Showing 1 - 3 of 3 results
1.
Desensitisation of Notch signalling through dynamic adaptation in the nucleus.
Abstract:
During embryonic development, signalling pathways orchestrate organogenesis by controlling tissue-specific gene expression programmes and differentiation. Although the molecular components of many common developmental signalling systems are known, our current understanding of how signalling inputs are translated into gene expression outputs in real-time is limited. Here we employ optogenetics to control the activation of Notch signalling during Drosophila embryogenesis with minute accuracy and follow target gene expression by quantitative live imaging. Light-induced nuclear translocation of the Notch Intracellular Domain (NICD) causes a rapid activation of target mRNA expression. However, target gene transcription gradually decays over time despite continuous photo-activation and nuclear NICD accumulation, indicating dynamic adaptation to the signalling input. Using mathematical modelling and molecular perturbations, we show that this adaptive transcriptional response fits to known motifs capable of generating near-perfect adaptation and can be best explained by state-dependent inactivation at the target cis-regulatory region. Taken together, our results reveal dynamic nuclear adaptation as a novel mechanism controlling Notch signalling output during tissue differentiation.
2.
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 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.
3.
Principles and applications of optogenetics in developmental biology.
Abstract:
The development of multicellular organisms is controlled by highly dynamic molecular and cellular processes organized in spatially restricted patterns. Recent advances in optogenetics are allowing protein function to be controlled with the precision of a pulse of laser light in vivo, providing a powerful new tool to perturb developmental processes at a wide range of spatiotemporal scales. In this Primer, we describe the most commonly used optogenetic tools, their application in developmental biology and in the nascent field of synthetic morphogenesis.
