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Calcineurin sets the bandwidth for discrimination of signals during thymocyte development

Nature volume 450, pages 731–735 (2007)Cite this article

Abstract

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the αβ T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide–MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection)1,2. Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf–MEK–ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to ‘weak’ positive selecting signals but not in response to ‘strong’ negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.

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Figure 1: Specific and severe defect in Raf–MEK–ERK activation in Cnb1 -deficient thymocytes.
Figure 2: Developmental but not direct requirement for calcineurin/NFAT activity for proper activation of ERK.
Figure 3: Reconstitution of Raf–MEK–ERK signalling partly rescues positive selection in the absence of calcineurin activity.
Figure 4: Transition to the ‘high ERK competence’ state is required to respond functionally to positively selecting ligands.

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Acknowledgements

We thank P. Ebert for helping with calcium flux studies, and K. A. Hogquist and C. Wilson for providing mice and reagents. E.M.G., M.M.W. and A.N.R. were supported by Stanford Graduate Fellowships. M.M.W. was additionally supported by a Howard Hughes Medical Institute predoctoral fellowship. A.N.R. was also supported by a National Science Foundation Graduate Research Fellowship. K.C.B. was supported by the Boehringer Ingelheim Fonds. L.H. was also supported by Agency for Science, Technology and Research Singapore. This work was supported by grants from Howard Hughes Medical Institute and the National Institute of Heath to G.R.C.

Author Contributions E.M.G., M.M.W. and G.R.C. generated the hypotheses, designed the experiments and wrote the manuscript. E.M.G. performed the experiments and generated the figures. K.C.-B. generated the NFATc3 conditional knockout mice, maintained this line in the NFATc2-null background and contributed to the experiments in Fig. 4. A.N.R. and L.H. contributed to pilot experiments and experiments shown in Fig. 2 and Supplementary Fig. 8. J.R.N. generated the Cnb1 conditional knockout mice, conducted pilot experiments and contributed to experimental rationale. L.M. contributed to experiments shown in Supplementary Fig. 5. B.I. provided the Raf-CAAX transgenic mice.

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Author notes

  1. Monte M. Winslow, Kirsten Canté-Barrett & Joel R. Neilson

    Present address: Present addresses: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA (M.M.W., J.R.N.); Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden 2300 RC, The Netherlands (K.C.-B.)., Leiden

Authors and Affiliations

  1. Howard Hughes Medical Institute and the Departments of Pathology and of Developmental Biology, Stanford University, Stanford, California 94305, USA, California

    Elena M. Gallo, Monte M. Winslow, Kirsten Canté-Barrett, Amy N. Radermacher, Lena Ho, Lisa McGinnis, Joel R. Neilson & Gerald R. Crabtree

  2. Program in Immunology, Stanford University, Stanford University, Stanford, California 94305, USA , California

    Elena M. Gallo, Monte M. Winslow, Kirsten Canté-Barrett, Amy N. Radermacher, Lena Ho, Lisa McGinnis & Gerald R. Crabtree

  3. Department of Comparative Medicine, School of Medicine, University of Washington, Seattle, Washington 98195, USA, Washington

    Brian Iritani

  4. Department of Microbiology and Immunology, Stanford University, Stanford, California 94305, USA, California

    Joel R. Neilson

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Correspondence to Gerald R. Crabtree.

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The file contains Supplementary Figures 1-9 and Supplementary Table 1. (PDF 610 kb)

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Gallo, E., Winslow, M., Canté-Barrett, K. et al. Calcineurin sets the bandwidth for discrimination of signals during thymocyte development. Nature 450, 731–735 (2007). https://doi.org/10.1038/nature06305

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