A conserved network of eye field transcription factors (EFTFs) underlies the development of the eye in vertebrates and invertebrates 1 . To direct eye development, Pax6, a key gene in this network 2,3 , interacts with genes encoding other EFTFs such as . However, the mechanisms that control expression of the EFTFs remain unclear 7 . 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 ADP 9 , 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 ADP 10,11 , 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 release 12-14 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 purinemediated 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 microphthalmia [15][16][17][18] . Our results suggest a new mechanism for the initiation of eye development.To assess the developmental functions of the E-NTPDases, we simultaneously injected the mRNA for the closely related ENTPDases1-3 (ref. 8) with lineage tracer into a dorsal animal blastomere at the eight-cell stage to target overexpression of this gene to one side of the nervous system. Overexpression of E-NTPDase2 affected eye development in 27 of 41 embryos, causing in some cases complete duplication of the eye on the injected side (Fig. 1A, a). In contrast, overexpression of E-NTPDase1 decreased eye size (11 of 44 embryos; Fig. 1A, b), whereas overexpression of E-NTPDase3 gave a weaker phenotype somewhat similar to that of E-NTPDase2 (4 of 42 embryos, Fig. 1A, c, Supplementary Tables 1a and 2). E-NTPDases differ in their catalytic activity. Like their mammalian orthologues, E-NTPDase1 can metabolize ATP and ADP with roughly equal efficacy, E-NTPDase2 is highly selective for ATP and hardly metabolizes ADP, and E-NTPDase3 has intermediate selectivity for ATP and ADP ( Supplementary Fig. 2). The phenotypes elicited by overexpression of these membrane-bound E-NTPDases correlate with their capacity to metabolize ADP.The eye phenotypes caused by overexpression of E-NTPDase2 (Supplementary Table 1b) included the following: disrupted eye development (Fig. 1A, d); ectopic retinal pigment epithelium (RPE) (Fig. 1A, e); RPE extensions (Fig. 1A, f); and ectopic...

No abstract