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 SIX2/CITED1-positive 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. Inductive signaling in MM progenitors that results in MET can be mediated by a variety of factors, including Wnt4, which is essential for nephron formation. We previously reported that Wnt4 induces MET by a calcium-dependent mechanism and not by canonical Wnt signaling as thought. We also determined that the cytokine leukemia inhibitory factor (LIF) can similarly induce tubule formation (MET) in renal progenitors, as can small molecule GSK3beta inhibitors. The common thread among these inducers is their ability to activate calcium signaling. LIF, for example, induces phosphorylation of PLCgamma, which activates calcium signaling and subsequently NFAT-mediated transcription. In addition to its role in the induction of MET in MM, LIF also appears to function in the maintenance and expansion of the MM progenitor population. LIF induces MET in MM at 30-50 ng/ml through activation of calcium signaling in these progenitors. However, LIF also maintains and expands the progenitor population at levels that fail to induce MET (1 ng/ml) or activate PLCgamma. It is known to function principally through STAT activation, and the lower levels of LIF activate STATs 1, 3 and 5 in renal progenitors without simultaneously activating calcium signaling. At 1 ng/ml, LIF increases proliferation and the number of cells expressing renal stem cell marker and critical maintenance and self-renewal factor Six2. This response is further facilitated with the concurrent addition of a Rho kinase inhibitor (ROCKi). Importantly, LIF/ROCKi-treated cells retain their ability to undergo MET in culture, thus producing a powerful tool for studying this process. Cells from these cultures have now been passaged multiple times without a loss of ability to undergo MET. 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. Ectopic expression of a series of dominant-negative mutant constructs for YAP confirmed that nuclear YAP is sufficient to sustain MM cell 'stemness'and increase their rate of proliferation. Since YAP interacts with Tead transcription factors, we also knocked down members of the Tead family using siRNA and determined that Tead2 and/or Tead3 are required for YAP-dependent transcription and MM progenitor cell maintenance. Taken together, our results demonstrate that LIF/ROCK signaling controls MM cell maintenance and self-renewal by regulating YAP/Tead2/Tead3-dependent transcription. This culture system of MM provides unique opportunities to comprehensively address key mechanisms involved in renal progenitor maintenance and differentiation. As part of a collaboration with the Carroll lab/UTexas, we characterized the role of Yap and Fat4 in the regulation of the aggregate Six2-expressing progenitor population during metanephric development. Our studies demonstrated that the Six2+ population is growth limited by the surrounding cortical stroma and that loss of stromal cells causes a dramatic expansion of Six+ stem cells. Furthermore, the studies found that Fat4 in stromal cells is responsible for the suppression of Yap in regulating the Six2+ population. These findings suggest a possible role for dysregulated Fat4/Yap signaling in the pathogenensis of Wilms tumors, since tumors characteristically contain a massively expanded Six2+ population. Immunostaining for Yap in Wilms tumor cells also revealed nuclear localization of the co-activator, which of course is consistent with transcriptional activation. We are currently evaluating the significance of this observation. We have continued our examination of the role of STATs in the developing metanephros, where we have found STATs 1, 3, 5, and 6 to be highly expressed and phosphorylated/activated. Given the responsiveness of MM cells to LIF treatment in inducing progenitor marker expression and MM cell proliferation as described above, we believe that Stat signaling plays a fundamental role in stem cell maintenance in the metanephros and possibly other mesodermal tissues. 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 but no obvious alterations in the kidney other than possible size differences. In collaboration with colleagues in CDBL, we have found that Stat3 is required for maintenance of the trabecular bone, and the loss of Stat3 results in the loss of mineralization in this tissue. 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. 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 continuiing our examination of Stat redundancy in the metanephros through the acquisition of a conditional Stat3/5 mouse line. This line will also allow us to generate a Stat1/3/5 triple mutant and assess the role of their combined effects on renal development. Finally, in collaboration with CDBL PI Terry Yamaguchi, we continue to investigate the role of Wnt5a in metanephric development. We have found that its specific inactivation in mesoderm using T/Brachyury-Cre results in duplex kidneys and double ureter formation bilaterally, a common malformation in the overall population. 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. Since Wnt5a is expressed in both the IM and the adjacent paraxial mesoderm (PM), which directs axis extension, its loss from either or both tissues may cause the WD malformation. To address this directly, we have acquired IM and PM specific Cre lines to establish each tissue's contribution to formation of the WD. We are also investigating the mechanism by which Wnt5a signals to regulate WD formation. To this end, we have demonstrated that the Wnt receptor ROR2 is linked genetically to the Wnt5a duplex ureter phenotype.
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