Loss of ICAT gene function leads to arrest of ureteric bud branching and renal agenesis

ICAT, inhibitor of beta-catenin and T cell factor, or Ctnnbip1, is a negative regulator of the Wnt signaling pathway that interferes with the interaction between beta-catenin and T cell factor. Some ICAT-deficient (ICAT-/-) embryos exhibit unilateral or bilateral renal agenesis. In this study, we investigated developmental processes in the ICAT-/- kidney. ICAT was highly expressed in both the ureteric bud (UB) and the surrounding metanephric mesenchymal (MM) cells in the metanephros of embryonic day E11.5-E13.5 wild-type (ICAT+/+) mouse. In the E12.5-ICAT-/- metanephros, UB branching was delayed, and a T-shaped, bifurcated UB was frequently seen; this was never seen in the E12.5-ICAT+/+ metanephros. More apoptotic MM cells were detected in the ICAT-/- metanephros than in the ICAT+/+ metanephros. These results suggest that the loss of ICAT gene function causes the arrest of UB branching and the apoptotic death of MM cells, resulting in renal agenesis.     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Comparative immunohistochemical analysis of the expression of cytokeratins, vimentin and alpha-smooth muscle actin in human foetal mesonephros and metanephros

The human mesonephros is currently regarded as a simplified version of the foetal metanephros, primarily due to the close morphological resemblance between these two structures. The aim of the present study was to define whether human mesonephric and foetal metanephric nephrons share immunophenotypical traits in their corresponding structures (glomeruli, proximal and distal tubules). For this purpose we first investigated immunohistochemically the overall expression and topographical distribution of cytokeratins 7, 8, 18, 19, and 20, vimentin and -smooth muscle actin in mature mesonephric nephrons and compared the results with those obtained in maturing-stage foetal metanephric nephrons. No expression of cytokeratins 7 and 20 was found. Cytokeratins 8, 18, and 19 and vimentin showed a restricted and basically coincident expression along the different components of both mesonephric and metanephric nephrons. These findings indicate that the intermediate filament protein profile of human mature mesonephric nephrons closely recapitulates that observed in developing metanephros and thereby strengthens the concept that human mesonephros, a transient ontogenic structure, is largely similar to the foetal metanephros.The sole difference between human mesonephros and foetal metanephros was the divergent expression of -smooth muscle actin. This protein exhibited an increasingly accentuated mesangial expression paralleling the morphological maturation of metanephric glomerulus, whereas it was absent from the mesonephric one. This would suggest that the mesangial cells in these two renal structures have a different function during the foetal life.     

Role of Wnt5a-Ror2 Signaling in Morphogenesis of the Metanephric Mesenchyme during Ureteric Budding

Development of the metanephric kidney begins with the induction of a single ureteric bud (UB) on the caudal Wolffian duct (WD) in response to GDNF (glial cell line-derived neurotrophic factor) produced by the adjacent metanephric mesenchyme (MM). Mutual interaction between the UB and MM maintains expression of GDNF in the MM, thereby supporting further outgrowth and branching morphogenesis of the UB, while the MM also grows and aggregates around the branched tips of the UB. Ror2, a member of the Ror family of receptor tyrosine kinases, has been shown to act as a receptor for Wnt5a to mediate noncanonical Wnt signaling. We show that Ror2 is predominantly expressed in the MM during UB induction and that Ror2- and Wnt5a-deficient mice exhibit duplicated ureters and kidneys due to ectopic UB induction. During initial UB formation, these mutant embryos show dysregulated positioning of the MM, resulting in spatiotemporally aberrant interaction between the MM and WD, which provides the WD with inappropriate GDNF signaling. Furthermore, the numbers of proliferating cells in the mutant MM are markedly reduced compared to the wild-type MM. These results indicate an important role of Wnt5a-Ror2 signaling in morphogenesis of the MM to ensure proper epithelial tubular formation of the UB required for kidney development.     

Down-regulated Six2 by knockdown of neurofibromin results in apoptosis of metanephric mesenchyme cells in vitro

Embryonic Six2-positive nephron progenitor cells adjacent to ureteric bud tips ultimately give rise to nephron structures, including proximal and distal tubules, podocytes, Bowman’s capsules, and the glomeruli. This process requires an internal balance between self-renew and differentiation of the nephron progenitor cells, which is mediated by numerous molecules. Recent studies have shown that the neurofibromin (Nf1) null mutant mouse embryos have an 18- to 24-h developmental delay in metanephros manifesting retardation in its cephalad repositioning and reduction number of glomeruli. However, the underlying inter-/intracellular signaling mechanisms responsible for reducing number of glomeruli during nephrogenesis remain to be fully elucidated. Here, we originally detected the Nf1 expression in developing kidney and metanephric mesenchyme cells. Surprisingly, Nf1 knockdown by small interfering RNAs in the metanephric mesenchyme cells (mK3) resulted in a decreased expression of Six2, the key marker of renal progenitor cells, while the ratio of apoptotic cells was significantly increased. Furthermore, overexpression of Six2 in mk3 cells partially rescued apoptosis phenotype. Collectively, these results implied that knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro probably through down-regulation of Six2 expression. Collectively, we demonstrated that down-regulated Six2 by knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro. These results implied that inhibition of Nf1 may delay metanephros development via down-regulation of Six2.     

Expression of Stem Cell Markers in the Human Fetal Kidney

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34^th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms'' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms'' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAM^neg and EpCAM^dim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24⁺CD133⁺ cells comprise >50% of HFK cells and predominantly co-express EpCAM^bright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM⁺EpCAM^- and to a lesser extent in NCAM⁺EpCAM⁺ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM⁺EpCAM⁺FZD7⁺), MM stem cells (NCAM⁺EpCAM^-FZD7⁺) or both (NCAM⁺FZD7⁺). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.     

Low Ambient O2 Enhances Ureteric Bud Branching In Vitro

Hypoxia exists widely in developing embryos where it may regulate blood vessel formation. VEGF and FGF2 produced in developing renal primordia (metanephroi) stimulate microvessel formation from embryonic thoracic aorta cultured under hypoxic conditions (HC) relative to room air (RA). The aim of the present study was to provide insight into the participation of hypoxia in a process that occurs concomitant with metanephros vascularization in vivo, ureteric bud (UB) branching. To this end, the arborization of the UB and growth of metanephroi were measured in metanephroi grown in serum-free organ culture for two days under RA or HC. When metanephroi were cultured under HC the arborization of UB was stimulated relative to RA. In the presence of anti-VEGF neutralizing antibody (αmVEGF), or anti-FGF2 neutralizing antibody (αhFGF2) UB branching was inhibited under both RA and HC. When both αmVEGF and αhFGF2 were added, the inhibition was enhanced. Addition of exogenous VEGF or FGF2 to cultures stimulated UB branching under RA and HC and addition of both stimulated it further. These findings provide evidence for roles of hypoxia and metanephric VEGF and FGF2, as regulators not only for vascularization but also for UB bud branching during renal organogenesis.Key Words: metanephroi, embryogenesis, fibroblast growth factor, vascular endothelial growth factor     

Frizzled-4 expression during chick kidney development

Wnts have been implicated in metanephric kidney development. To determine whether Frizzleds, the genes that encode Wnt receptors, are present at early stages of nephrogenesis, we examined the expression of several recently identified Frizzled genes in the chick by in situ hybridization. Here we report the cloning and characterization of chick Frizzled-4 (cFz-4), which we found to be expressed in the developing chick kidney. cFz-4 was first expressed in the pronephros caudal to the third somite at Hamburger and Hamilton stage 10. Its expression increased with maturation, becoming restricted to the newly induced glomeruli and tubules in the mesonephros and metanephros. Within the metanephros, cFz-4 and Wnt-4 expression patterns were similar, whereas Wnt-11 was expressed solely in the tips of the branching ureteric bud. cFz-4 expression was compared with that of known kidney markers. It preceded that of Lmx-1, but was similarly restricted to developing glomeruli and tubules. In contrast, Pax-2 expression and Lim 1/2 antibody labeling occurred in intermediate mesoderm caudal to the fifth somite in the early pronephros, and each persisted in both the tubules and nephric ducts throughout further development.     

Hoxc8 downregulates Mgl1 tumor suppressor gene expression and reduces its concomitant function on cell adhesion

Hoxc8 is a homeobox gene family member, which is essential for growth and differentiation. Mgl1, a mouse homologue of the Drosophila tumor suppressor gene lgl, was previously identified as a possible target of Hoxc8. However, the biological effects and underlying molecular mechanism of Hoxc8 regulation on Mgl1 has not been fully established. The endogenous expression patterns of Hoxc8 were inversely correlated with those of Mgl1 in different types of cells and tissues. Here we showed that Hoxc8 overexpression downregulated the Mgl1 mRNA expression. Characterization of the ∼2 kb Mgl1 promoter region revealed that the upstream sequence contains several putative Hox core binding sites and chromatin immunoprecipitation assay confirmed that Hoxc8 directly binds to the 5′ upstream region of Mgl1. The promoter activity of this region was diminished by Hoxc8 expression but resumed by knockdown of Hoxc8 using siRNA against Hoxc8. Functional study of Mgl1 in C3H10T1/2 cells revealed a significant reduction in cell adhesion upon expression of Hoxc8. Taken together, our data suggest that Hoxc8 downregulates Mgl1 expression via direct binding to the promoter region, which in turn reduces cell adhesion and concomitant cell migration.     

Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system

Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.     

Similar developmental patterns in immunolocalisation of stem cell factor and KIT in bovine meso- and metanephros

The mesonephros is often regarded as a simplified version of the terminal renal organ, the metanephros. Both renal organs result from an epithelio-mesenchymal interaction between the Wolffian duct and the nephrogenic ridge. It appears that the epithelio-mesenchymal interaction makes use of similar signal cascades for both renal organs and that key events required for the development of the metanephros occur at earlier stages. In murine metanephroi, the stem cell factor (SCF)/-KIT-signal transduction pathway has recently been shown to regulate ureteric bud branching and epithelial cell differentiation. We immunohistochemically defined the time-sequence of KIT and SCF presence in both renal organs using bovine embryos/foetuses with crown rump length (CRL) of 1.7–24 cm. In the mesonephroi, epithelial cells with strong KIT staining were scattered in distal tubules, and SCF was expressed in the epithelial wall of corpuscles and proximal tubules. KIT positivity occurred in the metanephroi of embryos prior to SCF; KIT was predominantly localised at the ureteric bud tips in the nephrogenic zone. In foetuses of 13 cm and more CRL, the SCF/KIT profile of developmentally advanced nephrons mirrored the situation in the mesonephros. Epithelial cells with strong KIT staining were scattered in the cortical areas of distal tubules, while SCF was expressed in the epithelial wall of corpuscles and proximal tubules. Our morphological findings agree with a potential role of KIT at the ureteric bud tips and demonstrate a similar expression of KIT and SCF along the areas of developmentally advanced mesonephric and metanephric nephrons.     

Stepwise renal lineage differentiation of mouse embryonic stem cells tracing in vivo development

The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0-2, A plus BMP-4 (4) during days 2-4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4-6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6-8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24(+) cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III.     

Tight regulation of p53 activity by Mdm2 is required for ureteric bud growth and branching

Mdm2 (Murine Double Minute-2) is required to control cellular p53 activity and protein levels. Mdm2 null embryos die of p53-mediated growth arrest and apoptosis at the peri-implantation stage. Thus, the absolute requirement for Mdm2 in organogenesis is unknown. This study examined the role of Mdm2 in kidney development, an organ which develops via epithelial–mesenchymal interactions and branching morphogenesis. Mdm2 mRNA and protein are expressed in the ureteric bud (UB) epithelium and metanephric mesenchyme (MM) lineages. We report here the results of conditional deletion of Mdm2 from the UB epithelium. UB^mdm2−/− mice die soon after birth and uniformly display severe renal hypodysplasia due to defective UB branching and underdeveloped nephrogenic zone. Ex vivo cultured UB^mdm2−/− explants exhibit arrested development of the UB and its branches and consequently develop few nephron progenitors. UB^mdm2−/− cells have reduced proliferation rate and enhanced apoptosis. Although markedly reduced in number, the UB tips of UB^mdm2−/−metanephroi continue to express c-ret and Wnt11; however, there was a notable reduction in Wnt9b, Lhx-1 and Pax-2 expression levels. We further show that the UB^mdm2−/− mutant phenotype is mediated by aberrant p53 activity because it is rescued by UB-specific deletion of the p53 gene. These results demonstrate a critical and cell autonomous role for Mdm2 in the UB lineage. Mdm2-mediated inhibition of p53 activity is a prerequisite for renal organogenesis.     

Low Ambient O2 Enhances Ureteric Bud Branching In Vitro

Hypoxia exists widely in developing embryos where it may regulate blood vessel formation. VEGF and FGF2 produced in developing renal primordia (metanephroi) stimulate microvessel formation from embryonic thoracic aorta cultured under hypoxic conditions (HC) relative to room air (RA). The aim of the present study was to provide insight into the participation of hypoxia in a process that occurs concomitant with metanephros vascularization in vivo, ureteric bud (UB) branching. To this end, the arborization of the UB and growth of metanephroi were measured in metanephroi grown in serum-free organ culture for 2 days under RA or HC. When metanephroi were cultured under HC the arborization of UB was stimulated relative to RA. In the presence of anti-VEGF neutralizing antibody (amVEGF), or anti-FGF2 neutralizing antibody (ahFGF2) UB branching was inhibited under both RA and HC. When both amVEGF and ahFGF2 were added, the inhibition was enhanced. Addition of exogenous VEGF or FGF2 to cultures stimulated UB branching under RA and HC and addition of both stimulated it further. These findings provide evidence for roles of hypoxia and metanephric VEGF and FGF2, as regulators not only for vascularization but also for UB bud branching during renal organogenesis.     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Role of fibroblast growth factor receptor signaling in kidney development

Fibroblast growth factor receptors (Fgfrs) consist of four signaling family members and one nonsignaling "decoy" receptor, Fgfr-like 1 (Fgfrl1), all of which are expressed in the developing kidney. Several studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB) in cultured tissues. Transgenic and conditional knockout approaches in whole animals have shown that Fgfr1 and Fgfr2 (predominantly the IIIc isoform) in kidney mesenchyme are critical for early MM and UB formation. Conditional deletion of the ligand, Fgf8, in nephron precursors or global deletion of Fgfrl1 interrupts nephron formation. Fgfr2 (likely the IIIb isoform signaling downstream of Fgf7 and Fgf10) is critical for ureteric morphogenesis. Moreover, Fgfr2 appears to act independently of Frs2α (the major signaling adapter for Fgfrs) in regulating UB branching. Loss of Fgfr2 in the MM leads to many kidney and urinary tract anomalies, including vesicoureteral reflux. Thus Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.     

Ontogeny of calbindin-D28K and calretinin in developing chick kidney

The ontogeny of two calcium-binding proteins (calbindin-D_28k and calretinin) was studied by immunohistochemical techniques in developing chick kidney. This study showed the presence of calbindin on the 5th incubation day and calretinin on the 7th incubation day in mesonephric distal and connecting tubules, and in the medial wall of the Wolffian duct. At later stages, immunostaining for these two proteins, in particular for calretinin, was also demonstrated in some metanephric proximal tubules. Glomeruli and Bowman's capsules were negative both in the mesonephros and metanephros. The presence of calretinin in the developing kidney has thus been demonstrated for the first time. The early expression of calbindin and calretinin in mesonephric distal tubules suggests their role in regulating the final excretion of calcium. The different patterns of immunoreactivity of the walls of the Wolffian duct can be correlated with their different histogenetic and histological features.