Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development

AbstractThe respective role of Pax2 and Pax8 in early kidney development in vertebrates is poorly understood. In this report, we have studied the roles of Pax8 and Pax2 in Xenopus pronephros development using a loss-of-function approach. Our results highlight a differential requirement of these two transcription factors for proper pronephros formation. Pax8 is necessary for the earliest steps of pronephric development and its depletion leads to a complete absence of pronephric tubule. Pax2 is required after the establishment of the tubule pronephric anlage, for the expression of several terminal differentiation markers of the pronephric tubule. Neither Pax2 nor Pax8 is essential to glomus development. We further show that Pax8 controls hnf1b, but not lhx1 and Osr2, expression in the kidney field as soon as the mid-neurula stage. Pax8 is also required for cell proliferation of pronephric precursors in the kidney field. It may exert its action through the wnt/beta-catenin pathway since activation of this pathway can rescue MoPax8 induced proliferation defect and Pax8 regulates expression of the wnt pathway components, dvl1 and sfrp3. Finally, we observed that loss of pronephros in Pax8 morphants correlates with an expanded vascular/blood gene expression domain indicating that Pax8 function is important to delimit the blood/endothelial genes expression domain in the anterior part of the dorso-lateral plate

Etude du rôle de la signalisation calcique dépendante du canal TRPP2 au cours de la spécification du pronéphros chez Xenopus laevis

Chez le xénope, le pronéphros constitue le rein fonctionnel du têtard. Le sang est filtré au niveau du glomus et l urine, ainsi formée, est modifiée lors de son transit dans le tubule. Il a été montré que le territoire contenant les cellules à l origine du pronéphros, le champ pronéphrique (CP), est le siège d une augmentation de la concentration en Ca2+ cytoplasmique ( Ca2+ c), culminant au stade jeune neurula. La région présentant le maximum d augmentation de Ca2+ c correspond à la zone où se différencieront les tubules. Grâce à l injection ciblée d un chélateur de Ca2+ photoactivable, nous avons mis en évidence que l inhibition de la signalisation calcique avant la formation du CP, provoque une inhibition de l expression d un gène clef requis pour les premières étapes de la formation du pronéphros, pax8. De manière intéressante, l expression d une forme phosphorylée du canal TRPP2 (codé par le gène pkd2) atteint un maximum à la fin de la gastrulation, au moment où la vague calcique est mise en place au niveau du CP. Au même moment, elle est détectée, dans des explants de CP, au niveau de la membrane plasmique et de structures vésiculaires au sein du cytoplasme. De plus, nous avons montré que la perte de fonction de pkd2 (Mopkd2) entraine à la fois une inhibition des transitoires calciques et une inhibition de l expression du gène pax8 dans des explants de CP. Par contre, elle n affecte, pas les autres gènes du CP (lhx1, osr1 et osr2). Plus tard au cours du développement rénal, la perte de fonction de pkd2 perturbe la différenciation du glomus et du tubule. Nous avons identifié des partenaires de TRPP2 au stade gastrula âgée/jeune neurula, régulant potentiellement sont activité: la protéine kinase PRKD1 et la protéine GolginA2, impliquée dans le transport vésiculaire au niveau de l appareil de Golgi.Pronephros is the functional kidney of Xenopus leavis at tadpole stage. It consists of a single nephron subdivided into three structural and functional parts: the glomus, the tubule and the collecting tube. The pronephric field (PF) is defined at neurula stage as the mesodermique region able to give rise ex vivo to the pronéphros. It has been shown that a [Ca2+]i wave occurs in in the PF. The maximum is reached at early neurula stage. Furthermore, it has been demonstrated that the area where the strongest [Ca2+]i increase is recorded eventually differentiate into pronephric tubule. Using diazo-2 (photoactivable Ca2+chelator) injections in early embryos we show that pax8 expression in the PF is strongly inhibited. This is supporting the idea that Ca2+signaling is necessary for PF formation. TRPP2 (encoded by gene pkd2) is highly expressed at the end of gastrulation/beginning of neurulation in the PF, when the [Ca2+]i wave occurs. Then we studied the effect of pkd2 knockdown (Mopkd2) on calcium transients in PF explants and on pronephric genes expression. Pkd2 knockdown leads to the strong inhibition of calcium transients in PF explants. Furthermore, at early neurula stage our data strongly suggest that pkd2 knockdown leads to a defective PF formation by impeding pax8 expression. Later development of both glomus and tubule is also disturbed. Futhermore, we have identified two TRPP2 partners : the kinase PRKD1 and GolginA2, a protein of the Gogi apparatus involved in vesicular trafficking.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Characterization of potential TRPP2 regulating proteins in early Xenopus embryos

International audienceTransient receptor potential cation channel‐2 (TRPP2) is a nonspecific Ca2+‐dependent cation channel with versatile functions including control of extracellular calcium entry at the plasma membrane, release of intracellular calcium ([Ca2+]i) from internal stores of endoplasmic reticulum, and calcium‐dependent mechanosensation in the primary cilium. In early Xenopus embryos, TRPP2 is expressed in cilia of the gastrocoel roof plate (GRP) involved in the establishment of left‐right asymmetry, and in nonciliated kidney field (KF) cells, where it plays a central role in early specification of nephron tubule cells dependent on [Ca2+]i signaling. Identification of proteins binding to TRPP2 in embryo cells can provide interesting clues about the mechanisms involved in its regulation during these various processes. Using mass spectrometry, we have therefore characterized proteins from late gastrula/early neurula stage embryos coimmunoprecipitating with TRPP2. Binding of three of these proteins, golgin A2, protein kinase‐D1, and disheveled‐2 has been confirmed by immunoblotting analysis of TRPP2‐coprecipitated proteins. Expression analysis of the genes, respectively, encoding these proteins, golga2, prkd1, and dvl2 indicates that they are likely to play a role in these two regions. Golga2 and prkd1 are expressed at later stage in the developing pronephric tubule where golgin A2 and protein kinase‐D1 might also interact with TRPP2. Colocalization experiments using exogenously expressed fluorescent versions of TRPP2 and dvl2 in GRP and KF reveal that these two proteins are generally not coexpressed, and only colocalized in discrete region of cells. This was observed in KF cells, but does not appear to occur in the apical ciliated region of GRP cells