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 identified several growth factors and small molecule pathway agonists that preserve and propagate long-term the nephronic progenitor. 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 principally acts through the JAK/STAT pathway and up regulates the expression of several renal stem cell markers, e.g., Six2 and Pax2. Mechanistically, 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. Our conditions have also proven successful in promoting the growth of mouse and human Six2+ nephronic progenitors. However, this required further culture optimization with a Wnt agonist and Bmp family member in addition to LIF/ROCKi. In this case, Six2+ 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 are now collaborating with another lab that studies renal damage in mice in efforts to determine if our cultured progenitors can facilitate kidney repair following injury. We have now also applied progenitor culture conditions to the propagation of human Wilms tumor (WT) cells from several different patients and have determined that these same factors selectively expand the Six2+ progenitor from tumor tissues. These cells also retain the expression of several other stemness and WT-associated genes, such as NCAM and PAX2. These cells are stable for several passages and retain a tumorigenic phenotype based upon their ability for anchorage-independent growth. Cultured cells from these tumors have now been xenografted into NSG mice to further assess tumorigenic behavior. Notably, WT cells readily form organoids when placed in nonadherent culture dishes. Given the belief that these cells provide the driving force for WTs, the massive expansion of this population in culture witnessed in these studies may permit the development of personalized therapeutic screening for individual WT patients. This is a major advance in the field, as heretofore, failed efforts to propagate WT cells in culture have led some to speculate that they cannot be sustained except in xenografts. 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, we have found that Stat3 is required for maintenance of the trabecular bone, and loss of Stat3 results in shortening and bending of the long bones and downregulation of the osteochondro master regulator Sox9. We further identified two Stat-dependent DNA response elements in the Sox9 promoter. 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 now attempting to clarify the role of Stat3 in the osteoblast lineage as well, since the bones that form in Stat3 mutants are severely demineralized. We have now also found that knockdown of Stats in nephronic progenitors attenuates Sox9 expression in cultured cells and are therefore targeting Stat3 ablation specifically to the Six2+ progenitor in vivo in order to understand its role during development or in response to renal tubular injury. Sox9 has been implicated in the repair of ischemic tubular injury, and, based upon our work, this could be initiated by Stat activation. We continue to investigate two aberrant phenotypes in the urinary tract associated with Wnt5a ablation. The first involves duplex kidney formation, which we have determined is caused by a bilateral duplication of the Wolffian duct, a common malformation in humans. We are also evaluating a second renal phenotype in Wnt5a mutants, i.e., hydronephrosis. 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. The blockage occurs at the interface between the ureter and bladder when cells in the common nephric duct fail to apoptose. Gene expression analysis revealed that Shh was increased in Wnt5a mutants, and when only one Shh allele was expressed, we could correct the aberrant phenotype. This work demonstrates for the first time that Wnt5a modulates Shh levels during development and possibly also during tumorigenesis, since Wnt5a can also function as a tumor suppressor. Finally, we have continued our efforts to identify a novel genetic locus associated with Wilms tumor/nephroblastoma. The Noble rat strain is exquisitely sensitive to transplacental induction of nephroblastoma at high incidence; whereas the Fisher rat is completely insensitive to nephroblastoma induction. Cross breeding between these two rat strains suggests inheritance occurs as an incomplete dominant trait. To map the responsible locus, we have backcrossed F1 hybrids to the Fisher strain and then whole-genome sequenced the DNAs from all rats that developed nephroblastoma (25) as well as several controls. George Nelson is responsible for coordinating and assessing all sequencing data. Since the rat genome is not well annotated, we are experiencing technical issues with alignments, but are now sorting through those problems.
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