Single-cell multiomics reveals ENL mutation perturbs kidney developmental trajectory by rewiring gene regulatory landscape.

How disruptions to normal cell differentiation link to tumorigenesis remains incompletely understood. Wilms tumor, an embryonal tumor associated with disrupted organogenesis, often harbors mutations in epigenetic regulators, but their role in kidney development remains unexplored. Here, we show at single-cell resolution that a Wilms tumor-associated mutation in the histone acetylation reader ENL disrupts kidney differentiation in mice by rewiring the gene regulatory landscape. Mutant ENL promotes nephron progenitor commitment while restricting their differentiation by dysregulating transcription factors such as Hox clusters. It also induces abnormal progenitors that lose kidney-associated chromatin identity. Furthermore, mutant ENL alters the transcriptome and chromatin accessibility of stromal progenitors, resulting in hyperactivation of Wnt signaling. The impacts of mutant ENL on both nephron and stroma lineages lead to profound kidney developmental defects and postnatal mortality in mice. Notably, a small molecule inhibiting mutant ENL's histone acetylation binding activity largely reverses these defects. This study provides insights into how mutations in epigenetic regulators disrupt kidney development and suggests a potential therapeutic approach.

Isthmin-1: A critical regulator of branching morphogenesis and metanephric mesenchyme condensation during early kidney development.

Isthmin-1 (Ism1) was first described to be syn-expressed with Fgf8 in Xenopus. However, its biological role has not been elucidated until recent years. Despite of accumulated evidence that Ism1 participates in angiogenesis, tumor invasion, macrophage apoptosis, and glucose metabolism, the cognate receptors for Ism1 remain largely unknown. Ism1 deficiency in mice results in renal agenesis (RA) with a transient loss of Gdnf transcription and impaired mesenchyme condensation at E11.5. Ism1 binds to and activates Integrin α8ß1 to positively regulate Gdnf/Ret signaling, thus promoting mesenchyme condensation and ureteric epithelium branching morphogenesis. Here, we propose the hypothesis underlying the mechanism by which Ism1 regulates branching morphogenesis during early kidney development.

Whole genome sequencing and novel candidate genes for CAKUT and altered nephrogenesis in the HSRA rat.

The HSRA rat is a model of congenital abnormalities of the kidney and urogenital tract (CAKUT). Our laboratory has used this model to investigate the role of nephron number (functional unit of the kidney) in susceptibility to develop kidney disease as 50-75% offspring are born with a single kidney (HSRA-S), while 25-50% are born with two kidneys (HSRA-C). HSRA-S rats develop increased kidney injury and hypertension with age compared with nephrectomized two-kidney animals (HSRA-UNX), suggesting that even slight differences in nephron number can be an important driver in decline in kidney function. The HSRA rat was selected and inbred from a family of outbred heterogeneous stock (NIH-HS) rats that exhibited a high incidence of CAKUT. The HS model was originally developed from eight inbred strains (ACI, BN, BUF, F344, M520, MR, WKY, and WN). The genetic make-up of the HSRA is therefore a mosaic of these eight inbred strains. Interestingly, the ACI progenitor of the HS model exhibits CAKUT in 10-15% of offspring with the genetic cause being attributed to the presence of a long-term repeat (LTR) within exon 1 of the c-Kit gene. Our hypothesis is that the HSRA and ACI share this common genetic cause, but other alleles in the HSRA genome contribute to the increased penetrance of CAKUT (75% HSRA vs. 15% in ACI). To facilitate genetic studies and better characterize the model, we sequenced the whole genome of the HSRA to a depth of ~50×. A genome-wide variant analysis of high-impact variants identified a number of novel genes that could be linked to CAKUT in the HSRA model. In summary, the identification of new genes/modifiers that lead to CAKUT/loss of one kidney in the HSRA model will provide greater insight into association between kidney development and susceptibility to develop cardiovascular disease later in life.

Kidney Regeneration in Later-Stage Mouse Embryos via Transplanted Renal Progenitor Cells.

BACKGROUND: The limited availability of donor kidneys for transplantation has spurred interest in investigating alternative strategies, such as regenerating organs from stem cells transplanted into animal embryos. However, there is no known method for transplanting cells into later-stage embryos, which may be the most suitable host stages for organogenesis, particularly into regions useful for kidney regeneration. METHODS: We demonstrated accurate transplantation of renal progenitor cells expressing green fluorescent protein to the fetal kidney development area by incising the opaque uterine muscle layer but not the transparent amniotic membrane. We allowed renal progenitor cell-transplanted fetuses to develop for 6 days postoperatively before removal for analysis. We also transplanted renal progenitor cells into conditional kidney-deficient mouse embryos. We determined growth and differentiation of transplanted cells in all cases. RESULTS: Renal progenitor cell transplantation into the retroperitoneal cavity of fetuses at E13-E14 produced transplant-derived, vascularized glomeruli with filtration function and did not affect fetal growth or survival. Cells transplanted to the nephrogenic zone produced a chimera in the cap mesenchyme of donor and host nephron progenitor cells. Renal progenitor cells transplanted to conditional kidney-deficient fetuses induced the formation of a new nephron in the fetus that is connected to the host ureteric bud. CONCLUSIONS: We developed a cell transplantation method for midstage to late-stage fetuses. In vivo kidney regeneration from renal progenitor cells using the renal developmental environment of the fetus shows promise. Our findings suggest that fetal transplantation methods may contribute to organ regeneration and developmental research.

Loss of miR-17~92 results in dysregulation of Cftr in nephron progenitors.

We have previously demonstrated that loss of miR-17~92 in nephron progenitors in a mouse model results in renal hypodysplasia and chronic kidney disease. Clinically, decreased congenital nephron endowment because of renal hypodysplasia is associated with an increased risk of hypertension and chronic kidney disease, and this is at least partly dependent on the self-renewal of nephron progenitors. Here, we present evidence for a novel molecular mechanism regulating the self-renewal of nephron progenitors and congenital nephron endowment by the highly conserved miR-17~92 cluster. Whole transcriptome sequencing revealed that nephron progenitors lacking this cluster demonstrated increased Cftr expression. We showed that one member of the cluster, miR-19b, is sufficient to repress Cftr expression in vitro and that perturbation of Cftr activity in nephron progenitors results in impaired proliferation. Together, these data suggest that miR-19b regulates Cftr expression in nephron progenitors, with this interaction playing a role in appropriate nephron progenitor self-renewal during kidney development to generate normal nephron endowment.

Evolution, kidney development, and chronic kidney disease.

There is a global epidemic of chronic kidney disease (CKD) characterized by a progressive loss of nephrons, ascribed in large part to a rising incidence of hypertension, metabolic syndrome, and type 2 diabetes mellitus. There is a ten-fold variation in nephron number at birth in the general population, and a 50% overall decrease in nephron number in the last decades of life. The vicious cycle of nephron loss stimulating hypertrophy by remaining nephrons and resulting in glomerulosclerosis has been regarded as maladaptive, and only partially responsive to angiotensin inhibition. Advances over the past century in kidney physiology, genetics, and development have elucidated many aspects of nephron formation, structure and function. Parallel advances have been achieved in evolutionary biology, with the emergence of evolutionary medicine, a discipline that promises to provide new insight into the treatment of chronic disease. This review provides a framework for understanding the origins of contemporary developmental nephrology, and recent progress in evolutionary biology. The establishment of evolutionary developmental biology (evo-devo), ecological developmental biology (eco-devo), and developmental origins of health and disease (DOHaD) followed the discovery of the hox gene family, the recognition of the contribution of cumulative environmental stressors to the changing phenotype over the life cycle, and mechanisms of epigenetic regulation. The maturation of evolutionary medicine has contributed to new investigative approaches to cardiovascular disease, cancer, and infectious disease, and promises the same for CKD. By incorporating these principles, developmental nephrology is ideally positioned to answer important questions regarding the fate of nephrons from embryo through senescence.

Embryonal Origin of Metanephric Adenoma and its Differential Diagnosis.

BACKGROUND/AIM: The association of Wilms' tumor (WT), papillary renal cell tumor (PRCT) and mucinous tubular and spindle cell carcinoma (MTSCC) with embryonal rests has already been documented, but the cellular origin of metanephric adenoma (MA) is not yet known. The aim of this study was to understand their developmental evolution and find diagnostic markers. MATERIALS AND METHODS: CD57, KRT7, AMACR, SCEL, WT1 and CDH17 expression was analysed by immunohistochemistry in the four types of tumors and the associated pre-neoplastic lesions. RESULTS: Immunohistochemistry was able to differentiate WT, MA, MTSCC and PRCT. A phenotypic correlation between MA and perilobar nephrogenic rest associated with WT was identified. CONCLUSION: Perilobar nephrogenic rest and MA arise from differentiation arrested cells of the proximal domain of the S-shape body. We propose that WT1, MA, MTSCC and PRCT derive from different forms of maturation arrested embryonal rests.

Hedgehog-GLI signaling in Foxd1-positive stromal cells promotes murine nephrogenesis via TGFß signaling.

Normal kidney function depends on the proper development of the nephron: the functional unit of the kidney. Reciprocal signaling interactions between the stroma and nephron progenitor compartment have been proposed to control nephron development. Here, we show that removal of hedgehog intracellular effector smoothened (Smo-deficient mutants) in the cortical stroma results in an abnormal renal capsule, and an expanded nephron progenitor domain with an accompanying decrease in nephron number via a block in epithelialization. We show that stromal-hedgehog-Smo signaling acts through a GLI3 repressor. Whole-kidney RNA sequencing and analysis of FACS-isolated stromal cells identified impaired TGFß2 signaling in Smo-deficient mutants. We show that neutralization and knockdown of TGFß2 in explants inhibited nephrogenesis. In addition, we demonstrate that concurrent deletion of Tgfbr2 in stromal and nephrogenic cells in vivo results in decreased nephron formation and an expanded nephrogenic precursor domain similar to that observed in Smo-deficient mutant mice. Together, our data suggest a mechanism whereby a stromal hedgehog-TGFß2 signaling axis acts to control nephrogenesis.

Histone deacetylases 1 and 2 regulate the transcriptional programs of nephron progenitors and renal vesicles.

Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a, as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles.

Disparate levels of beta-catenin activity determine nephron progenitor cell fate.

Formation of a functional kidney depends on the balance between renewal and differentiation of nephron progenitors. Failure to sustain this balance can lead to kidney failure or stem cell tumors. For nearly 60 years, we have known that signals from an epithelial structure known as the ureteric bud were essential for maintaining this balance. More recently it was discovered that one molecule, Wnt9b, was necessary for both renewal and differentiation of the nephron progenitor cells. How one ligand signaling through one transcription factor promoted two seemingly contradictory cellular processes was unclear. In this study, we show that Wnt9b/beta-catenin signaling alone is sufficient to promote both renewal and differentiation. Moreover, we show that discrete levels of beta-catenin can promote these two disparate fates, with low levels fostering progenitor renewal and high levels driving differentiation. These results provide insight into how Wnt9b regulates distinct target genes that balance nephron progenitor renewal and differentiation.

Conserved and Divergent Features of Mesenchymal Progenitor Cell Types within the Cortical Nephrogenic Niche of the Human and Mouse Kidney.

Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2+ nephron progenitor cells (NPCs) and Foxd1+ interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1, were readily detected within SIX2+ NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2+ NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2, are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs.

Cross-platform single cell analysis of kidney development shows stromal cells express Gdnf.

The developing kidney provides a useful model for study of the principles of organogenesis. In this report we use three independent platforms, Drop-Seq, Chromium 10x Genomics and Fluidigm C1, to carry out single cell RNA-Seq (scRNA-Seq) analysis of the E14.5 mouse kidney. Using the software AltAnalyze, in conjunction with the unsupervised approach ICGS, we were unable to identify and confirm the presence of 16 distinct cell populations during this stage of active nephrogenesis. Using a novel integrative supervised computational strategy, we were able to successfully harmonize and compare the cell profiles across all three technological platforms. Analysis of possible cross compartment receptor/ligand interactions identified the nephrogenic zone stroma as a source of GDNF. This was unexpected because the cap mesenchyme nephron progenitors had been thought to be the sole source of GDNF, which is a key driver of branching morphogenesis of the collecting duct system. The expression of Gdnf by stromal cells was validated in several ways, including Gdnf in situ hybridization combined with immunohistochemistry for SIX2, and marker of nephron progenitors, and MEIS1, a marker of stromal cells. Finally, the single cell gene expression profiles generated in this study confirmed and extended previous work showing the presence of multilineage priming during kidney development. Nephron progenitors showed stochastic expression of genes associated with multiple potential differentiation lineages.

Haploinsufficiency for the Six2 gene increases nephron progenitor proliferation promoting branching and nephron number.

The regulation of final nephron number in the kidney is poorly understood. Cessation of nephron formation occurs when the self-renewing nephron progenitor population commits to differentiation. Transcription factors within this progenitor population, such as SIX2, are assumed to control expression of genes promoting self-renewal such that homozygous Six2 deletion results in premature commitment and an early halt to kidney development. In contrast, Six2 heterozygotes were assumed to be unaffected. Using quantitative morphometry, we found a paradoxical 18% increase in ureteric branching and final nephron number in Six2 heterozygotes, despite evidence for reduced levels of SIX2 protein and transcript. This was accompanied by a clear shift in nephron progenitor identity with a distinct subset of downregulated progenitor genes such as Cited1 and Meox1 while other genes were unaffected. The net result was an increase in nephron progenitor proliferation, as assessed by elevated EdU (5-ethynyl-2'-deoxyuridine) labeling, an increase in MYC protein, and transcriptional upregulation of MYC target genes. Heterozygosity for Six2 on an Fgf20-/- background resulted in premature differentiation of the progenitor population, confirming that progenitor regulation is compromised in Six2 heterozygotes. Overall, our studies reveal a unique dose response of nephron progenitors to the level of SIX2 protein in which the role of SIX2 in progenitor proliferation versus self-renewal is separable.

Troy/TNFRSF19 marks epithelial progenitor cells during mouse kidney development that continue to contribute to turnover in adult kidney.

During kidney development, progressively committed progenitor cells give rise to the distinct segments of the nephron, the functional unit of the kidney. Similar segment-committed progenitor cells are thought to be involved in the homeostasis of adult kidney. However, markers for most segment-committed progenitor cells remain to be identified. Here, we evaluate Troy/TNFRSF19 as a segment-committed nephron progenitor cell marker. Troy is expressed in the ureteric bud during embryonic development. During postnatal nephrogenesis, Troy+ cells are present in the cortex and papilla and display an immature tubular phenotype. Tracing of Troy+ cells during nephrogenesis demonstrates that Troy+ cells clonally give rise to tubular structures that persist for up to 2 y after induction. Troy+ cells have a 40-fold higher capacity than Troy- cells to form organoids, which is considered a stem cell property in vitro. In the adult kidney, Troy+ cells are present in the papilla and these cells continue to contribute to collecting duct formation during homeostasis. The number of Troy-derived cells increases after folic acid-induced injury. Our data show that Troy marks a renal stem/progenitor cell population in the developing kidney that in adult kidney contributes to homeostasis, predominantly of the collecting duct, and regeneration.

Generation of interspecies limited chimeric nephrons using a conditional nephron progenitor cell replacement system.

Animal fetuses and embryos may have applications in the generation of human organs. Progenitor cells may be an appropriate cell source for regenerative organs because of their safety and availability. However, regenerative organs derived from exogenous lineage progenitors in developing animal fetuses have not yet been obtained. Here, we established a combination system through which donor cells could be precisely injected into the nephrogenic zone and native nephron progenitor cells (NPCs) could be eliminated in a time- and tissue-specific manner. We successfully achieved removal of Six2+ NPCs within the nephrogenic niche and complete replacement of transplanted NPCs with donor cells. These NPCs developed into mature glomeruli and renal tubules, and blood flow was observed following transplantation in vivo. Furthermore, this artificial nephron could be obtained using NPCs from different species. Thus, this technique enables in vivo differentiation from progenitor cells into nephrons, providing insights into nephrogenesis and organ regeneration.

Role of CD34 family members in lumen formation in the developing kidney.

Previous studies have shown CD34 family member Podocalyxin is required for epithelial lumen formation in vitro. We demonstrate that Endoglycan, a CD34 family member with homology to Podocalyxin, is produced prior to lumen formation in developing nephrons. Endoglycan localizes to Rab11-containing vesicles in nephron progenitors, and then relocalizes to the apical surface as progenitors epithelialize. Once an apical/luminal surface is formed, Endoglycan (and the actin-binding protein Ezrin) localize to large, intraluminal structures that may be vesicles/exosomes. We generated mice lacking Endoglycan and found mutants had timely initiation of lumen formation and continuous lumens, similar to controls. Mice with conditional deletion of both Endoglycan and Podocalyxin in developing nephrons also had normal tubular lumens. Despite this, Endoglycan/Podocalyxin is required for apical recruitment of the adaptor protein NHERF1, but not Ezrin, in podocyte precursors, a subset of the epithelia. In summary, while CD34 family members appear dispensable for lumen formation, our data identify Endoglycan as a novel pre-luminal marker and suggest lumen formation occurs via vesicular trafficking of apical cargo that includes Endoglycan.

Little fish, big catch: zebrafish as a model for kidney disease.

The zebrafish, Danio rerio, is a relevant vertebrate model for biomedical research and translational studies because of its broad genetic conservation with humans. In recent years, scientists have formulated a growing list of zebrafish kidney disease paradigms, the study of which has contributed a multitude of insights into the basic biology of human conditions and even identified potential therapeutic agents. Conversely, there are also distinctive aspects of zebrafish biology lacking in higher vertebrates, such as the capacity to heal without lasting scar formation after tissue damage and the ability to generate nephrons throughout their lifespan, which makes the zebrafish uniquely suited to study regeneration in the context of the kidney. Here, we review several informative zebrafish models of kidney disease and discuss their future applications in nephrology.

Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish kidney development.

Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a "salt-and-pepper" fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA treatment expanded the etv5a domain within the renal progenitor field and RA inhibition blocked etv5a in this populace, indicating that etv5a acts downstream of RA. Additionally, treatment with exogenous RA partially rescued the reduced MCC phenotype after loss of etv5a. Further, abrogation of Notch with the small molecule inhibitor DAPT increased the renal progenitor etv5a expression domain as well as MCC density in etv5a deficient embryos, suggesting Notch acts upstream to inhibit etv5a. In contrast, etv4 levels in renal progenitors were unaffected by changes in RA or Notch signaling levels, suggesting a possible non-cell autonomous role during pronephros formation. Taken together, these findings have revealed new insights about the genetic mechanisms of epithelial cell development during nephrogenesis.

Renal dysplasia in the neonate.

PURPOSE OF REVIEW: Renal dysplasia is classically described as a developmental disorder whereby the kidneys fail to undergo appropriate differentiation, resulting in the presence of malformed renal tissue elements. It is the commonest cause of chronic kidney disease and renal failure in the neonate. Although several genes have been identified in association with renal dysplasia, the underlying molecular mechanisms are often complex and heterogeneous in nature, and remain poorly understood. RECENT FINDINGS: In this review, we describe new insights into the fundamental process of normal kidney development, and how the renal cortex and medulla are patterned appropriately during gestation. We review the key genes that are indispensable for this process, and discuss how patterning of the kidney is perturbed in the absence of these signaling pathways. The recent use of whole exome sequencing has identified genetic mutations in patients with renal dysplasia, and the results of these studies have increased our understanding of the pathophysiology of renal dysplasia. SUMMARY: At present, there are no specific treatments available for patients with renal dysplasia. Understanding the molecular mechanisms of normal kidney development and the pathogenesis of renal dysplasia may allow for improved therapeutic options for these patients.

Urogenital development in Pallister-Hall syndrome is disrupted in a cell-lineage-specific manner by constitutive expression of GLI3 repressor.

Pallister-Hall syndrome (PHS) is a rare disorder caused by mutations in GLI3 that produce a transcriptional repressor (GLI3R). Individuals with PHS present with a variably penetrant variety of urogenital system malformations, including renal aplasia or hypoplasia, hydroureter, hydronephrosis or a common urogenital sinus. The embryologic mechanisms controlled by GLI3R that result in these pathologic phenotypes are undefined. We demonstrate that germline expression of GLI3R causes renal hypoplasia, associated with decreased nephron number, and hydroureter and hydronephrosis, caused by blind-ending ureters. Mice with obligate GLI3R expression also displayed duplication of the ureters that was caused by aberrant common nephric duct patterning and ureteric stalk outgrowth. These developmental abnormalities are associated with suppressed Hedgehog signaling activity in the cloaca and adjacent vesicular mesenchyme. Mice with conditional expression of GLI3R were utilized to identify lineage-specific effects of GLI3R. In the ureteric bud, GLI3R expression decreased branching morphogenesis. In Six2-positive nephrogenic progenitors, GLI3R decreased progenitor cell proliferation reducing the number of nephrogenic precursor structures. Using mutant mice with Gli3R and Gli3 null alleles, we demonstrate that urogenital system patterning and development is controlled by the levels of GLI3R and not by an absence of full-length GLI3. We conclude that the urogenital system phenotypes observed in PHS are caused by GLI3R-dependent perturbations in nephric duct patterning, renal branching morphogenesis and nephrogenic progenitor self-renewal.