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Automated high-throughput mapping of promoter-enhancer interactions in zebrafish embryos

Nature Methods volume 6, pages 911–916 (2009)Cite this article

Abstract

Zebrafish embryos offer a unique combination of high-throughput capabilities and the complexity of the vertebrate animal for a variety of phenotypic screening applications. However, there is a need for automation of imaging technologies to exploit the potential of the transparent embryo. Here we report a high-throughput pipeline for registering domain-specific reporter expression in zebrafish embryos with the aim of mapping the interactions between cis-regulatory modules and core promoters. Automated microscopy coupled with custom-built embryo detection and segmentation software allowed the spatial registration of reporter activity for 202 enhancer-promoter combinations, based on images of thousands of embryos. The diversity of promoter-enhancer interaction specificities underscores the importance of the core promoter sequence in cis-regulatory interactions and provides a promoter resource for transgenic reporter studies. The technology described here is also suitable for the spatial analysis of fluorescence readouts in genetic, pharmaceutical or toxicological screens.

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Figure 1: A pipeline for automated spatial registration of tissue-specific reporter gene activity in zebrafish embryos.
Figure 2: Two-dimensional embryo image warping tool to detect mosaic reporter expression patterns in groups of embryos.
Figure 3: Registration of domain-specific expression in transgenic embryos.
Figure 4: Activities of enhancer-promoter combinations.
Figure 5: Detection of domain specific changes of reporter gene expression in a stable transgenic line.

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Acknowledgements

We thank N. Borel for fish care, K. Straatman, N. Groebner and P. Kobor for technical support, T. Becker (Brain and Mind Research Institute, University of Sydney) for the dre-mir9-1 enhancer and R. Sanges and E. Stupka for unpublished promoter analysis. This work was supported by FP6 projects EUTRACC and TRANSCODE, by the European Commission and the Deutsche Forschungsgemeinschaft to F.M. and programme grant by the Helmholtz Association of German Research Centres (HGF) to U.L. We thank the technical support team of Olympus Germany and Olympus UK, U. Strähle for general support and N. Foulkes and A. Cullinane for advice and critical reading of the manuscript.

Author information

Author notes

  1. Chengyi Song

    Present address: Present address: College of Animal Science and Technology, Yangzhou University, Jiangsu, China.,

  2. Jochen Gehrig, Markus Reischl and Éva Kalmár: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany

    Jochen Gehrig, Éva Kalmár, Marco Ferg, Yavor Hadzhiev, Andreas Zaucker, Chengyi Song, Simone Schindler, Urban Liebel & Ferenc Müller

  2. Department of Medical and Molecular Genetics, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

    Jochen Gehrig, Yavor Hadzhiev, Andreas Zaucker & Ferenc Müller

  3. Institute of Applied Computer Science, Forschungszentrum Karlsruhe, Eggenstein Leopoldshafen, Germany

    Markus Reischl

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Contributions

E.K. created the constructs, E.K., J.G. led the screening and imaging of embryos, M.R. designed and created automation software and automated image processing, F.M. and U.L. supervised the screen, M.F., Y.H., A.Z., C.S. and S.S. participated in the embryo screen. J.G., M.R., E.K. and F.M. analyzed data, U.L. designed and constructed the screening platform, F.M., E.K., J.G., M.R. and U.L. designed the study and J.G., M.R., E.K. and F.M. wrote the manuscript.

Corresponding authors

Correspondence to Urban Liebel or Ferenc Müller.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–3, 5–7, Supplementary Note (PDF 3433 kb)

Supplementary Table 4

Number of embryos and domain specific Venus signal intensity data of enhancer-promoter combinations assayed in this study (XLS 67 kb)

Supplementary Software

Zebrafish 2D reporter gene expression profiler (ZIP 132 kb)

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Gehrig, J., Reischl, M., Kalmár, É. et al. Automated high-throughput mapping of promoter-enhancer interactions in zebrafish embryos. Nat Methods 6, 911–916 (2009). https://doi.org/10.1038/nmeth.1396

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