Development, regeneration and tumorigenesis of the urothelium.

The urothelium of the bladder functions as a waterproof barrier between tissue and outflowing urine. Largely quiescent during homeostasis, this unique epithelium rapidly regenerates in response to bacterial or chemical injury. The specification of the proper cell types during development and injury repair is crucial for tissue function. This Review surveys the current understanding of urothelial progenitor populations in the contexts of organogenesis, regeneration and tumorigenesis. Furthermore, we discuss pathways and signaling mechanisms involved in urothelial differentiation, and consider the relevance of this knowledge to stem cell biology and tissue regeneration.

Mouse and human studies support DSTYK loss of function as a low-penetrance and variable expressivity risk factor for congenital urinary tract anomalies.

PURPOSE: Previous work identified rare variants in DSTYK associated with human congenital anomalies of the kidney and urinary tract (CAKUT). Here, we present a series of mouse and human studies to clarify the association, penetrance, and expressivity of DSTYK variants. METHODS: We phenotypically characterized Dstyk knockout mice of 3 separate inbred backgrounds and re-analyzed the original family segregating the DSTYK c.654+1G>A splice-site variant (referred to as "SSV" below). DSTYK loss of function (LOF) and SSVs were annotated in individuals with CAKUT, epilepsy, or amyotrophic lateral sclerosis vs controls. A phenome-wide association study analysis was also performed using United Kingdom Biobank (UKBB) data. RESULTS: Results demonstrate ∼20% to 25% penetrance of obstructive uropathy, at least, in C57BL/6J and FVB/NJ Dstyk-/- mice. Phenotypic penetrance increased to ∼40% in C3H/HeJ mutants, with mild-to-moderate severity. Re-analysis of the original family segregating the rare SSV showed low penetrance (43.8%) and no alternative genetic causes for CAKUT. LOF DSTYK variants burden showed significant excess for CAKUT and epilepsy vs controls and an exploratory phenome-wide association study supported association with neurological disorders. CONCLUSION: These data support causality for DSTYK LOF variants and highlights the need for large-scale sequencing studies (here >200,000 cases) to accurately assess causality for genes and variants to lowly penetrant traits with common population prevalence.

Apoptosis induced by vitamin A signaling is crucial for connecting the ureters to the bladder.

Removal of toxic substances from the blood depends on patent connections between the kidney, ureters and bladder that are established when the ureter is transposed from its original insertion site in the male genital tract to the bladder. This transposition is thought to occur as the trigone forms from the common nephric duct and incorporates into the bladder. Here we re-examine this model in the context of normal and abnormal development. We show that the common nephric duct does not differentiate into the trigone but instead undergoes apoptosis, a crucial step for ureter transposition controlled by vitamin A-induced signals from the primitive bladder. Ureter abnormalities occur in 1-2% of the human population and can cause obstruction and end-stage renal disease. These studies provide an explanation for ureter defects underlying some forms of obstruction in humans and redefine the current model of ureter maturation.

Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.

Almost 1% of human infants are born with urogenital abnormalities, many of which are linked to irregular connections between the distal ureters and the bladder. During development, ureters migrate by an unknown mechanism from their initial integration site in the Wolffian ducts up to the base of the bladder in a process that we call ureter maturation. Rara(-/-) Rarb2(-/-) mice display impaired vitamin A signaling and develop syndromic urogenital malformations similar to those that occur in humans, including renal hypoplasia, hydronephrosis and mega-ureter, abnormalities also seen in mice with mutations in the proto-oncogene Ret. Here we show that ureter maturation depends on formation of the 'trigonal wedge', a newly identified epithelial outgrowth from the base of the Wolffian ducts, and that the distal ureter abnormalities seen in Rara(-/-) Rarb2(-/-) and Ret(-/-) mutant mice are probably caused by a failure of this process. Our studies indicate that formation of the trigonal wedge may be essential for correct insertion of the distal ureters into the bladder, and that these events are mediated by the vitamin A and Ret signaling pathways.

FGFR3b Extracellular Loop Mutation Lacks Tumorigenicity In Vivo but Collaborates with p53/pRB Deficiency to Induce High-grade Papillary Urothelial Carcinoma.

Missense mutations of fibroblast growth factor receptor 3 (FGFR3) occur in up to 80% of low-grade papillary urothelial carcinoma of the bladder (LGP-UCB) suggesting that these mutations are tumor drivers, although direct experimental evidence is lacking. Here we show that forced expression of FGFR3b-S249C, the most prevalent FGFR3 mutation in human LGP-UCB, in cultured urothelial cells resulted in slightly reduced surface translocation than wild-type FGFR3b, but nearly twice as much proliferation. When we expressed a mouse equivalent of this mutant (FGFR3b-S243C) in urothelia of adult transgenic mice in a tissue-specific and inducible manner, we observed significant activation of AKT and MAPK pathways. This was, however, not accompanied by urothelial proliferation or tumorigenesis over 12 months, due to compensatory tumor barriers in p16-pRB and p19-p53-p21 axes. Indeed, expressing FGFR3b-S249C in cultured human urothelial cells expressing SV40T, which functionally inactivates pRB/p53, markedly accelerated proliferation and cell-cycle progression. Furthermore, expressing FGFR3b-S243C in transgenic mouse urothelium expressing SV40T converted carcinoma-in-situ to high-grade papillary urothelial carcinoma. Together, our study provides new experimental evidence indicating that the FGFR3 mutations have very limited urothelial tumorigenicity and that these mutations must collaborate with other genetic events to drive urothelial tumorigenesis.

A mucosal imprint left by prior Escherichia coli bladder infection sensitizes to recurrent disease.

Recurrent bacterial infections are a significant burden worldwide, and prior history of infection is often a significant risk factor for developing new infections. For urinary tract infection (UTI), a history of two or more episodes is an independent risk factor for acute infection. However, mechanistic knowledge of UTI pathogenesis has come almost exclusively from studies in naive mice. Here we show that, in mice, an initial Escherichia coli UTI, whether chronic or self-limiting, leaves a long-lasting molecular imprint on the bladder tissue that alters the pathophysiology of subsequent infections, affecting host susceptibility and disease outcome. In bladders of previously infected versus non-infected, antibiotic-treated mice, we found (1) an altered transcriptome and defects in cell maturation, (2) a remodelled epithelium that confers resistance to intracellular bacterial colonization, and (3) changes to cyclooxygenase-2-dependent inflammation. Furthermore, in mice with a history of chronic UTI, cyclooxygenase-2-dependent inflammation allowed a variety of clinical E. coli isolates to circumvent intracellular colonization resistance and cause severe recurrent UTI, which could be prevented by cyclooxygenase-2 inhibition or vaccination. This work provides mechanistic insight into how a history of infection can impact the risk for developing recurrent infection and has implications for the development of therapeutics for recurrent UTI.

Pathways of vitamin A delivery to the embryo: insights from a new tunable model of embryonic vitamin A deficiency.

Circulating retinoids (vitamin A and its derivatives) are found predominantly as retinol bound to retinol-binding protein (RBP), which transports retinol from liver stores to target tissues, or as retinyl ester incorporated in lipoproteins of dietary origin. The transport of retinoids from maternal to fetal circulation is poorly understood, especially under conditions of inadequate dietary vitamin A intake. Here we present RBP-/- mice as a tunable model of embryonic vitamin A deficiency. This model has enabled us to analyze metabolic links between maternal nutrition and retinoid delivery to the fetus. Our data show that retinol-RBP is the primary contributor to fetal development, whereas retinyl ester are largely responsible for accumulation of fetal retinoid stores. Furthermore, these studies indicate the importance of embryonic RBP in distributing vitamin A to certain developing tissues under restrictive diets. We also show differences among developing tissues in their dependency on the embryonic retinol-RBP pathway. Finally, we demonstrate that accumulation of embryonic vitamin A stores does not depend on the expression of RBP in the fetal liver.

Organotypic culture of the urogenital tract.

Organotypic culture is an invaluable technique that allows researchers with the tool to analyze a tissue development in an isolated and well-defined environment. This technique also permits one to study the roles of different signaling systems/signaling molecules and to take advantage of the modern real-time imaging techniques, including confocal microscopy. With great success, our lab has used organotypic culture of the urogenital tract (UGT) to study growth and extension of the mesonephric (Wolffian) duct and its cloaca connection, ureter maturation, and bladder urothelium development (Batourina et al. Nat Genet 32:109, 2002; Batourina et al. Nat Genet 37:1082, 2005; Mendelsohn Organogenesis 5:306, 2009).

A high-resolution anatomical ontology of the developing murine genitourinary tract.

Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17-27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.

c-kit delineates a distinct domain of progenitors in the developing kidney.

Early inductive events in mammalian nephrogenesis depend on an interaction between the ureteric bud and the metanephric mesenchyme. However, mounting evidence points towards an involvement of additional cell types--such as stromal cells and angioblasts--in growth and patterning of the nephron. In this study, through analysis of the stem cell factor (SCF)/c-kit ligand receptor pair, we describe an additional distinct cell population in the early developing kidney. While SCF is restricted to the ureteric bud, c-kit-positive cells are located within the renal interstitium, but are negative for Foxd1, an established marker of stromal cells. In fact, the c-kit-positive domain is continuous with a central mesodermal cell mass ventral and lateral to the dorsal aorta, while Foxd1-expressing stromal cells are continuous with a dorsal perisomitic cell population suggesting distinct intraembryonic origins for these cell types. A subset of c-kit-positive cells expresses Flk-1 and podocalyxin, suggesting that this cell population includes angioblasts and their progenitors. c-kit activation is not required for the survival of these cells in vivo, because white spotting (c-kit(W/W)) mice, carrying a natural inactivating mutation of c-kit, display normal intrarenal distribution of the c-kit-positive cells at E13.5. In addition, early kidney development in these mutants is preserved up to the stage when anemia compromises global embryonic development. In contrast, under defined conditions in organ cultures of metanephric kidneys, c-kit-positive cells, including the Flk-1-positive subset, undergo apoptosis after treatment with STI-571, an inhibitor of c-kit tyrosine phosphorylation. This is associated with reductions in ureteric bud branching and nephron number. Conversely, exogenous SCF expands the c-kit-positive population, including Flk-1-positive angioblasts, and accelerates kidney development in vitro. These data suggest that ureteric bud-derived SCF elicits growth-promoting effects in the metanephric kidney by expanding one or more components of the interstitial c-kit-positive progenitor pool.

Foxd1-dependent signals control cellularity in the renal capsule, a structure required for normal renal development.

Development of the metanephric kidney involves the establishment of discrete zones of induction and differentiation that are crucial to the future radial patterning of the organ. Genetic deletion of the forkhead transcription factor, Foxd1, results in striking renal abnormalities, including the loss of these discrete zones and pelvic fused kidneys. We have investigated the molecular and cellular basis of the kidney phenotypes displayed by Foxd1-null embryos and report here that they are likely to be caused by a failure in the correct formation of the renal capsule. Unlike the single layer of Foxd1-positive stroma that comprises the normal renal capsule, the mutant capsule contains heterogeneous layers of cells, including Bmp4-expressing cells, which induce ectopic phospho-Smad1 signaling in nephron progenitors. This missignaling disrupts their early patterning, which, in turn, causes mispatterning of the ureteric tree, while delaying and disorganizing nephrogenesis. In addition, the defects in capsule formation prevent the kidneys from detaching from the body wall, thus explaining their fusion and pelvic location. For the first time, functions have been ascribed to the renal capsule that include delineation of the organ and acting as a barrier to inappropriate exogenous signals, while providing a source of endogenous signals that are crucial to the establishment of the correct zones of induction and differentiation.

Distinct and sequential tissue-specific activities of the LIM-class homeobox gene Lim1 for tubular morphogenesis during kidney development.

Kidney organogenesis requires the morphogenesis of epithelial tubules. Inductive interactions between the branching ureteric buds and the metanephric mesenchyme lead to mesenchyme-to-epithelium transitions and tubular morphogenesis to form nephrons, the functional units of the kidney. The LIM-class homeobox gene Lim1 is expressed in the intermediate mesoderm, nephric duct, mesonephric tubules, ureteric bud, pretubular aggregates and their derivatives. Lim1-null mice lack kidneys because of a failure of nephric duct formation, precluding studies of the role of Lim1 at later stages of kidney development. Here, we show that Lim1 functions in distinct tissue compartments of the developing metanephros for both proper development of the ureteric buds and the patterning of renal vesicles for nephron formation. These observations suggest that Lim1 has essential roles in multiple steps of epithelial tubular morphogenesis during kidney organogenesis. We also demonstrate that the nephric duct is essential for the elongation and maintenance of the adjacent Mullerian duct, the anlage of the female reproductive tract.