Requirement of Wnt/β-catenin signaling in pronephric kidney development

The pronephric kidney controls water and electrolyte balance during early fish and amphibian embryogenesis. Many Wnt signaling components have been implicated in kidney development. Specifically, in Xenopus pronephric development as well as the murine metanephroi, the secreted glycoprotein Wnt-4 has been shown to be essential for renal tubule formation. Despite the importance of Wnt signals in kidney organogenesis, little is known of the definitive downstream signaling pathway(s) that mediate their effects. Here we report that inhibition of Wnt/β-catenin signaling within the pronephric field of Xenopus results in significant losses to kidney epithelial tubulogenesis with little or no effect on adjoining axis or somite development. We find that the requirement for Wnt/β-catenin signaling extends throughout the pronephric primordium and is essential for the development of proximal and distal tubules of the pronephros as well as for the development of the duct and glomus. Although less pronounced than effects upon later pronephric tubule differentiation, inhibition of the Wnt/β-catenin pathway decreased expression of early pronephric mesenchymal markers indicating it is also needed in early pronephric patterning. We find that upstream inhibition of Wnt/β-catenin signals in zebrafish likewise reduces pronephric epithelial tubulogenesis. We also find that exogenous activation of Wnt/β-catenin signaling within the Xenopus pronephric field results in significant tubulogenic losses. Together, we propose Wnt/β-catenin signaling is required for pronephric tubule, duct and glomus formation in Xenopus laevis, and this requirement is conserved in zebrafish pronephric tubule formation.     

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.     

The specification and growth factor inducibility of the pronephric glomus in Xenopus laevis

We report a study on the specification of the glomus, the filtration device of the amphibian pronephric kidney, using an explant culturing strategy in Xenopus laevis. Explants of presumptive pronephric mesoderm were dissected from embryos of mid-gastrula to swimming tadpole stages. These explants were cultured within ectodermal wraps and analysed by RT-PCR for the presence of the Wilm's Tumour-1 gene, xWT1, a marker specific for the glomus at the stages analysed, together with other mesodermal markers. We show that the glomus is specified at stage 12.5, the same stage at which pronephric tubules are specified. We have previously shown that pronephric duct is specified somewhat later, at stage 14. Furthermore, we have analysed the growth factor inducibility of the glomus in the presence or absence of retinoic acid (RA) by RT-PCR. We define for the first time the conditions under which these growth factors induce glomus tissue in animal cap tissue. Activin together with high concentrations of RA can induce glomus tissue from animal cap ectoderm. Unlike the pronephric tubules, the glomus can also be induced by FGF and RA.     

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.     

Notch regulates cell fate in the developing pronephros

The mechanisms that regulate cell fate within the pronephros are poorly understood but are important for the subsequent development of the urogenital system and show many similarities to nephrogenesis in the definitive kidney. Dynamic expression of Notch-1, Serrate-1, and Delta-1 in the developing Xenopus pronephros suggests a role for this pathway in cell fate segregation. Misactivation of Notch signaling using conditionally active forms of either Notch-1 or RBP-J/Su(H) proteins prevented normal duct formation and the proper expression of genetic markers of duct cell differentiation. Inhibition of endogenous Notch signaling elicited the opposite effect. Taken together with the mRNA expression patterns, these data suggest that endogenous Notch signaling functions to inhibit duct differentiation in the dorsoanterior region of the anlage where cells are normally fated to form tubules. In addition, elevated Notch signaling in the pronephric anlage both perturbed the characteristic pattern of the differentiated tubule network and increased the expression of early markers of pronephric precursor cells, Pax-2 and Wilms' tumor suppressor gene (Wt-1). We propose that Notch signaling plays a previously unrecognized role in the early selection of duct and tubule cell fates as well as functioning subsequently to control tubule cell patterning and development.     

Amino acid cotransporter SLC3A2 is selectively expressed in the early proximal segment of Xenopus pronephric kidney nephrons

The transport of amino acids across membranes is critical to all cells. As amino acids freely pass through the glomerular filtration barrier of the kidney, they must be efficiently resorbed to avoid depletion of circulating amino acid reserves. Not only do defects in amino acid resorption lead to costly wastage, they also cause congenital aminoacidurias. A clone encoding Xenopus SLC3A2 was identified and shown to be expressed at high levels in the early segment of the pronephric proximal tubules in developing tadpoles. The type II membrane glycoprotein encoded by this gene can associate with a wide variety of protein partners and participates in a broad spectrum of biological processes. In this report, the first whole-mount analysis of SLC3A2 during early embryonic development is presented. The expression pattern of SLC3A2 in the early proximal segment of the Xenopus pronephros is analogous to that of a previously described SLC7A8/XAA2 amino acid transporter. In mammals, SLC3A2 and SLC7A8/XAA2 associate to form a functional neutral amino acid transporter complex and coexpression of these two genes in a small domain within the pronephric tubules indicates that this is also the situation in the developing Xenopus kidney.     

The pronephros of the early ammocoete larva of lampreys (Cyclostomata,Petromyzontes): Fine structure of the renal tubules

Summary The renal tubules of the paired pronephros in early larvae (ammocoetes) of two lamprey species, Lampetra fluviatilis and Petromyzon marinus, were studied by use of light-, scanning- and transmission electron microscopy. They consist of (1) a variable number of pronephric tubules (3 to 6), and (2) an excretory duct. By fine-structural criteria, the renal tubules can be divided into 6 segments. Each pronephric tubule is divided into (1) the nephrostome and (2) the proximal tubule, the excretory duct consisting of (3) a common proximal tubule followed by (4) a short intermediate segment, and then by a pronephric duct composed of (5) a cranial and (6) a caudal section. The epithelium of the nephrostome displays bundles of cilia. The cells of the proximal tubule possess a brush border, many endocytotic organelles and a system of canaliculi (tubular invaginations of the basolateral plasmalemma). The same characteristics are encountered in the epithelium of the common proximal tubule; however, the number of these specific organelles decreases along the course of this segment in a posterior direction. In the intermediate segment, the epithelium appears structurally nonspecialized. The cells of the cranial pronephric duct lack a brush border; they have an extensive system of canaliculi and numerous mitochondria. The caudal pronephric duct is lined by an epithelium composed of light and dark cells; the latter are filled with mitochondria and the former contain mucus granules beneath the luminal plasmalemma. The tubular segments found in the pronephros are the same in structure and sequence as in the lamprey opisthonephroi. However, only the nephrostomes and proximal tubules occur serially in the pronephros, while the common proximal tubule, the intermediate segment and the cranial pronephric duct form portions of a single excretory duct.This paper is dedicated to the memory of Professor W. Bargmann, long-time editor of Cell and Tissue Research, the author of a splendid review on the structure of the vertebrate kidney and a master of German scientific writing.     

In vitro induction of the pronephric duct in Xenopus explants

The earliest form of embryonic kidney, the pronephros, consists of three components: glomus, tubule and duct. Treatment of the undifferentiated animal pole ectoderm of Xenopus laevis with activin A and retinoic acid (RA) induces formation of the pronephric tubule and glomus. In this study, the rate of induction of the pronephric duct, the third component of the pronephros, was investigated in animal caps treated with activin A and RA. Immunohistochemistry using pronephric duct-specific antibody 4A6 revealed that a high proportion of the treated explants contained 4A6-positive tubular structures. Electron microscopy showed that the tubules in the explants were similar to the pronephric ducts of normal larvae, and they also expressed Gremlin and c-ret, molecular markers for pronephric ducts. These results suggest that the treatment of Xenopus ectoderm with activin A and RA induces a high rate of differentiation of pronephric ducts, in addition to the differentiation of the pronephric tubule and glomus, and that this in vitro system can serve as a simple and effective model for analysis of the mechanism of pronephros differentiation.     

Organization of the pronephric kidney revealed by large-scale gene expression mapping

Background

The pronephros, the simplest form of a vertebrate excretory organ, has recently become an important model of vertebrate kidney organogenesis. Here, we elucidated the nephron organization of the Xenopus pronephros and determined the similarities in segmentation with the metanephros, the adult kidney of mammals.

Results

We performed large-scale gene expression mapping of terminal differentiation markers to identify gene expression patterns that define distinct domains of the pronephric kidney. We analyzed the expression of over 240 genes, which included members of the solute carrier, claudin, and aquaporin gene families, as well as selected ion channels. The obtained expression patterns were deposited in the searchable European Renal Genome Project Xenopus Gene Expression Database. We found that 112 genes exhibited highly regionalized expression patterns that were adequate to define the segmental organization of the pronephric nephron. Eight functionally distinct domains were discovered that shared significant analogies in gene expression with the mammalian metanephric nephron. We therefore propose a new nomenclature, which is in line with the mammalian one. The Xenopus pronephric nephron is composed of four basic domains: proximal tubule, intermediate tubule, distal tubule, and connecting tubule. Each tubule may be further subdivided into distinct segments. Finally, we also provide compelling evidence that the expression of key genes underlying inherited renal diseases in humans has been evolutionarily conserved down to the level of the pronephric kidney.

Conclusion

The present study validates the Xenopus pronephros as a genuine model that may be used to elucidate the molecular basis of nephron segmentation and human renal disease.     

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.     

Jagged2a-notch signaling mediates cell fate choice in the zebrafish pronephric duct

Pronephros, a developmental model for adult mammalian kidneys (metanephros) and a functional kidney in early teleosts, consists of glomerulus, tubule, and duct. These structural and functional elements are responsible for different kidney functions, e.g., blood filtration, waste extraction, salt recovery, and water balance. During pronephros organogenesis, cell differentiation is a key step in generating different cell types in specific locations to accomplish designated functions. However, it is poorly understood what molecules regulate the differentiation of different cell types in different parts of the kidney. Two types of epithelial cells, multi-cilia cells and principal cells, are found in the epithelia of the zebrafish distal pronephric duct. While the former is characterized by at least 15 apically localized cilia and expresses centrin2 and rfx2, the latter is characterized by a single primary cilium and sodium pumps. Multi-cilia cells and principal cells differentiate from 17.5 hours post-fertilization onwards in a mosaic pattern. Jagged2a-Notch1a/Notch3-Her9 is responsible for specification and patterning of these two cell types through a lateral inhibition mechanism. Furthermore, multi-cilia cell hyperplasia was observed in mind bomb mutants and Mind bomb was shown to interact with Jagged2a and facilitate its internalization. Taken together, our findings add a new paradigm of Notch signaling in kidney development, namely, that Jagged2a-Notch signaling modulates cell fate choice in a nephric segment, the distal pronephric duct.