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Purine-mediated signalling triggers eye development

Nature volume 449, pages 1058–1062 (2007)Cite this article

Abstract

A conserved network of eye field transcription factors (EFTFs) underlies the development of the eye in vertebrates and invertebrates1. To direct eye development, Pax6, a key gene in this network2,3, interacts with genes encoding other EFTFs such as Rx1 and Six3 (refs 4–6). However, the mechanisms that control expression of the EFTFs remain unclear7. Here we show that purine-mediated signalling triggers both EFTF expression and eye development in Xenopus laevis. Overexpression of ectonucleoside triphosphate diphosphohydrolase 2 (E-NTPDase2)8, an ectoenzyme that converts ATP to ADP9, caused ectopic eye-like structures, with occasional complete duplication of the eye, and increased expression of Pax6, Rx1 and Six3. In contrast, downregulation of endogenous E-NTPDase2 decreased Rx1 and Pax6 expression. E-NTPDase2 therefore acts upstream of these EFTFs. To test whether ADP (the product of E-NTPDase2) might act to trigger eye development through P2Y1 receptors, selective in Xenopus for ADP10,11, we simultaneously knocked down expression of the genes encoding E-NTPDase2 and the P2Y1 receptor. This could prevent the expression of Rx1 and Pax6 and eye formation completely. We next measured ATP release12,13,14 in the presumptive eye field, demonstrating a transient release of ATP at a time that could plausibly trigger (once converted to ADP) expression of the EFTFs. This surprising role for transient purine-mediated signalling in eye development may be widely conserved, because alterations to the locus of E-NTPDase2 on human chromosome 9 cause severe head and eye defects, including microphthalmia15,16,17,18. Our results suggest a new mechanism for the initiation of eye development.

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Figure 1: E-NTPDase2 induces formation of ectopic eye-like structures.
Figure 2: Mis-expression of E-NTPDase2 alters EFTF expression.
Figure 3: Both E-NTPDase2 and P2Y1 receptors are necessary for EFTF expression.
Figure 4: E-NTPDase2 and P2Y1 receptors synergistically affect eye development.
Figure 5: Pax6 overexpression rescues loss of eye phenotype induced by knockdown of E-NTPDase2 and P2Y1.
Figure 6: All components of purinergic signalling are present for the initiation of eye development.

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Acknowledgements

We thank P. Jarrett for the maintenance of the frogs and E. Llaudet for the production of biosensors. We thank M. Andreazzoli, G. Guidetti, W. Harris, M. Hodgkin, H. Isaacs, A. Philpott, D. Sakaguchi, C. Smith and P. Stanfield for constructs and antibodies used in this work. This work was supported by the Wellcome Trust.

Author Contributions K.M. performed all molecular biology. K.M. and E.A.J. performed the mis-expression studies and phenotype analysis. E.A.J. performed all microinjections and dissection. S.B. performed in situ hybridization and sectioning, R.E. performed enzymatic activity assays, N.D. performed the ATP biosensor studies and the statistical analysis with K.M. E.A.J. and N.D. supervised the research project. K.M., N.D. and E.A.J. wrote the manuscript.

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  1. Nicholas Dale and Elizabeth A. Jones: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Warwick University, Coventry CV4 7AL, UK

    Karine Massé, Surinder Bhamra, Robert Eason, Nicholas Dale & Elizabeth A. Jones

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  1. Karine Massé
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Correspondence to Nicholas Dale or Elizabeth A. Jones.

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Competing interests

N.D. is a director and shareholder in Sarissa Biomedical Ltd., a company that makes biosensors similar to those used in this study.

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Supplementary Information

The file contains Supplementary Tables 1-5 and Supplementary Figures 1-9 with Legends. (PDF 2602 kb)

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Massé, K., Bhamra, S., Eason, R. et al. Purine-mediated signalling triggers eye development. Nature 449, 1058–1062 (2007). https://doi.org/10.1038/nature06189

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