Poster Sessions

Dev-10

Myoung Shin Kim

M. S. Kim, J. Kitagaki, J. Y. Park, A. O. Perantoni, D. W. Bell, S. B. Lee

WID, a Novel Negative Regulator of the WNT/beta-catenin Signaling Pathway, is Important for Kidney Development

The Wilms tumor suppressor gene, WT1, encodes a developmentally regulated transcription factor that is inactivated in a subset of Wilms tumors, a childhood kidney cancer. WT1 null mouse studies have shown that WT1 plays critical roles in renal and gonadal development, but the target genes critical for organogenesis have not been fully identified. Thus, we performed expression-profiling analysis using a cell line with inducible WT1 expression and identified a number of putative WT1 target genes. Among them, we identified a previously uncharacterized gene, containing a CXXC motif, which we have named WID (WT1-induced Inhibitor of Dishevelled). Using real time PCR, we confirmed that endogenous WID transcript is rapidly induced in response to WT1 expression. Chromatin-IP and luciferase reporter assays demonstrated that WT1 directly activate transcription of WID. Sequence analysis using BLAST search revealed that C-terminal region of WID is highly homologous to CXXC4 (45% similarity). CXXC4, also known as IDAX (inhibitor of Dvl and Axin complex), contains a DVL binding domain which is perfectly conserved in WID. IDAX has been shown to inhibit WNT signaling pathway by directly binding to DVL and preventing DVL-AXIN complex formation. To determine whether WID functions as an inhibitor of WNT signaling pathway, we performed WNT-reporter assay by transfecting cells with WID and Super8TOPFLASH-reporter plasmids. In cells treated with Wnt3a conditioned medium, expression of WID led to a potent inhibition of WNT signaling. Furthermore, we evaluated the in vivo role of WID in WNT signaling pathway using Zebrafish model. The ectopic Wnt8-induced headless phenotype was also suppressed by coexpression of WID, demonstrating that WID functions as a negative regulator of the WNT pathway in vivo. In the developing mouse kidney, WID is expressed in renal vesicles and in the precursors of podocytes, overlapping precisely with WT1 expression. To study the physiological role of Wid in kidney development, we generated Wid-null mice by gene targeting. Homozygous inactivation of Wid in the mouse resulted in defective kidney development, with reduced nephron and renal tubule formation. Remarkably, ablation of wid expression in zebrafish with an antisense morpholino oligonucleotide resulted in perturbation of pronephros development, demonstrating that the function of wid in kidney development is evolutionarily conserved. Taken together, these results demonstrate that the novel WT1 target gene WID plays an important role in kidney development and further point to a direct role for WT1 in the negative regulation of WNT signaling during nephrogenesis.