Desensitisation of Notch signalling through dynamic adaptation in the nucleus.

Ranjith Viswanathan,Cristina Pallares Cartes,Jonas Hartmann,Stefano De Renzis

doi:10.15252/embj.2020107245

Ranjith Viswanathan, Cristina Pallares Cartes + Show 2 more

Open Access

https://doi.org/10.15252/embj.2020107245

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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.

Highlights

Signalling dynamics have emerged as fundamental to cellular information processing in the context of tissue differentiation and organogenesis (Freeman & Gurdon, 2002; Sonnen & Aulehla, 2014)
We employed LANS (Niopek et al, 2014; Guntas et al, 2015) to cage the nuclear localisation signal (NLS) of Notch Intracellular Domain (NICD) with a photo-sensitive LOV domain and LOVTRAP (Wang et al, 2016) to anchor NICD-LOV to mitochondria through a Zdark tag, which recognises the dark conformation of the LOV domain and was itself localised to mitochondria by a TOM20 tag
Optogenetic activation at the beginning of cellularisation caused a premature activation of sim transcription, appearing as a bright nuclear dot from5 min after NICD began translocating into the nucleus (Fig 1F and G, Movie EV2)

Summary

Introduction

Signalling dynamics have emerged as fundamental to cellular information processing in the context of tissue differentiation and organogenesis (Freeman & Gurdon, 2002; Sonnen & Aulehla, 2014). Understanding how cells integrate and translate dynamic signalling information into specific gene expression outputs in vivo has proven challenging, as it requires methods for precise spatio-temporal control of the input and quantification of output levels in real time (Sako et al, 2016; Huang et al, 2017; Krueger et al, 2019; McDaniel et al, 2019; Hartmann et al, 2020; Johnson et al, 2020; Rogers & Mu€ller, 2020). Notch signalling does not involve any signal amplification step, and its strength is thought to be directly related to the level of NICD production

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