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. This culture system of MM provides unique opportunities to comprehensively address key mechanisms involved in renal progenitor maintenance and differentiation. In order to better understand the role that LIF-induced Stat signaling has in kidney development, we are generating mouse line to unravel the redundant functioning of this family of transcription factors. 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 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. Further, a rapid depletion of the osteoblast lineage coinciding with elevation of the osteoclast population results in wide-spread osteoporotic lesions soon after birth. 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. 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 are currently looking at the simultaneous ablation of other Stat molecules in the kidney to confirm our in culture findings that Stat activation sustains the Six+ nephronic progenitor. 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 have found 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 grouping of significant congenital abnormalities called CAKUT (Congenital Anomalies of the Kidney and Urogenital Tract). Interestingly though,Wnt5a expression is already lost in the region where bud outgrowth occurs by the time it is initiated. This suggested that the events/interactions responsible for dysmorphogenesis of the metanephros likely precede its development. Thus, we temporally ablated Wnt5a and found that inactivation at E7.5 but not E8.5 resulted in duplex collecting systems. Since Wnt5a is expressed in the primitive streak and neural plate at these earlier times, it is likely that dysregulation in those tissues is responsible for the phenotype. Consistent with this hypothesis is the fact that the entire length of the nephric duct throughout the mesonephros is aberrantly formed, i.e., it is wider and truncated in the mutant embryo. Furthermore, at its caudal end, it appears as a fused doublet, consistent with double ureter outgrowth. Finally, we have also examined the ablation of Wnt5a using several different tissue-specific Cre lines to isolate the source and timing of Wnt5a in the proper patterning of the kidney. Thus far we have determined that simultaneous inactivation of Wnt5a in the nephric duct and surrounding nephrogenic cord is insufficient to cause the abnormality; whereas, early removal in the primitive streak does result in aberrant development. In addition to duplex kidney and ureter formation, we observed that the Wnt5a mutation also causes another phenotype in the urogenital system - a severe secondary vesicoureteral reflux. In the Wnt5a mouse, the ureter intersects inappropriately with the bladder leading to a failed connectivity and resulting in the improper termination of the ureter. The malformation leads either to the inadvertent release of urine into the peritoneum or to back pressure in the kidney, causing hydronephrosis. Unlike duplex kidney formation, this abnormality is dependent upon proper signaling by Wnt5a later in urogenital development, i.e., E9.5. We are currently developing tools that will allow us to visualize the mutant structures with 3-dimensional imaging.
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