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Cis-interactions between Notch and Delta generate mutually exclusive signalling states

Abstract

The Notch–Delta signalling pathway allows communication between neighbouring cells during development1. It has a critical role in the formation of ‘fine-grained’ patterns, generating distinct cell fates among groups of initially equivalent neighbouring cells and sharply delineating neighbouring regions in developing tissues2,3,4,5. The Delta ligand has been shown to have two activities: it transactivates Notch in neighbouring cells and cis-inhibits Notch in its own cell. However, it remains unclear how Notch integrates these two activities and how the resulting system facilitates pattern formation. Here we report the development of a quantitative time-lapse microscopy platform for analysing Notch–Delta signalling dynamics in individual mammalian cells, with the aim of addressing these issues. By controlling both cis- and trans-Delta concentrations, and monitoring the dynamics of a Notch reporter, we measured the combined cistrans input–output relationship in the Notch–Delta system. The data revealed a striking difference between the responses of Notch to trans- and cis-Delta: whereas the response to trans-Delta is graded, the response to cis-Delta is sharp and occurs at a fixed threshold, independent of trans-Delta. We developed a simple mathematical model that shows how these behaviours emerge from the mutual inactivation of Notch and Delta proteins in the same cell. This interaction generates an ultrasensitive switch between mutually exclusive sending (high Delta/low Notch) and receiving (high Notch/low Delta) signalling states. At the multicellular level, this switch can amplify small differences between neighbouring cells even without transcription-mediated feedback. This Notch–Delta signalling switch facilitates the formation of sharp boundaries and lateral-inhibition patterns in models of development, and provides insight into previously unexplained mutant behaviours.

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Figure 1: System for analysing signal integration in the Notch–Delta pathway.
Figure 2: Transactivation of Notch occurs in a graded fashion.
Figure 3: Cis trans signal integration by Notch.
Figure 4: The mutual-inactivation model in multicellular patterning.

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Acknowledgements

We would like to thank I. Bernstein for the IgG-Deltaext, U. Lendahl for the 12xCSL reporter construct, J. Aster for human NOTCH1 and other constructs, and G. Weinmaster for the rat DLL1 construct and advice. We also thank R. Tsien and K. Thorn for mCherry, S. Megason and S. Fraser for H2B–citrine and other constructs, R. Diamond and D. Perez for assistance with FACS, and F. Tan and J. Yong for help with cloning some of the constructs. We thank A. Eldar, J. Locke, G. Seelig, R. Kishony, B. Shraiman, A. C. Oates and members of the Elowitz laboratory for discussions and advice. This work was supported by the US National Institutes of Health Fellowship F32GM77014 (D.S.), the Caltech Center for Biological Circuit Design and the Packard Foundation. A.L. acknowledges support from the Fannie and John Hertz Foundation and the UCLA/Caltech Medical Scientist Training Program (NIH GM08042). J.G.O. acknowledges support from the Ministerio de Ciencia e Innovacion (Spain, project FIS2009-13360 and the I3 programme).

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Authors and Affiliations

Authors

Contributions

D.S. and M.B.E. designed the research. D.S., L.A.S., M.E.F. and G.A.A. built cell lines and performed experiments. D.S., A.L., L.L., J.G.-O. and M.B.E. performed data analysis and mathematical modelling. D.S. and M.B.E. wrote the manuscript with substantial input from the other authors.

Corresponding author

Correspondence to Michael B. Elowitz.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1-S17 with legends, Supplementary Tables S1-S3, Supplementary Methods and Data Analysis and Data and References for the Supplementary Material. (PDF 3625 kb)

Supplementary Movie 1

This movie shows the trans-activation of hN1G4esn by plate bound Delta. Movie used to generate filmstrip in Figure 2B. (AVI 10479 kb)

Supplementary Movie 2

This movie shows the effect of cis-Delta on hN1G4esn activation. Movie used to generate filmstrip in Figure 3B. (AVI 22000 kb)

Supplementary Movie 3

This movie shows the trans-activation of hN1G4esn-No-Delta by co-culture with Delta-expressing cells. Movie used to generate filmstrip in Fig S5. (AVI 5340 kb)

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Sprinzak, D., Lakhanpal, A., LeBon, L. et al. Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature 465, 86–90 (2010). https://doi.org/10.1038/nature08959

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