool to spatially perturb signalling, to study Notch signalling during neuroblast delamination, and for adaptation to mammalian cell-culture systems.
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