Notch activates Wnt-4 signalling to control medio-lateral patterning of the pronephros

Previous studies have highlighted a role for the Notch signalling pathway during pronephrogenesis in the amphibian Xenopus laevis, and in nephron development in the mammalian metanephros, yet a mechanism for this function remains elusive. Here, we further the understanding of how Notch signalling patterns the early X. laevis pronephros anlagen, a function that might be conserved in mammalian nephron segmentation. Our results indicate that early phase pronephric Notch signalling patterns the medio-lateral axis of the dorso-anterior pronephros anlagen, permitting the glomus and tubules to develop in isolation. We show that this novel function acts through the Notch effector gene hrt1 by upregulating expression of wnt4. Wnt-4 then patterns the proximal pronephric anlagen to establish the specific compartments that span the medio-lateral axis. We also identified pronephric expression of lunatic fringe and radical fringe that is temporally and spatially appropriate for a role in regulating Notch signalling in the dorso-anterior region of the pronephros anlagen. On the basis of these results, along with data from previous publications, we propose a mechanism by which the Notch signalling pathway regulates a Wnt-4 function that patterns the proximal pronephric anlagen

Normal levels of p27Xic1 are necessary for somite segmentation and determining pronephric organ size

The Xenopus laevis cyclin dependent kinase inhibitor p27Xic1 has been shown to be involved in exit from the cell cycle and differentiation of cells into a quiescent state in the nervous system, muscle tissue, heart and retina. We show that p27Xic1 is expressed in the developing kidney in the nephrostomal regions. Using over-expression and morpholino oligonucleotide (MO) knock-down approaches we show normal levels of p27Xic1 regulate pronephros organ size by regulating cell cycle exit. Knock-down of p27Xic1 expression using a MO prevented myogenesis, as previously reported; an effect that subsequently inhibits pronephrogenesis. Furthermore, we show that normal levels of p27Xic1 are required for somite segmentation also through its cell cycle control function. Finally, we provide evidence to suggest correct paraxial mesoderm segmentation is not necessary for pronephric induction in the intermediate mesoderm. These results indicate novel developmental roles for p27Xic1, and reveal its differentiation function is not universally utilised in all developing tissues

Heat-shock mediated overexpression of HNF1β mutations has differential effects on gene expression in the Xenopus pronephric kidney.

The transcription factor HNF1B, encoded by the TCF2 gene, plays an important role in the organogenesis of vertebrates. In humans, heterozygous mutations of HNF1B are associated with several diseases, such as pancreatic β-cell dysfunction leading to maturity-onset diabetes of the young (MODY5), defective kidney development, disturbed liver function, pancreas atrophy, and malformations of the genital tract. The African claw frog Xenopus laevis is an excellent model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a series of methods. In the present study, we overexpressed HNF1β mutants in the developing Xenopus embryo to assess their roles during organogenesis, particularly in the developing pronephric kidney. Towards this goal, we developed a heat-shock inducible binary Cre/loxP system with activator and effector strains. Heat-shock activation of the mutant HNF1B variants P328L329del and A263insGG resulted in malformations of various organs and the affected larvae developed large edemas. Defects in the pronephros were primarily confined to malformed proximal tubules. Furthermore, the expression of the proximal tubule marker genes tmem27 and slc3a1, both involved in amino acid transport, was affected. Both P328L329del and A263insGG downregulated expression of slc3a1. In addition, P328L329del reduced tmem27 expression while A263insGG overexpression decreased expression of the chloride channel clcnk and the transcription factor pax2. Overexpression of two mutant HNF1B derivatives resulted in distinct phenotypes reflected by either a reduction or an enlargement of pronephros size. The expression of selected pronephric marker genes was differentially affected upon overexpression of HNF1B mutations. Based on our findings, we postulate that HNF1B mutations influence gene regulation upon overexpression in specific and distinct manners. Furthermore, our study demonstrates that the newly established Cre/loxP system for Xenopus embryos is an attractive alternative to examine the gene regulatory potential of transcription factors in developing pronephric kidney as exemplified here for HNF1B

Requirement for a Uroplakin 3a-like protein in the development of zebrafish pronephric tubule epithelial cell function, morphogenesis, and polarity

Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na+/K+-ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression. © 2012 Mitra et al

Kidney regeneration: common themes from the embryo to the adult

The vertebrate kidney has an inherent ability to regenerate following acute damage. Successful regeneration of the injured kidney requires the rapid replacement of damaged tubular epithelial cells and reconstitution of normal tubular function. Identifying the cells that participate in the regeneration process as well as the molecular mechanisms involved may reveal therapeutic targets for the treatment of kidney disease. Renal regeneration is associated with the expression of genetic pathways that are necessary for kidney organogenesis, suggesting that the regenerating tubular epithelium may be “reprogrammed” to a less-differentiated, progenitor state. This review will highlight data from various vertebrate models supporting the hypothesis that nephrogenic genes are reactivated as part of the process of kidney regeneration following acute kidney injury (AKI). Emphasis will be placed on the reactivation of developmental pathways and how our understanding of the resulting regeneration process may be enhanced by lessons learned in the embryonic kidney.Fil: Cirio, Maria Cecilia. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Groh, Eric D.. University of Pittsburgh; Estados UnidosFil: de Caestecker, Mark P.. Vanderbilt University; Estados UnidosFil: Davidson, Alan J.. The University of Auckland; Nueva ZelandaFil: Hukriede, Neil A.. University of Pittsburgh; Estados Unido

Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localisation of polycystin-2 in vivo and in vitro

PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 (PC2), the PKD2 protein, is a nonselective Ca2 + -permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser812) has been previously reported. Since we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser812 or all 5 predicted phosphorylation sites in the C-terminus, we hypothesised that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser76) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK-3). The consensus recognition sequence for GSK-3 (Ser76/Ser80) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK-3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without a change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK-3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology

Sublethal concentrations of waterborne copper induce cellular stress and cell death in zebrafish embryos and larvae

http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602011000100002&lng=es&nrm=isoCopper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals

Developmental Basis of Pronephric Defects in Xenopus Body Plan Phenotypes

AbstractWe have used monoclonal antibodies that recognize the pronephric tubules or pronephric duct to explore the induction of the embryonic kidney in developing Xenopus embryos. Morphogenesis of the pronephros was examined in UV-ventralized and lithium-dorsalized embryos. We find that the pronephric tubules are present in all but the strongest UV-induced phenotypes, but absent from relatively moderate lithium phenotypes. Interestingly the pronephric duct, which develops from the ventroposterior portion of the pronephric anlage, is missing from more of the mild UV phenotypes than are pronephric tubules. The loss of the capacity to form pronephroi in UV-ventralized embryos is caused by the loss of tissues capable of inducing the pronephric mesoderm, as marginal zone explants from ventralized embryos are still competent to respond to pronephric-inductive signals. Explant recombination experiments indicate that the tissue responsible for both the loss of pronephroi in UV-ventralized embryos and the induction of pronephroi during normal development is the anterior somites. The absence of pronephroi in relatively mild lithium phenotypes has a developmental basis different from that of the UV phenotype, as explants from lithium-treated embryos are effective inducers of pronephroi in recombinants with competent mesoderm, even though they themselves do not form pronephroi in isolation. Together these data indicate that dorsal tissues, especially the anterior somites, are responsible for the establishment of the intermediate mesoderm and the induction of the embryonic kidneys and that even mild dorsalization destroys the capacity to form cells competent to receive this signal

Identification and functional characterization of an N-terminal oligomerization domain for polycystin-2*

Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of kidney failure, is caused by mutations in either PKD1 (85%) or PKD2 (15%). The PKD2 protein, polycystin-2 (PC2 or TRPP2), is a member of the transient receptor potential (TRP) superfamily and functions as a non-selective calcium channel. PC2 has been found to form oligomers in native tissues suggesting that it may form functional homo- or heterotetramers with other subunits, similar to other TRP channels. Our experiments unexpectedly revealed that PC2 mutant proteins lacking the known C-terminal dimerization domain were still able to form oligomers and co-immunoprecipitate full-length PC2, implying the possible existence of a proximal dimerization domain. Using yeast two-hybrid and biochemical assays, we have mapped an alternative dimerization domain to the N terminus of PC2 (NT2-1-223, L224X). Functional characterization of this domain demonstrated that it was sufficient to induce cyst formation in zebrafish embryos and inhibit PC2 surface currents in mIMCD3 cells probably by a dominant-negative mechanism. In summary, we propose a model for PC2 assembly as a functional tetramer which depends on both C- and N-terminal dimerization domains. These results have significant implications for our understanding of PC2 function and disease pathogenesis in ADPKD and provide a new strategy for studying PC2 function

MDM2 prevents spontaneous tubular epithelial cell death and acute kidney injury

Murine double minute-2 (MDM2) is an E3-ubiquitin ligase and the main negative regulator of tumor suppressor gene p53. MDM2 has also a non-redundant function as a modulator of NF-kB signaling. As such it promotes proliferation and inflammation. MDM2 is highly expressed in the unchallenged tubular epithelial cells and we hypothesized that MDM2 is necessary for their survival and homeostasis. MDM2 knockdown by siRNA or by genetic depletion resulted in demise of tubular cells in vitro. This phenotype was completely rescued by concomitant knockdown of p53, thus suggesting p53 dependency. In vivo experiments in the zebrafish model demonstrated that the tubulus cells of the larvae undergo cell death after the knockdown of mdm2. Doxycycline-induced deletion of MDM2 in tubular cell-specific MDM2-knockout mice Pax8rtTa-cre; MDM2f/f caused acute kidney injury with increased plasma creatinine and blood urea nitrogen and sharp decline of glomerular filtration rate. Histological analysis showed massive swelling of renal tubular cells and later their loss and extensive tubular dilation, markedly in proximal tubules. Ultrastructural changes of tubular epithelial cells included swelling of the cytoplasm and mitochondria with the loss of cristae and their transformation in the vacuoles. The pathological phenotype of the tubular cell-specific MDM2-knockout mouse model was completely rescued by co-deletion of p53. Tubular epithelium compensates only partially for the cell loss caused by MDM2 depletion by proliferation of surviving tubular cells, with incomplete MDM2 deletion, but rather mesenchymal healing occurs. We conclude that MDM2 is a non-redundant survival factor for proximal tubular cells by protecting them from spontaneous p53 overexpression-related cell death