Optogenetic inhibition of Delta reveals digital Notch signaling output during tissue differentiation

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 cisinhibition 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 tissuelevel 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.We thank all members of the De Renzis laboratory for helpful discussion. We thank C. Tischer for helping with image analysis and the data pipeline developed in Cell Profiler for quantifying Delta clustering kinetics. We thank A. Aulehla, J.Crocker, J. Hartmann, and T. Hiiragi for helpful discussions and critical reading of the manuscript. We thank the advanced light microscopy for their advice and assistance. We thank the Bloomington Drosophila Stock Center and the Drosophila Genomics Resource Center for providing fly stocks and cDNAs. We thank M. Levine for providing fly stocks. Author contributionsThe experiments were conceived and designed by R.V., A.N., P.N. and S.D.R.

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