Karine Massé

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.

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.

Assessed Online Discussion Groups in Biology Education

Abstract Sophisticated software such as Virtual Learning Environments (VLEs) are rapidly being deployed by universities. Despite widespread use of such systems, experience shows that there is frequently poor pedagogic development, leading primarily to use of VLEs as electronic document repositories rather than as online learning systems in which the available suite of tools are used to their full potential. Online assessment is the major potential efficiency gain of such systems, but most staff do not scratch the surface of the full capabilities of the software. Based on our experience, we describe practical guidelines for a model of online assessment which promotes deep versus superficial learning, encourages higher level learning competencies and inclusivity.

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.

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 characterised by their ability to interact with membranes in a calcium-dependent manner. Defined as a marker for polarised 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.

Roles of enpp4 during vertebrate kidney development

The rodent P2X7k splice variant, which utilizes a different exon 1 and is expressed in both wild type and the Glaxo P2X7−/−mice, has at least a 10-fold greater sensitivity to agonists ATP and BzATP than the full length mouse P2X7a variant, as determined from patch clamp experiments. ThemP2X7k receptor also coupled to the rapid uptake of the cationic dye ethidium, and this process was unaffected by the SNP, P451L, that previously was shown to inhibit mP2X7a receptor-mediated dye uptake. In HEK293 cells, activation of mP2X7k also triggered the uptake of the anionic dye Lucifer yellow, the rate of which was dramatically increased by a 5-min pre-incubation with the pannexin-1 inhibitor, carbenoxolone (CBX) at 37°C. CBX also increased mP2X7k-mediated ethidium uptake but the Ca2+ signal evoked by activation of mP2X7k was unaffected, suggesting that CBX selectively affects ‘large pore’ formation. Themechanismof action of CBX is unclear but the results are not consistentwith pannexin-1 being the dye uptake pathway. P2X7k is expressed in mice spleen T-lymphocytes where NAD is reported to be a physiologically important agonist. In HEK293 cells, 10 uM NAD stimulated mP2X7k-mediated ethidium uptake whereas mP2X7a did not respond to concentrations up to 300 uM NAD. NAD also stimulated a rapid Ca2+ response in HEK293 cells expressing mP2X7k. The coexpression of mP2X7a and mP2X7k in HEK cells reduced the amplitude of the NAD response compared to cells expressing mP2X7k alone. Our results suggest the expression of P2X7k increases the diversity of P2X7 receptor signaling in rodents.

09-P068 Functional analysis of enpp4 and enpp6 in Xenopus pronephros development

we find that that expression of claudin 5, a component of endothelial tight junctions, is dramatically down-regulated in Tal1-deficient embryos. Consistent with a defect in tight junction formation, localization of junctional components is disorganized in the Tal1-deficient endocardium. Therefore, we propose that Tal1 transcriptional targets regulate endocardial migration and tube extension through establishment of tight junctions.