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.
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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).
The authors declare no competing financial interests.
Supplementary Figures 1–17, Supplementary Tables 1–4 and Supplementary Notes 1–4 (PDF 16979 kb)
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 (ZIP 206908 kb)
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 (ZIP 67846 kb)
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 (ZIP 98717 kb)
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 (ZIP 4288 kb)
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 (ZIP 4664 kb)
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. (ZIP 1 kb)
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. (AVI 9703 kb)
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. (AVI 2203 kb)
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. (AVI 2389 kb)
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. (AVI 13290 kb)
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. (AVI 24116 kb)
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. (AVI 18142 kb)
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. (AVI 16198 kb)
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. (AVI 14998 kb)
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. (AVI 16852 kb)
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. (AVI 16167 kb)
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. (AVI 20870 kb)
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. (AVI 19143 kb)
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. (AVI 17317 kb)
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Ronneberger, O., Liu, K., Rath, M. et al. ViBE-Z: a framework for 3D virtual colocalization analysis in zebrafish larval brains. Nat Methods 9, 735–742 (2012). https://doi.org/10.1038/nmeth.2076
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