Kidney Mesenchyme Research Articles

Single-Cell RNA Sequencing Reveals mRNA Splice Isoform Switching during Kidney Development.

During mammalian kidney development, nephron progenitors undergo a mesenchymal-to-epithelial transition and eventually differentiate into the various tubular segments of the nephron. Recently, Drop-seq single-cell RNA sequencing technology for measuring gene expression from thousands of individual cells identified the different cell types in the developing kidney. However, that analysis did not include the additional layer of heterogeneity that alternative mRNA splicing creates. Full transcript length single-cell RNA sequencing characterized the transcriptomes of 544 individual cells from mouse embryonic kidneys. Gene expression levels measured with full transcript length single-cell RNA sequencing identified each cell type. Further analysis comprehensively characterized splice isoform switching during the transition between mesenchymal and epithelial cellular states, which is a key transitional process in kidney development. The study also identified several putative splicing regulators, including the genes Esrp1/2 and Rbfox1/2. Discovery of the sets of genes that are alternatively spliced as the fetal kidney mesenchyme differentiates into tubular epithelium will improve our understanding of the molecular mechanisms that drive kidney development.

Monitoring and maintaining the differentiation status of human ipsc derived kidney organoids

Background & Aim Human induced pluripotent stem cell (iPSC) derived kidney organoids resemble first trimester fetal kidney and offer a great model for studying kidney development and disease. During early renal development, the inductive interaction between two intermediate mesoderm (IM)-derived progenitor cell populations, the ureteric bud and the kidney mesenchyme, promotes nephron and collecting duct formation. Protocols are developed to in vitro differentiate human iPSC into ureteric epithelium and metanephric mesenchyme simultaneously, and then aggregate them to generate a complex multicellular kidney organoids. However, this is a complex procedure with unpredictable outcomes. Furthermore, after 25 days of culture, kidney organoids start to degenerate and form fibrotic and cartilage tissue. We investigated how kidney organoid differentiation can be monitored and how the survival of kidney organoids can be prolonged. Methods, Results & Conclusion We treated five human iPSC with CHIR99021 and followed by FGF9 to induce IM for 7 days. These IM like cells were formed into cell pellets and transferred to air-liquid interface for a further 18 up to 32 days. Successfully differentiated organoids demonstrate glomerular and tubular structures in H&E section, while failed differentiation results in amorphous fibrotic tissue. The same iPSC line had variable results among batch to batch. Along the differentiation process, LDH activity was measured to monitor organoid metabolism, which reached a peak on days 7∼9. LDH peaks higher than 50 milliunits/ml indicated excessive necrosis and resulted in abnormal organoid formation. We observed that iPSC with elevated OCT4 expression showed induced capacity to differentiate into kidney organoids. Furthermore high TGFβ expression at day 7 was associated with fibrosis in the kidney organoids. As shortage of oxygen supply to kidney organoids due to lack of functional vasculature limits their long term stability, we split the organoids on day 8 and day 21 and placed the organoids on a shaker on day 21 to allow for better oxygen supply. Using this method, it was possible to preserve the kidney organoids for 2 additional weeks with maintenance of glomerular and tubular structures. Taken together, these data will help us to monitor the differentiation and prolong the culture time of kidney organoids.

Simple 3D culture of dissociated kidney mesenchyme mimics nephron progenitor niche and facilitates nephrogenesis Wnt-independently

Kidney mesenchyme (KM) and nephron progenitors (NPs) depend on WNT activity, and their culture in vitro requires extensive repertoire of recombinant proteins and chemicals. Here we established a robust, simple culture of mouse KM using a combination of 3D Matrigel and growth media supplemented with Fibroblast Growth Factor 2 (FGF2) and Src inhibitor PP2. This allows dissociated KM to spontaneously self-organize into spheres. To reassess the requirement of WNT activity in KM self-organization and NPs maintenance, cells were cultured with short pulse of high-dose GSK3β inhibitor BIO, on a constant low-dose or without BIO. Robust proliferation at 48 hours and differentiation at 1 week were observed in cultures with high BIO pulse. Importantly, dissociated KM cultured without BIO, similarly to that exposed to constant low dose of BIO, maintained NPs up to one week and spontaneously differentiated into nephron tubules at 3 weeks of culture. Our results show that KM is maintained and induced to differentiate in a simple culture system. They also imply that GSK3β/WNT-independent pathways contribute to the maintenance and induction of mouse KM. The robust and easy 3D culture enables further characterization of NPs, and may facilitate disease modeling when applied to human cells.

Identification of biomarkers, pathways and potential therapeutic agents for white adipocyte insulin resistance using bioinformatics analysis

ABSTRACTFor the better understanding of insulin resistance (IR), the molecular biomarkers in IR white adipocytes and its potential mechanism, we downloaded two mRNA expression profiles from Gene Expression Omnibus (GEO). The white adipocyte samples in two databases were collected from the human omental adipose tissue of IR obese (IRO) subjects and insulin-sensitive obese (ISO) subjects, respectively. We identified 86 differentially expressed genes (DEGs) between the IRO and ISO subjects using limma package in R software. Gene Set Enrichment Analysis (GSEA) provided evidence that the most gene sets enriched in kidney mesenchyme development in the ISO subjects, as compared with the IRO subjects. The Gene Ontology (GO) analysis indicated that the most significantly enriched in cellular response to interferon-gamma. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the DEGs were most significantly enriched in cytokine-cytokine receptor interaction. Protein–Protein Interaction (PPI) network was performed with the STRING, and the top 10 hub genes were identified with the Cytohubba. CMap analysis found several small molecular compounds to reverse the altered DEGs, including dropropizine, aceclofenac, melatonin, and so on. Our outputs could empower the novel potential targets to treat omental white adipocyte insulin resistance, diabetes, and diabetes-related diseases.

Transcription factor Wilms' tumor 1 regulates developmental RNAs through 3' UTR interaction.

Wilms' tumor 1 (WT1) is essential for the development and homeostasis of multiple mesodermal tissues. Despite evidence for post-transcriptional roles, no endogenous WT1 target RNAs exist. Using RNA immunoprecipitation and UV cross-linking, we show that WT1 binds preferentially to 3' untranslated regions (UTRs) of developmental targets. These target mRNAs are down-regulated upon WT1 depletion in cell culture and developing kidney mesenchyme. Wt1 deletion leads to rapid turnover of specific mRNAs. WT1 regulates reporter gene expression through interaction with 3' UTR-binding sites. Combining experimental and computational analyses, we propose that WT1 influences key developmental and disease processes in part through regulating mRNA turnover.

Inactivation of Tsc2 in Mesoderm-Derived Cells Causes Polycystic Kidney Lesions and Impairs Lung Alveolarization

The tuberous sclerosis complex (TSC) proteins are critical negative regulators of the mammalian/mechanistic target of rapamycin complex 1 pathway. Germline mutations of TSC1 or TSC2 cause TSC, affecting multiple organs, including the kidney and lung, and causing substantial morbidity and mortality. The mechanisms of organ-specific disease in TSC remain incompletely understood, and the impact of TSC inactivation on mesenchymal lineage cells has not been specifically studied. We deleted Tsc2 specifically in mesoderm-derived mesenchymal cells of multiple organs in mice using the Dermo1-Cre driver. The Dermo1-Cre-driven Tsc2 conditional knockout mice had body growth retardation and died approximately 3 weeks after birth. Significant phenotypes were observed in the postnatal kidney and lung. Inactivation of Tsc2 in kidney mesenchyme caused polycystic lesions starting from the second week of age, with increased cell proliferation, tubular epithelial hyperplasia, and epithelial-mesenchymal transition. In contrast, Tsc2 deletion in lung mesenchyme led to decreased cell proliferation, reduced postnatal alveolarization, and decreased differentiation with reduced numbers of alveolar myofibroblast and type II alveolar epithelial cells. Two major findings thus result from this model: inactivation of Tsc2 in mesoderm-derived cells causes increased cell proliferation in the kidneys but reduced proliferation in the lungs, and inactivation of Tsc2 in mesoderm-derived cells causes epithelial-lined renal cysts. Therefore, Tsc2-mTOR signaling in mesenchyme is essential for the maintenance of renal structure and for lung alveolarization.

Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.

The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.

Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.

The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.

Mechanisms of gene activation and repression by Pax proteins in the developing kidney

During embryonic development, DNA binding proteins help specify and restrict the fates of pluripotent stem cells. In the developing kidney, Pax2 proteins are among the earliest markers for the renal epithelial cell lineage, with expression in the mesenchyme and in proliferating epithelia. The Pax2 protein is essential for interpreting inductive signals emanating from the ureteric bud such that the kidney mesenchyme can convert to epithelia. The biochemistry of Pax protein function is being studied in a variety of model systems. Through interactions with the adaptor Pax transactivation-domain interacting protein (PTIP), Pax proteins can recruit members of the Trithorax family of histone methyltransferases to imprint activating epigenetic marks on chromatin. However, interactions with the corepressor Groucho-related gene-4 (Grg4) protein can inhibit activation and instead recruit Polycomb repressor complexes to promote target-gene silencing. We present a model whereby the regulated interactions of Pax proteins with available cofactor-mediated activation or gene silencing at different stages of development. The implications for establishing and maintaining the epigenome are discussed.

Conditional Expression of Wnt9b in Six2-Positive Cells Disrupts Stomach and Kidney Function

During kidney development, canonical Wnt signaling activates differentiation, while the transcription factor Six2 maintains the progenitor pool. These opposing signals help to regulate nephron formation and ensure the full complement of nephrons are formed. Since these two factors control differing fates in kidney mesenchyme, we hypothesized that overexpression of Wnt9b in Six2-expressing cells would disrupt kidney formation and may alter cell differentiation decisions in other tissues. We created a transgenic mouse that conditionally expressed the canonical Wnt ligand in the developing kidney, Wnt9b. The transgene is activated by cre recombinase and expresses GFP. We first tested its biological activity using Hoxb7-cre and found that transgenic Wnt9b was capable of inducing differentiation genes and of rescuing kidney development in Wnt9b−/− homozygous deficient mice. In contrast, expression of Wnt9b in cells using Six2-cre caused gastrointestinal distress and severe renal failure in adult mice. Transgenic kidneys had numerous cystic tubules and elevated creatinine values (0.652±0.044) compared to wild-type mice (0.119±0.002). These animals also exhibited a malformed pyloric sphincter, duodenogastric reflux, and a transformation of the distal stomach into proximal fate. The gene expression changes observed for the Wnt9b:EGFP transgene were compared to a stabilized β-catenin allele to determine that Wnt9b is activating the canonical Wnt pathway in the tissues analyzed. These results demonstrate that expression of Wnt9b in Six2-positive cells disrupts cell fate decisions in the kidney and the gastrointestinal tract.

Invasion of Distal Nephron Precursors Associates with Tubular Interconnection during Nephrogenesis

Formation of a functional renal network requires the interconnection of two epithelial tubes: the nephron, which arises from kidney mesenchyme, and the collecting system, which originates from the branching ureteric epithelium. How this connection occurs, however, is incompletely understood. Here, we used high-resolution image analysis in conjunction with genetic labeling of epithelia to visualize and characterize this process. Although the focal absence of basal lamina from renal vesicle stages ensures that both epithelial networks are closely apposed, we found that a patent luminal interconnection is not established until S-shaped body stages. Precursor cells of the distal nephron in the interconnection zone exhibit a characteristic morphology consisting of ill-defined epithelial junctional complexes but without expression of mesenchymal markers such as vimentin and Snai2. Live-cell imaging revealed that before luminal interconnection, distal cells break into the lumen of the collecting duct epithelium, suggesting that an invasive behavior is a key step in the interconnection process. Furthermore, loss of distal cell identity, which we induced by activating the Notch pathway, prevented luminal interconnection. Taken together, these data support a model in which establishing the distal identity of nephron precursor cells closest to the nascent collecting duct epithelium leads to an active cell invasion, which in turn contributes to a patent tubular interconnection between the nephron and collecting duct epithelia.

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.

A Wt1-Controlled Chromatin Switching Mechanism Underpins Tissue-Specific Wnt4 Activation and Repression

Wt1 regulates the epithelial-mesenchymal transition (EMT) in the epicardium and the reverse process (MET) in kidney mesenchyme. The mechanisms underlying these reciprocal functions are unknown. Here, we show in both embryos and cultured cells that Wt1 regulates Wnt4 expression dichotomously. In kidney cells, Wt1 recruits Cbp and p300 as coactivators; in epicardial cells it enlists Basp1 as a corepressor. Surprisingly, in both tissues, Wt1 loss reciprocally switches the chromatin architecture of the entire Ctcf-bounded Wnt4 locus, but not the flankingregions; we term this mode of action "chromatin flip-flop." Ctcf and cohesin are dispensable for Wt1-mediated chromatin flip-flop but essential for maintaining the insulating boundaries. This work demonstrates that a developmental regulator coordinates chromatin boundaries with the transcriptional competence of the flanked region. These findings also have implications for hierarchical transcriptional regulation in development and disease.

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.

Fgfr1 and the IIIc isoform of Fgfr2 play critical roles in the metanephric mesenchyme mediating early inductive events in kidney development

Fibroblast growth factor receptors (Fgfrs) have critical roles in kidney development. FgfrIIIb is thought to act in epithelium, while FgfrIIIc functions in mesenchyme. We aimed to determine roles of Fgfr2IIIc in kidney development. Mice with deletion of Fgfr2IIIc (Fgfr2IIIc-/-) had normal kidneys. Combination of Fgfr2IIIc-/- with conditional deletion of Fgfr1 in metanephric mesenchyme (MM) (Fgfr1(Mes-/-)Fgfr2IIIc-/-) had small but identifiable MM at embryonic day (E) 10.5, expressing mesenchymal markers including Eya1, Six2, Pax2, and Gdnf (unlike Fgfr1/2(Mes-/-) mice that have no obvious MM). E11.5 Fgfr1(Mes-/-)Fgfr2IIIc-/- mice had rudimentary MM expressing only Eya1. Control, Fgfr2IIIc-/-, and Fgfr1(Mes-/-)Fgfr2IIIc-/- kidney mesenchymal tissues also express Fgfr2IIIb. In ureteric lineages, E10.5 Fgfr1(Mes-/-)Fgfr2IIIc-/- embryos had ureteric outgrowth (sometimes multiple buds); however, by E11.5 Gdnf absence lead to no ureteric elongation or branching (similar to Fgfr1/2(Mes-/-) mice). Beyond E12.5, Fgfr1(Mes-/-)Fgfr2IIIc-/- mice had no renal tissue. In conclusion, Fgfr2IIIc and Fgfr1 in kidney mesenchyme (together) are critical for normal early renal development.

Kif26b , a kinesin family gene, regulates adhesion of the embryonic kidney mesenchyme

The kidney develops through reciprocal interactions between two precursor tissues: the metanephric mesenchyme and the ureteric bud. We previously demonstrated that the zinc finger protein Sall1 is essential for ureteric bud attraction toward the mesenchyme. Here, we show that Kif26b, a kinesin family gene, is a downstream target of Sall1 and that disruption of this gene causes kidney agenesis because of impaired ureteric bud attraction. In the Kif26b-null metanephros, compact adhesion between mesenchymal cells adjacent to the ureteric buds and the polarized distribution of integrin alpha8 were impaired, resulting in failed maintenance of Gdnf, a critical ureteric bud attractant. Overexpression of Kif26b in vitro caused increased cell adhesion through interactions with nonmuscle myosin. Thus, Kif26b is essential for kidney development because it regulates the adhesion of mesenchymal cells in contact with ureteric buds.

A mouse line expressing Sall1‐driven inducible Cre recombinase in the kidney mesenchyme

Sall1 is expressed in the metanephric mesenchyme in the developing kidney, and mice deficient in Sall1 show kidney agenesis or dysgenesis. Sall1 is also expressed elsewhere, including in the limb buds, anus, heart, and central nervous system. Dominant-negative mutations of Sall1 in mice and humans lead to developmental defects in these organs. Here, we generated a mouse line expressing tamoxifen-inducible Cre recombinase (CreER(T2)) under the control of the endogenous Sall1 promoter. Upon tamoxifen treatment, these mice showed genomic recombination in the tissues where endogenous Sall1 is expressed. When CreER(T2) mice were crossed with the floxed Sall1 allele, tamoxifen administration during gestation led to a significant decrease in Sall1 expression and small kidneys at birth, suggesting that Sall1 functions were disrupted. Furthermore, Sall1 expression in the kidney was significantly reduced by neonatal tamoxifen treatment. The Sall1CreER(T2) mouse is a valuable tool for in vivo time-dependent and region-specific knockout and overexpression studies.

Hs2st mediated kidney mesenchyme induction regulates early ureteric bud branching

Heparan sulfate proteoglycans (HSPGs) are central modulators of developmental processes likely through their interaction with growth factors, such as GDNF, members of the FGF and TGFβ superfamilies, EGF receptor ligands and HGF. Absence of the biosynthetic enzyme, heparan sulfate 2-O-sulfotransferase (Hs2st) leads to kidney agenesis. Using a novel combination of in vivo and in vitro approaches, we have reanalyzed the defect in morphogenesis of the Hs2st−/− kidney. Utilizing assays that separately model distinct stages of kidney branching morphogenesis, we found that the Hs2st−/− UB is able to undergo branching and induce mesenchymal-to-epithelial transformation when recombined with control MM, and the isolated Hs2st null UB is able to undergo branching morphogenesis in the presence of exogenous soluble pro-branching growth factors when embedded in an extracellular matrix, indicating that the UB is intrinsically competent. This is in contrast to the prevailing view that the defect underlying the renal agenesis phenotype is due to a primary role for 2-O sulfated HS in UB branching. Unexpectedly, the mutant MM was also fully capable of being induced in recombination experiments with wild-type tissue. Thus, both the mutant UB and mutant MM tissue appear competent in and of themselves, but the combination of mutant tissues fails in vivo and, as we show, in organ culture. We hypothesized a 2OS-dependent defect in the mutual inductive process, which could be on either the UB or MM side, since both progenitor tissues express Hs2st. In light of these observations, we specifically examined the role of the HS 2-O sulfation modification on the morphogenetic capacity of the UB and MM individually. We demonstrate that early UB branching morphogenesis is not primarily modulated by factors that depend on the HS 2-O sulfate modification; however, factors that contribute to MM induction are markedly sensitive to the 2-O sulfation modification. These data suggest that key defect in Hs2st null kidneys is the inability of MM to undergo induction either through a failure of mutual induction or a primary failure of MM morphogenesis. This results in normal UB formation but affects either T-shaped UB formation or iterative branching of the T-shaped UB (possibly two separate stages in collecting system development dependent upon HS). We discuss the possibility that a disruption in the interaction between HS and Wnts (e.g. Wnt 9b) may be an important aspect of the observed phenotype. This appears to be the first example of a defect in the MM preventing advancement of early UB branching past the first bifurcation stage, one of the limiting steps in early kidney development.

Lumbo-sacral neural crest derivatives fate mapped with the aid of Wnt-1 promoter integrate but are not essential to kidney development

Neural crest (NC) cells may be involved in kidney organogenesis by providing inductive signals and contributing to cells of the renal stroma. We show here that the lumbo-sacral NC cells fate mapped with the aid of Wnt-1 promoter in the mouse migrate close to the metanephros at the initiation of organogenesis but these cells remain superficial to the condensed Pax2-expressing mesenchymal cells. NC-derived cells enter later into the kidney proper from the midline region. The NC cells contribute also to development of the extra-adrenal para-aortic bodies, Zuckerkandl's bodies and the nerve cord of the sympathetic nervous system. Splotch ( Sp 2H / Sp 2H ) embryos, having a NC defect in the lumbo-sacral region, develop a normal metanephros even though the kidney does not express the NC markers Sox10, Phox2b and tyrosine hydroxylase. Consistent with the histological findings, the kidneys of Sp 2H / Sp 2H embryos also express the stromal genes Foxd1, Hoxa10 and RARβ normally. Wnt-1 promoter-marked wild-type LacZ NC cells migrate intensely from the heterologous inducer tissue of the embryonic dorsal spinal cord (SPC) to the kidney mesenchyme, but tubule induction does not depend on NC migration, since the Sp 2H / Sp 2H SPC also induces tubulogenesis. The Sp 2H / Sp 2H mesenchyme also remains competent for tubulogenesis. We conclude that the NC cells fate mapped with the aid of Wnt-1 promoter migrate to the close to the metanephros and form later derivatives integrating with the kidney, but they may not be essential to the development of the stromal cells nor they may provide critical morphogenetic signals to regulate early kidney development in vivo.

Antagonism of BMP4 Signaling Disrupts Smooth Muscle Investment of the Ureter and Ureteropelvic Junction

Congenital ureteropelvic junction obstruction has been associated with aberrant ureteral smooth muscle organization. Recent evidence has shown that BMP4 may be involved in ureteral morphogenesis. We determined whether the disruption of BMP4 signaling results in abnormal smooth muscle investment of the ureter and ureteropelvic junction. We used a Cre mediated Bmp4 knockout system to conditionally excise the Bmp4 gene in developing mouse embryos. Kidney rudiments were isolated from embryos at varying gestational ages from WT and conditional knockout mice. Metanephric kidney explants were cultured in the presence or absence of the BMP antagonist Noggin. Agarose beads pre-incubated with Gremlin, another BMP antagonist, were used for localized disruption of BMP signaling. Frozen sections and whole metanephric explants were then analyzed by immunofluorescence. Bmp4 gene excision resulted in a dose dependent loss of ureteral smooth muscle. Antagonism of BMP signaling inhibited ureteral smooth muscle investment in a dose dependent manner and was paralleled by a dose dependent decrease in the immediate downstream targets of BMP signaling, phosphorylated Smad1, 5 and 8. Localized antagonism of BMP resulted in the focal disruption of ureteral smooth muscle investment. We report that decreased BMP signaling, whether by the loss of BMP4 in vivo or direct antagonism in vitro, results in a gradual reduction of the normal, well organized coat of smooth muscle surrounding the ureter. Our results also suggest that this occurs via a direct Smad dependent pathway. This raises the possibility that abnormalities in BMP4 signaling may have a role in the development of congenital ureteropelvic junction obstruction.