Retinoic acid signalling is required for specification of pronephric cell fate

The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.     

Essential function of Wnt-4 for tubulogenesis in the Xenopus pronephric kidney

In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. We therefore investigated the expression of Wnt genes during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but was absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincided with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, we performed gain- and loss-of-function experiments. Misexpression of Wnt4 in the intermediate and lateral mesoderm caused abnormal morphogenesis of the pronephric tubules, but was not sufficient to initiate ectopic tubule formation. We used a morpholino antisense oligonucleotide-based gene knockdown strategy to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos developed normally, but marker gene and morphological analysis revealed a complete absence of pronephric tubules. Pronephric duct development was largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. Our results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs.     

Sequential gene expression during pronephric tubule formation in vitro in Xenopus ectoderm

The kidney has been used as a model organ to analyze organogenesis. In in vitro experiments using Xenopus blastula ectoderm, the development of pronephric tubules (the prototype of the kidney) may be induced by treatment with activin A and retinoic acid (RA). The present study examined whether pronephric tubules induced in ectodermal explants exhibited similar characteristics to those of normal embryos at the molecular level. The experimental conditions required for high frequency induction (100%) of pronephric tubule formation from presumptive ectoderm without the development of muscle and notochord were determined. The developmental expression of the pronephros marker genes Xlim-1 and Xlcaax-1 was examined in induced pronephric tubules. After treatment with 10 ng/mL activin A and 10⁻⁴ mol/L RA, only pronephric tubules were induced at a high frequency. Induced pronephric tubules showed the same timing and patterns of expression for the marker genes Xlim-1 and Xlcaax-1 as normal embryos. These results suggest that the in vitro development of pronephric tubules induced in the presumptive ectoderm by activin A and RA parallels normal development at the molecular level.     

A role for Xlim-1 in pronephros development in Xenopus laevis

Xlim-1, a LIM class homeobox gene expressed in Xenopus laevis, is one of the earliest known marker genes of pronephros development and is expressed in pronephros rudiment. In this study, we examined the role of Xlim-1 in pronephros development. Temporal expression of Xlim-1 in explants was analyzed in a series of induction assays using RT-PCR analysis. Xlim-1 was expressed 9 to 15 h after activin/retinoic acid treatment, corresponding to pronephros differentiation in explants. We further examined the role of Xlim-1 using a series of microinjection experiments. Presumptive pronephric anlagen of embryos were injected with various Xlim-1 mutants, and effects of these Xlim-1 mutants on pronephrogenesis in embryos and in explants were analyzed by RT-PCR and immunohistochemistry. Dominant-negative Xlim-1 inhibited differentiation of pronephros in activin/retinoic acid-treated animal caps. In embryos injected with a dominant-negative form of Xlim-1, development of pronephric tubules was inhibited at the late tail-bud stage. Our results suggest that Xlim-1 may not initiate differentiation of the pronephros, but that it is necessary for growth and elongation in the development of pronephric tubules.     

Expression of Pax258 in the gastropod statocyst: insights into the antiquity of metazoan geosensory organs

Most animals have sensory systems that allow them to balance and orient relative to the pull of gravity. Structures responsible for these functions range from very simple statocysts found in many aquatic invertebrates to the complex inner ear of mammals. Previous studies suggest that the specialized mechanosensory structures responsible for balance in vertebrates and insects may be homologous based on the requirement and expression of group II Pax genes (i.e., Pax-2/5/8 genes). Here we report the expression of a Pax-258 gene in the statocysts and other chemosensory and mechanosensory cells during the development of the gastropod mollusk Haliotis asinina, a member of the Lophotrochozoa. Based on the phylogenetic distribution of geosensory systems and the consistent expression of Pax-258 in the cells that form these systems, we propose that Pax-258, along with POU-III and -IV genes, has an ancient and conserved role in the formation of structures responsible for balance and geotaxis in eumetazoans.     

Zebrafish no isthmus reveals a role for pax2.1 in tubule differentiation and patterning events in the pronephric primordia

Pax genes are important developmental regulators and function at multiple stages of vertebrate kidney organogenesis. In this report, we have used the zebrafish pax2.1 mutant no isthmus to investigate the role for pax2.1 in development of the pronephros. We demonstrate a requirement for pax2.1 in multiple aspects of pronephric development including tubule and duct epithelial differentiation and cloaca morphogenesis. Morphological analysis demonstrates that noi(- )larvae specifically lack pronephric tubules while glomerular cell differentiation is unaffected. In addition, pax2.1 expression in the lateral cells of the pronephric primordium is required to restrict the domains of Wilms' tumor suppressor (wt1) and vascular endothelial growth factor (VEGF) gene expression to medial podocyte progenitors. Ectopic podocyte-specific marker expression in pronephric duct cells correlates with loss of expression of the pronephric tubule and duct-specific markers mAb 3G8 and a Na(+)/K(+) ATPase (&agr;)1 subunit. The results suggest that the failure in pronephric tubule differentiation in noi arises from a patterning defect during differentiation of the pronephric primordium and that mutually inhibitory regulatory interactions play an important role in defining the boundary between glomerular and tubule progenitors in the forming nephron.     

An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice

We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline-dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor beta-1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice.     

Pax: a murine multigene family of paired box-containing genes.

A murine multigene family has been identified that shares a conserved sequence motif, the paired box, with developmental control and tissue-specific genes of Drosophila. To date five murine paired box-containing genes (Pax genes) have been described and one, Pax-1, has been associated with the developmental mutant phenotype undulated. Here we describe the paired boxes of three novel Pax genes, Pax-4, Pax-5, and Pax-6. Comparison of the eight murine paired domains of the mouse, the five Drosophila paired domains, and the three human paired domains shows that they fall into six distinct classes: class I comprises Pox meso, Pax-1, and HuP48; class II paired, gooseberry-proximal, gooseberry-distal, Pax-3, Pax-7, HuP1, and HuP2; class III Pax-2, Pax-5, and Pax-8; class IV Pax-4; class V Pox neuro; and class VI Pax-6. Pax-1 and the human gene HuP48 have identical paired domains, as do Pax-3 and HuP2 as well as Pax-7 and HuP1, and are likely to represent homologous genes in mouse and man. Identical intron-exon structure and extensive sequence homology of their paired boxes suggest that several Pax genes represent paralogs. The chromosomal location of all novel Pax genes and of Pax-3 and Pax-7 has been determined and reveals that they are not clustered.     

Betaglycan is required for the establishment of nephron endowment in the mouse

Betaglycan is an accessory receptor for the transforming growth factor-β (TGFβ) superfamily, many members of which play key roles in kidney development. The purpose of this study was to define the role of this co-receptor on fetal murine kidney development. Stereological examination of embryonic and adult betaglycan heterozygous kidneys revealed augmented nephron number relative to littermate controls. Fetal heterozygous kidneys exhibited accelerated ureteric branching, which correlated with augmented nephron development at embryonic day (e) 15.5. In contrast, betaglycan null kidneys exhibited renal hypoplasia from e13.5 and reduced nephron number at e15.5. Quantitative real-time PCR analysis of e11.5-e14.5 kidneys demonstrated that heterozygous kidneys exhibited a transient decrease in Bmp4 expression at e11.5 and a subsequent cascade of changes in the gene regulatory network that governs metanephric development, including significant increases in Pax2, Eya1, Gdnf, Ret, Wnt4, and Wt1 expression. Conversely, gene expression in null kidneys was normal until e13.5, when significant reductions were detected in the expression of Bmp4 as well as other key metanephric regulatory genes. Tgfb1 and Tgfb2 mRNA expression was down-regulated in both nulls and heterozygotes at e13.5 and e14.5. The opposing morphological and molecular phenotypes in betaglycan heterozygote and null mutants demonstrate that the levels of betaglycan must be tightly regulated for optimal kidney development.     

Pax-5 is expressed at the midbrain-hindbrain boundary during mouse development.

The murine paired-box-containing gene 5, Pax-5, is highly homologous to two other Pax genes, Pax-2 and Pax-8. The expression pattern of Pax-5 during mouse embryogenesis was examined by in situ RNA hybridization and compared to those of Pax-2 and Pax-8. Beginning at day 9.5 postcoitum (p.c.), Pax-5 was expressed in the developing brain, predominantly at the midbrain-hindbrain boundary, and in the neural tube. While the neural tube expression pattern overlapped completely with Pax-2 and Pax-8, the expression pattern in the brain was only partially overlapping. Unlike Pax-2 and Pax-8, Pax-5 was not expressed in the developing excretory system, thyroid, eye or ear. Our data suggest that Pax-5 has a role in the development of the central nervous system.     

FGF is essential for both condensation and mesenchymal-epithelial transition stages of pronephric kidney tubule development

The pronephros is a transient embryonic kidney that is essential for the survival of aquatic larvae. It is also absolutely critical for adult kidney development, as the pronephric derivative the wolffian duct forms the ductal system of the adult kidney and also triggers the condensation of metanephric mesenchyme into the adult nephrons. While exploring Xenopus pronephric patterning, we observed that epidermally delivered hedgehog completely suppresses pronephric kidney tubule development but does not effect development of the pronephric glomus, the equivalent of the mammalian glomerulus or corpuscle. This effect is not mediated by apoptosis. Microarray analysis of microdissected primordia identified FGF8 as one of the potential mediators of hedgehog action. Further investigation demonstrated that SU5402-sensitive FGF signaling plays a critical role in the very earliest stages of pronephric tubule development. Modulation of FGF8 activity using a morpholino has a later effect that blocks condensation of pronephric mesenchyme into the pronephric tubule. Together, these data show that FGF signaling plays a critical role at two stages of embryonic kidney development, one in the condensation of the pronephric primordium from the intermediate mesoderm and a second in the later epithelialization of this mesenchyme into the pronephric nephron. The data also show that in Xenopus, development of the glomus/glomerulus can be uncoupled from nephron formation via ectopic hedgehog expression and provides an experimental avenue for investigating glomerulogenesis in the complete absence of tubules.