Inactivation of Wt1 causes pre-granulosa cell to steroidogenic cell transformation and defect of ovary development†.

Wt1 gene encodes a nuclear transcription factor which is specifically expressed in ovarian granulosa cells and testicular Sertoli cells. Our previous studies demonstrated that Wt1 is required for the lineage specification of supporting cells and inactivation of Wt1 results in Sertoli cells to Leydig-like cells transformation. To test whether Wt1 is also involved in lineage maintenance of granulosa cells during ovary development, Wt1 was specifically deleted in pre-granulosa cells using Foxl2-cre. We found that the female Wt1-/flox; Foxl2-cre mice were infertile with atrophic ovaries and no growing follicles with multiple layers of granulosa cells were observed. A large number of 3ß-HSD-positive steroidogenic cells were detected in ovaries of Wt1-/flox; Foxl2-cre mice during embryonic stage and these cells were derived from Foxl2-expressing pre-granulosa cells. The quantitative results showed the expression of granulosa cell marker genes (Foxl2, Follistatin) was downregulated and steroidogenic cell marker genes (3ß-HSD, Cyp11a1, Star and Sf1) was dramatically increased in Wt1-/flox; Foxl2-cre ovaries. We also found that the meiosis of germ cells in Wt1-/flox; Foxl2-cre ovaries was delayed but not arrested. This study demonstrates that Wt1 is required for lineage maintenance of granulosa cells and inactivation of Wt1 results in pre-granulosa cells to steroidogenic cells transformation which in turn causes the defect of ovary development.

Mesothelial mobilization in the developing lung and heart differs in timing, quantity, and pathway dependency.

The mesothelial lining of the lung, the visceral pleura, and of the heart, the epicardium, derive from a common multipotent precursor tissue, the mesothelium of the embryonic thoracic cavity that also contributes to organ-specific mesenchymal cell types. Insight into mesothelial mobilization and differentiation has prevailedin the developing heart while the mesenchymal transition and fate of the visceral pleura are poorly understood. Here, we use the fact that the early mesothelium of both the lung and the heart expresses the transcription factor gene Wt1, to comparatively analyze mesothelial mobilization in the two organs by a genetic cre-loxP-based conditional approach. We show that epicardial cells are mobilized in a large number between E12.5 and E14.5, whereas pleural mobilization occurs only sporadically and variably in few regions of the lung in a temporally highly confined manner shortly after E12.5. Mesothelium-specific inactivation of unique pathway components using a Wt1creERT2 line excluded a requirement for canonical WNT, NOTCH, HH, TGFB, PDGFRA, and FGFR1/FGFR2 signaling in the mesenchymal transition of the visceral pleura but indicated a deleterious effect of activated WNT, NOTCH, and HH signaling on lung development. Epicardial mobilization was negatively impacted on by loss of HH, PDGFRA, FGFR1/2 signaling. Epicardial overactivation of WNT, NOTCH, and HH disturbed epicardial and myocardial integrity. We conclude that mesothelial mobilization in the developing lung and heart differs in timing, quantity and pathway dependency, indicating the organ specificity of the program.

WT1 loss attenuates the TP53-induced DNA damage response in T-cell acute lymphoblastic leukemia.

Loss-of-function mutations and deletions in Wilms tumor 1 (WT1) gene are present in approximately 10% of T-cell acute lymphoblastic leukemia. Clinically, WT1 mutations are enriched in relapsed series and are associated to inferior relapse-free survival in thymic T-cell acute lymphoblastic leukemia cases. Here we demonstrate that WT1 plays a critical role in the response to DNA damage in T-cell leukemia. WT1 loss conferred resistance to DNA damaging agents and attenuated the transcriptional activation of important apoptotic regulators downstream of TP53 in TP53-competent MOLT4 T-leukemia cells but not in TP53-mutant T-cell acute lymphoblastic leukemia cell lines. Notably, WT1 loss positively affected the expression of the X-linked inhibitor of apoptosis protein, XIAP, and genetic or chemical inhibition with embelin (a XIAP inhibitor) significantly restored sensitivity to γ-radiation in both T-cell acute lymphoblastic leukemia cell lines and patient-derived xenografts. These results reveal an important role for the WT1 tumor suppressor gene in the response to DNA damage, and support the view that anti-XIAP targeted therapies could have a role in the treatment of WT1-mutant T-cell leukemia.

A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS.

FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.

WT1 regulates murine hematopoiesis via maintenance of VEGF isoform ratio.

Mutations in the Wilms tumor suppressor 1 (WT1) gene are as frequent in acute myeloid leukemia (AML) as in nephroblastma and predict poor prognosis. However, the role of WT1 in hematopoiesis remains unclear. We show that Wt1-deficient mouse embryonic stem cells exhibit reduced hematopoietic potential caused by vascular endothelial growth factor A (Vegf-a)-dependent apoptosis of hematopoietic progenitor cells associated with overproduction of the Vegf-a120 isoform. We demonstrate that Wt1 promotes exon inclusion using a Vegf-a minigene-based splicing assay. These data identify a critical role for Wt1 in hematopoiesis and Vegf-a as a cellular RNA whose splicing is potentially regulated by Wt1. The correction of Wt1 deficiency by treatment with exogenous Vegf-a protein indicates that the Wt1/Vegf-a axis is a molecular pathway that could be exploited for the management/treatment of poor prognosis AMLs.

The transcription factors Tbx18 and Wt1 control the epicardial epithelial-mesenchymal transition through bi-directional regulation of Slug in murine primary epicardial cells.

During cardiac development, a subpopulation of epicardial cells migrates into the heart as part of the epicardial epithelial-mesenchymal transition (EMT) and differentiates into smooth muscle cells and fibroblasts. However, the roles of transcription factors in the epicardial EMT are poorly understood. Here, we show that two transcription factors expressed in the developing epicardium, T-box18 (Tbx18) and Wilms' tumor 1 homolog (Wt1), bi-directionally control the epicardial EMT through their effects on Slug expression in murine primary epicardial cells. Knockdown of Wt1 induced the epicardial EMT, which was accompanied by an increase in the migration and expression of N-cadherin and a decrease in the expression of ZO-1 as an epithelial marker. By contrast, knockdown of Tbx18 inhibited the mesenchymal transition induced by TGFß1 treatment and Wt1 knockdown. The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1. Knockdown of Slug also attenuated the epicardial EMT induced by TGFß1 treatment and Wt1 knockdown. Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression. Chromatin immunoprecipitation and promoter assay revealed that Tbx18 and Wt1 directly bound to the Slug promoter region and regulated Slug expression. These results provide new insights into the regulatory mechanisms that control the epicardial EMT.

Partial absence of pleuropericardial membranes in Tbx18- and Wt1-deficient mice.

The pleuropericardial membranes are fibro-serous walls that separate the pericardial and pleural cavities and anchor the heart inside the mediastinum. Partial or complete absence of pleuropericardial membranes is a rare human disease, the etiology of which is poorly understood. As an attempt to better understand these defects, we wished to analyze the cellular and molecular mechanisms directing the separation of pericardial and pleural cavities by pleuropericardial membranes in the mouse. We found by histological analyses that both in Tbx18- and Wt1-deficient mice the pleural and pericardial cavities communicate due to a partial absence of the pleuropericardial membranes in the hilus region. We trace these defects to a persisting embryonic connection between these cavities, the pericardioperitoneal canals. Furthermore, we identify mesenchymal ridges in the sinus venosus region that tether the growing pleuropericardial membranes to the hilus of the lung, and thus, close the pericardioperitoneal canals. In Tbx18-deficient embryos these mesenchymal ridges are not established, whereas in Wt1-deficient embryos the final fusion process between these tissues and the body wall does not occur. We suggest that this fusion is an active rather than a passive process, and discuss the interrelation between closure of the pericardioperitoneal canals, lateral release of the pleuropericardial membranes from the lateral body wall, and sinus horn development.

Wilms' tumour protein Wt1 stimulates transcription of the gene encoding vascular endothelial cadherin.

The Wilms' tumour gene, Wt1, encodes a zinc finger protein, which is mutated in a subset of paediatric renal carcinomas known as Wilms' tumours (nephroblastomas). Recent findings indicate that Wt1, beside its role in genitourinary development, is also necessary for normal vascularisation of the embryonic heart, and may even be involved in tumour angiogenesis. The original purpose of this study was to decipher potential downstream signalling pathways of Wt1 for blood vessel formation. We found that the Wt1(-KTS) protein, which functions as a transcription factor, stimulated the expression of cadherin 5 (CDH5, vascular endothelial (VE) cadherin) and other vascular genes, i.e. those encoding vascular endothelial growth factor receptors 1 and 2, and angiopoietin-2. Furthermore, an enhancer element was identified in the first intron of the CDH5 gene, which bound to the Wt1(-KTS) protein and was necessary for reporter gene activation by Wt1(-KTS) in transiently transfected cell lines. Wt1 and VE-cadherin proteins could be co-localised by double immunofluorescence staining in maturating glomeruli of embryonic murine kidneys. VE-cadherin transcripts were reduced in some but not all tissues of Wt1-deficient mouse embryos. These results indicate that Wt1 can stimulate vascular gene transcription. By demonstrating that Wt1(-KTS) protein trans-activates an enhancer element in the first intron we identified CDH5 as a novel target gene of Wt1. It is suggested that transcriptional activation of CDH5 by Wt1 fulfils regulatory functions during vascular development and kidney formation.

Wt1 and retinoic acid signaling in the subcoelomic mesenchyme control the development of the pleuropericardial membranes and the sinus horns.

RATIONALE: The cardiac venous pole is a common focus of congenital malformations and atrial arrhythmias, yet little is known about the cellular and molecular mechanisms that regulate its development. The systemic venous return myocardium (sinus node and sinus horns) forms only late in cardiogenesis from a pool of pericardial mesenchymal precursor cells. OBJECTIVE: To analyze the cellular and molecular mechanisms directing the formation of the fetal sinus horns. METHODS AND RESULTS: We analyzed embryos deficient for the Wt1 (Wilms tumor 1) gene and observed a failure to form myocardialized sinus horns. Instead, the cardinal veins become embedded laterally in the pleuropericardial membranes that remain tethered to the lateral body wall by the persisting subcoelomic mesenchyme, a finding that correlates with decreased apoptosis in this region. We show by expression analysis and lineage tracing studies that Wt1 is expressed in the subcoelomic mesenchyme surrounding the cardinal veins, but that this Wt1-positive mesenchyme does not contribute cells to the sinus horn myocardium. Expression of the Raldh2 (aldehyde dehydrogenase family 1, subfamily A2) gene was lost from this mesenchyme in Wt1(-/-) embryos. Phenotypic analysis of Raldh2 mutant mice rescued from early cardiac defects by retinoic acid food supply revealed defects of the venous pole and pericardium highly similar to those of Wt1(-/-) mice. CONCLUSIONS: Pericardium and sinus horn formation are coupled and depend on the expansion and correct temporal release of pleuropericardial membranes from the underlying subcoelomic mesenchyme. Wt1 and downstream Raldh2/retinoic acid signaling are crucial regulators of this process. Thus, our results provide novel insight into the genetic and cellular pathways regulating the posterior extension of the mammalian heart and the formation of its coelomic lining.

A cell-autonomous role for WT1 in regulating Sry in vivo.

Human patients with Frasier syndrome express reduced levels of the +KTS isoforms of the developmental regulator WT1 and exhibit complete XY gonadal dysgenesis and male-to-female sex reversal. Mice with a targeted mutation that blocks production of these isoforms show a reduction in Sry mRNA in the gonad, but the molecular and cellular basis of this reduction has not been established. Using immunofluorescence analysis, we found a significantly lower level of SRY protein per cell in XY Wt1(+KTS)-null mouse gonads. We also found a reduced number of SRY-expressing cells, correlating with a decrease in cell proliferation at and near the coelomic epithelium at 11.5 dpc. No reduction in somatic cell numbers was seen in XX Wt1(+KTS)-null gonads, indicating that the effect of WT1 on cell proliferation is mediated by Sry. Sertoli cell differentiation was blocked in XY Wt1(+KTS)-null mouse gonads, as indicated by the loss of SOX9 and Fgf9 expression, but the addition of recombinant FGF9 to ex vivo gonad cultures rescued the mutant phenotype, as indicated by the induction of the Sertoli-cell specific marker anti-Müllerian hormone. Our data suggest that WT1(+KTS) is involved in the cell-autonomous regulation of Sry expression, which in turn influences cell proliferation and Sertoli cell differentiation via FGF9. Thus, sex reversal in Wt1(+KTS)-null mice and Frasier syndrome patients results from a failure of Sertoli cells both to fully differentiate and to reach sufficient numbers to direct testis development.

The Wilms' tumor suppressor Wt1 activates transcription of the erythropoietin receptor in hematopoietic progenitor cells.

The Wilms' tumor protein Wt1 is required for embryonic development and has been implicated in hematologic disorders. Since Wt1 deficiency may compromise the proliferation and differentiation of erythroid progenitor cells, we analyzed the possible role of the transcriptionally active Wt1 isoform, Wt1(-KTS), in regulating the expression of the erythropoietin receptor (EpoR). Wt1 and EpoR were coexpressed in CD117(+) hematopoietic progenitor cells and in several hematopoietic cell lines. CD117(+) cells of Wt1-deficient murine embryos (Wt1(-/-)) exhibited a significantly lower proliferation response to recombinant erythropoietin than CD117(+) cells of heterozygous (Wt1(+/-)) and wild-type littermates (Wt1(+/+)). EpoR expression was significantly diminished in hematopoietic progenitors (CD117(+)) that lacked Wt1, and the erythroid colony-forming capacity was reduced by more than 50% in fetal liver cells of Wt1-deficient embryonic mice. Wt1(-KTS) significantly increased endogenous EpoR transcripts in transfected cells. The proximal EpoR promoter of human and mouse was stimulated more than 10-fold by Wt1(-KTS) in transiently cotransfeced K562 erythroleukemia cells. A responsible cis-element, which is highly conserved in the EpoR promoter of human and mouse, was identified by mutation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. In conclusion, activation of the EpoR gene by Wt1 may represent an important mechanism in normal hematopoiesis.

Wt1 and retinoic acid signaling are essential for stellate cell development and liver morphogenesis.

Previous studies of knock-out mouse embryos have shown that the Wilms' tumor suppressor gene (Wt1) is indispensable for the development of kidneys, gonads, heart, adrenals and spleen. Using OPT (Optical Projection Tomography) we have found a new role for Wt1 in mouse liver development. In the absence of Wt1, the liver is reduced in size, and shows lobing abnormalities. In normal embryos, coelomic cells expressing Wt1, GATA-4, RALDH2 and RXRalpha delaminate from the surface of the liver, intermingle with the hepatoblasts and incorporate to the sinusoidal walls. Some of these cells express desmin, suggesting a contribution to the stellate cell population. Other cells, keeping high levels of RXRalpha immunoreactivity, are negative for stellate or smooth muscle cell markers. However, coelomic cells lining the liver of Wt1-null embryos show decreased or absent RALDH2 expression, the population of cells expressing high levels of RXRalpha is much reduced and the proliferation of hepatoblasts and RXRalpha-positive cells is significantly decreased. On the other hand, the expression of smooth muscle cell specific alpha-actin increases throughout the liver, suggesting an accelerated and probably anomalous differentiation of stellate cell progenitors. We describe a similar retardation of liver growth in RXRalpha-null mice as well as in chick embryos after inhibition of retinoic acid synthesis. We propose that Wt1 expression in cells delaminating from the coelomic epithelium is essential for the expansion of the progenitor population of liver stellate cells and for liver morphogenesis. Mechanistically, at least part of this effect is mediated via the retinoic acid signaling pathway.

Wt1 is not essential for hematopoiesis in the mouse.

WT1 has been implicated in human leukemia and hematopoiesis, but its role in stem cell differentiation is not yet fully defined. We show that Wt1-null murine fetal liver cells are capable of reconstituting functional hematopoiesis following transplantation into irradiated recipients. There was also no significant difference between the in vitro colony-forming ability of wild-type and Wt1-null cells. Using a reporter gene assay in a transgenic mouse system, expression from the WT1 promoter was detectable in adult bone marrow, but undetectable in subsets of different hematopoietic cells. We conclude that Wt1 is not essential for murine hematopoiesis and that there may be significant differences in its role between mouse and man.

Upregulation of Wilms' tumor gene 1 (WT1) in desmoid tumors.

Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors in which beta-catenin/TCF3 mediated Wnt signaling is activated. More than 80% of desmoid tumors contain activating mutations in beta-catenin. It has been shown that the Wnt signaling pathway interacts with Wilms' tumor gene 1 (WT1) in normal kidney development and plays a role in the genesis of some Wilms' tumors. About 15% of Wilms' tumors contain WT1 mutations and of these, about 50% contain beta-catenin mutations. This overlap in mutation pattern of WT1 and beta-catenin in Wilms' tumor suggests that these 2 genes may collaborate in the genesis of a subset of Wilms' tumors. To investigate whether this hypothesis could be extended to other Wnt-dependent tumor types, we searched for WT1 mutations and studied WT1 expression in beta-catenin mutant desmoid tumors. We investigated the expression of WT1 mRNA and protein in desmoid tumors. Medium to high abundant levels of WT1 mRNA were detected by TaqMan quantitative PCR in all tested desmoid cells, whereas adjacent normal fibroblasts showed less expression of WT1. Western blot analysis and immunohistochemistry confirmed this overexpression at the protein level. A mutational screen of the WT1 zinc-finger region by sequence analysis did not identify any mutations. Finally, we investigated a possible role of beta-catenin on WT1 regulation and vice versa. Overexpression of different beta-catenin mutants in the HEK293T cell line did not modulate WT1 promoter activity and WT1 did not affect beta-catenin /TCF transcriptional activity in this cell line. These results show that the wild-type WT1 gene is strongly overexpressed in beta-catenin mutant desmoid tumors and may play a role in tumorigenesis of desmoid tumors, similar to what has been suggested in some epithelial malignancies.

Genetic interactions between the Wilms' tumor 1 gene and the p53 gene.

In recent years, a number of proteins have been identified that can modify the activities of the Wilms' Tumor 1 (WT1) proteins. One of these modifiers is the p53 protein. To investigate a genetic interaction between the p53 gene and the wt1 gene, we have crossed their respective knockout mice. The absence of p53 appears to have no gross effect on the phenotype of wt1-null mice. Both wt1-null and double-null embryos develop pericardial bleeding and die in utero. In adult p53-null mice, wt1-heterozygosity (wt1het) predisposes to an earlier onset of lymphomagenesis and the development of kidney abnormalities resembling oncocytoma in humans. wt1-heterozygosity alone predisposes to the development of glomerular sclerosis.