Permissive ureter specification by TBX18-mediated repression of metanephric gene expression.

The murine kidney and ureter develop in a regionalized fashion from the ureteric bud and its surrounding mesenchyme. Whereas the factors that establish the metanephric cell lineages have been well characterized, much less is known about the molecular cues that specify the ureter. Here, we have identified a crucial patterning function in this process for Tbx18, a T-box transcription factor gene specifically expressed in the mesenchymal primordium of the ureter. Using misexpression and loss-of-function mice combined with molecular profiling approaches, we show that Tbx18 is required and sufficient to repress metanephric mesenchymal gene programs. We identify Wt1 as a functional target of TBX18. Our work suggests that TBX18 acts as a permissive factor in ureter specification by generating a mesenchymal domain around the distal ureteric bud where SHH and BMP4 signaling can occur.

GATA6 is a crucial factor for Myocd expression in the visceral smooth muscle cell differentiation program of the murine ureter.

Smooth muscle cells (SMCs) are a crucial component of the mesenchymal wall of the ureter, as they account for the efficient removal of the urine from the renal pelvis to the bladder by means of their contractile activity. Here, we show that the zinc-finger transcription factor gene Gata6 is expressed in mesenchymal precursors of ureteric SMCs under the control of BMP4 signaling. Mice with a conditional loss of Gata6 in these precursors exhibit a delayed onset and reduced level of SMC differentiation and peristaltic activity, as well as dilatation of the ureter and renal pelvis (hydroureternephrosis) at birth and at postnatal stages. Molecular profiling revealed a delayed and reduced expression of the myogenic driver gene Myocd, but the activation of signaling pathways and transcription factors previously implicated in activation of the visceral SMC program in the ureter was unchanged. Additional gain-of-function experiments suggest that GATA6 cooperates with FOXF1 in Myocd activation and SMC differentiation, possibly as pioneer and lineage-determining factors, respectively.

Notch signaling is a novel regulator of visceral smooth muscle cell differentiation in the murine ureter.

The contractile phenotype of smooth muscle cells (SMCs) is transcriptionally controlled by a complex of the DNA-binding protein SRF and the transcriptional co-activator MYOCD. The pathways that activate expression of Myocd and of SMC structural genes in mesenchymal progenitors are diverse, reflecting different intrinsic and extrinsic signaling inputs. Taking the ureter as a model, we analyzed whether Notch signaling, a pathway previously implicated in vascular SMC development, also affects visceral SMC differentiation. We show that mice with a conditional deletion of the unique Notch mediator RBPJ in the undifferentiated ureteric mesenchyme exhibit altered ureter peristalsis with a delayed onset, and decreased contraction frequency and intensity at fetal stages. They also develop hydroureter 2 weeks after birth. Notch signaling is required for precise temporal activation of Myocd expression and, independently, for expression of a group of late SMC structural genes. Based on additional expression analyses, we suggest that a mesenchymal JAG1-NOTCH2/NOTCH3 module regulates visceral SMC differentiation in the ureter in a biphasic and bimodal manner, and that its molecular function differs from that in the vascular system.

Genetic Variants in ARHGEF6 Cause Congenital Anomalies of the Kidneys and Urinary Tract in Humans, Mice, and Frogs.

BACKGROUND: About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant. METHODS: To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models. RESULTS: We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. CONCLUSIONS: Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.

Inflammation-like changes in the urothelium of Lifr-deficient mice and LIFR-haploinsufficient humans with urinary tract anomalies.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of end-stage kidney disease in children. While the genetic aberrations underlying CAKUT pathogenesis are increasingly being elucidated, their consequences on a cellular and molecular level commonly remain unclear. Recently, we reported rare heterozygous deleterious LIFR variants in 3.3% of CAKUT patients, including a novel de novo frameshift variant, identified by whole-exome sequencing, in a patient with severe bilateral CAKUT. We also demonstrated CAKUT phenotypes in Lifr-/- and Lifr+/- mice, including a narrowed ureteric lumen due to muscular hypertrophy and a thickened urothelium. Here, we show that both in the ureter and bladder of Lifr-/- and Lifr+/- embryos, differentiation of the three urothelial cell types (basal, intermediate and superficial cells) occurs normally but that the turnover of superficial cells is elevated due to increased proliferation, enhanced differentiation from their progenitor cells (intermediate cells) and, importantly, shedding into the ureteric lumen. Microarray-based analysis of genome-wide transcriptional changes in Lifr-/- versus Lifr+/+ ureters identified gene networks associated with an antimicrobial inflammatory response. Finally, in a reverse phenotyping effort, significantly more superficial cells were detected in the urine of CAKUT patients with versus without LIFR variants indicating conserved LIFR-dependent urinary tract changes in the murine and human context. Our data suggest that LIFR signaling is required in the epithelium of the urinary tract to suppress an antimicrobial response under homeostatic conditions and that genetically induced inflammation-like changes underlie CAKUT pathogenesis in Lifr deficiency and LIFR haploinsufficiency.

Loss of Anks6 leads to YAP deficiency and liver abnormalities.

ANKS6 is a ciliary protein that localizes to the proximal compartment of the primary cilium, where it regulates signaling. Mutations in the ANKS6 gene cause multiorgan ciliopathies in humans, which include laterality defects of the visceral organs, renal cysts as part of nephronophthisis and congenital hepatic fibrosis (CHF) in the liver. Although CHF together with liver ductal plate malformations are common features of several human ciliopathy syndromes, including nephronophthisis-related ciliopathies, the mechanism by which mutations in ciliary genes lead to bile duct developmental abnormalities is not understood. Here, we generated a knockout mouse model of Anks6 and show that ANKS6 function is required for bile duct morphogenesis and cholangiocyte differentiation. The loss of Anks6 causes ciliary abnormalities, ductal plate remodeling defects and periportal fibrosis in the liver. Our expression studies and biochemical analyses show that biliary abnormalities in Anks6-deficient livers result from the dysregulation of YAP transcriptional activity in the bile duct-lining epithelial cells. Mechanistically, our studies suggest, that ANKS6 antagonizes Hippo signaling in the liver during bile duct development by binding to Hippo pathway effector proteins YAP1, TAZ and TEAD4 and promoting their transcriptional activity. Together, this study reveals a novel function for ANKS6 in regulating Hippo signaling during organogenesis and provides mechanistic insights into the regulatory network controlling bile duct differentiation and morphogenesis during liver development.

Uridine diphosphate-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase deletion in mice leads to lethal intracerebral hemorrhage during embryonic development.

Among the enzymes of the biosynthesis of sialoglycoconjugates, uridine diphosphate-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), catalyzing the first essential step of the sialic acid (Sia) de novo biosynthesis, and cytidine monophosphate (CMP)-Sia synthase (CMAS), activating Sia to CMP-Sia, are particularly important. The knockout of either of these enzymes in mice is embryonically lethal. While the lethality of Cmas-/- mice has been attributed to a maternal complement attack against asialo fetal placental cells, the cause of lethality in Gne-deficient embryos has remained elusive. Here, we advanced the significance of sialylation for embryonic development through detailed histological analyses of Gne-/- embryos and placentae. We found that Gne-/- embryonic and extraembryonic tissues are hyposialylated rather than being completely deficient of sialoglycans, which holds true for Cmas-/- embryos. Residual sialylation of Gne-/- cells can be explained by scavenging free Sia from sialylated maternal serum glycoconjugates via the lysosomal salvage pathway. The placental architecture of Gne-/- mice was unaffected, but severe hemorrhages in the neuroepithelium with extensive bleeding into the cephalic ventricles were present at E12.5 in the mutants. At E13.5, the vast majority of Gne-/- embryos were asystolic. This phenotype persisted when Gne-/- mice were backcrossed to a complement component 3-deficient background, confirming distinct pathomechanisms of Cmas-/- and Gne-/- mice. We conclude that the low level of sialylation observed in Gne-/- mice is sufficient both for immune homeostasis at the fetal-maternal interface and for embryonic development until E12.5. However, formation of the neural microvasculature is the first critical process, depending on a higher degree of sialylation during development of the embryo proper.

A 3D iPSC-differentiation model identifies interleukin-3 as a regulator of early human hematopoietic specification.

Hematopoietic development is spatiotemporally tightly regulated by defined cell-intrinsic and extrinsic modifiers. The role of cytokines has been intensively studied in adult hematopoiesis; however, their role in embryonic hematopoietic specification remains largely unexplored. Here, we used induced pluripotent stem cell (iPSC) technology and established a 3-dimensional, organoid-like differentiation system (hemanoid) maintaining the structural cellular integrity to evaluate the effect of cytokines on embryonic hematopoietic development. We show, that defined stages of early human hematopoietic development were recapitulated within the generated hemanoids. We identified KDR+/CD34high/CD144+/CD43-/CD45- hemato-endothelial progenitor cells (HEPs) forming organized, vasculature-like structures and giving rise to CD34low/CD144-/CD43+/CD45+ hematopoietic progenitor cells. We demonstrate that the endothelial to hematopoietic transition of HEPs is dependent on the presence of interleukin 3 (IL-3). Inhibition of IL-3 signalling blocked hematopoietic differentiation and arrested the cells in the HEP stage. Thus, our data suggest an important role for IL-3 in early human hematopoiesis by supporting the endothelial to hematopoietic transition of hemato-endothelial progenitor cells and highlight the potential of a hemanoid-based model to study human hematopoietic development.

Expansion of the renal capsular stroma, ureteric bud branching defects and cryptorchidism in mice with Wilms tumor 1 gene deletion in the stromal compartment of the developing kidney.

Development of the mammalian kidney is orchestrated by reciprocal interactions of stromal and nephrogenic mesenchymal cells with the ureteric bud epithelium. Previous work showed that the transcription factor Wilms tumor 1 (WT1) acts in the nephrogenic lineage to maintain precursor cells, to drive the epithelial transition of aggregating precursors into a renal vesicle and to specify and maintain the podocyte fate. However, WT1 is expressed not only in the nephrogenic lineage but also transiently in stromal progenitors in the renal cortex. Here we report that specific deletion of Wt1 in the stromal lineage using the Foxd1cre driver line results at birth in cryptorchidism and hypoplastic kidneys that harbour fewer and enlarged ureteric bud tips and display an expansion of capsular stroma into the cortical region. In vivo and ex vivo analysis at earlier stages revealed that stromal loss of Wt1 reduces stromal proliferation, and delays and alters branching morphogenesis, resulting in a variant architecture of the collecting duct tree with an increase of single at the expense of bifurcated ureteric bud tips. Molecular analysis identified a transient reduction of Aldh1a2 expression and of retinoic acid signalling activity in stromal progenitors, and of Ret in ureteric bud tips. Administration of retinoic acid partly rescued the branching defects of mutant kidneys in culture. We propose that WT1 maintains retinoic acid signalling in the cortical stroma, which, in turn, assures proper levels and dynamics of Ret expression in the ureteric bud tips, and thus normal ramification of the ureteric tree. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Growth differentiation factor 11 attenuates liver fibrosis via expansion of liver progenitor cells.

OBJECTIVE: Liver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of ageing organs, but its role in chronic liver diseases has remained unknown. Here, we investigated the expression and function of GDF11 in liver fibrosis, a common feature of most chronic liver diseases. DESIGN: We analysed the expression of GDF11 in patients with liver fibrosis, in a mouse model of liver fibrosis and in hepatic stellate cells (HSCs) as well as in other liver cell types. The functional relevance of GDF11 in toxin-induced and cholestasis-induced mouse models of liver fibrosis was examined by in vivo modulation of Gdf11 expression using adeno-associated virus (AAV) vectors. The effect of GDF11 on leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)+ liver progenitor cells was studied in mouse and human liver organoid culture. Furthermore, in vivo depletion of LGR5+ cells was induced by injecting AAV vectors expressing diptheria toxin A under the transcriptional control of Lgr5 promoter. RESULTS: We showed that the expression of GDF11 is upregulated in patients with liver fibrosis and in experimentally induced murine liver fibrosis models. Furthermore, we found that therapeutic application of GDF11 mounts a protective response against fibrosis by increasing the number of LGR5+ progenitor cells in the liver. CONCLUSION: Collectively, our findings uncover a protective role of GDF11 during liver fibrosis and suggest a potential application of GDF11 for the treatment of chronic liver disease.

Delayed onset of smooth muscle cell differentiation leads to hydroureter formation in mice with conditional loss of the zinc finger transcription factor gene Gata2 in the ureteric mesenchyme.

The establishment of the peristaltic machinery of the ureter is precisely controlled to cope with the onset of urine production in the fetal kidney. Retinoic acid (RA) has been identified as a signal that maintains the mesenchymal progenitors of the contractile smooth muscle cells (SMCs), while WNTs, SHH, and BMP4 induce their differentiation. How the activity of the underlying signalling pathways is controlled in time, space, and quantity to activate coordinately the SMC programme is poorly understood. Here, we provide evidence that the Zn-finger transcription factor GATA2 is involved in this crosstalk. In mice, Gata2 is expressed in the undifferentiated ureteric mesenchyme under control of RA signalling. Conditional deletion of Gata2 by a Tbx18cre driver results in hydroureter formation at birth, associated with a loss of differentiated SMCs. Analysis at earlier stages and in explant cultures revealed that SMC differentiation is not abrogated but delayed and that dilated ureters can partially regain peristaltic activity when relieved of urine pressure. Molecular analysis identified increased RA signalling as one factor contributing to the delay in SMC differentiation, possibly caused by reduced direct transcriptional activation of Cyp26a1, which encodes an RA-degrading enzyme. Our study identified GATA2 as a feedback inhibitor of RA signalling important for precise onset of ureteric SMC differentiation, and suggests that in a subset of cases of human congenital ureter dilatations, temporary relief of urine pressure may ameliorate the differentiation status of the SMC coat. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

BMP4 uses several different effector pathways to regulate proliferation and differentiation in the epithelial and mesenchymal tissue compartments of the developing mouse ureter.

Heterozygous loss of Bmp4 results both in humans and mice in severe malformation of the urinary tract. These defects have at least partially been attributed to loss of expression of Bmp4 in the ureteric mesenchyme, yet the cellular and molecular function of this signal as well as its effector pathways in this tissue have remained incompletely resolved. Here, we show that mice with a conditional deletion of Bmp4 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional and physical ureter obstruction. Proliferation in both the mesenchymal and epithelial progenitor pools was severely reduced and smooth muscle cell and urothelial differentiation programs were not activated. Epithelial expression of P-ERK1/2, P-AKT and P-P38, and mesenchymal expression of P-SMAD1/5/9, P-P38 and P-AKT were abrogated. Pharmacological inhibition and activation experiments in ureter cultures defined AKT as the most relevant downstream effector for epithelial and mesenchymal proliferation as well as for epithelial differentiation. Epithelial proliferation and differentiation were also influenced by P-38 and ERK1/2, while SMAD signaling, together with AKT and P-38, were required for smooth muscle cell differentiation. Our analysis suggests that BMP4 is the signal that couples the proliferation and differentiation programs in the epithelial and mesenchymal tissue compartments of the developing ureter by different downstream effectors, most importantly AKT and SMAD.

Mutations in the leukemia inhibitory factor receptor (LIFR) gene and Lifr deficiency cause urinary tract malformations.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. As CAKUT is a genetically heterogeneous disorder and most cases are genetically unexplained, we aimed to identify new CAKUT causing genes. Using whole-exome sequencing and trio-based de novo analysis, we identified a novel heterozygous de novo frameshift variant in the leukemia inhibitory factor receptor (LIFR) gene causing instability of the mRNA in a patient presenting with bilateral CAKUT and requiring kidney transplantation at one year of age. LIFR encodes a transmembrane receptor utilized by IL-6 family cytokines, mainly by the leukemia inhibitory factor (LIF). Mutational analysis of 121 further patients with severe CAKUT yielded two rare heterozygous LIFR missense variants predicted to be pathogenic in three unrelated patients. LIFR mutants showed decreased half-life and cell membrane localization resulting in reduced LIF-stimulated STAT3 phosphorylation. LIFR showed high expression in human fetal kidney and the human ureter, and was also expressed in the developing murine urogenital system. Lifr knockout mice displayed urinary tract malformations including hydronephrosis, hydroureter, ureter ectopia, and, consistently, reduced ureteral lumen and muscular hypertrophy, similar to the phenotypes observed in patients carrying LIFR variants. Additionally, a form of cryptorchidism was detected in all Lifr-/- mice and the patient carrying the LIFR frameshift mutation. Altogether, we demonstrate heterozygous novel or rare LIFR mutations in 3.3% of CAKUT patients, and provide evidence that Lifr deficiency and deactivating LIFR mutations cause highly similar anomalies of the urogenital tract in mice and humans.

Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life. Identification of single-gene mutations that cause CAKUT permits the first insights into related disease mechanisms. However, for most cases the underlying defect remains elusive. We identified a kindred with an autosomal-dominant form of CAKUT with predominant ureteropelvic junction obstruction. By whole exome sequencing, we identified a heterozygous truncating mutation (c.1010delG) of T-Box transcription factor 18 (TBX18) in seven affected members of the large kindred. A screen of additional families with CAKUT identified three families harboring two heterozygous TBX18 mutations (c.1570C>T and c.487A>G). TBX18 is essential for developmental specification of the ureteric mesenchyme and ureteric smooth muscle cells. We found that all three TBX18 altered proteins still dimerized with the wild-type protein but had prolonged protein half life and exhibited reduced transcriptional repression activity compared to wild-type TBX18. The p.Lys163Glu substitution altered an amino acid residue critical for TBX18-DNA interaction, resulting in impaired TBX18-DNA binding. These data indicate that dominant-negative TBX18 mutations cause human CAKUT by interference with TBX18 transcriptional repression, thus implicating ureter smooth muscle cell development in the pathogenesis of human CAKUT.

Diversification of Cell Lineages in Ureter Development.

The mammalian ureter consists of a mesenchymal wall composed of smooth muscle cells and surrounding fibrocytes of the tunica adventitia and the lamina propria and an inner epithelial lining composed of layers of basal, intermediate, and superficial cells. How these cell types arise from multipotent progenitors is poorly understood. Here, we performed marker analysis, cell proliferation assays, and genetic lineage tracing to define the lineage relations and restrictions of the mesenchymal and epithelial cell types in the developing and mature mouse ureter. At embryonic day (E) 12.5, the mesenchymal precursor pool began to subdivide into an inner and outer compartment that began to express markers of smooth muscle precursors and adventitial fibrocytes, respectively, by E13.5. Smooth muscle precursors further diversified into lamina propria cells directly adjacent to the ureteric epithelium and differentiated smooth muscle cells from E16.5 onwards. Uncommitted epithelial progenitors of the ureter differentiated into intermediate cells at E14.5. After stratification into two layers at E15.5 and three cell layers at E18.5, intermediate cells differentiated into basal cells and superficial cells. In homeostasis, proliferation of all epithelial and mesenchymal cell types remained low but intermediate cells still gave rise to basal cells, whereas basal cells divided only into basal cells. These studies provide a framework to further determine the molecular mechanisms of cell differentiation in the tissues of the developing ureter.

A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene (NRIP1) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RARα, and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RARα RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease.

Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.

The vesico-ureteric junction (VUJ) forms through a complex developmental program that connects the primordium of the upper urinary tract [the nephric duct (ND)] with that of the lower urinary tract (the cloaca). The signals that orchestrate the various tissue interactions in this program are poorly understood. Here, we show that two members of the EphA subfamily of receptor tyrosine kinases, EphA4 and EphA7, are specifically expressed in the mesenchyme surrounding the caudal ND and the cloaca, and that Epha4(-/-);Epha7(+/-) and Epha4(-/-);Epha7(-/-) (DKO) mice display distal ureter malformations including ureterocele, blind and ectopically ending ureters with associated hydroureter, megaureter and hydronephrosis. We trace these defects to a late or absent fusion of the ND with the cloaca. In DKO embryos, the ND extends normally and approaches the cloaca but the tip subsequently looses its integrity. Expression of Gata3 and Lhx1 and their downstream target Ret is severely reduced in the caudal ND. Conditional deletion of ephrin B2 from the ND largely phenocopies these changes, suggesting that EphA4/EphA7 from the pericloacal mesenchyme signal via ephrin B2 to mediate ND insertion. Disturbed activity of this signaling module may entail defects of the VUJ, which are frequent in the spectrum of congenital anomalies of the kidney and the urinary tract (CAKUT) in human newborns.

Whole-exome sequencing identifies mutations of TBC1D1 encoding a Rab-GTPase-activating protein in patients with congenital anomalies of the kidneys and urinary tract (CAKUT).

Congenital anomalies of the kidneys and urinary tract (CAKUT) are genetically highly heterogeneous leaving most cases unclear after mutational analysis of the around 30 causative genes known so far. Assuming that phenotypes frequently showing dominant inheritance, such as CAKUT, can be caused by de novo mutations, de novo analysis of whole-exome sequencing data was done on two patient-parent-trios to identify novel CAKUT genes. In one case, we detected a heterozygous de novo frameshift variant in TBC1D1 encoding a Rab-GTPase-activating protein regulating glucose transporter GLUT4 translocation. Sequence analysis of 100 further CAKUT cases yielded three novel or rare inherited heterozygous TBC1D1 missense variants predicted to be pathogenic. TBC1D1 mutations affected Ser237-phosphorylation or protein stability and thereby act as hypomorphs. Tbc1d1 showed widespread expression in the developing murine urogenital system. A mild CAKUT spectrum phenotype, including anomalies observed in patients carrying TBC1D1 mutations, was found in kidneys of some Tbc1d1 (-/-) mice. Significantly reduced Glut4 levels were detected in kidneys of Tbc1d1 (-/-) mice and the dysplastic kidney of a TBC1D1 mutation carrier versus controls. TBC1D1 and SLC2A4 encoding GLUT4 were highly expressed in human fetal kidney. The patient with the truncating TBC1D1 mutation showed evidence for insulin resistance. These data demonstrate heterozygous deactivating TBC1D1 mutations in CAKUT patients with a similar renal and ureteral phenotype, and provide evidence that TBC1D1 mutations may contribute to CAKUT pathogenesis, possibly via a role in glucose homeostasis.

Inhibition of Sox2-dependent activation of Shh in the ventral diencephalon by Tbx3 is required for formation of the neurohypophysis.

Tbx2 and Tbx3 are two highly related members of the T-box transcription factor gene family that regulate patterning and differentiation of a number of tissue rudiments in the mouse. Both genes are partially co-expressed in the ventral diencephalon and the infundibulum; however, a functional requirement in murine pituitary development has not been reported. Here, we show by genetic lineage tracing that Tbx2(+) cells constitute the precursor population of the neurohypophysis. However, Tbx2 is dispensable for neurohypophysis development as revealed by normal formation of this organ in Tbx2-deficient mice. By contrast, loss of Tbx3 from the ventral diencephalon results in a failure to establish the Tbx2(+) domain in this region, and a lack of evagination of the infundibulum and formation of the neurohypophysis. Rathke's pouch is severely hypoplastic, exhibits defects in dorsoventral patterning, and degenerates after E12.5. In Tbx3-deficient embryos, the ventral diencephalon is hyperproliferative and displays an abnormal cellular architecture, probably resulting from a failure to repress transcription of Shh. We further show that Tbx3 and Tbx2 repress Shh by sequestering the SRY box-containing transcription factor Sox2 away from a Shh forebrain enhancer (SBE2), thus preventing its activation. These data suggest that Tbx3 is required in the ventral diencephalon to establish a Shh(-) domain to allow formation of the infundibulum.