Abstract
Spatio‐temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. Here, we employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto‐Delta), which replaces both copies of the endogenous wild‐type locus. Using clonal analysis, we show that optogenetic activation blocks Notch activation through cis‐inhibition in signal‐receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue‐ and cell‐scale regulatory modes. 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 acts through a switch‐like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.
Highlights
Notch signalling is central to most developmental decision-making events in animals, and its misregulation is implicated in many diseases, including cancer [1]
We employed optogenetics to modulate Notch signalling during Drosophila embryonic development in order to characterize its dynamic regulation and input–output relationship during tissue differentiation in real time
We focused on mesectoderm specification at the onset of gastrulation, which is defined by the expression of the transcription factor sim in two parallel single rows of cells flanking the mesoderm [20,21]
Summary
Notch signalling is central to most developmental decision-making events in animals, and its misregulation is implicated in many diseases, including cancer [1]. In agreement with the general paradigm of Notch signalling activation, Delta is internalized from the surface of mesodermal cells along with the Notch extracellular domain (NECD) in response to the expression of the ubiquitin ligase neuralized [8,9]. We developed an optogenetic strategy to inhibit endogenous Delta activity with subminute temporal precision and simultaneously follow sim transcription in real time using the MS2-MCP system [22] Using this approach, we show that while at the tissue-level Notch functions in an analog manner controlling both the timing and the frequency at which individual nuclei express sim, at the level of individual cells, Notch acts as a switch, with a minimum threshold of Notch activity determining whether sim is expressed or not. These results are consistent with a model in which Notch signalling performs digital time-integration during tissue differentiation
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