The Renal Differentiation and Neoplasia Section studies inductive signaling in tissue development/morphogenesis and, in parallel, its dysregulation in tumorigenesis with emphasis on the ligands that mediate normal tissue interactions and the pathways and targets that are activated in response to signaling. Our focus has been on development of the urogenital tract, which features reciprocal interactions between two distinct mesodermal progenitors, highly coordinated tissue movements, mesenchymal-epithelial transition (MET), integration of structures from different lineages, reiterative cycles of development, and a tumor that caricatures nephrogenesis. More specifically we are interested in the signaling mechanisms that direct metanephric mesenchyme (MM) to convert to the epithelia of the nephron. Wilms tumor (WT) is characterized by an expanded blastemal/progenitor population with a restricted capacity for epithelial conversion (MET). It is our long-term goal to identify targets on which WT cells depend for survival or dysregulated signaling that can be reprogrammed to allow tumor cells to differentiate to a more benign phenotype. We have determined that the cytokine leukemia inhibitory factor (LIF) in combination with Rho kinase inhibitor (ROCKi) maintains and selectively expands the Six2+ nephronic stem cell population in culture. Moreover, these propagated stem cells retain their capacity to convert to all segments of the nephron, demonstrating that they are multipotent progenitors. LIF functions principally through activation of STATs 1, 3 and 5 and up regulates the expression of several renal stem cell markers, e.g., Six2 and Pax2. Mechanistically, we have now found that LIF stimulates JNK activation, which induces MM proliferation and enhances cell competence to differentiate. The Rho kinase inhibitor (ROCKi) attenuates the LIF-induced Jnk activation thus inhibiting the differentiation of the progenitor. An investigation into the mechanism(s) mediated by LIF/ROCKi in these cells revealed that our conditions facilitate the nuclear localization of Yes-associated protein (YAP), a transcriptional co-activator and component of the Hippo signaling pathway. Furthermore, silencing Yap gene expression by siRNA knockdown in MM cells decreased the expression of progenitor markers and increased levels of MET markers, suggesting that YAP maintains MM cells in an undifferentiated state. Since YAP interacts with Tead transcription factors, we also determined that canonical Yap signaling through Tead activation is required for YAP-dependent transcription and MM progenitor cell maintenance. Since last year, we have now reported optimized niche conditions for the propagation of mouse Six2+ nephronic progenitors. This involved treatment of FACs sorted GFP-Six2+ cells with a Wnt agonist and Bmp7 in addition to LIF/ROCKi. These cells could be maintained for several passages and induced to form all segments of the nephronic epithelia. This culture system of MM provides unique opportunities to comprehensively address key mechanisms involved in renal progenitor maintenance and differentiation and raises the possibility that they may be applied to models of tissue repair/regeneration. We have now also applied similar conditions to the propagation of human Wilms tumor cells and determined that these same factors selectively expand the Six2+ progenitor from tumor tissues. Given the belief that these cells provide the driving force for Wilms tumors, expansion of this population in culture may provide an important tool for developing individual treatments for Wilms tumor patients. In order to better understand the role that LIF-induced Stat signaling has in kidney development, we are evaluating Stat3 mutant mice, which develop kidneys that are dramatically reduced in size. During the course of our mouse genetic studies we discovered a significant role for Stat3 in bone development. We have determined that the conditional loss of Stat3 causes a phenotype typical of two bent bone disorders, campomelic dysplasia and Stuve-Wiedemann syndrome. Using conditional loss-of-function (LOF) mouse models, a preliminary assessment of a LOF mutant for Stat3 has revealed extensive defects in the skeletal system, which appear to tie these two congenital abnormalities together. Specifically, we have found that Stat3 is required for maintenance of the trabecular bone, and the loss of Stat3 results in shortening and bending of the long bones and their improper mineralization. Signatures consistent with interrupted endochondral bone formation were evident in the expansion of hypertrophic chondrocytes and the observed downregulation of the osteochondro master regulator Sox9. To interrogate the mechanism, we analyzed the Sox9 proximal promoter region and discovered several potential Stat DNA response elements (DRE). Indeed, we found that a Sox9 promoter-driven reporter is activated in cells by oncostatin M in a Stat3-dependent fashion. Further, reporter activation is mediated by the Stat DREs, and Stat3 physically binds the promoter of Sox9. We have mutated these sites and demonstrated that cells lose their ability to activate a Sox9 reporter. Moreover, knockdown of Stat3 in chondrogenic progenitors attenuates Sox9 expression. These findings demonstrate a critical role for Stat3 in the proper patterning of the mammalian skeleton and implicate Sox9 as a downstream target of Stat3 signaling in this process. We have now also found that knockdown of Stats in nephronic progenitors attenuates Sox9 expression as well and that loss of Drosophila's Stat92E similarly ablates the fly's Sox9 homolog, suggesting that Stats regulation of Sox gene expression is conserved in evolution. Finally, in collaboration with CDBL PI Terry Yamaguchi, we continue to investigate the role of Wnt5a in metanephric development. Normally the ureteric bud, which forms the collecting ducts and ureter, extends as a single outgrowth from the Wolffian duct (WD) in the intermediate mesoderm (IM) at E10.5 in the mouse. However, we reported that inactivation of Wnt5a in mesoderm using T/Brachyury-Cre results in duplex kidneys and double ureter formation bilaterally, a common malformation in the overall population and part of a major group of significant congenital abnormalities called CAKUT (Congenital Anomalies of the Kidney and Urogenital Tract). We have now also evaluated a second renal phenotype in the Wnt5a mutants, i.e., hydronephrosis. Using a series of different Cre lines, we identified lines that genetically isolated this phenotype from duplex kidney formation and that also eliminated the axis extension phenotype. In these studies we show that hydronephrosis occurs as the result of a blockage in urine flow at the time renal function becomes active, causing apoptosis of the medullary region in the kidney. We then show that the blockage occurs at the interface between the ureter and bladder and that, as a result of the failure of the common nephric duct to undergo apoptosis, there is ureterocele formation in the wall of the bladder. An analysis of gene expression patterns revealed that Shh was dramatically increased in Wnt5a mutants. In efforts to rescue the hydronephrosis phenotype, we generated a mouse with only one allele of Shh and found that reduction in Shh was sufficient to significantly reduce the incidence of hydronephrosis. This work describes a new Wnt5a-mediated regulatory mechanism in development.
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