Purine-mediated signalling triggers eye development

Abstract

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...

Hyperglycemia alters E-NTPDases, ecto-5′-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio)

“…While we do not have specific information about the catalytic properties of ecto-nucleotidase isoforms expressed in zebrafish, assuming data from mammals and amphibian, the decrease in ATP hydrolysis seems here could involve NTPDase 2, due to hydrolysis ratio 30:1 (ATP:ADP) that contributes more effectively to ATP hydrolysis when compared with other NTPDases [15,49,50]. In fact, the expression of E-NTPDase 2 isoforms (entpd2a.1 and entpd2a) was significantly decreased in glucose-treated zebrafish.…”

Structural insight into signal conversion and inactivation by NTPDase2 in purinergic signaling

“…This behavior would potentially support platelet aggregation. Control of purinergic signaling by NTPDase2 has been implicated in the differentiation of stem cells in the nervous system (3,21) and was recently shown to be responsible for initiation of embryonic eye development (5). Both the processivity of NTPDase1 and the high ATPase:ADPase ratio of NTPDase2 depend on native interactions of the transmembrane domains (19,(22)(23)(24).…”

“…Cellular responses are induced by binding to two classes of P2 receptors: G proteincoupled P2Y receptors and ligand-gated P2X ion channels (1)(2)(3)(4)(5)(6). The ''purinergic signaling'' effects are converted and terminated via successive dephosphorylation of the nucleotides by a cascade of membrane-bound enzymes (7)(8)(9)(10)(11).…”

Measurement of purine release with microelectrode biosensors

“…However, a role for ATP signalling is now evident [64]. Masse et al reported that overexpression of NTPDase2 in the anterior part of the embryo could induce ectopic eye formation-an effect that was increased by simultaneous overexpression of the P2Y 1 receptor.…”

“…Note that ATP release does not occur at this time in the posterior location, although at later times, there is a clearly substantial ATP release in this location that may be involved in the control of the development of the future nervous system. Reproduced with permission from [64] simple sequential chain of gene activation, but involves both positive feedback from downstream genes to activate upstream genes and positive self-regulation of gene expression. Pax6 for example activates Six3, Lhx2 and tll, but these genes in turn upregulate expression of Pax6, which is also able to reinforce its own expression.…”