Purine Biosynthesis Pathways Are Required for Myogenesis in Xenopus laevis.

Purines are required for fundamental biological processes and alterations in their metabolism lead to severe genetic diseases associated with developmental defects whose etiology remains unclear. Here, we studied the developmental requirements for purine metabolism using the amphibian Xenopus laevis as a vertebrate model. We provide the first functional characterization of purine pathway genes and show that these genes are mainly expressed in nervous and muscular embryonic tissues. Morphants were generated to decipher the functions of these genes, with a focus on the adenylosuccinate lyase (ADSL), which is an enzyme required for both salvage and de novo purine pathways. adsl.L knockdown led to a severe reduction in the expression of the myogenic regulatory factors (MRFs: Myod1, Myf5 and Myogenin), thus resulting in defects in somite formation and, at later stages, the development and/or migration of both craniofacial and hypaxial muscle progenitors. The reduced expressions of hprt1.L and ppat, which are two genes specific to the salvage and de novo pathways, respectively, resulted in similar alterations. In conclusion, our data show for the first time that de novo and recycling purine pathways are essential for myogenesis and highlight new mechanisms in the regulation of MRF gene expression.

The enpp4 ectonucleotidase regulates kidney patterning signalling networks in Xenopus embryos.

The enpp ectonucleotidases regulate lipidic and purinergic signalling pathways by controlling the extracellular concentrations of purines and bioactive lipids. Although both pathways are key regulators of kidney physiology and linked to human renal pathologies, their roles during nephrogenesis remain poorly understood. We previously showed that the pronephros was a major site of enpp expression and now demonstrate an unsuspected role for the conserved vertebrate enpp4 protein during kidney formation in Xenopus. Enpp4 over-expression results in ectopic renal tissues and, on rare occasion, complete mini-duplication of the entire kidney. Enpp4 is required and sufficient for pronephric markers expression and regulates the expression of RA, Notch and Wnt pathway members. Enpp4 is a membrane protein that binds, without hydrolyzing, phosphatidylserine and its effects are mediated by the receptor s1pr5, although not via the generation of S1P. Finally, we propose a novel and non-catalytic mechanism by which lipidic signalling regulates nephrogenesis.

Animal models of pain: Diversity and benefits.

Chronic pain is a maladaptive neurological disease that remains a major health problem. A deepening of our knowledge on mechanisms that cause pain is a prerequisite to developing novel treatments. A large variety of animal models of pain has been developed that recapitulate the diverse symptoms of different pain pathologies. These models reproduce different pain phenotypes and remain necessary to examine the multidimensional aspects of pain and understand the cellular and molecular basis underlying pain conditions. In this review, we propose an overview of animal models, from simple organisms to rodents and non-human primates and the specific traits of pain pathologies they model. We present the main behavioral tests for assessing pain and investing the underpinning mechanisms of chronic pathological pain. The validity of animal models is analysed based on their ability to mimic human clinical diseases and to predict treatment outcomes. Refine characterization of pathological phenotypes also requires to consider pain globally using specific procedures dedicated to study emotional comorbidities of pain. We discuss the limitations of pain models when research findings fail to be translated from animal models to human clinics. But we also point to some recent successes in analgesic drug development that highlight strategies for improving the predictive validity of animal models of pain. Finally, we emphasize the importance of using assortments of preclinical pain models to identify pain subtype mechanisms, and to foster the development of better analgesics.

Developmental Expression of Ectonucleotidase and Purinergic Receptors Detection by Whole-Mount In Situ Hybridization in Xenopus Embryos.

Xenopus embryos are one of the most used animal models in developmental biology and are well suited for apprehending functions of signaling pathways during embryogenesis. To do so, it is necessary to be able to detect expression pattern of the key genes of these signaling pathways. Here we describe the whole-mount in situ hybridization technique to investigate the expression pattern of ectonucleotidases and purinergic receptors during embryonic development.

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

P2X receptors are ATP-gated cations channels formed by the homo or hetero-trimeric association from the seven cloned subunits (P2X1-7). P2X receptors are widely distributed in different organs and cell types throughout the body including the nervous system and are involved in a large variety of physiological but also pathological processes in adult mammals. However, their expression and function during embryogenesis remain poorly understood. Here, we report the cloning and the comparative expression map establishment of the entire P2X subunit family in the clawed frog Xenopus. Orthologous sequences for 6 mammalian P2X subunits were identified in both X. laevis and X. tropicalis, but not for P2X3 subunit, suggesting a potential loss of this subunit in the Pipidae family. Three of these genes (p2rx1, p2rx2, and p2rx5) exist as homeologs in the pseudoallotetraploid X. laevis, making a total of 9 subunits in this species. Phylogenetic analyses demonstrate the high level of conservation of these receptors between amphibian and other vertebrate species. RT-PCR revealed that all subunits are expressed during the development although zygotic p2rx6 and p2rx7 transcripts are mainly detected at late organogenesis stages. Whole mount in situ hybridization shows that each subunit displays a specific spatio-temporal expression profile and that these subunits can therefore be grouped into two groups, based on their expression or not in the developing nervous system. Overlapping expression in the central and peripheral nervous system and in the sensory organs suggests potential heteromerization and/or redundant functions of P2X subunits in Xenopus embryos. The developmental expression of the p2rx subunit family during early phases of embryogenesis indicates that these subunits may have distinct roles during vertebrate development, especially embryonic neurogenesis.

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

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.

Purines as potential morphogens during embryonic development.

Components of purinergic signalling are expressed in the early embryo raising the possibility that ATP, ADP and adenosine may contribute to the mechanisms of embryonic development. We summarize the available data from four developmental models-mouse, chick, Xenopus and zebrafish. While there are some notable examples where purinergic signalling is indeed important during development, e.g. development of the eye in the frog, it is puzzling that deletion of single components of purinergic signalling often results in rather minor developmental phenotypes. We suggest that a key step in further analysis is to perform combinatorial alterations of expression of purinergic signalling components to uncover their roles in development. We introduce the concept that purinergic signalling could create novel morphogenetic fields to encode spatial location via the concentration of ATP, ADP and adenosine. We show that using minimal assumptions and the known properties of the ectonucleotidases, complex spatial patterns of ATP and adenosine can be set up. These patterns may provide a new way to assess the potential of purinergic signalling in developmental processes.

The lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) receptor gene families: cloning and comparative expression analysis in Xenopus 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-protein-coupled 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.

Ectophosphodiesterase/nucleotide phosphohydrolase (Enpp) nucleotidases: cloning, conservation and developmental restriction.

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.

Database of queryable gene expression patterns for Xenopus.

The precise localization of gene expression within the developing embryo, and how it changes over time, is one of the most important sources of information for elucidating gene function. As a searchable resource, this information has up until now been largely inaccessible to the Xenopus community. Here, we present a new database of Xenopus gene expression patterns, queryable by specific location or region in the embryo. Pattern matching can be driven either from an existing in situ image, or from a user-defined pattern based on development stage schematic diagrams. The data are derived from the work of a group of 21 Xenopus researchers over a period of 4 days. We used a novel, rapid manual annotation tool, XenMARK, which exploits the ability of the human brain to make the necessary distortions in transferring data from the in situ images to the standard schematic geometry. Developmental Dynamics 238:1379-1388, 2009. (c) 2009 Wiley-Liss, Inc.

The lmx1b gene is pivotal in glomus development in Xenopus laevis.

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.

A functional screen for genes involved in Xenopus pronephros development.

In Xenopus, the pronephros is the functional larval kidney and consists of two identifiable components; the glomus, the pronephric tubules, which can be divided into four separate segments, based on marker gene expression. The simplicity of this organ, coupled with the fact that it displays the same basic organization and function as more complex mesonephros and metanephros, makes this an attractive model to study vertebrate kidney formation. In this study, we have performed a functional screen specifically to identify genes involved in pronephros development in Xenopus. Gain-of-function screens are performed by injecting mRNA pools made from a non-redundant X. tropicalis full-length plasmid cDNA library into X. laevis eggs, followed by sib-selection to identify the single clone that caused abnormal phenotypes in the pronephros. Out of 768 egg and gastrula stage cDNA clones, 31 genes, approximately 4% of the screened clones, affected pronephric marker expression examined by whole mount in situ hybridization or antibody staining. Most of the positive clones had clear expression patterns in pronephros and predicted/established functions highly likely to be involved in developmental processes. In order to carry out a more detailed study, we selected Sox7, Cpeb3, P53csv, Mecr and Dnajc15, which had highly specific expression patterns in the pronephric region. The over-expression of these five selected clones indicated that they caused pronephric abnormalities with different temporal and spatial effects. These results suggest that our strategy to identify novel genes involved in pronephros development was highly successful, and that this strategy is effective for the identification of novel genes involved in late developmental events.

Purine-mediated signalling triggers eye development.

A conserved network of eye field transcription factors (EFTFs) underlies the development of the eye in vertebrates and invertebrates. To direct eye development, Pax6, a key gene in this network, interacts with genes encoding other EFTFs such as Rx1 and Six3 (refs 4-6). However, the mechanisms that control expression of the EFTFs remain unclear. 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), an ectoenzyme that converts ATP to ADP, 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, 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 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 microphthalmia. Our results suggest a new mechanism for the initiation of eye development.

Anxa4 Genes are Expressed in Distinct Organ Systems in Xenopus laevis and tropicalis But are Functionally Conserved.

Anxa4 belongs to the multigenic annexin family of proteins which are characterized by their ability to interact with membranes in a calcium-dependent manner. Defined as a marker for polarized epithelial cells, Anxa4 is believed to be involved in many cellular processes but its functions in vivo are still poorly understood. Previously, we cloned Xanx4 in Xenopus laevis (now referred to as anxa4a) and demonstrated its role during organogenesis of the pronephros, providing the first evidence of a specific function for this protein during the development of a vertebrate. Here, we describe the strict conservation of protein sequence and functional domains of anxa4 during vertebrate evolution. We also identify the paralog of anxa4a, anxa4b and show its specific temporal and spatial expression pattern is different from anxa4a. We show that anxa4 orthologs in X. laevis and tropicalis display expression domains in different organ systems. Whilst the anxa4a gene is mainly expressed in the kidney, Xt anxa4 is expressed in the liver. Finally, we demonstrate Xt anxa4 and anxa4a can display conserved function during kidney organogenesis, despite the fact that Xt anxa4 transcripts are not expressed in this domain. This study highlights the divergence of expression of homologous genes during Xenopus evolution and raises the potential problems of using X. tropicalis promoters in X. laevis.

Comparative genomic and expression analysis of the conserved NTPDase gene family in Xenopus.

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.

Developmental expression of Pod 1 in Xenopus laevis.

The basic helix-loop-helix transcription factor, Pod 1, has been shown to be expressed in the mesenchyme of many developing mouse organs, including the heart, lungs and gut. In the kidneys of developing mice, Pod 1 is highly expressed in the condensing metanephric mesenchyme, differentiating and late stromal cells and in developing podocytes. We have obtained an EST (CF270487) which contains the Xenopus laevis Pod 1 sequence. Conceptual translation of the Xenopus laevis Pod 1 sequence shows approximately 85% similarity to other vertebrate homologues. RT-PCR indicates that expression is initiated at stage 13 and increases differentially in the developing pronephros compared to the whole embryo. RT-PCR of a kidney dissection at stage 42 shows higher expression in the glomus than in the tubule or duct. In situ hybridisation analysis at tail bud stages shows the anterior-most branchial arch and pronephric glomus are intensely stained. At stage 40, staining persists in the glomus and in the epicardium region of the heart. Adult organ analysis shows expression is highest in the rectum and the spleen, with significant expression in the duodenum, heart, kidney, lungs, pancreas, skin, liver and muscle.

X-epilectin: a novel epidermal fucolectin regulated by BMP signalling.

This paper reports the cloning and characterisation of a new posterior epidermal marker, X-epilectin, in Xenopus laevis. This gene encodes for a fucolectin, which belongs to the lectin superfamily of carbohydrate binding proteins and specifically binds fucose residues. RT-PCR and in situ hybridisation show that the expression of this gene is switched on during gastrulation and up-regulated during neurula stages and found expressed ubiquitously throughout the epidermis. From tailbud stages, the expression is limited to the dorsal posterior region of the embryo, suggesting that X-epilectin expression is regulated along anteroposterior and dorsoventral gradients during development. In the adult, X-epilectin is mainly expressed in intestinal components, kidney, spinal cord and skin. The effects of growth factors on the regulation of X-epilectin were studied. Change of the fate of animal caps into cement gland or dorsal mesoderm induces a down-regulation of X-epilectin expression in explants treated respectively with ammonium chloride and activin A. We also show that X-epilectin expression is down-regulated by Noggin and tBR and that this effect is inhibited by BMP4 over-expression, suggesting X-epilectin expression is mediated by the BMP signalling pathway.

Nm23-M2/NDP kinase B induces endogenous c-myc and nm23-M1/NDP kinase A overexpression in BAF3 cells. Both NDP kinases protect the cells from oxidative stress-induced death.

The nm23 gene family encodes nucleoside diphosphate kinases (NDPKs) which supply the cell with (d)NTPs. The human NDPKB, also known as the PuF protein, binds the c-myc promoter and transactivates the c-myc protooncogene. We have now studied the effects of mouse NDPKA and NDPKB overexpression on endogenous c-myc transactivation in the mouse BAF3 and the rat PC12 cell lines. c-myc transcripts were found to be up-regulated by NDPKB only in the BAF3 line. This suggests that c-myc transcriptional control via NDPKB depends on the presence of cell-specific co-factors. Unexpectedly, NDPKB also induced NDPKA expression. This new effect was found in both cell lines, suggesting that NDPKB-dependent nm23-M1 gene transactivation requires cis and/or trans elements different from those involved in c-myc transactivation. Moreover, the BAF3 cell proliferation capacities were found to be independent of NDPKA or B cell contents. Interestingly, cell death induced by c-myc overexpression or H(2)O(2) exposure was decreased in nm23-transfected compared to control BAF3 cells. These data collectively suggest that NDPKs might improve cell survival by a mechanism coupling DNA repair and transcriptional regulation of genes involved in DNA damage response.

Cloning and characterisation of the immunophilin X-CypA in Xenopus laevis.

This paper reports the cloning of Xenopus laevis, cyclophilin A gene, X-CypA. This study is the first developmental and functional characterisation in vivo of cyclophilin A in a vertebrate. X-CypA belongs to the superfamily of the immunophilin/PPIase proteins that can bind the immunosuppressant drug Cyclosporin A. Sequence analysis showed that X-CypA is highly conserved during evolution. RT-PCR and in situ hybridisation analysis showed that X-CypA expression is regulated during development and its transcripts are found in three major expression domains: nervous system, sensory organs and pronephros. Over-expression of X-CypA in embryos, analysed by in situ hybridisation and RT-PCR, leads to an expansion and disorganisation of the neural crest domain.

Isolation and growth factor inducibility of the Xenopus laevis Lmx1b gene.

This paper reports the cloning of the full length Xenopus laevis Lmx1b gene, Xlmx1b. Xlmx1b is a LIM homeodomain protein with high conservation to homologues identified in human, mouse, hamster and chick. In situ hybridisation and RT-PCR analysis showed that Xlmx1b has a specific temporal expression pattern which can be separated into three main spatial domains. An Xlmx1b probe hybridized to regions of the nervous system from stage 13 onwards; these regions included the placodes and otic vesicles, the eye and specific sets of neurons. Sectioning of in situ hybridised embryos confirmed the location of transcripts as discreet regions of staining in ventrolateral regions of the neural tube. From stage 27, transcripts could be detected in the capsule of pronephric glomus. Finally, transcripts were detected by Northern blot analysis in the developing fore and hind limbs. Xlmx1b transcripts were also detected by Northern blot analysis in eye, brain, muscle and mesonephros tissue in metamorphosing tadpoles. RT-PCR analysis showed that zygotic expression of Xlmx1b is initiated at stage 10.5 and the temporal sequence of Xlmx1b expression is identical in both neural and presumptive pronephros regions. The effects of the growth factors activin A, retinoic acid (RA) and basic fibroblast growth factor (bFGF) on the regulation of Xlmx1b were also studied. Xlmx1b was found to be upregulated by activin A and RA inhibited this upregulation in a concentration dependant manner. In contrast, bFGF had no effect on the regulation of Xlmx1b.