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Visualization of gene expression in whole mouse retina by in situ hybridization

Nature Protocols volume 7, pages 1086–1096 (2012)Cite this article

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Abstract

The mouse retinal vasculature provides a powerful model system for studying development and pathologies of the vasculature. Because it forms as a two-dimensional flat plexus, it is easily imaged in its entirety in whole-mount retinal preparations. In order to study molecular signaling mechanisms, it is useful to visualize the expression of specific genes in the entire vascular plexus and retina. However, in situ hybridization on whole-mount retinal preparations is problematic because isolated retinas have a tendency to curl up during hybridization and are difficult to stain. Here we provide a detailed protocol that overcomes these difficulties and visualizes the mRNA distribution of one or two genes in the context of the counterstained retinal vasculature. The protocol takes 3–4 d for single-probe stains, with an additional 2 d for immunohistochemistry co-labeling. In situ hybridization with two probes adds a further 3 d.

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Figure 1: Flow diagram of the protocol.
Figure 2: Instruments and retinal dissection.
Figure 3: Digestion and hybridization.
Figure 4: Examples of in situ hybridization stains.

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References

  1. Fruttiger, M. Development of the retinal vasculature. Angiogenesis 10, 77–88 (2007).

    Google Scholar 

  2. Gerhardt, H. et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell Biol. 161, 1163–1177 (2003).

    Google Scholar 

  3. Geudens, I. & Gerhardt, H. Coordinating cell behaviour during blood vessel formation. Development 138, 4569–4583 (2011).

    Google Scholar 

  4. Pitulescu, M.E., Schmidt, I., Benedito, R. & Adams, R.H. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat. Protoc. 5, 1518–1534 (2010).

    Google Scholar 

  5. Claxton, S. et al. Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis 46, 74–80 (2008).

    Google Scholar 

  6. Connor, K.M. et al. Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat. Protoc. 4, 1565–1573 (2009).

    Google Scholar 

  7. Scott, A. & Fruttiger, M. Oxygen-induced retinopathy: a model for vascular pathology in the retina. Eye (Lond.) 24, 416–421 (2010).

    Google Scholar 

  8. Sawamiphak, S., Ritter, M. & Acker-Palmer, A. Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses. Nat. Protoc. 5, 1659–1665 (2010).

    Google Scholar 

  9. Fruttiger, M. Development of the mouse retinal vasculature: angiogenesis versus vasculogenesis. Invest. Ophthalmol. Vis. Sci. 43, 522–527 (2002).

    Google Scholar 

  10. Claxton, S. & Fruttiger, M. Periodic Delta-like 4 expression in developing retinal arteries. Gene Expr. Patterns 5, 123–127 (2004).

    Google Scholar 

  11. Hellstrom, M. et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445, 776–780 (2007).

    Google Scholar 

  12. Benedito, R. et al. The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137, 1124–1135 (2009).

    Google Scholar 

  13. Lobov, I.B. et al. Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting. Proc. Natl. Acad. Sci. USA 104, 3219–3224 (2007).

    Google Scholar 

  14. Jensen, A.M. & Wallace, V.A. Expression of Sonic hedgehog and its putative role as a precursor cell mitogen in the developing mouse retina. Development 124, 363–371 (1997).

    Google Scholar 

  15. Hindges, R., McLaughlin, T., Genoud, N., Henkemeyer, M. & O′Leary, D.D. EphB forward signaling controls directional branch extension and arborization required for dorsal-ventral retinotopic mapping. Neuron 35, 475–487 (2002).

    Google Scholar 

  16. Birgbauer, E., Cowan, C.A., Sretavan, D.W. & Henkemeyer, M. Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina. Development 127, 1231–1241 (2000).

    Google Scholar 

  17. Herrington, C.S. Demystified in situ hybridisation. Mol. Pathol. 51, 8–13 (1998).

    Google Scholar 

  18. Jin, L. & Lloyd, R.V. In situ hybridization: methods and applications. J. Clin. Lab Anal. 11, 2–9 (1997).

    Google Scholar 

  19. Sambrook, J. & Russell, D.W. Protocol 6: synthesis of single-stranded RNA probes by in vitro transcription. in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001).

  20. Thisse, C. & Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat. Protoc. 3, 59–69 (2008).

    Google Scholar 

  21. David, R. & Wedlich, D. PCR-based RNA probes: a quick and sensitive method to improve whole mount embryo in situ hybridizations. Biotechniques 30, 769–772, 74 (2001).

    Google Scholar 

  22. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R.R. Techniques for visualizing gene products, cells, tissues and organ systems. in Manipulating the Mouse Embryo (eds. Nagy, A. et al.) (Cold Spring Harbor Laboratory Press, 2003).

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Acknowledgements

We are grateful to N. Pringle for many helpful tips during the development of this protocol. This work was supported by grants from the MRC (G0501711), The Wellcome Trust (WT086511MA) and the Lowy Medical Research Institute.

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Authors and Affiliations

  1. UCL Institute of Ophthalmology, University College London, London, UK

    Michael B Powner, Kristis Vevis, Jenny A G McKenzie, Pranita Gandhi, Shalini Jadeja & Marcus Fruttiger

Authors
  1. Michael B Powner
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  2. Kristis Vevis
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  3. Jenny A G McKenzie
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  4. Pranita Gandhi
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  5. Shalini Jadeja
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  6. Marcus Fruttiger
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Contributions

All authors contributed to the development of the protocol and M.B.P., K.V., J.A.G.M. and M.F. wrote the manuscript.

Corresponding author

Correspondence to Marcus Fruttiger.

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The authors declare no competing financial interests.

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Powner, M., Vevis, K., McKenzie, J. et al. Visualization of gene expression in whole mouse retina by in situ hybridization. Nat Protoc 7, 1086–1096 (2012). https://doi.org/10.1038/nprot.2012.050

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