Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system

Nature volume 450, pages 56–62 (2007)Cite this article

Abstract

Detailed analysis of neuronal network architecture requires the development of new methods. Here we present strategies to visualize synaptic circuits by genetically labelling neurons with multiple, distinct colours. In Brainbow transgenes, Cre/lox recombination is used to create a stochastic choice of expression between three or more fluorescent proteins (XFPs). Integration of tandem Brainbow copies in transgenic mice yielded combinatorial XFP expression, and thus many colours, thereby providing a way to distinguish adjacent neurons and visualize other cellular interactions. As a demonstration, we reconstructed hundreds of neighbouring axons and multiple synaptic contacts in one small volume of a cerebellar lobe exhibiting approximately 90 colours. The expression in some lines also allowed us to map glial territories and follow glial cells and neurons over time in vivo. The ability of the Brainbow system to label uniquely many individual cells within a population may facilitate the analysis of neuronal circuitry on a large scale.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Brainbow-1 : stochastic recombination using incompatible lox variants.
Figure 2: Brainbow-2 : stochastic recombination using Cre-mediated inversion.
Figure 3: XFP expression in Brainbow transgenic mice.
Figure 4: Combinatorial XFP expression results from tandem copy integration.
Figure 5: Cerebellar circuit tracing and colour analysis.
Figure 6: Brainbow expression in glial cells and time-lapse imaging.

Similar content being viewed by others

References

  1. Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004)

    Article  Google Scholar 

  2. Briggman, K. L. & Denk, W. Towards neural circuit reconstruction with volume electron microscopy techniques. Curr. Opin. Neurobiol. 16, 562–570 (2006)

    Article  CAS  Google Scholar 

  3. Micheva, K. D. & Smith, S. J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25–36 (2007)

    Article  CAS  Google Scholar 

  4. Gan, W. B., Grutzendler, J., Wong, W. T., Wong, R. O. & Lichtman, J. W. Multicolor “DiOlistic” labeling of the nervous system using lipophilic dye combinations. Neuron 27, 219–225 (2000)

    Article  CAS  Google Scholar 

  5. Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nature Methods 2, 905–909 (2005)

    Article  CAS  Google Scholar 

  6. Hadjantonakis, A. K., Macmaster, S. & Nagy, A. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnol. 2, 11 (2002)

    Article  Google Scholar 

  7. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000)

    Article  CAS  Google Scholar 

  8. Kasthuri, N. & Lichtman, J. W. The role of neuronal identity in synaptic competition. Nature 424, 426–430 (2003)

    Article  ADS  CAS  Google Scholar 

  9. Zong, H., Espinosa, J. S., Su, H. H., Muzumdar, M. D. & Luo, L. Mosaic analysis with double markers in mice. Cell 121, 479–492 (2005)

    Article  CAS  Google Scholar 

  10. Branda, C. S. & Dymecki, S. M. Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28 (2004)

    Article  CAS  Google Scholar 

  11. Lam, K. P. & Rajewsky, K. Rapid elimination of mature autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity in vivo . Proc. Natl Acad. Sci. USA 95, 13171–13175 (1998)

    Article  ADS  CAS  Google Scholar 

  12. Lee, G. & Saito, I. Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216, 55–65 (1998)

    Article  CAS  Google Scholar 

  13. Caroni, P. Overexpression of growth-associated proteins in the neurons of adult transgenic mice. J. Neurosci. Methods 71, 3–9 (1997)

    Article  CAS  Google Scholar 

  14. Guo, C., Yang, W. & Lobe, C. G. A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis 32, 8–18 (2002)

    Article  CAS  Google Scholar 

  15. Metzger, D., Clifford, J., Chiba, H. & Chambon, P. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc. Natl Acad. Sci. USA 92, 6991–6995 (1995)

    Article  ADS  CAS  Google Scholar 

  16. Garrick, D., Fiering, S., Martin, D. I. & Whitelaw, E. Repeat-induced gene silencing in mammals. Nature Genet. 18, 56–59 (1998)

    Article  CAS  Google Scholar 

  17. Palmiter, R. D. & Brinster, R. L. Germ-line transformation of mice. Annu. Rev. Genet. 20, 465–499 (1986)

    Article  CAS  Google Scholar 

  18. Rodriguez, C. I. & Dymecki, S. M. Origin of the precerebellar system. Neuron 27, 475–486 (2000)

    Article  CAS  Google Scholar 

  19. Rowan, S. & Cepko, C. L. Genetic analysis of the homeodomain transcription factor Chx10 in the retina using a novel multifunctional BAC transgenic mouse reporter. Dev. Biol. 271, 388–402 (2004)

    Article  CAS  Google Scholar 

  20. Eccles, J. C., Ito, M. & Szentágothai, J. in The Cerebellum as a Neuronal Machine (Springer, Berlin, 1967)

    Book  Google Scholar 

  21. Wu, H. S., Sugihara, I. & Shinoda, Y. Projection patterns of single mossy fibers originating from the lateral reticular nucleus in the rat cerebellar cortex and nuclei. J. Comp. Neurol. 411, 97–118 (1999)

    Article  CAS  Google Scholar 

  22. Palay, S. L. & Chan-Palay, V. in Cerebellar Cortex (Springer, New York, 1974)

    Book  Google Scholar 

  23. Bushong, E. A., Martone, M. E., Jones, Y. Z. & Ellisman, M. H. Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains. J. Neurosci. 22, 183–192 (2002)

    Article  CAS  Google Scholar 

  24. Ogata, K. & Kosaka, T. Structural and quantitative analysis of astrocytes in the mouse hippocampus. Neuroscience 113, 221–233 (2002)

    Article  CAS  Google Scholar 

  25. Silver, D. P. & Livingston, D. M. Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Mol. Cell 8, 233–243 (2001)

    Article  CAS  Google Scholar 

  26. Gong, S. et al. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425, 917–925 (2003)

    Article  ADS  CAS  Google Scholar 

  27. Lein, E. S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007)

    Article  ADS  CAS  Google Scholar 

  28. Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002)

    Article  ADS  CAS  Google Scholar 

  29. Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnol. 22, 445–449 (2004)

    Article  CAS  Google Scholar 

  30. Karasawa, S., Araki, T., Nagai, T., Mizuno, H. & Miyawaki, A. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem. J. 381, 307–312 (2004)

    Article  CAS  Google Scholar 

  31. Campbell, R. E. et al. A monomeric red fluorescent protein. Proc. Natl Acad. Sci. USA 99, 7877–7882 (2002)

    Article  ADS  CAS  Google Scholar 

  32. Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004)

    Article  CAS  Google Scholar 

  33. Kay, J. N. et al. Transient requirement for ganglion cells during assembly of retinal synaptic layers. Development 131, 1331–1342 (2004)

    Article  CAS  Google Scholar 

  34. Buffelli, M. et al. Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition. Nature 424, 430–434 (2003)

    Article  ADS  CAS  Google Scholar 

  35. Zuo, Y. et al. Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination. J. Neurosci. 24, 10999–11009 (2004)

    Article  CAS  Google Scholar 

  36. Fiala, J. C. Reconstruct: a free editor for serial section microscopy. J. Microsc. 218, 52–61 (2005)

    Article  MathSciNet  CAS  Google Scholar 

  37. Kakoki, M. et al. Altering the expression in mice of genes by modifying their 3′ regions. Dev. Cell 6, 597–606 (2004)

    Article  CAS  Google Scholar 

  38. Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001)

    Article  CAS  Google Scholar 

  39. Marquardt, T. et al. Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105, 43–55 (2001)

    Article  CAS  Google Scholar 

  40. Gorski, J. A. et al. Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J. Neurosci. 22, 6309–6314 (2002)

    Article  CAS  Google Scholar 

  41. Ringrose, L., Chabanis, S., Angrand, P. O., Woodroofe, C. & Stewart, A. F. Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. EMBO J. 18, 6630–6641 (1999)

    Article  CAS  Google Scholar 

  42. Zheng, B., Sage, M., Sheppeard, E. A., Jurecic, V. & Bradley, A. Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications. Mol. Cell. Biol. 20, 648–655 (2000)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank members of the Lichtman and Sanes laboratories for discussions; H. Nishimune for insights; A. Mendelsohn and A. Bruskin for cell culture and PCR; S.-H. Shieu and D. Mou for imaging software development; S. Haddad, D. Pelusi, D. Malkowski, K. Mahoney and the Harvard MCB BRI facility for animal care and genotyping; M. Wallace, the WUSTL Mouse Genetic Core and the MCB Genome Manipulation Facility for pronuclei injections; and R. Hellmiss for help with graphics. We thank J. C. Fiala for assistance with Reconstruct; M. L. Nonet, J. S. Mumm and R. O. Wong for advice and reagents; D. W. Piston for mCerulean; R. Y. Tsien for tdimer2 and mCherry; and A. Miyawaki for Kusabira. This work was supported by the James S. McDonnell foundation and grants from NIH.

Author Contributions J.L., J.R.S. and J.W.L. conceived the Brainbow strategies. J.R.S. and J.W.L. supervised the project. J.L. built initial constructs and validated them in vitro and in vivo. T.A.W. performed all cerebellar axonal tracing and colour profile analysis with programs developed with J. Lu. H.K. performed all live imaging experiments. R.W.D. generated Brainbow-1.0 lines expressing cytoplasmic XFPs, and R.A.B. generated Brainbow-1.1 constructs and lines. J.L., T.A.W. and R.W.D. screened mouse lines.

Author information

Authors and Affiliations

  1. Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA,

    Jean Livet, Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, Robyn A. Bennis, Joshua R. Sanes & Jeff W. Lichtman

Authors
  1. Jean Livet
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Tamily A. Weissman
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Hyuno Kang
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Ryan W. Draft
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Ju Lu
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Robyn A. Bennis
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Joshua R. Sanes
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Jeff W. Lichtman
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence to Jeff W. Lichtman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-6 with Legends and Supplementary Tables 1-2. (PDF 10125 kb)

Supplementary Movie 1

The file contains Supplementary Movie 1 which shows rotation of the cerebellar reconstruction. (MOV 39667 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Livet, J., Weissman, T., Kang, H. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007). https://doi.org/10.1038/nature06293

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06293

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing