Comparative genomic and expression analysis of the adenosine signaling pathway members in Xenopus

Abstract

Adenosine is an endogenous molecule that regulates many physiological processes via the activation of four specific G-protein-coupled ADORA receptors. Extracellular adenosine may originate either from the hydrolysis of released ATP by the ectonucleotidases or from cellular exit via the equilibrative nucleoside transporters (SLC29A). Adenosine extracellular concentration is also regulated by its successive hydrolysis into uric acid by membrane-bound enzymes or by cell influx via the concentrative nucleoside transporters (SLC28A). All of these members constitute the adenosine signaling pathway and regulate adenosine functions. Although the roles of this pathway are quite well understood in adults, little is known regarding its functions during vertebrate embryogenesis. We have used Xenopus laevis as a model system to provide a comparative expression map of the different members of this pathway during vertebrate development. We report the characterization of the different enzymes, receptors, and nucleoside transporters in both X. laevis and X. tropicalis, and we demonstrate by phylogenetic analyses the high level of conservation of these members between amphibians and mammals. A thorough expression analysis of these members during development and in the adult frog reveals that each member displays distinct specific expression patterns. These data suggest potentially different developmental roles for these proteins and therefore for extracellular adenosine. In addition, we show that adenosine levels during amphibian embryogenesis are very low, confirming that they must be tightly controlled for normal development.

Purine biosynthesis pathways are required for myogenesis inXenopus laevis

“…This muscle alteration was even more exacerbated by increasing the dose of MO, with severe reduction, and in some cases, absence of 12/101 positive domain and no somitic-like structure observed on the injected side in more than 80% of the injected 20 ng MO1 embryos (Figure 3C). This dose- gene encoding the Hypoxanthine-PhosphoRybosylTransferase (Hprt) in the salvage pathway (Figure 1A), the enzyme allowing metabolization of hypoxanthine, the major purine source in X. laevis embryos [46]. Comparable muscle alterations were obtained with independent knockdown of these genes, i.e.…”

“…However, from the early organogenesis phase (stage 22), all the 17 tested genes are expressed. We have previously shown that the unique reserve of purine in embryos, hypoxanthine, is depleted at stage 22 [46], so this embryonic stage is placed as a key turning point of purine source for the embryo. From this stage onwards and until stage 45 (stage where embryos start to feed), purines can only be synthesized de novo or recycled within the embryo, and zygotic expression of purine biosynthesis genes becomes then crucial.…”

“…Hypoxanthine, the main source of purine in X. laevis embryo [46], would have been the best candidate metabolite for the MO rescue but it cannot be tested here since its metabolization requires the enzymatic activity of Adsl.L (Figure 1A). Rescue experiments were therefore carried out by adding adenine to the Adsl.L morphant culture medium in concentrations at which this purine precursor was soluble in the medium (50 µM) but without any success.…”

Leukemia inhibitory factor signaling in Xenopus embryo: Insights from gain of function analysis and dominant negative mutant of the receptor

“…cDNA was synthesized from 1µg of RNA in 20µL final volume with Promega reverse transcriptase. Semi quantitative PCR was performed with the PCR primers listed in Table S1 and analysed on agarose gel as previously described (Tocco et al, 2015). Real time PCR was performed in a final volume of 20µL with the Mx3000P QPCR system (Stratagene-Agilent).…”

Comparative Embryonic Spatio-Temporal Expression Profile Map of the Xenopus P2X Receptor Family

“…We have previously established the embryonic expression pattern of enpp and entpd ectonucleotidases and adenosine signaling pathway members in Xenopus embryos. The specific spatio-temporal expression profile of each member of ectonucleotidases and adenosine receptors, suggested that ATP, ADP and adenosine may have distinct and specific functions during vertebrate embryogenesis (Massé et al, 2006, 2010; Tocco et al, 2015). Indeed, we demonstrated that ADP, via the P2Y1 receptor, triggers eye development by regulating the expression of the eye field transcription factors, such as pax6 (Massé et al, 2007).…”