Cis-interactions between Notch and Delta generate mutually exclusive signalling states
The Notch–Delta signalling pathway allows communication between neighbouring cells during development^1. 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 tissues. 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 cis–trans 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.
Additional Information© 2010 Macmillan Publishers Limited. Received 27 April 2009; accepted 26 February 2010. Published online 25 April 2010. 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). Author 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.
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