Calcium signaling induces partial EMT and renal fibrosis in a Wnt4mCherry knock-in mouse model.

The renal tubular epithelial cells (TEC) have a strong capacity for repair after acute injury, but when this mechanism becomes uncontrollable, it leads to chronic kidney diseases (CKD). Indeed, in progress toward CKDs, the TECs may dedifferentiate, undergo epithelial-to-mesenchyme transition (EMT), and promote inflammation and fibrosis. Given the critical role of Wnt4 signaling in kidney ontogenesis, we addressed whether changes in this signaling are connected to renal inflammation and fibrosis by taking advantage of a knock-in Wnt4mCh/mCh mouse. While the Wnt4mCh/mCh embryos appeared normal, the corresponding mice, within one month, developed CKD-related phenotypes, such as pro-inflammatory responses including T-cell/macrophage influx, expression of fibrotic markers, and epithelial cell damage with a partial EMT. The Wnt signal transduction component ß-catenin remained unchanged, while calcium signaling is induced in the injured TECs involving Nfat and Tfeb transcription factors. We propose that the Wnt4 signaling pathway is involved in repairing the renal injury, and when the signal is overdriven, CKD is established.

Deletion of hypoxia-inducible factor prolyl 4-hydroxylase 2 in FoxD1-lineage mesenchymal cells leads to congenital truncal alopecia.

Hypoxia-inducible factors (HIFs) induce numerous genes regulating oxygen homeostasis. As oxygen sensors of the cells, the HIF prolyl 4-hydroxylases (HIF-P4Hs) regulate the stability of HIFs in an oxygen-dependent manner. During hair follicle (HF) morphogenesis and cycling, the location of dermal papilla (DP) alternates between the dermis and hypodermis and results in varying oxygen levels for the DP cells. These cells are known to express hypoxia-inducible genes, but the role of the hypoxia response pathway in HF development and homeostasis has not been studied. Using conditional gene targeting and analysis of hair morphogenesis, we show here that lack of Hif-p4h-2 in Forkhead box D1 (FoxD1)-lineage mesodermal cells interferes with the normal HF development in mice. FoxD1-lineage cells were found to be mainly mesenchymal cells located in the dermis of truncal skin, including those cells composing the DP of HFs. We found that upon Hif-p4h-2 inactivation, HF development was disturbed during the first catagen leading to formation of epithelial-lined HF cysts filled by unorganized keratins, which eventually manifested as truncal alopecia. Furthermore, the depletion of Hif-p4h-2 led to HIF stabilization and dysregulation of multiple genes involved in keratin formation, HF differentiation, and HIF, transforming growth factor ß (TGF-ß), and Notch signaling. We hypothesize that the failure of HF cycling is likely to be mechanistically caused by disruption of the interplay of the HIF, TGF-ß, and Notch pathways. In summary, we show here for the first time that HIF-P4H-2 function in FoxD1-lineage cells is essential for the normal development and homeostasis of HFs.

Wnt4, a pleiotropic signal for controlling cell polarity, basement membrane integrity, and antimüllerian hormone expression during oocyte maturation in the female follicle.

Wnt4 is a key signal that channels the developmental fate of the indifferent mammalian gonad toward the ovary, but whether Wnt4 has later roles during ovary development remains unknown. To investigate this, we inactivated the Wnt4 gene by crossing Amhr2Cre and doxycycline-inducible Rosa(rtTA)-knock-in Cre mice with mice carrying a floxed Wnt4 allele and used a novel Wnt4(mCherry)-knock-in mouse. In these models, ovarian folliculogenesis was compromised, and female fertility was severely reduced, and Wnt4 deficiency eventually led to premature ovarian failure. These anomalies were associated with cell polarity defects in the follicle. Within the follicle, laminin and type IV collagen assembled ectopic basement membrane-like structures, the cell adherens junction components N-cadherin and ß-catenin lost their polarized expression pattern, and expression of the gap junction protein connexin 43 was reduced by ~30% when compared with that of the controls. Besides these changes, expression of antimüllerian hormone (Amh) was inhibited in the absence of Wnt4 signaling in vivo. Consistent with this, Wnt4 signaling up-regulated Amh gene expression in KK1 cells in vitro. Thus, Wnt4 signaling is necessary during maturation of the ovarian follicles, where it coordinates expression of Amh, cell survival, and polarized organization of the follicular cells.

ErbB4 modulates tubular cell polarity and lumen diameter during kidney development.

ErbB4 receptor tyrosine kinase contributes to the development of the heart, the central nervous system, and the lactating mammary gland, but whether it has a role in the development of the kidney epithelium is unknown. Here, we found that expression of Erbb4 isoforms JM-a CYT-1 and JM-a CYT-2 was first detectable around embryonic day 13 in the mouse, mainly in the collecting ducts and both the proximal and distal tubules. In vitro, overexpression of a relevant ErbB4 isoform promoted proliferation and disturbed polarization of kidney epithelial cells when cultured as three-dimensional structures. We examined ErbB4 function in developing kidney tubules in vivo with Pax8-Cre-mediated conditional overexpression of Rosa26 locus-targeted ERBB4 and with conditional Erbb4 knock-out mice. The Pax8-Cre-driven ERBB4 overexpression enhanced proliferation in the collecting ducts, reduced the size of epithelial duct lumens, and promoted formation of cortical tubular cysts. These defects were associated with changes in the subcellular distribution of markers of epithelial cell polarity. Similarly, the Pax8-Cre-mediated Erbb4 knock-out mice manifested dysfunctional kidneys with larger duct lumens and epithelial cell mispolarization. Taken together, these data suggest that ErbB4 signaling modulates proliferation and polarization, cellular functions critical for the development of epithelial ducts in the kidney.

A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development.

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.

Secreted Wnt antagonist Dickkopf-1 controls kidney papilla development coordinated by Wnt-7b signalling.

Wnt signalling regulates several aspects of kidney development such as nephrogenesis, ureteric bud branching and organisation of the collecting duct cells. We addressed the potential involvement of Dickkopf-1 (Dkk1), a secreted Wnt pathway antagonist. Dkk1 is expressed in the developing mouse kidney by pretubular cell aggregates and the nephrons derived from them. Besides the mesenchyme cells, the epithelial ureteric bud and more mature ureteric bud derivatives in the medulla and the papilla tip express the Dkk1 gene. To reveal the potential roles of Dkk1, we generated a floxed allele and used three Cre lines to inactivate Dkk1 function in the developing kidney. Interestingly, Dkk1 deficiency induced by Pax8Cre in the kidneys led in newborn mice to an overgrown papilla that was generated by stimulated proliferation of the collecting duct and loop of Henle cells, implying a role for Dkk1 in the collecting duct and/or loop of Henle development. Since Pax8Cre-induced Dkk1 deficiency reduced marker gene expression, Scnn1b in the collecting duct and Slc12a1 in the loop of Henle, these results together with the extended papilla phenotype are likely reasons for the decreased amount of ions and urine produced by Dkk1-deficient kidneys in the adult. Recombinant Dkk1 protein in cultured cells inhibited Wnt-7b-induced canonical Wnt signalling, which is critical for collecting duct and loop of Henle development. Moreover, Dkk1 deficiency led to an increase in the expression of canonical Wnt signalling of target Lef-1 gene expression in the stromal cells of the developing papilla. Based on the results, we propose that Dkk1 controls the degree of Wnt-7b signalling in the papilla to coordinate kidney organogenesis.

Wnt-11 signalling controls ventricular myocardium development by patterning N-cadherin and beta-catenin expression.

AIMS: The stage-dependent organization of the cardiomyocytes during formation of the different layers of the developing ventricular wall is critical for the establishment of a functional heart, but the instructive signals involved are still poorly known. We have addressed the potential role of Wnt-11 in the control of early ventricular myocardium assembly. METHODS AND RESULTS: We demonstrate by means of expression analysis and a mouse model in which Wnt-11 function has been inactivated that Wnt-11 is expressed by the embryonic ventricular cardiomyocytes and serves as one important signal for ventricular wall development. In the absence of Wnt-11, the coordinated organization, intercellular contacts, co-localized expression of the cell adhesion components N-cadherin and beta-catenin, and the cytoskeleton of the differentiating ventricular cardiomyocytes are all disturbed. Moreover, the ventricular wall lacking Wnt-11 signalling is thinner and the expression of the Gata-4, Nkx2.5, Mef2c, ANP, and BNP genes is down-regulated relative to controls. These defects lie behind disturbed embryonic cardiac functional development, marked by an increase in the ventricular relaxation time during the early diastole. CONCLUSION: We conclude that Wnt-11 signalling serves as a critical cell adhesion cue for the organization of the cardiomyocytes in the developing ventricular wall, which is essential for the establishment of a functional heart.

Genomic response to Wnt signalling is highly context-dependent--evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets.

Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-beta-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of beta-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

Wnt-11 signaling leads to down-regulation of the Wnt/beta-catenin, JNK/AP-1 and NF-kappaB pathways and promotes viability in the CHO-K1 cells.

The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical beta-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical beta-catenin mediated Wnt signaling but also JNK/AP-1 and NF-kappaB signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-kappaB pathway. Consistent with the central role of Akt, JNK and NF-kappaB in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways.

Polyamines are involved in murine kidney development controlling expression of c-ret, E-cadherin, and Pax2/8 genes.

Polyamines play an important role in cell growth and differentiation. We studied changes in morphogenesis and the expression of the developmental control genes in the embryonic mouse kidney in response to polyamine depletion, using a kidney organ culture approach and reducing the polyamine pools with alpha-difluoromethylornithine (DFMO), an irreversible suicide inhibitor of ornithine decarboxylase (ODC). We found that inhibition of ODC results in a systematic kidney organogenesis phenotype, in that the DFMO-treated kidney specimens were of smaller size, had less epithelial ureteric bud branches, and their mesenchymal-derived tubule formation was retarded. These dysmorphologies were shown to be associated with changes in cell proliferation. Whole-mount in situ experiments revealed that inhibition of ODC causes increases in epithelial c-ret and E-cadherin and a decrease in mesenchymal Pax-8 expression, whereas levels of epithelial Wnt-11, mesenchymal GDNF, FoxD1, and Pax-2 transcripts remain unchanged. We studied regulation of the Pax-2 gene by analyzing a mouse line in which lacZ was driven by an 8.5 kb Pax-2 enhancer in the epithelial ureteric bud, and found that Pax-2 expression, as indicated by lacZ expression, increased after DFMO treatment. Transient transfection experiments in HEK 293 cells with the minimal Pax-2 promoter showed enhanced transcription upon reduction of the polyamine pools. We propose that ODC and polyamines have an important role in kidney organogenesis, being involved in the regulation of the expression of genes implicated in epithelial-mesenchymal tissue interactions.

Murine Wnt-1 with an internal c-myc tag recombinantly produced in Escherichia coli can induce intracellular signaling of the canonical Wnt pathway in eukaryotic cells.

Wnt-1 belongs to the Wnt family of secreted glycoproteins inducing an intracellular signaling pathway involved in cell proliferation, differentiation, and pattern formation. The canonical branch is one of three known branches. This is also valid in vitro, and Wnts can be considered beneficial for culturing primary cells from organs, provided Wnts are available and applicable even with cells of different species. It was shown here that internally c-myc-tagged murine Wnt-1 produced in the heterologous host Escherichia coli was appropriate for inducing intracellular signaling of the canonical Wnt pathway in eukaryotic cells via stabilization of cytosolic beta-catenin. The pioneering injection of the protein into the blastocoels of Xenopus laevis embryos led to axis duplication and suppression of head formation. Applying the recombinant murine Wnt-1 to metanephric mesenchyme activated the tubulogenic program. The signal-inducing activity of the recombinant protein was also positively demonstrated in the TOP-flash reporter assay. Although Wnts were purified recently from the growth media of stably transfected eukaryotic cell lines, the production of active Wnt proteins in pro- or eukaryotic microorganisms reportedly has never been successful. Here soluble production in E. coli and translocation into the oxidizing environment of the periplasm were achieved. The protein was purified using the internal c-myc tag. The effect on the eukaryotic cells implies that activity was retained. Thus, this approach could make recombinant murine Wnt-1 available as a good starting point for other Wnts needed, for example, for maintaining and differentiating stem cells, organ restoration therapy, and tissue engineering.