The formation of intramyocardial blood vessels is critical for normal heart development and tissue repair after infarction. We report here expression of the Wilms' tumor gene-1, Wt1, in coronary vessels, which could contribute to the defective cardiac vascularization in Wt1 −/− mice. Furthermore, the high-affinity neurotrophin receptor TrkB, which is expressed in the epicardium and subepicardial blood vessels, was nearly absent from Wt1-deficient hearts. Activation of Wt1 in an inducible cell line significantly enhanced TrkB expression. The promoter of NTRK2, the gene encoding TrkB, was stimulated ∼10-fold by transient cotransfection of a Wt1 expression construct. The critical DNA-binding site for activation of the NTRK2 promoter by Wt1 was delineated by DNase I footprint analysis and electrophoretic mobility shift assay. Transgenic experiments revealed that the identified Wt1 consensus motif in the NTRK2 promoter was necessary to direct expression of a reporter gene to the epicardium and the developing vasculature of embryonic mouse hearts. Finally, mice with a disrupted Ntrk2 gene lacked a significant proportion of their intramyocardial blood vessels. These findings demonstrate that transcriptional activation of the TrkB neurotrophin receptor gene by the Wilms' tumor suppressor Wt1 is a crucial mechanism for normal vascularization of the developing heart.

Highlights d p16 High senescence is a slow process that manifests around 10-12 months of age d p16 High cells in the liver of 12-month-old mice are LSECs and macrophages d Senescent p16 High LSECs are structurally and functionally important d Elimination of p16 High senescent cells induces liver and perivascular tissue fibrosis

Abstract. NPHS1 encodes the structural protein nephrin, which has a crucial role in the filtration barrier of the glomerular podocyte. Mutations or deregulation of NPHS1 are associated with a variety of renal diseases, including the Finnish type congenital nephrotic syndrome. This study analyzed a potential regulation of nephrin by the Wilms' tumor protein, Wt1. Using an inducible U2OS osteosarcoma cell line, it is shown that upon Wt1 induction, endogenous nephrin mRNA becomes highly upregulated. Co-transfection studies demonstrate that Wt1 can activate the nephrin promoter Ͼ10-fold. DNase footprinting and mutation analysis identify a Wt1 responsive element in the nephrin promoter, which is required for the binding of Wt1 protein. Mutations or deletion of this Wt1 responsive element completely abolished transactivation of the nephrin promoter by Wt1. Moreover, transgenic analysis demonstrates the requirement of the identified binding site to direct podocyte-specific expression of a reporter gene in transgenic mice, thus confirming the importance of this site for the regulation of nephrin in vivo. Finally, it is shown that nephrin expression is lowest in kidneys of mice that lack specifically the Wt1(ϪKTS) splice variant, but in comparison with wild-type littermates, it is also reduced in animals with disruption of the Wt1(ϩKTS) splice variant. Taken together, these data identify nephrin as a direct transcriptional target for Wt1 and underline the importance of Wt1 as a key regulator in podocyte function.

contributed equally to this workThe Wilms' tumor gene Wt1 is known for its important functions during genitourinary and mesothelial formation. Here we show that Wt1 is necessary for neuronal development in the vertebrate retina. Mouse embryos with targeted disruption of Wt1 exhibit remarkably thinner retinas than age-matched wildtype animals. A large fraction of retinal ganglion cells is lost by apoptosis, and the growth of optic nerve ®bers is severely disturbed. Strikingly, expression of the class IV POU-domain transcription factor Pou4f2 (formerly Brn-3b), which is critical for the survival of most retinal ganglion cells, is lost in Wt1 ±/± retinas. Forced expression of Wt1 in cultured cells causes an up-regulation of Pou4f2 mRNA. Moreover, the Wt1(±KTS) splice variant can activate a reporter construct carrying 5¢-regulatory sequences of the human POU4F2. The lack of Pou4f2 and the ocular defects in Wt1 ±/± embryos are rescued by transgenic expression of a 280 kb yeast arti®cial chromosome carrying the human WT1 gene. Taken together, our ®ndings demonstrate a continuous requirement for Wt1 in normal retina formation with a critical role in Pou4f2-dependent ganglion cell differentiation.

No abstract

Expression of the Wilms' tumor gene Wt1 in the epicardium is critical for normal heart development. Mouse embryos with inactivated Wt1 gene have extremely thin ventricles, which can result in heart failure and death. Here, we demonstrate that Wt1 can be activated in adult hearts by local ischemia. Wt1 mRNA was increased more than twofold in the left ventricular myocardium of rats between 1 day and 9 wk after infarction. Wt1 expression was localized by means of mRNA in situ hybridization and immunohistochemistry to vascular endothelial and vascular smooth muscle cells in the border zone of the infarcted tissue. A strikingly similar distribution was seen for vascular endothelial growth factor and two different cell proliferation markers in the coronary vessels of the ischemic heart. No Wt1 could be detected in the vasculature of the noninfarcted right ventricles. Wt1 expression in the coronary vessels of the ischemic heart was mimicked by exposure of rats to normobaric hypoxia (8% O2) and 0.1% CO, respectively. These findings demonstrate that Wt1 is expressed in the vasculature of the heart in response to local ischemia and hypoxia. They suggest that Wt1 has a role in the growth of coronary vessels after myocardial infarction.

Telomeric repeat binding factor 2 (TRF2), which plays a central role in telomere capping, is frequently increased in human tumors. We reveal here that TRF2 is expressed in the vasculature of most human cancer types, where it colocalizes with the Wilms' tumor suppressor WT1. We further show that TRF2 is a transcriptional target of WT1 and is required for proliferation, migration, and tube formation of endothelial cells. These angiogenic effects of TRF2 are uncoupled from its function in telomere capping. Instead, TRF2 binds and transactivates the promoter of the angiogenic tyrosine kinase platelet-derived growth factor receptor β (PDGFRβ). These findings reveal an unexpected role of TRF2 in neoangiogenesis and delineate a distinct function of TRF2 as a transcriptional regulator.

Vascularization is an important step in tumour growth. Although a variety of molecules, for example, VEGF, ETS-1 or nestin have been implicated in tumour angiogenesis, the molecular mechanisms of vessel formation are not fully characterized. We showed that the Wilms' tumour suppressor WT1 activates nestin during development. Here we tested whether WT1 might also be involved in tumour angiogenesis. Endothelial WT1 expression was detected in 95% of 113 tumours of different origin. To analyse the function of WT1 in endothelial cells, we used an RNAi approach in vitro and showed that inhibition of WT1 reduces cell proliferation, migration and endothelial tube formation. On a molecular level, WT1 silencing diminished expression of the ETS-1 transcription factor. WT1 and ETS-1 shared an overlapping expression in tumour endothelia. The ETS-1 promoter was stimulated approximately 10-fold by transient co-transfection of a WT1 expression construct and WT1 bound to the ETS-1 promoter in chromatin immunoprecipitation and electrophoretic mobility shift assays. Deletion of the identified WT1-binding site abolished stimulation of the ETS-1 promoter by WT1. These findings suggest that transcriptional activation of ETS-1 by the Wilms' tumour suppressor WT1 is a crucial step in tumour vascularization via regulation of endothelial cell proliferation and migration.