Mutation of the transcription factor and tumor suppressor gene WT1 results in a range of genitourinary anomalies in humans, including 46,XY gonadal dysgenesis, indicating that WT1 plays a critical role in sex determination. However, because knockout of Wt1 in mice results in apoptosis of the genital ridge, it is unknown whether WT1 is required for testis development after the initial steps of sex determination. To address this question, we generated a mouse strain carrying a Wt1 conditional knockout allele and ablated Wt1 function specifically in Sertoli cells by embryonic day 14.5, several days after testis determination. Wt1 knockout resulted in disruption of developing seminiferous tubules and subsequent progressive loss of Sertoli cells and germ cells such that postnatal mutant testes were almost completely devoid of these cell types and were severely hypoplastic. Thus, Wt1 is essential for the maintenance of Sertoli cells and seminiferous tubules in the developing testes. Of particular note, expression of the testisdetermining gene Sox9 in mutant Sertoli cells was turned off at embryonic day 14.5 after Wt1 ablation, suggesting that WT1 regulates Sox9, either directly or indirectly, after Sry expression ceases. Our data, along with previous work demonstrating the role of Wt1 at early stages of gonadal development, thus indicate that Wt1 is essential at multiple steps in testicular development.Sertoli cell ͉ testicular cord ͉ Mü llerian duct ͉ Amh ͉ Sox8

β-Catenin, as an important effector of the canonical Wnt signaling pathway and as a regulator of cell adhesion, has been demonstrated to be involved in multiple developmental processes and tumorigenesis. β-Catenin expression was found mainly on the Sertoli cell membrane starting from embryonic day 15.5 in the developing testes. However, its potential role in Sertoli cells during testis formation has not been examined. To determine the function of β-catenin in Sertoli cells during testis formation, we either deleted β-catenin or expressed a constitutively active form of β-catenin in Sertoli cells. We found that deletion caused no detectable abnormalities. However, stabilization caused severe phenotypes, including testicular cord disruption, germ cell depletion and inhibition of Müllerian duct regression. β-Catenin stabilization caused changes in Sertoli cell identity and misregulation of inter-Sertoli cell contacts. As Wt1 conditional knockout in Sertoli cells causes similar phenotypes to our stabilized β-catenin mutants, we then investigated the relationship of Wt1 and β-catenin in Sertoli cells and found Wt1 inhibits β-catenin signaling in these cells during testis development. Wt1 deletion resulted in upregulation of β-catenin expression in Sertoli cells both in vitro and in vivo. Our study indicates that Sertoli cell expression of β-catenin is dispensable for testis development. However, the suppression of β-catenin signaling in these cells is essential for proper testis formation and Wt1 is a negative regulator of β-catenin signaling during this developmental process.

Wilms tumor (WT) is a genetically heterogeneous childhood kidney tumor. Several genetic alterations have been identified in WT patients, including inactivating mutations in WT1 and loss of heterozygosity or loss of imprinting at 11p15, which results in biallelic expression of IGF2. However, the mechanisms by which one or a combination of genetic alterations results in tumorigenesis has remained challenging to determine, given the lack of a mouse model of WT. Here, we engineered mice to sustain mosaic, somatic ablation of Wt1 and constitutional Igf2 upregulation, mimicking a subset of human tumors. Mice with this combination of genetic alterations developed tumors at an early age. Mechanistically, Wt1 ablation blocked mesenchyme differentiation, and increased Igf2 expression upregulated ERK1/2 phosphorylation. Importantly, a subset of human tumors similarly displayed upregulation of ERK1/2 phosphorylation, which suggests ERK signaling might contribute to WT development. Thus, we have generated a biologically relevant mouse model of WT and defined one combination of driver alterations for WT. This mouse model will provide a powerful tool to study the biology of WT initiation and progression and to investigate therapeutic strategies for cancers with IGF pathway dysregulation. IntroductionWilms tumor (WT) is a childhood kidney tumor that is thought to arise from undifferentiated metanephric mesenchyme. WT is genetically heterogeneous. Mutations that occur in tumors include inactivation of WT1 (~20% of tumors), somatic stabilizing CTNNB1 mutations (~15%), somatic deletion of WTX (~20%), and p53 mutations (~5%) that occur specifically in the subset of anaplastic WT (1-5). Overall, only one-third of tumors have mutations in 1 or more of these 4 genes (6). Additionally, loss of heterozygosity (LOH) or loss of imprinting (LOI) at the chromosomal region 11p15, which harbors a cluster of imprinted genes, is observed in approximately 70% of tumors (7,8), resulting in biallelic expression of IGF2. However, the mechanism by which one or a combination of alterations results in tumorigenesis is not known. Children heterozygous for germline WT1 mutations are predisposed to WT, and these tumors have invariably sustained mutation of the wild-type WT1 allele. However, inactivation of WT1 is also observed in premalignant lesions (9), which suggests that one or more additional, rate-limiting genetic alterations is required for progression to a malignant phenotype.The cellular pathways dysregulated in WTs as a result of WT1 ablation or IGF2 upregulation are unknown, and identifying such pathways in human tumors is challenging because of the genetic heterogeneity of the disease and the biologic complexity of primary human tumors. While animal models can be powerful tools for dissecting the biology of human tumors, the development of a mouse model for WT has been elusive. Wt1 -/-mice lack kidneys

Neddylation has an important role in ubiquitin-mediated protein degradation through modification of cullins, which are the main substrates for NEDD8 modification. Here, we show that breast cancer-associated protein 3 (BCA3) is a NEDD8 substrate. BCA3 suppressed NFkappaB-dependent transcription through its ability to bind to p65 and the cyclin D1 promoter in a neddylation-dependent manner. Transcriptional suppression mediated by BCA3 may be attributed to the ability of neddylated BCA3 to recruit SIRT1, a class III histone deacetylase. Silencing of endogenous BCA3 in DU145 and MCF7 cells enhanced NFkappaB transcription and inhibited tumour necrosis factor (TNF)alpha-induced apoptosis. Conversely, BCA3 silencing could be reversed by over-expression of wild-type BCA3 and SENP8, a NEDD8-specific protease, but not by neddylation-deficient BCA3 or a SENP8 mutant. These results provide a crucial link between neddylation and transcriptional regulation by SIRT1, a NAD-dependent histone deacetylase that prolongs life span in yeast and worms.