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...

Ectonucleotidase proteins occupy a central role in purine signalling regulation by sequentially hydrolysing ATP to ADP and to adenosine. The ENPP ( or PDNP) gene family, which encodes ectophosphodiesterase/nucleotide phosphohydrolases, is a subfamily of these enzymes, which consists of 7 members in mammals. These proteins catalyse the generation of bioactive lipids, placing the ENPP enzymes as key regulators of major physiological signalling pathways and also important players in several pathological conditions. Here we report the cloning of all the members, except enpp5, of the enpp family in Xenopus laevis and tropicalis. Phylogenetic analyses demonstrate the high level of conservation of these proteins between amphibian and other vertebrate species. During development and in the adult frog, each gene displays a distinct specific expression pattern, suggesting potentially different functions for these proteins during amphibian embryogenesis. This is the first complete developmental analysis of gene expression of this gene family in vertebrates.

The purines, ATP and adenosine, are important signaling molecules in the nervous system. ATP is sequentially degraded to adenosine by the ectonucleotidase proteins. The NTPDase (or CD39) family is a subfamily of these enzymes, which consists of nine members in mammals. In Xenopus embryos, we have shown that ATP, and its antagonist adenosine, regulate the rundown of swimming and we therefore proposed that ectonucleotidase proteins are key regulators of locomotor activity. Here, we report the cloning of all nine members of the NTPDase family in Xenopus laevis and Xenopus tropicalis. Our phylogenetic analysis shows that this family is highly conserved between the frog species and also during vertebrate evolution. In the adult frog, NTPDase genes are broadly expressed. During development, all NTPDase genes, except for NTPDase8, are expressed and display a distinct specific expression pattern, suggesting potentially different functions of these proteins during embryogenesis of X. laevis.

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are endogenous bioactive lipids which mediate a variety of biological cell responses such as cell proliferation, migration, differentiation and apoptosis. Their actions are mediated by binding to the G-proteincoupled endothelial differentiation gene (Edg) receptor subfamily, referred to as S1P1-5 and LPA1-5, and regulate a variety of signalling pathways involved in numerous physiological processes and pathological conditions. Their importance during embryogenesis has been demonstrated by the generation of knock-out mice and specific roles have been assigned to these receptors. However, potential functional redundancy and the lethality of some mutants have complicated functional analysis in these models. Here we report the cloning of the S1P and LPA receptors in Xenopus laevis and tropicalis. Phylogenetic analyses demonstrate the high level of conservation of these receptors between amphibian and other vertebrate species. We have conducted a comparative expression analysis of these receptors during development and in the adult frog, by both RT-PCR and whole mount in situ hybridisation. In particular, we show that S1P1, 2 and 5 display distinct embryonic specific expression patterns, suggesting potentially different developmental roles for these receptors, and therefore for their ligands, during amphibian embryogenesis.

We have previously shown that lmx1b, a LIM homeodomain protein, is expressed in the pronephric glomus. We now show temporal and spatial expression patterns of lmx1b and its potential binding partners in both dissected pronephric anlagen and in individual dissected components of stage 42 pronephroi. Morpholino oligonucleotide knock-down of lmx1b establishes a role for lmx1b in the development of the pronephric components. Depletion of lmx1b results in the formation of a glomus with reduced size. Pronephric tubules were also shown to be reduced in structure and/or coiling whereas more distal tubule structure was unaffected. Over-expression of lmx1b mRNA resulted in no significant phenotype. Given that lmx1b protein is known to function as a heterodimer, we have over-expressed lmx1b mRNA alone or in combination with potential interacting molecules and analysed the effects on kidney structures. Phenotypes observed by over-expression of lim1 and ldb1 are partially rescued by co-injection with lmx1b mRNA. Animal cap experiments confirm that co-injection of lmx1b with potential binding partners can up-regulate pronephric molecular markers suggesting that lmx1b lies upstream of wt1 in the gene network controlling glomus differentiation. This places lmx1b in a genetic hierarchy involved in pronephros development and suggests that it is the balance in levels of binding partners together with restricted expression domains of lmx1b and lim1 which influences differentiation into glomus or tubule derivatives in vivo.

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