Article | Published:

ViBE-Z: a framework for 3D virtual colocalization analysis in zebrafish larval brains

Nature Methods volume 9, pages 735742 (2012) | Download Citation

  • An Erratum to this article was published on 27 September 2012

This article has been updated

Abstract

Precise three-dimensional (3D) mapping of a large number of gene expression patterns, neuronal types and connections to an anatomical reference helps us to understand the vertebrate brain and its development. We developed the Virtual Brain Explorer (ViBE-Z), a software that automatically maps gene expression data with cellular resolution to a 3D standard larval zebrafish (Danio rerio) brain. ViBE-Z enhances the data quality through fusion and attenuation correction of multiple confocal microscope stacks per specimen and uses a fluorescent stain of cell nuclei for image registration. It automatically detects 14 predefined anatomical landmarks for aligning new data with the reference brain. ViBE-Z performs colocalization analysis in expression databases for anatomical domains or subdomains defined by any specific pattern; here we demonstrate its utility for mapping neurons of the dopaminergic system. The ViBE-Z database, atlas and software are provided via a web interface.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Change history

  • 09 July 2012

    In the version of this article initially published, two items in the Online Methods section were incorrect. The MATLAB code in the 'ViBE-Z database file' section contained an extraneous semicolon, which appeared in the HTML only and has been corrected. In the section 'Stitching and dorsal-ventral alignment', two formulas had a 'mapsto' symbol instead of an arrow. These errors have been corrected in the HTML and PDF versions of the article.

References

  1. 1.

    et al. Analysis of cell fate from single-cell gene expression profiles in C. elegans. Cell 139, 623–633 (2009).

  2. 2.

    , , , & A 3D digital atlas of C. elegans and its application to single-cell analyses. Nat. Methods 6, 667–672 (2009).

  3. 3.

    et al. Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution. Curr. Biol. 21, 1–11 (2011).

  4. 4.

    et al. BrainAligner: 3D registration atlases of Drosophila brains. Nat. Methods 8, 493–500 (2011).

  5. 5.

    et al. Visualization of image data from cells to organisms. Nat. Methods 7, S26–S41 (2010).

  6. 6.

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

  7. 7.

    et al. Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Methods Cell Biol. 77, 505–519 (2004).

  8. 8.

    et al. Expression of the zebrafish genome during embryogenesis. ZFIN Zebrafish Model Organism Database <> (2001).

  9. 9.

    & Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

  10. 10.

    et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14, 1481–1488 (2011).

  11. 11.

    , , , & Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

  12. 12.

    et al. Automated high-throughput mapping of promoter-enhancer interactions in zebrafish embryos. Nat. Methods 6, 911–916 (2009).

  13. 13.

    et al. Attenuation correction in confocal laser microscopes: a novel two-view approach. J. Microsc. 211, 67–79 (2003).

  14. 14.

    , , , & Adaptive correction technique for 3D reconstruction of fluorescence microscopy images. Microsc. Res. Tech. 71, 146–157 (2008).

  15. 15.

    et al. Fast rotation invariant 3D feature computation utilizing efficient local neighborhood operators. IEEE Trans. Pattern Anal. Mach. Intell. published online, doi:10.1109/TPAMI.2011.263 (20 December 2011).

  16. 16.

    , & Invariants and covariants in pattern recognition and image analysis in Pattern Recognition and Machine Vision (ed. Wang, P.S.-P.) Ch. 5, 71–85 (River, 2010).

  17. 17.

    et al. Harmonic Filters for 3D multi-channel data: rotation invariant detection of mitoses in colorectal cancer. IEEE Trans. Med. Imaging 29, 1485–1495 (2010).

  18. 18.

    , , , & Dense image registration through MRFs and efficient linear programming. Med. Image Anal. 12, 731–741 (2008).

  19. 19.

    & Atlas of Early Zebrafish Brain Development: a Tool for Molecular Neurogenetics (Elsevier, 2005).

  20. 20.

    , , , & Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nat. Commun. 2, 171 (2011).

  21. 21.

    , , , & Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish. J. Neurosci. 26, 13400–13410 (2006).

  22. 22.

    , , , & Emx3 is required for the differentiation of dorsal telencephalic neurons. Dev. Dyn. 238, 1984–1998 (2009).

  23. 23.

    et al. Orthopedia homeodomain protein is essential for diencephalic dopaminergic neuron development. Curr. Biol. 17, 873–880 (2007).

  24. 24.

    et al. Active suppression of interneuron programs within developing motor neurons revealed by analysis of homeodomain factor HB9. Neuron 23, 675–687 (1999).

  25. 25.

    et al. Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. Dev. Biol. 314, 84–92 (2008).

  26. 26.

    , , , & Arcuate nucleus expression of NKX2.1 and DLX and lineages expressing these transcription factors in neuropeptide Y(+), proopiomelanocortin(+), and tyrosine hydroxylase(+) neurons in neonatal and adult mice. J. Comp. Neurol. 517, 37–50 (2009).

  27. 27.

    et al. In vivo protein trapping produces a functional expression codex of the vertebrate proteome. Nat. Methods 8, 506–515 (2011).

  28. 28.

    The Zebrafish Book: a Guide for the Laboratory Use of Zebrafish (Danio rerio) 4th edn (University of Oregon Press, 2000).

  29. 29.

    , & Migration and function of a glial subtype in the vertebrate peripheral nervous system. Neuron 34, 577–588 (2002).

  30. 30.

    , & Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer. J. Neurosci. 20, 206–218 (2000).

  31. 31.

    , & Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo. Mech. Dev. 120, 211–218 (2003).

  32. 32.

    , , , & Neural cell fate analysis in zebrafish using olig2 BAC transgenics. Methods Cell Sci. 25, 7–14 (2003).

  33. 33.

    , , & Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations. Development 132, 4471–4481 (2005).

  34. 34.

    et al. A highly conserved enhancer in the Dlx5/Dlx6 intergenic region is the site of cross-regulatory interactions between Dlx genes in the embryonic forebrain. J. Neurosci. 20, 709–721 (2000).

  35. 35.

    , , , & Function and regulation of zebrafish nkx2.2a during development of pancreatic islet and ducts. Dev. Biol. 304, 875–890 (2007).

  36. 36.

    et al. Noradrenergic neurons in the zebrafish hindbrain are induced by retinoic acid and require tfap2a for expression of the neurotransmitter phenotype. Development 130, 5741–5754 (2003).

  37. 37.

    et al. The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity. Cell 61, 157–170 (1990).

  38. 38.

    et al. Expression and function of nr4a2, lmx1b, and pitx3 in zebrafish dopaminergic and noradrenergic neuronal development. BMC Dev. Biol. 7, 135 (2007).

  39. 39.

    , , & Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo. Mech. Dev. 101, 237–243 (2001).

  40. 40.

    , , & Spatially and temporally-restricted expression of two T-box genes during zebrafish embryogenesis. Mech. Dev. 80, 219–221 (1999).

  41. 41.

    , & Distribution of prospective glutamatergic, glycinergic, and GABAergic neurons in embryonic and larval zebrafish. J. Comp. Neurol. 480, 1–18 (2004).

  42. 42.

    et al. XuvTools: free, fast and reliable stitching of large 3D datasets. J. Microsc. 233, 42–60 (2009).

  43. 43.

    Principal warps: thin-plate splines and the decomposition of deformations. IEEE Trans. Pattern Anal. Mach. Intell. 11, 567–585 (1989).

  44. 44.

    , & Performance vs computational efficiency for optimizing single and dynamic mrfs: setting the state of the art with primal-dual strategies. Comput. Vis. Image Underst. 112, 14–29 (2008).

  45. 45.

    et al. Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A 58, 167–176 (2004).

Download references

Acknowledgements

The authors would like to thank S. Lin (University of California, Los Angeles), Z. Varga (University of Oregon), D. Gilmour (EMBL), M. Manoli (University of Freiburg), H. Okamoto (RIKEN), U. Strähle (Karlsruhe Institute of Technology), B. Appel (University of Colorado, Denver), D. Meyer (University of Innsbruck), J. Schweitzer (University of Freiburg), E. Zecchin and F. Argenton (University of Padova) and M. Ekker (University of Ottawa) for sharing transgenic zebrafish lines, and S. Götter for excellent fish care. Special thanks to the staff of the Center for Systems Biology Life Imaging Center for excellent confocal microscopy resources. We are grateful to A. Arrenberg, D. Onichtchouck and J. Schweitzer for critical discussion. This work was funded by the Excellence Initiative of the German Federal and State Governments (Centre for Biological Signalling Studies EXC 294; Freiburg Institute for Advanced Studies) as well as by the European Commission projects 223744 (ZF-HEALTH), 222999 (mesDANEURODEV) and 223744 (DOPAMINET).

Author information

Affiliations

  1. Computer Science Department, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

    • Olaf Ronneberger
    • , Kun Liu
    • , Dominik Rueβ
    • , Henrik Skibbe
    • , Benjamin Drayer
    • , Thorsten Schmidt
    • , Thomas Brox
    •  & Hans Burkhardt
  2. BIOSS Centre for Biological Signalling Studies, Freiburg, Germany.

    • Olaf Ronneberger
    • , Kun Liu
    • , Benjamin Drayer
    • , Thorsten Schmidt
    • , Roland Nitschke
    • , Thomas Brox
    • , Hans Burkhardt
    •  & Wolfgang Driever
  3. Department of Developmental Biology, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

    • Meta Rath
    • , Thomas Mueller
    • , Alida Filippi
    •  & Wolfgang Driever
  4. Freiburg Institute for Advanced Studies, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

    • Thomas Mueller
    •  & Wolfgang Driever
  5. Zentrum für Biosystemanalyse, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

    • Roland Nitschke
    •  & Wolfgang Driever

Authors

  1. Search for Olaf Ronneberger in:

  2. Search for Kun Liu in:

  3. Search for Meta Rath in:

  4. Search for Dominik Rueβ in:

  5. Search for Thomas Mueller in:

  6. Search for Henrik Skibbe in:

  7. Search for Benjamin Drayer in:

  8. Search for Thorsten Schmidt in:

  9. Search for Alida Filippi in:

  10. Search for Roland Nitschke in:

  11. Search for Thomas Brox in:

  12. Search for Hans Burkhardt in:

  13. Search for Wolfgang Driever in:

Contributions

O.R. designed and implemented the ViBE-Z platform and performed analyses. W.D. designed the biological experiments and performed analyses. O.R. developed the variational absorption correction. K.L., D.R., H.S. and O.R. developed the landmark detection; B.D. and O.R. developed the elastic registration. T.S. contributed software and libraries. M.R. and A.F. prepared the samples and acquired confocal images. W.D. and H.B. initiated the project. T.M. generated the brain segmentation. R.N. contributed to confocal microscopy analysis. O.R., K.L., T.B. and W.D. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Olaf Ronneberger or Wolfgang Driever.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–17, Supplementary Tables 1–4 and Supplementary Notes 1–4

Zip files

  1. 1.

    Supplementary Data 1

    The ViBE-Z database for 72-h.p.f. larvae. The HDF5 file contains the following data sets (All data sets are of type uint8, have 800 × 500 × 500 voxel and an element size of 1 × 1 × 1 μm3):  /anatomy/average_brain  /anatomy/reference_larvae  /anatomy/segment_lines  /anatomy/segment_regions  /expression/3A10  /expression/AcTub  /expression/TH  /expression/dlx5a_6a_GFP  /expression/emx3_YFP  /expression/foxd3_GFP  /expression/glyt2_WISH  /expression/hb9_GFP  /expression/hcrt_EGFP  /expression/isl1_GFP  /expression/neurod_GFP  /expression/nkx2.2a_GFP  /expression/olig2_EGFP  /expression/otpb_GFP  /expression/tbr1b_WISH  /expression/th_WISH  /expression/vmat2_GFP

  2. 2.

    Supplementary Data 2

    The ViBE-Z database for 48-h.p.f. larvae. The HDF5 file contains the following data sets (All data sets are of type uint8, have 800 × 500 × 500 voxel and an element size of 1 × 1 × 1 μm3):  /anatomy/average_brain  /anatomy/reference_larvae  /anatomy/segment_lines  /anatomy/segment_regions

  3. 3.

    Supplementary Data 3

    The ViBE-Z database for 96-h.p.f. larvae. The HDF5 file contains the following data sets (All data sets are of type uint8, have 1,000 × 500 × 500 voxel and an element size of 1 × 1 × 1 μm3):  /anatomy/average_brain  /anatomy/reference_larvae  /anatomy/segment_lines  /anatomy/segment_regions

  4. 4.

    Supplementary Software 1

    HDF5 plugin for ImageJ for 32 bit Systems (Linux, Windows and MacOS). Unzip this file in the ImageJ installation directory and restart ImageJ. Check for updates on http://lmb.informatik.uni-freiburg.de/resources/opensource/imagej_plugins/hdf5.html

  5. 5.

    Supplementary Software 2

    HDF5 plugin for ImageJ for 64 bit Systems (Linux, Windows and MacOS). Unzip this file in the ImageJ installation directory and restart ImageJ. Check for updates on http://lmb.informatik.uni-freiburg.de/resources/opensource/imagej_plugins/hdf5.html

  6. 6.

    Supplementary Software 3

    Zebrafish Atlas color lookup table for ImageJ. The color lookup table to view the anatomical segments of the ViBE-Z database in the same colors as used for this paper. Copy this file into the “luts” directory in your ImageJ installation, and restart ImageJ.

Videos

  1. 1.

    Supplementary Video 1

    Computation of an average brain nuclear stain intensity. Overlay of the nuclear stain data for all 71 larvae in the database. The average nucleus channel was computed from all larvae in the database. The movie shows the stack from dorsal to ventral.

  2. 2.

    Supplementary Video 2

    Grouped anatomical segmentation of reference larva 3D volume. Surface rendering of each anatomical part, grouped by major brain regions, in movie sequence. Dorsoanterior view; for color code see Supplementary Table 2.

  3. 3.

    Supplementary Video 3

    Individual anatomical segmentation of reference larva 3D volume. Surface rendering of each anatomical part individually in a movie sequence. Dorsoanterior view; for color code see Supplementary Table 2.

  4. 4.

    Supplementary Video 4

    Anatomical segmentation of reference larva 3D volume with color-coded anatomical regions. For color code, see Supplementary Table 2. The movie shows the stack from dorsal to ventral.

  5. 5.

    Supplementary Video 5

    Anti-TH immunofluorescence combination of data stacks with the anatomical model in white lines. This representation identifies anatomical domains of gene expression stain of e138, e139 and e141 combined into anatomical standard model. The movie shows the stack from dorsal to ventral. The intensities have been assigned the ImageJ Fire-LUT colors (color code: see Figure 4b). The data stacks are combined with the anatomical model segment borders in white lines to identify anatomical domains of gene expression.

  6. 6.

    Supplementary Video 6

    Anti-TH immunofluorescence combination of data stacks with the anatomical model in color representation. This representation identifies anatomical domains of gene expression stain of e138, e139 and e141 combined into anatomical standard model. The movie shows the stack from dorsal to ventral. The intensities have been assigned the ImageJ Fire-LUT colors (color code: see Figure 4b). The data stacks are combined with the anatomical model in color, for color code see Supplementary Table 2.

  7. 7.

    Supplementary Video 7

    TH and Vmat2 colocalization. Colocalization analysis of TH (green, anti-TH immunofluorescence, three-larva overlay) and Vmat2 (red, anti-GFP immunofluorescence in vmat2:GFP, three-larva overlay) revealing colocalization of TH and Vmat2 expression in catecholaminergic neurons. The movie shows the stack from dorsal to ventral.

  8. 8.

    Supplementary Video 8

    TH, Vmat2, and Otpb colocalization. Colocalization analysis of TH (red, anti-TH immunofluorescence, three-larvae overlay), Vmat2 (blue, anti-GFP immunofluorescence in vmat2:GFP, three-larva overlay) and Otpb (green, anti-GFP immunofluorescence in otpb:GFP, three-larva overlay) revealing colocalization of TH, Vmat2 and Otpb expression in posterior tubercular dopaminergic neurons. The movie shows the stack from dorsal to ventral.

  9. 9.

    Supplementary Video 9

    TH, AcTub, and 3A10 colocalization. Analysis of axon bundles containing acetylated tubulin (blue, anti-AcTub immunofluorescence, three-larva overlay), TH (red, anti-TH immunofluorescence, three-larva overlay) and surface antigen 3A10 (green, 3A10 immunofluorescence in otpb:GFP, three-larva overlay). The movie shows the stack from dorsal to ventral.

  10. 10.

    Supplementary Video 10

    Dlx5a/6a, Foxd3 and Emx3 colocalization. Analysis of the relative positioning of the expression domains of transcription factors involved in brain patterning and differentiation: Dlx5a/6a domain (blue, anti-GFP immunofluorescence in dlx5a/6a:GFP, three-larva overlay), Foxd3 (red, anti-GFP immunofluorescence in foxd3:GFP, three-larva overlay) and Emx3 (green, anti-GFP immunofluorescence in emx3:GFP, three-larva overlay), revealing exclusion expression domains and localization of boundaries between domains. The movie shows the stack from dorsal to ventral.

  11. 11.

    Supplementary Video 11

    Multi-expression analysis. Virtual qualitative colocalization analysis of 11 different expression patterns representing 33 stained larvae. The color code is shown in Figure 4i. The movie shows the stack from dorsal to ventral.

  12. 12.

    Supplementary Video 12

    Protein and mRNA signal colocalization. Registration of tyrosine hydroxylase mRNA (th WISH, red; three larvae) and protein (anti-TH immunofluorescence, green; three larvae) detection reveals colocalization of catecholaminergic somata. The movie shows a stack from dorsal to ventral.

  13. 13.

    Supplementary Video 13

    WISH data registration. Registration of Tbr1b transcription factor (green, WISH, three larvae) and Glyt2 marker for glycinergic neuron (red, WISH, three larvae) mRNA expression to the anatomical model (blue lines). In this stack the nuclear stain calculated for the 3-dpf average larvae is shown for reference. The movie shows a stack from dorsal to ventral.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.2076

Further reading