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INSERM U423 Hôpital Necker Enfants Malades, Université René Descartes, Paris, France.
WT1 was initially identified as a Wilms tumour suppressor gene through the study of children presenting the contiguous gene syndrome WAGR (Wilms tumor, Aniridia, Genitourinary malformations, mental Retardation) due to large constitutional deletions of DNA in band 11p13 (1,2). This 10-exon gene produces a 52- to 54-kD protein with the structure of a transcription factor. Exons 7 to 10 encode four zinc fingers (ZF) that are able to bind RNA and DNA with high affinity, whereas the first 6 exons encode a proline/glutamine-rich region that is involved in transcriptional activation and repression, nuclear localization, RNA recognition, and homodimerization of the protein (3–5). It quickly became clear that WT1 plays a major role not only in tumorigenesis but also in kidney and gonad development and in the maintenance of the podocyte function, as demonstrated by its strong expression throughout life from early stages of embryogenesis (6), the results of targeted gene disruption in mice, and the occurrence of gonad and kidney disorders in humans with constitutional mutation of the gene. However, many questions remained unsolved due to the high complexity of the gene and the lack of precise identification of its in vivo target genes.
WT1 is a complex gene that can encode at least 24 isoforms as a result of alternative start sites, alternative splicing (3,4), and RNA editing. There are two alternative splice donor sites, the first one leading to the presence/absence of the 17 amino acids encoded by exon 5 and the second including/excluding a short sequence of three amino acids, lysine, threonine, and serine (KTS) between zinc fingers 3 and 4. The KTS motif is conserved in all vertebrates, whereas exon 5 is conserved only in mammals (7). The four resulting isoforms are expressed in stable and definite proportion, the +KTS variants corresponding to 80% of the transcripts (4,8). Most studies have been devoted to these spliced isoforms and especially to the analysis of the function of the +KTS and -KTS variants, the -KTS forms being active in transcriptional regulation and the +KTS variants playing a putative role in RNA processing. On the other hand, numerous potential target genes regulated, most often negatively, by WT1 have been identified by in vitro studies (8,9). However, the physiologic significance of these findings remains to be determined, as their relevance has not been established in vivo (10). Until recently, it was not known if the presence of exon 5 was required for normal development.
Input about the role of WT1 and the specific functions of the different isoforms during development came from the study of patients with WT1 mutation and the analysis of genetically modified mice. Targeted gene disruption in mice conclusively established the role of WT1in renal and extrarenal development (11). Homozygous null mice may survive to birth, but they had no gonads and no kidneys and severe heart, lung, and mesothelium anomalies. The ureteric bud did not form, and the metanephric blastema was unable to differentiate into nephrons. No anomaly was initially reported in heterozygous mice. However, more recent observations suggest an increased rate of mortality in Wt1+/- mice due to the progressive development of renal lesions leading to end-stage renal disease (ESRD) (12). In the same way, combining Wt1-knockout and inducible yeast artificial chromosome transgenic mouse models, Guo et al. (12) could demonstrate that reduced expression levels of WT1 result in glomerulonephritis, the severity of which depending of the gene dosage. The human counterpart of these observations has been recently reported on the basis of the study of a large cohort of patients with Wilms tumor: the cumulative risk of renal failure, 20 yr after the diagnosis of tumor, was 38% in WAGR patients with deletion of one WT1 allele compared with 1% in patients with isolated unilateral nephroblastoma (13). All these data indicate that WT1plays a major role in the induction of the ureteric bud, the survival and the epithelial differentiation of the mesenchymal blastema, the progression of nephrogenesis, and the maintenance of podocyte function. Because of the risk linked to the haploinsufficiency, WAGR patients should be closely followed throughout life for signs of glomerulopathy or renal failure.
In the Denys-Drash syndrome (DDS), which is characterized by the triad of severe glomerulopathy progressing rapidly to ESRD, male pseudohermaphroditism, and Wilms tumor, the specific kidney lesion associates diffuse mesangial sclerosis and severe alterations of the podocytes (14). WT1 lesions affecting the third or the second zinc finger of the protein result in loss or alteration of its DNA binding ability, as demonstrated by in vitro studies (15). Furthermore, by dimerization with the wild-type protein, the mutated protein may act in a dominant negative fashion (16). In patients, the absence or reduction of the nuclear expression of WT1 within the podocytes is consistent with the reduced binding capacities of the mutated protein (17). In addition, the de novo expression of PAX2 and transforming growth factor-
1 (TGF-1) and the hyperexpression of PDGFA are indirect confirmation of their regulation by WT1 (17,18). Animal models of DDS have been generated by gene targeting, which truncated ZF3 (19). One sterile heterozygous male and chimeric mice developed mesangial sclerosis, but the puzzling observation, questioning the dominant negative mechanism, is that a relatively low level of mutant protein (5%) is sufficient to induce glomerular disease in mice.
Specific mutations have been identified in all patients with the Frasier syndrome, which is characterized by progressive glomerulopathy with focal and segmental glomerulosclerosis and male-to-female sex reversal. They affect the splice donor site of WT1 intron 9 and result in the marked reduction of the +KTS variants, indicating that a strict equilibrium between the different WT1 isoforms is required for normal renal and testicular development (20). Moreover, the absence of Wilms tumor in Frasier patients suggests that the tumor-suppressor activity of WT1 depends on the -KTS isoforms. To further determine the respective functions of these proteins, Hammes et al. generated mice expressing only +KTS or only -KTS isoforms (21). Heterozygous mice develop normally, but contrarily to the +KTS mice, which did not develop any renal disorder, mice with a reduced expression of +KTS isoforms develop a severe renal phenotype similar to the one in Frasier patients, suggesting that +KTS isoforms are more important for the maintenance of the podocyte function. Homozygous mutants of both strains survive to birth, indicating some partial functional redundancy of +KTS and -KTS forms. But they die soon after birth due to kidney defects, especially severe in +KTS mice lacking the -KTS variants, who have hypoplastic kidneys and streak gonads. From these results, it has been proposed that -KTS isoforms are required for the survival of the embryonic kidney and also of the gonadal primordium.
Until recently, nothing was known about the role of exon 5 in urogenital development. Because of the normal formation of kidneys in lower vertebrates in the absence of this exon and the strong expression of WT1 containing the 17-amino acid sequence in the uterine stroma and the mammary glands, the exon 5+ isoforms were supposed to play a special role in embryonic implantation and lactation in mammals. To test this hypothesis, mice lacking exon 5 have been generated by gene targeting. They develop normally, have normal kidneys, and surprisingly, they are fertile and females are capable of lactation (22). However, long-term survival studies are still necessary to determine if exon 5-containing isoforms play a role in the maintenance of organ function. Another approach to the same question is presented in this issue of the JASN. Natoli et al. (23) used the nephrin promotor to express in podocytes a truncated form of WT1 CDNA known to result in DDS in humans. Interestingly, they constructed two versions of the transgene, one containing and the other omitting exon 5. Analysis of the resulting transgenic mice confirms the prominent role of WT1 lacking exon 5. The transgene containing exon 5 did not interfere with mouse renal development or function. In contrast, poor postnatal survival and glomerular abnormalities were observed in transgenic mice if the transgene did not contain exon 5, due to a possible dominant-negative action of the mutant WT1. In these mice, the reduced number of glomerular capillary loops and their dilation associated with the reduced endothelial cell expression of PECAM suggest that WT1 may play a role in the control of the expression of growth factors involved in glomerular capillary branching. Further studies are, however, necessary to fully decipher the molecular mechanisms regulated by WT1 and leading to the normal development of podocytes.
References
1 in diffuse mesangial sclerosis. Pediatr Nephrol 16: 429–438, 2001[CrossRef][Medline]
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