WT1 proteins : functions in growth and differentiation

Citation for published version (APA): Scharnhorst, V., Eb, van der, A. J., & Jochemsen, A. G. (2001). WT1 proteins : functions in growth and differentiation. Gene, 273(2), 141-161. https://doi.org/10.1016/S0378-1119(01)00593-5

DOI: 10.1016/S0378-1119(01)00593-5

Document status and date: Published: 01/01/2001

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Download date: 29. Sep. 2021 Gene 273 ,2001) 141±161 www.elsevier.com/locate/gene Review WT1 proteins: functions in growth and differentiation

Volkher Scharnhorst, Alex J. van der Eb, Aart G. Jochemsen*

Department of Molecular and Cellular Biology and Center for Biomedical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Received 23 March 2001; received in revised form 11 June 2001; accepted 2 July 2001 Received by A.J. van Wijnen

Abstract The Wilms' tumor 1 gene ,WT1) has been identi®ed as a tumor suppressor gene involved in the etiology of Wilms' tumor. Approximately 10% of all Wilms' tumors carry mutations in the WT1 gene. Alterations in the WT1 gene have also been observed in other tumor types, such as leukemia, mesothelioma and desmoplastic small round cell tumor. Dependent on the tumor type, WT1 proteins might either function as tumor suppressor proteins or as survival factors. Mutations in the WT1 gene can also result in congenital abnormalities as observed in Denys± Drash and Frasier syndrome patients. Mouse models have proven the critical importance of WT1 expression for the development of several organs, including the kidneys, the gonads and the spleen. The WT1 proteins seem to perform two main functions. They regulate the transcription of a variety of target genes and may be involved in post-transcriptional processing of RNA. The WT1 gene encodes at least 24 protein forms. These isoforms have partially distinct biological functions and effects, which in many cases are also speci®c for the model system in which WT1 is studied. This review discusses the molecular mechanisms by which the various WT1 isoforms exert their functions in normal development and how alterations in WT1 may lead to developmental abnormalities and tumor growth. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Wilms' tumor 1 protein; Tumor suppressor; Transcriptional regulation; Splicing factors; Differentiation

1. Introduction nephrectomy, the biology of Wilms' tumors has for several reasons received a great deal of attention. A major reason is Wilms' tumor or nephroblastoma is a pediatric kidney that pediatric tumors often arise through erroneous develop- malignancy that was ®rst described by Max Wilms in ment and studying these tumors may thus offer insight into 1899. This primitive tumor affects about 1:10,000 children, embryonic development. Wilms' tumor is thought to arise usually below the age of 5 years, and accounts for approxi- from mesenchymal blastema cells that fail to differentiate mately 7.5% of all childhood tumors. into metanephric structures but continue to proliferate Although patients presenting a unilateral tumor can in ,Hastie, 1994; Machin and McCaughey, 1984). A second most cases be successfully treated with chemotherapy and reason is that Wilms' tumor is often found in association with other congenital abnormalities, the WAGR ,Wilms' tumor, aniridia, genitourinary abnormalities, mental retar- Abbreviations: AdBRK cells, adenovirus-transformed baby rat kidney dation), the Denys±Drash and the Beckwith±Wiedemann cells; CSF-I, colony stimulating factor I; CTGF, connective tissue growth syndromes, suggesting an overlap in the pathogenesis of factor; Cu,Zn-SOD, Cu,Zn-superoxide dismutase; DDS, Denys±Drash syndrome; DSRCT, desmoplastic small round cell tumor; EGF, epidermal these syndromes and Wilms' tumor ,Call et al., 1990; Gess- growth factor; EGFR, epidermal growth factor receptor; FS, Frasier ler et al., 1990; Koufos et al., 1989; Pelletier et al., 1991a). syndrome; IGF-II, insulin-like growth factor-II; IGF-IR, insulin-like Indeed, studies of the WAGR and the Beckwith±Wiede- growth factor-I receptor; MDR-1, multidrug resistance-1; MIS, MuÈllerian mann syndromes facilitated the mapping of two minimal inhibitory substance; ODC, ornithine decarboxylase; PDGF-A, platelet- critical regions on chromosome 11p13 and 11p15, respec- derived growth factor-A chain; PKA, protein kinase A; PKC, protein kinase C; RAR-a, retinoic acid receptor-a; SF-1, steroidogenic factor-1; TGF-b, tively, involved in the development of sporadic Wilms' transforming growth factor-b; TPA, 12-O-tetradecanoylphorbol-13-acet- tumor ,Bickmore et al., 1989; Call et al., 1990; Francke et ate; WAGR, Wilms' tumor, aniridia, genitourinary abnormalities and al., 1979; Gessler et al., 1990; Glaser et al., 1989; Koufos et mental retardation; WT1, Wilms' tumor 1; WTAP, Wilms' tumor 1 asso- al., 1989; Reeve et al., 1989). Third, a locus for familial ciated protein Wilms' tumor, which accounts for only 1% of all Wilms' * Corresponding author. Tel.: 131-71-5276136; fax: 131-71-5276284. E-mail address: [email protected] ,A.G. Jochemsen). tumors, is not linked to chromosome 11 ,Grundy et al.,

0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S0378-1119,01)00593-5 142 V. Scharnhorst et al. / Gene 273 .2001) 141±161 1988), but instead maps to other chromosomal regions native splice donor site at the end of exon 9 results in ,Rahman et al., 1996, 1997; Slater and Mannens, 1992), incorporation of three additional amino acids, lysine, threo- demonstrating that Wilms' tumors are genetically heteroge- nine and serine ,KTS), between the third and fourth zinc neous and that at least three candidate genes are implicated ®ngers. The ratio of the different WT1 splice variants was in their development. found to be conserved between normal fetal kidney, Wilms' So far, the Wilms' tumor 1 gene ,WT1) at 11p13 is the tumors and several tissues of the murine genitourinary only gene involved in development of Wilms' tumor that system. The mRNA isoform containing both splice inserts has been cloned ,Call et al., 1990; Gessler et al., 1990) and is the most prevalent variant in both human and mouse, classi®ed as a tumor suppressor gene. It is now recognized whereas the least common is the transcript missing both that WT1 is homozygously mutated in 5±10% of Wilms' inserts ,Haber et al., 1991). These ®ndings were later tumors ,Gessler et al., 1994; Little and Wells, 1997; Little extended by a report describing that splicing of exon 5 is et al., 1992). differentially regulated in a species-, tissue- and develop- mental stage-speci®c manner, while the 1KTS/2KTS ratio is maintained in all cell types tested ,Renshaw et al., 1997). 2. The WT1 gene, mRNAs and proteins Additional WT1 mRNAs are generated through RNA edit- ing at nucleotide 839 of the WT1 mRNA that replaces The human WT1 gene spans about 50 kb at chromosome leucine 280 in WT1 proteins by proline ,Sharma et al., locus 11p13 ,Call et al., 1990; Gessler et al., 1990). It 1994), although the frequency of RNA editing might be comprises ten exons and encodes mRNAs of approximately signi®cantly lower than initially published ,Mrowka and 3 kb ,Call et al., 1990; Gessler et al., 1992). Schedl, 2000). The WT1 gene may thus produce eight differ- In mammals, exons 5 and 9 of WT1 are alternatively ent mRNA isoforms, suggesting that each isoform has a spliced, giving rise to four different splice isoforms ,Fig. distinct contribution to the function of the WT1 gene and 1) ,Gessler et al., 1992; Haber et al., 1991). In all other that balanced expression of the isoforms is essential for vertebrates tested, exon 5 is not present in the WT1 gene, proper WT1 function. so that only two different mRNA transcripts are generated Depending on the absence or presence of the two splice ,Kent et al., 1995). Inclusion of exon 5 inserts 17 amino inserts, the WT1 proteins have molecular masses of 52±54 acids between the proline and glutamine-rich amino-termi- kDa ,Morris et al., 1991). The WT1,2/2) protein with a nus and the zinc-®nger domain of WT1. Usage of an alter- mass of 52 kDa lacks both splice inserts and the WT1,1/1) protein with a mass of 54 kDa contains both splice inserts. WT1 proteins containing the exon 5 splice insert only are referred to as WT1,1/2), and proteins including the three amino acid insert KTS only are referred to as WT1,2/1). In addition to the eight WT1 protein isoforms generated through translation initiation at the initiator AUG of the eight mRNAs described above, larger and smaller WT1 isoforms have been identi®ed ,see Fig. 1). Translation initia- tion at an in-frame CUG codon upstream of the initiator AUG results in WT1 protein isoforms with molecular masses of 60±62 kDa ,Bruening and Pelletier, 1996). Inter- nal translation initiation at an in-frame AUG127 codon downstream of the initiator AUG generates smaller WT1 isoforms with apparent molecular masses of 36±38 kDa ,Scharnhorst et al., 1999). Both the larger and the smaller WT1 isoforms could be detected in different mammalian tissues. Since the in-frame, downstream AUG is conserved in the WT1 genes of all species sequenced so far, it appears that it may function as an alternative translation initiation site in all of these species. When these novel isoforms are included, 24 WT1 protein forms have been described to Fig. 1. Schematic representation of the WT1 protein isoforms. Insertion or date, generating an enormous potential for regulation and exclusion of the two splice inserts ,17aa and KTS) generates the four main functioning of WT1 proteins. WT1 protein isoforms starting at the ®rst initiator AUG ,Met) of the WT1 Very recently a study by Dechsukhum et al. ,2000) has mRNAs. Translation initiation at an upstream, in-frame CUG codon gener- suggested another mechanism by which N-terminally trun- ates four larger WT1 proteins, while translation initiation at an in-frame AUG 127 codons downstream of the initiator AUG leads to four smaller cated WT1 proteins might be produced. RT-PCR and North- WT1 proteins. RNA editing at codon 280 replaces leucine by proline ,Leu ern blot studies indicated the presence of short WT1 to Pro), thereby raising the total number of WT1 protein isoforms to 24. transcripts, containing sequences downstream of exon 5. V. Scharnhorst et al. / Gene 273 .2001) 141±161 143 Since at the 50 end of the cDNAs intron ®ve sequences were Recently two reports from the same group described found, the activation of a cryptic promoter during tumori- NMR analyses of the structures of the zinc-®nger domains genesis within intron 5 was hypothesized. Further charac- of the 1KTS and 2KTS forms, either free in solution or terization of these aberrant WT1 transcripts and the products bound to a 14 base pair DNA duplex corresponding to the encoded is necessary before a putative function can be `extended' Egr-1 recognition sequence. Insertion of the assigned. However, these data indicate that the number of KTS tripeptide causes an increased ¯exibility of the linker proteins encoded by the WT1 gene most likely exceeds the between zinc ®ngers 3 and 4, which leads to absence of 24 characterized so far. binding of the fourth zinc ®nger to its cognate site in the Extensive structure and function analyses of the mamma- DNA major groove ,Laity et al., 2000a,b). lian WT1 protein have been performed by many groups. Another study identi®ed DNA sequences bound by all From the primary structure of the WT1 proteins, it was WT1 isoforms, regardless of whether they lack or contain predicted that they could function as transcription factors. the KTS splice insert, and DNA sequences bound exclu- The WT1 proteins contain a proline and glutamine-rich sively by either 2KTS or 1KTS isoforms ,Bickmore et region, which mediates transcriptional regulation and al., 1992). It is now known that the 2KTS and the 1KTS homodimerization, and four Cys2-His2-type zinc ®ngers isoforms of WT1 can bind overlapping DNA sequences in at their C-termini ,Fig. 1) ,Call et al., 1990; Gessler et al., the promoters of the IGF-II gene ,Drummond et al., 1994; 1990). The zinc-®nger domain can bind to several DNA Duarte et al., 1998; Wang et al., 1995a), the gene for the sequences ,Bickmore et al., 1992; Hamilton et al., 1995; platelet-derived growth factor-A ,PDGF-A) chain ,Wang Nakagama et al., 1995; Rauscher et al., 1990) and contains et al., 1995a), the WT1 gene ,Rupprecht et al., 1994) and two nuclear localization signals, one in zinc ®nger 1 and the PAX2 gene ,Ryan et al., 1995). Nucleotide sequences another in zinc ®ngers 2 and 3 ,Bruening et al., 1996). In bound by WT1 are generally GC-rich, for example the so- 1995, a ®rst report suggested that, in addition to its function called Egr-1 ,Rauscher et al., 1990) and WTE sites ,Naka- as a transcription factor, WT1 protein may also be involved gama et al., 1995), or contain ,TCC)n motifs ,Wang et al., in post-transcriptional processing of RNA ,Larsson et al., 1993b). However, WT1 2 KTS isoforms are thought to 1995). Structural modeling revealed a potential RNA recog- have a broader target site speci®city than WT1 1 KTS nition motive in the N-terminus of WT1 ,Kennedy et al., isoforms. 1996), and it has been demonstrated that the zinc-®nger The transcriptional regulatory properties of WT1 are less domain in the C-terminal part of WT1 can bind to IGF-II well understood. In transient transfection studies with repor- RNA ,Caricasole et al., 1996). ter constructs, WT1 may, depending on the expression Thus, WT1 may be involved in transcriptional and post- vector, the architecture of the reporter promoter and the transcriptional regulation of its target genes. cell lines used, activate or repress transcription ,Reddy et al., 1995b; for a thorough review see Menke et al., 1998). For example, WT1 2 KTS activates transcription from 3. DNA binding and transcription regulation by WT1 synthetic promoter constructs containing multimerized Egr-1 ,Reddy et al., 1995a), ,TCC)n ,Wang et al., 1993b) As described above, WT1 proteins contain a proline and or WTE,Scharnhorst et al., 1999) sites upstream of the glutamine-rich region and four contiguous zinc ®ngers. The transcription start site. Insertion of ,TCC)n motifs down- presence of these two structural motifs suggested that WT1 is stream of the transcription start site also leads to transcrip- a sequence-speci®c transcriptional regulator. Therefore, in tional activation ,Wang et al., 1993b). However, insertion of an initial study to investigate the biochemical activities of ,TCC)n sites both upstream and downstream of the tran- WT1, recombinant WT1 protein lacking the KTS insert scription start site results in ef®cient transcriptional repres- ,WT1 2 KTS) was used to select speci®c binding sequences sion by cotransfected WT1 ,Wang et al., 1993b). from a pool of degenerate oligonucleotides. The binding Furthermore, WT1,2/1) activates transcription from a sequences obtained were similar to the sequence recognized PDGF-A chain promoter fragment, whereas it suppresses by the early growth response-1 ,Egr-1) protein ,Rauscher et transcription from a longer fragment of the same promoter al., 1990). This was not surprising, since zinc ®ngers 2, 3 and ,Wang et al., 1995a). Finally, the promoter of the Egr-1 4 of WT1 exhibit 61% amino acid identity with the three zinc gene is activated by WT1 in the human tumor cell lines ®ngers of the transcription factor Egr-1 ,Rauscher, 1993). Saos-2 ,Maheswaran et al., 1993) and U2OS ,Englert et Compared to Egr-1 and Egr-1-like proteins, such as Sp1 al., 1995a), while, on the contrary, WT1 represses the and Egr-2, WT1 contains an additional zinc ®nger, and in same promoter in mouse NIH3T3 cells ,Maheswaran et some isoforms, the alternatively inserted tripeptide KTS al., 1993). A recent paper by Richard et al. ,2001) might between zinc ®ngers 3 and 4. Displacement of the fourth provide an explanation for the distinct regulation of tran- zinc ®nger by insertion of the three amino acids KTS prob- scription by WT1 in different cells. They show that a small ably prevents binding of WT1 1 KTS isoforms to the Egr-1 region in WT1 spanning the 17 amino acid ,17aa) insert sequence, while zinc ®nger 1 may be involved in binding to forms a new transcriptional activation domain whose activ- extended DNA sequences. ity is strongly dependent on the expression of the WT1- 144 V. Scharnhorst et al. / Gene 273 .2001) 141±161 interacting protein Par4. The functional interaction of WT1 les'. It is now recognized that pre-mRNA splicing is a and Par4 will be discussed in more detail in Section 5. predominantly cotranscriptional event ,Mattaj, 1994) and However, English and Licht ,1999) suggested that tran- that the `speckles' may represent storage sites from which scriptional activation and not repression by WT1 is the splicing factors are recruited to new sites of transcription critical transcriptional activity of the protein, because they ,Misteli et al., 1997). had found that N-terminal, tumor-associated missense More evidence for WT1 being involved in pre-mRNA mutations of WT1 render the protein defective for transcrip- splicing came from the ®nding that treatment of cells tional activation and fail to inhibit cell growth, but still expressing WT1,1/1) with RNase A after ®xation strongly retain their ability to repress several putative WT1 target decreases the number of nuclei with WT1 in `speckled' promoters. This ®nding ®ts with the observation that the patterns ,Larsson et al., 1995). Furthermore, the same WT1-associating protein Par4 inhibits both transcriptional group could show that WT1 co-immunoprecipitates with activation and growth suppression by WT1, but actually proteins of the splicing apparatus and that zinc ®ngers 3 enhances transcriptional repression ,Johnstone et al., and 4 of WT1 are not necessary for interaction with the 1996; see Section 5). splice complexes. Mutants lacking zinc ®ngers 3 and 4 WT1 comprises domains that function independently to even preferentially colocalize with splice complexes, repress or activate transcription when fused to the GAL4 suggesting that all four WT1 isoforms contain the domains DNA-binding domain. The repressor and activator domains necessary for these interactions, and that the KTS insert are located in amino acid residues 85±124 and 181±250 of determines the subnuclear localization of wild-type WT1. WT1, respectively ,Wang et al., 1995b). Similarly, the 17aa In line with this, a putative RNA recognition motif in amino splice insert is capable of repressing transcription when acids 11±72 of WT1 has been identi®ed ,Kennedy et al., fused to the DNA-binding domain of GAL4 ,Wang et al., 1996). As yet, it remains to be proven that this putative RNA 1995a), suggesting that inclusion of this domain in WT1 recognition motif in WT1 can complex with RNA. may modify the transcriptional regulatory properties of However, others have shown that both WT1 2 KTS and WT1. WT 1 KTS can bind to the murine IGF-II transcript in Table 1 summarizes the putative WT1 target genes iden- exon 2, with binding involving the zinc ®ngers ,Caricasole ti®ed so far. For several genes, the effect of WT1 on their et al., 1996). WT1 1 KTS binds to the IGF-II RNA with transcription rate has been assessed with transient transfec- greater af®nity than WT1 2 KTS, while zinc ®nger 1, which tion of reporter constructs. It is, therefore, uncertain whether has no counterpart in the transcription factor Egr-1 which they are bona ®de WT1 target genes in vivo. Some WT1 does not bind RNA, is required for RNA binding by either target genes, of which the physiological relevance has been WT1 isoform ,Caricasole et al., 1996). A more recent study ascertained, are marked with an asterisk and will be shows that WT1 directly associates with the constitutive discussed later. Although little is know about post-transla- splicing protein U2AF65, which binds to the 30 splice site tional modi®cations of WT1 that alter its DNA-binding and/ and is part of the splicing machinery ,Davies et al., 1998). or transcription-regulating properties, two reports demon- This interaction does not require the ®rst 180 amino acids strated that WT1 can be phosphorylated by PKA and nor zinc ®nger 1 of WT1, implying that RNA binding is not PKC, and that phosphorylation inhibits its DNA-binding essential for interaction with U2AF65. U2AF65 binding is activity ,Sakamoto et al., 1997; Ye et al., 1996). Accord- not a prerequisite for the `speckled' distribution of WT1, ingly, treatment of cells with activators of PKA decreased because WT1 mutants unable to bind to U2AF65 still colo- the transcriptional repression activity of WT1. Sakamoto et calize with splicing factors ,Davies et al., 1998). Impor- al. ,1997) mapped the PKA phosphorylation sites to two tantly, Davies and co-workers demonstrated that WT1 can serine residues in zinc ®ngers 2 and 3 and showed that become incorporated into spliceosomes in an in vitro spli- phosphorylation of these two sites is critical for inhibition cing assay. The indication that WT1 proteins are somehow of DNA binding. We recently found, by phospho-amino involved in splicing was further strengthened by the obser- acid analysis of endogenous WT1, that WT1 proteins are vations that WT1 proteins co-purify with nuclear phosphorylated on both threonine and serine in exponen- poly,A) 1 RiboNucleoProtein ,Ladomery et al., 1999). tially growing cells, whereas phosphorylation of tyrosine This complex, puri®ed by oligo,dT) chromatography, also could not be detected ,V. Scharnhorst and A.G. Jochemsen, contains, among others, the U2AF65 protein and p116, an unpublished data). essential splicing factor. In summary, WT1 has been shown to bind RNA through its zinc ®ngers, and additional data demonstrate that it 4. WT1 as a post-transcriptional regulator contains a potential RNA recognition motif in its N-termi- nus. WT1 has a distinct subnuclear expression pattern, with Larsson et al. ,1995) have shown that the subnuclear the 1KTS form being preferentially associated with `speck- localization of WT1 proteins is splice form-dependent. les'. Combined with the ®ndings that WT1 directly interacts WT1 1 KTS isoforms preferentially colocalize with mole- with U2AF65 and can be incorporated into spliceosomes, cules implicated in mRNA splicing to characteristic `speck- these data suggest that WT1 is involved in post-transcrip- V. Scharnhorst et al. / Gene 273 .2001) 141±161 145

Table 1 WT1 target genes

Genea References

Growth factor genes IGF-II ,Drummond et al., 1992; Duarte et al., 1998; Madden et al., 1991; Nichols et al., 1995; Ward et al., 1995) PDGF-A* ,Gashler et al., 1992; Wang et al., 1992, 1993a,b, 1995a) CSF-1 ,Harrington et al., 1993) TGF-b ,Dey et al., 1994) Amphiregulin* ,Lee et al., 1999) Inhibin-a ,Hsu et al., 1995) Midkine ,Adachi et al., 1996) MIS* ,Nachtigal et al., 1998) CTGF ,Stanhope-Baker and Williams, 2000)

Growth factor receptor genes InsulinR* ,Wang et al., 1993b; Webster et al., 1997) IGF-IR* ,Tajinda et al., 1999; Werner et al., 1993, 1994, 1995) EGFR* ,Englert et al., 1995a,b; Wang et al., 1993b; Liu et al., 2001) RAR-a ,Goodyer et al., 1995)

Transcription factor genes FREAC-4 ,Ernstsson et al., 1996) Egr-1 ,Madden et al., 1991; Reddy et al., 1995a) WT1 ,Hewitt et al., 1996; Hofmann et al., 1993; Malik et al., 1994; Rupprecht et al., 1994) c-Myb ,McCann et al., 1995) c-Myc ,Hewitt et al., 1995; Wang et al., 1993b) N-Myc ,Zhang et al., 1999) Pax2* ,Ryan et al., 1995) Dax-1* ,Kim et al., 1999) Sry* ,Hossain and Saunders, 2001)

Extracellular/secreted protein-encoding genes Syndecan-1* ,Cook et al., 1996) Thrombospondin 1 ,Dejong et al., 1999) NovH ,Martinerie et al., 1996) E-cadherin* ,Hosono et al., 2000)

Others

Ga i-2* ,Kinane et al., 1996) ODC ,Moshier et al., 1996) MDR-1 ,McCoy et al., 1999) Hsp70 ,Maheswaran et al., 1998) p21 ,Englert et al., 1997) Bcl-2* ,Hewitt et al., 1995; Mayo et al., 1999) Cu,Zn-SOD ,Minc et al., 1999) RbAp46 ,Guan et al., 1998) HTERT ,Oh et al., 1999)

a Genes marked with an asterisk are discussed in the text. tional processing of RNA. However, at present, a functional Reddy et al. ,1995a) have found that WT1-mediated tran- involvement of WT1 in splicing remains to be proven. scriptional activation is inhibited in a dominant-negative fashion by cotransfection of two WT1 mutants that can not bind to DNA. These mutants had been identi®ed in a 5. WT1 protein partners Wilms' tumor and in a Denys±Drash patient, with a wild- type allele also still present. In the same study, WT1 was In addition to its association with components of the spli- also shown to homodimerize in in vitro assays, with dimer- cing apparatus, WT1 is known to bind to several other ization requiring the ®rst 180 amino acids of WT1. This led proteins ,Table 2). In agreement with a role for WT1 in the authors to propose that dominant-negative mutants of transcriptional regulation, many of these proteins are also WT1 that can not bind to DNA may play a role in tumor- transcription factors and/or alter the transcriptional regula- igenesis and in the development of Denys±Drash syndrome tory properties of WT1. ,DDS) ,see Section 6.1) by associating with wild-type WT1 146 V. Scharnhorst et al. / Gene 273 .2001) 141±161

Table 2 WT1 protein partnersa

Protein Domain in WT1 bound Consequences/remarks References

WT1* First 180 aa Homodimerization proposed to be prerequisite for TR ,Englert et al., 1995a,b; Holmes et al., byWT1; mutant WT1 proteins inhibit TR by wt WT1 1997; Reddy et al., 1995a) proteins in a dominant-negative mode p53* Zn ®ngers Stabilization of p53 and increased DNA-binding by p53; ,Maheswaran et al., 1993, 1995; Zhan modulation of TR properties of WT1 and p53; inhibition et al., 1998; Scharnhorst et al., 2000b) of p53-mediated apoptosis p73* Zn ®ngers Inhibition of DNA-binding by WT1,2/2); modulation ,Scharnhorst et al., 2000b) of TR properties of WT1 and p73 p63* ND ,Scharnhorst et al., 2000b) SF-1* N-terminus Competition of WT1 2 KTS with Dax-1 for SF-1 ,Nachtigal et al., 1998) binding leads to elevated MIS transcription Par4* Zn ®ngers Inhibits WT1-dependent TA; augments transcriptional ,Johnstone et al., 1996) repression by WT1; rescues growth suppression caused by WT1 17aa Stimulates WT1 1 17aa TA; prevention of UV-induced ,Richard et al., 2001) apoptosis Ciao 1 N-terminus Decreases TA by WT1; no effect of transcriptional ,Johnstone et al., 1998) repression by WT1 UBC9 N-terminus ,Wang et al., 1996) Hsp70 N-terminus Promotes WT1 2 KTS-mediated growth arrest ,Maheswaran et al., 1998) Splicing ND Predominantly WT1 1 KTS isoforms colocalize with ,Larsson et al., 1995) complexes* splicing complexes ,U2-B00, U1- 70K, coilin) U2AF65* Several; Zn ®ngers essential WT1 1 KTS isoforms bind better ,Davies et al., 1998) WTAP C-terminus; all forms Partial colocalization with splicing factors ,Little et al., 2000)

a TR, transcriptional regulation; TA, transcriptional activation; wt, wild-type; Zn ®ngers, zinc ®ngers; ND, not determined; MIS, MuÈllerian inhibiting substance; asterisks mark proteins discussed in the text. proteins and decreasing their transcriptional activity. of binding of WT1 with p73, a recently identi®ed homolo- However, it has not been proven that association of mutant gue of p53 ,Kaghad et al., 1997), are more clear ,Scharn- with wild-type WT1 is responsible for the dominant-nega- horst et al., 2000b). Association between WT1 and p73 tive activity of mutant WT1. Titration of cofactors by diminishes their respective transcriptional activation prop- mutant WT1 molecules may also account for the observed erties, and p73 inhibits DNA binding by WT1 ,Scharnhorst dominant-negative effect. Interestingly, two studies have et al., 2000b). p73 and the product of another recently reported that mutant WT1 2 KTS isoforms, isolated from cloned p53 homologue, termed p63, p40, p51 or KET Denys±Drash patients, very ef®ciently colocalize with and ,Osada et al., 1998; Schmale and Bamberger, 1997; Trink bind to components of the splicing machinery ,Davies et al., et al., 1998; Yang et al., 1998), share sequence homology 1998; Larsson et al., 1995). This suggests that a mutant with the transactivation, DNA-binding and tetramerization WT1, which does not bind DNA, may acquire other, aber- domains of p53. In contrast to p53, which is dispensable for rant functions and thereby contribute to the Denys±Drash embryonic development ,Donehower et al., 1992), p73 and phenotype. p63 are critically involved in differentiation and develop- Other proteins that bind to WT1 include the product of ment ,Marin and Kaelin, 2000; Mills et al., 1999; Schmale the p53 tumor suppressor gene ,Maheswaran et al., 1993), and Bamberger, 1997; Yang et al., 1999a,b). Our ®nding an association which requires the zinc-®nger region of WT1 that WT1 not only binds to p53, but also to p73 and p63, ,Maheswaran et al., 1995; Scharnhorst et al., 2000b). Func- led us to propose a dual role for WT1 in which it is, through tionally, binding of WT1 stabilizes p53, enhances binding of its functional interaction with all p53-like proteins, involved p53 to its consensus sequence and inhibits p53-mediated in both stress response and development ,Scharnhorst et al., apoptosis triggered by ultraviolet irradiation without affect- 2000b). ing p53-mediated cell-cycle arrest ,Maheswaran et al., The last WT1 protein partner to be discussed here in some 1995). In a certain cellular setting, p53 can convert WT1 detail is the Par4 protein, already mentioned above. Initially from an activator to a repressor of a given reporter construct, identi®ed as a WT1-binding protein in a yeast two-hybrid while WT1 exerts a cooperative effect on transcription acti- assay, it was found to associate with the zinc-®nger domain vated by p53 ,Maheswaran et al., 1993). Under different of WT1. In cotransfection studies, Par4 was found to conditions, the effects of p53 on WT1 and vice versa are suppress the growth-inhibitory activity of WT1, correlating much less pronounced, while the functional consequences with inhibition of WT1 transactivation and stimulating WT1 V. Scharnhorst et al. / Gene 273 .2001) 141±161 147 transrepression ,Johnstone et al., 1996). Also, Par4 was shaped and S-shaped bodies into the mature nephrons. shown to inhibit the transactivation of the Dax-1 promoter During the process of nephrogenesis, the metanephric by WT1 ,see Section 6.2). Conversely, WT1 appeared to be mesenchyme differentiates into the epithelial components able to partly inhibit the sensitization of cells to thapsigar- of the nephrons, a process called `mesenchyme-to-epithe- gin-induced apoptosis by Par4 ,Sells et al., 1997). However, lium transition'. the functional assays in these studies were all performed WT1 is expressed during all stages of kidney develop- with the WT1,2/2) isoform. More recently, Par4 was ment. WT1 expression is low but detectable in the loose shown not only to bind to the zinc-®nger region of WT1, mesenchyme and its expression strongly increases during but also to a small domain spanning the 17aa insert ,Richard condensation. Consistent with a prominent role for WT1 et al., 2001). This region, amino acids 245±297, forms an in the differentiation of the metanephric mesenchyme, it autonomous transcription activation domain when fused to has been demonstrated that WT1 can induce features of the GAL4 DNA-binding domain, whose activity appears to renal epithelial differentiation in mesenchymal ®broblasts be strictly dependent on the association with Par4. Treat- ,Hosono et al., 1999). During subsequent nephrogenesis, ment of 293 cells with UV light strongly enhances transcrip- WT1 continues to be expressed in the posterior part of the tion activation by the 117aa construct, correlating with an nephron, while in the mature nephron WT1 protein expres- induction of endogenous Par4 expression. In the full-length sion is restricted to the podocytes ,Armstrong et al., 1992; WT1 context, WT1,1/2) but not WT1,2/2) was found to Pelletier et al., 1991c; Rackley et al., 1993; Sharma et al., enhance survival of UV-irradiated 293 cells. Unfortunately, 1992). Podocytes form a specialized single layer of epithe- the authors did not assess the effect of Par4 on transcrip- lial cells in the glomerulus, which surround the blood tional regulation by WT1 in a full-length WT1 context. vessels and are involved in ultra-®ltration of the primary However, the data suggest that, upon induction of certain urine. types of stress, the pro-apoptotic function of Par4 is modu- Wilms' tumor is thought to arise from the condensed lated by the stimulation of transcriptional activation by metanephric mesenchyme that is destined to differentiate WT1 1 17aa forms. This activity might provide an explana- into the epithelial components of the nephron but fails to tion for the proposed survival function of WT1 proteins, as do so properly and instead continues to proliferate ,Hastie, discussed in Section 7.1.5. 1994; Machin and McCaughey, 1984). Inactivation of WT1 The functional consequences of association between has been shown to occur in foci of primitive renal cells ,Park WT1 and steroidogenic factor-1 ,SF-1) will be discussed et al., 1993b), which are called nephrogenic rests. It appears in Section 6.2. that the tumors originate in the persistent clusters of condensed mesenchymal cells in the nephrogenic rests that have already started to differentiate in response to 6. WT1 in normal and abnormal development inductive signals from the ureteric bud. Therefore, the tumors are often of the so-called tri-phasic type, because The cloning of the WT1 gene was facilitated by the they consist of undifferentiated mesenchyme, stromal and mapping of deletions in chromosome 11p13 of patients epithelial cells. A small percentage of the tumors contain with the WAGR syndrome ,Bickmore et al., 1989; Call et ectopical mesodermal tissues, including bone, cartilage and al., 1990; Francke et al., 1979; Gessler et al., 1990; Glaser et skeletal muscle. Indeed, Miyagawa et al. ,1998) have shown al., 1989). Patients affected by this syndrome display conge- that loss of WT1 is associated with high levels of myogenic nital developmental abnormalities and sometimes develop gene expression in Wilms' tumors, and that WT1 suppresses Wilms' tumor, suggesting that WT1 is involved in embry- muscle differentiation in vitro, suggesting that loss of WT1 onal development. Initial clues to the roles of WT1 during in Wilms' tumors may lead to aberrant activation of development came from examination of WT1 expression myogenic differentiation. during mammalian embryogenesis ,Pritchard-Jones et al., The involvement of several developmentally regulated 1990; Armstrong et al., 1992; Park et al., 1993a; Pelletier proteins in nephrogenesis and their relation to WT1 have et al., 1991c; Rackley et al., 1993; Sharma et al., 1992). now been established ,Fig. 2). Pax2 and Pax8 can both 6.1. WT1 in kidney development, nephropathies and Wilms' activate transcription of the WT1 gene ,Dehbi et al., 1996; tumors Dehbi and Pelletier, 1996; Fraizer et al., 1997; McConnell et al., 1997; Torban and Goodyer, 1998), while WT1 can The metanephric kidney is formed through reciprocal repress transcription from the Pax2 promoter in transient inductive signals between the mesodermal mesenchyme transfection assays ,Ryan et al., 1995). A marked increase and the ureteric bud, an outgrowth of the Wolf®an duct in WT1 protein levels coincides precisely with downregula- ,Saxen and Sariola, 1987). Initially, the proliferating tion of Pax2 gene expression in the precursor cells of the mesenchyme condenses around the ureteric bud and, by glomerular epithelium, suggesting a direct effect of WT1 on unknown signals, induces bud branching necessary for the Pax2 promoter during nephrogenesis ,Ryan et al., 1995). nephrogenesis. Subsequent development of the metanephric In the condensing mesenchyme, WT1 2 KTS may kidneys proceeds via transitory structures called comma- promote kidney development by transcriptionally inducing 148 V. Scharnhorst et al. / Gene 273 .2001) 141±161

Fig. 2. WT1 in kidney development. PAX2 and PAX8 transcriptionally induce the WT1 gene, while WT1 proteins inhibit synthesis of PAX2. WT1 transcriptionally activates Bcl-2, which inhibits apoptosis, and amphiregulin, which may promote ureteric bud branching. A detailed discussion is given in Section 6.1. the Amphiregulin gene, encoding a member of the epider- mesenchyme undergo apoptosis. However, several markers mal growth factor ,EGF) family that stimulates epithelial of early metanephric differentiation, including Pax2, are branching in organ cultures of embryonic mouse kidney already present in the metanephric mesenchyme of WT1 ,Lee et al., 1999). As the expression pattern of Amphiregu- knock-out embryos, demonstrating that the metanephric lin in the fetal kidney mirrors the pattern of WT1 protein mesenchyme in mutant embryos has begun to differentiate expression ,Lee et al., 1999), it is possible that activation of towards the nephrogenic lineage, and that this early differ- Amphiregulin by WT1 facilitates ureteric bud branching at entiation does not require either WT1 or the presence of the early stages of nephrogenesis. Surprisingly, inactivation of ureteric bud ,Donovan et al., 1999). A recent review by the Amphiregulin gene does not result in aberrant kidney Schedl and Hastie ,2000) describes the different proteins development, suggesting that other factors can perform the and their functions in kidney development in more detail. same function ,Luetteke et al., 1999). WT1 is known to transcriptionally activate expression of Apart from Amphiregulin, also other genes of which tran- the Bcl-2 gene, which encodes an anti-apoptotic protein scription can be activated by WT1 might be involved in ,Hewitt et al., 1995; Mayo et al., 1999). WT1-expressing WT1-regulated kidney development. One example is the cells displaying upregulated Bcl-2 protein expression are syndecan-1 gene, identi®ed by Rauscher and co-workers resistant to apoptosis induced by several agents ,Mayo et as a WT1 target gene ,Cook et al., 1996). Expression of al., 1999). Thus, absence of WT1 in the mesenchymal blas- syndecan-1 protein re¯ects that of WT1 during kidney tema cells of homozygous knock-out mice may render these development. Syndecan-1 is supposed to function as a co- cells susceptible to apoptosis. In support of Bcl-2 being a receptor together with ligand-speci®c receptors and as such physiological target gene of WT1 during early kidney devel- to stimulate the translation of extracellular signals into intra- opment is the observation that Bcl-2 knock-out mice have cellular effects. It has been shown to be essential for the smaller kidneys, which contain fewer nephrons and display differentiated epithelial phenotype of cells and might also a greater susceptibility to apoptosis in the mesenchymal be involved in the mesenchyme-to-epithelial transformation blastema ,Novack and Korsmeyer, 1994). Since the major- event that occurs during nephrogenesis. ity of Wilms' tumors still express WT1 proteins ,Little and Secondly, the group of Licht recently showed that the E- Wells, 1997), upregulation of Bcl-2 by WT1 may provide a cadherin gene is a bona ®de target for WT1 proteins cell-survival advantage to tumor cells and explain the resis- ,Hosono et al., 2000). The transcription from the E-cadherin tance of anaplastic Wilms' tumors expressing WT1 to promoter can be activated by all WT1 forms, although the chemotherapeutic agents, particularly since Bcl-2 expres- 2KTS proteins have a stronger effect. In the kidney, E- sion coincides with WT1 protein levels in sporadic cadherin expression is induced in the condensing metaneph- Wilms' tumors ,Mayo et al., 1999). ric mesenchyme during the mesenchymal-epithelial transi- As mentioned above, heterozygous germ-line mutations tion, again re¯ecting the expression pattern of WT1 of WT1 are found in patients with DDS ,Pelletier et al., proteins. Importantly, E-cadherin might be a target gene 1991a). All 12 children with DDS tested had inherited for the tumor suppressor function of WT1, since loss of missense mutations in the region of the WT1 gene that the E-cadherin gene has been observed in multiple tumor encodes the zinc ®ngers. Eight of the 12 children had the types. same mutation, in which an amino acid in zinc ®nger 3 that The essential role of WT1 in kidney development was is essential for DNA-binding is substituted. Since the second unequivocally proven by the phenotype of homozygous WT1 allele in DDS patients is wild-type ,Pelletier et al., WT1 knock-out mice, which fail to develop metanephric 1991a) and WT1 proteins can homodimerize ,Englert et kidneys and die in utero ,Kreidberg et al., 1993). Speci®- al., 1995b; Holmes et al., 1997; Reddy et al., 1995a), it cally, at day 11 of gestation the ureteric bud fails to grow out has been suggested that the mutant WT1 proteins in DDS from the Wolf®an duct and the cells of the metanephric act in a dominant-negative fashion through association with V. Scharnhorst et al. / Gene 273 .2001) 141±161 149 the wild-type WT1 protein. Denys±Drash patient-derived 1998). Thus, the mere change in ratio of 1KTS/2KTS mutant WT1 proteins indeed interfere with the transcrip- must be responsible for the severe developmental defects. tional activation of WT1 reporter constructs, but these More recently, however, the situation has become more mutant WT1 proteins do not act in a dominant-negative complex because in two patients classi®ed as having FS, way at the level of DNA binding, since DNA binding by exon 9 mutations were found that did not change the wild-type WT1 is not inhibited by an excess of mutant WT1 1KTS/2KTS ratio ,Kohsaka et al., 1999). Actually, one ,Reddy et al., 1995a; Scharnhorst et al., 1999). Also, tran- of these mutations is very similar to a described DDS muta- scriptional repression by WT1 appears not to be inhibited, tion and the exact same mutation had been found before in a but might even be stimulated by DDS mutants ,Tajinda et Wilms' tumor. Conversely, although FS patients in general al., 1999). Thus, it can not be excluded at this time that the only develop glomerulopathy of the kidney and no Wilms' dominant-negative effect of DDS-derived WT1 mutants is tumor, a patient with a classical intronic FS point mutation brought about by an alternative mechanism. Evidence was found to have developed a Wilms' tumor ,Barbosa et against a `simple' dominant-negative model of mutant al., 1999). These data have started a controversy regarding WT1 is the observation that the urogenital abnormalities the classi®cation of DDS and FS. Thus, FS may be more of DDS are also found in a mouse carrying a heterozygous correctly classi®ed as an atypical subtype of DDS, as mutation in the WT1 gene that truncates the WT1 protein reviewed in more detail by Mrowka and Schedl ,2000). within zinc ®nger 3 at amino acid 396, although only 5% of Children with WAGR syndrome have microscopically total cellular WT1 protein was found to be derived from the visible deletions at 11p13, leading to loss of one copy of mutant WT1 allele ,Patek et al., 1999). the WT1 gene ,Brown et al., 1992; Fantes et al., 1992; Glomerular nephropathy is the most consistent ®nding in Gessler et al., 1993). Patients with WAGR and DDS DDS. The affected children suffer from hypertension caused syndromes develop glomerulopathy and, frequently, also by a collapse of the arteries in the glomerulus. Fibrotic Wilms' tumor, in contrast to FS patients ,Table 3). material produced by the mesangial cells that contact the As expected for a tumor suppressor gene, the wild-type arteries causes the hypertension ,Jadresic et al., 1990). In allele of WT1 is usually lost in tumors arising in children addition, the podocytes expressing WT1 are often underde- with the WAGR or DDS syndromes ,Baird et al., 1992; veloped in DDS ,Jadresic et al., 1990), suggesting that WT1 Brown et al., 1992; Gessler et al., 1993; Little et al., 1993; proteins are required for proper podocyte differentiation. Pelletier et al., 1991a). As mentioned earlier, homozygous Yang et al. ,1999) have recently demonstrated that expres- deletion of WT1 has been observed in approximately 10% of sion of mutant WT1 protein in DDS is associated with Wilms' tumors. Extending the ®ndings that alterations of abnormal podocyte expression of Pax2 mRNA and protein. the ratio between different isoforms might be suf®cient to Since WT1 can repress the Pax2 promoter ,Ryan et al., develop severe developmental defects are two reports 1995), persistent expression of Pax2 may result from the describing imbalanced expression of the WT1 mRNA loss of WT1-dependent repression and may contribute to isoforms in a high percentage of Wilms' tumors ,Liu et the etiology of glomerular dysfunction. al., 1999; Baudry et al., 2000). Both studies show a relative Two other congenital syndromes, the Frasier syndrome loss of WT1 1 17aa expression ,,55% of the cases), but, in ,FS) and the WAGR syndrome, are also linked to alterations addition, other changes were observed. The results of in the WT1 gene locus. Baudry et al. ,2000) suggest that, all together, nearly 90% FS was initially shown to be caused by mutations in the of the tested samples ,n ˆ 50) show aberrant expression of splice donor site in intron 9 of one WT1 allele, resulting in the WT1 mRNAs. the loss of expression of the WT1 1 KTS form of one allele Thus, the proper balance between the different WT1 ,Barbaux et al., 1997; Kikuchi et al., 1998; Klamt et al., isoforms is required for correct development, and distinct

Table 3 Congenital syndromes associated with mutations in WT1

Syndrome WT1 status Phenotype Mode

Denys±Drash Heterozygous point Diffuse mesangial sclerosis causes glomerular nephropathy, often Dominant-negative ,missense/ mutations ,in zinc ®ngers) Wilms' tumor; females: normal gonads; males: phenotype of truncation mutants) gonads varies: from streak gonads, female internal and external genitalia to mild hermaphroditism WAGR Heterozygous deletion at Wilms' tumor; aniridia; genitourinary abnormalities; mental WT1 gene dose; other genes involved chromosome 11p13 retardation; infrequently gonadoblastoma; milder overall ,PAX6) phenotype in kidneys and gonads than Denys±Drash and Frasier Frasier Heterozygous point Glomerulopathy, characterized by unspeci®c focal and segmental Gene dose ,50% less WT1 1 KTS mutation in splice donor glomerular sclerosis, one case of Wilms' tumor reported gonads: isoforms); truncation site in intron 9; occasional male-to-female sex reversal ,female external genitalia, streak mutation within exon 9 gonads, XY karyotype), frequently gonadoblastoma 150 V. Scharnhorst et al. / Gene 273 .2001) 141±161 imbalances in WT1 gene expression have different effects on dal ridge being signi®cantly smaller than in wild-type kidney development and tumorigenesis. embryos ,Kreidberg et al., 1993). These results prove that WT1 is necessary for early development of the mammalian 6.2. WT1 in the development of gonads and male intersex gonads, but do not explain why only male WAGR, Denys± disorders Drash and FS patients display intersex disorders. Several reports have demonstrated a direct functional In mammals, the gonads and the metanephric kidneys are involvement of the WT1 2 KTS isoforms in male sexual derived from a common precursor, called the urogenital development ,Kim et al., 1999; Nachtigal et al., 1998), ridge. During the indifferent stage of sexual development, which is shown in the simpli®ed scheme of mammalian the testes and ovaries cannot be distinguished and for that sex determination in Fig. 3. The orphan nuclear receptor reason are called indifferent or bipotential gonads. Simi- SF-1 regulates expression of MIS by binding to an upstream larly, the urogenital tracts are also indistinguishable in regulatory sequence in the MIS promoter ,Shen et al., 1994). female and male embryos. WT1 2 KTS isoforms clearly associate and synergize with Thickening of the epithelium of the urogenital ridge SF-1 to promote MIS expression, while the association of marks the ®rst stage of gonadal development, followed by WT1 1 KTS with SF-1 is weaker, and no synergistic acti- formation of the gonadal ridge. From the gonadal ridge, the vation of the MIS promoter is observed. The promoter MuÈllerian ducts are formed in the undifferentiated gonads of element necessary for synergistic activation of the MIS female and male embryos. The MuÈllerian ducts develop into promoter was mapped, and it was shown that DNA binding parts of the female reproductive tract, while another pair, the by WT1 2 KTS is required for WT1 to potentiate transcrip- Wolf®an ducts, is derived from the mesonephros and tional activation by SF-1. Consequently, DDS mutant WT1 becomes parts of the male reproductive tract. Consequently, proteins showed only a very weak cooperative effect for normal embryonal development, one duct system must together with SF-1. Finally, the same authors went on to differentiate while the other one must regress. show that Dax-1 interacts with SF-1 and represses SF-1/ Hormones produced by the developing fetus control male WT1-activated transcription of the MIS gene in a dose- and female development. Regression of the female ducts is dependent manner, suggesting that the relative levels of triggered by the MuÈllerian inhibitory substance ,MIS), a WT1 2 KTS and Dax-1 may determine the transcriptional TGF-b-like hormone, which is produced in the pre-Sertoli activation of the MIS gene by SF-1. The Dax-1 gene is cells of the testes. Then, testosterone, produced by the located on the X chromosome within the dosage-sensitive Leydig cells of the testes, triggers differentiation of the sex reversal locus and duplication of this locus in males Wolf®an ducts into the male reproductive organs. In the overrides male development, resulting in XY sex reversal absence of the testis-determining factor Sry, which is ,Muscatelli et al., 1994; Zanaria et al., 1994). The intersex located on the Y chromosome, the testis does not form disorders in male Denys±Drash patients may thus be and MIS and testosterone are not produced. As a conse- explained by a reduced expression of MIS caused by dimin- quence, the Wolf®an ducts passively regress and differentia- ished synergism between SF-1 and the remaining wild-type tion of the MuÈllerian ducts into the female reproductive WT1 2 KTS proteins in the presence of unaltered amounts organs occurs. Thus, MIS and testosterone impose male of Dax-1. The results of Nachtigal et al. ,1998), however, development upon an intrinsically female program ,for can not explain the intersex disorders in FS patients, since review see Parker et al., 1999a,b; Werner et al., 1996). these individuals have reduced expression of WT1 1 KTS Heterozygous mutations/deletions in WT1 are associated isoforms, which do not synergistically activate the MIS with intersex disorders in males ,Table 3). Analyses of WT1 promoter. expression during murine embryogenesis revealed that WT1 Another group has shown that WT1 2 KTS activates the is expressed in the urogenital ridge and then becomes loca- Dax-1 promoter ®ve- to seven-fold and that cotransfection lized to the Sertoli cells of the testis and granulosa and of Par4 or a DDS-derived mutant of WT1 inhibits this acti- epithelial cells of the ovary ,Armstrong et al., 1992; Pelle- vation ,Kim et al., 1999). Since Dax-1 antagonizes the tier et al., 1991c; Rackley et al., 1993). In humans, hetero- synergism between WT1,2/2) and SF-1 ,Nachtigal et al., zygous loss-of-function germ-line mutations are associated 1998), this ®nding ®ts poorly into the current model of with mild effects on sexual differentiation, hypospadias and gonadogenesis. However, the activation of the Dax-1 cryptorchidism ,Pelletier et al., 1991b). However, germ-line promoter by WT1 2 KTS ,®ve- to seven-fold) is much mutations that produce dominant-negative WT1 proteins or lower than the activatory synergism between WT1 2 KTS alter the ratio between the different WT1 isoforms are asso- and SF-1 on the MIS promoter ,30±40-fold synergistic acti- ciated with a severe effect on mammalian sex differentia- vation over transcription activated by SF-1 alone). tion, e.g. male pseudohermaphroditism ,Barbaux et al., A very recent report suggests that WT1 is also stimulating 1997; Kikuchi et al., 1998; Klamt et al., 1998; Pelletier et the expression of the Sry gene, which would mean that WT1 al., 1991a). Homozygous WT1 knock-out mice fail to proteins regulate the cascade of events even upstream of SF- develop gonads, with the thickening of the epithelium of 1 and MIS ,Hossain and Saunders, 2001). As mentioned the urogenital ridge being markedly reduced and the gona- above, Sry is the main testis-determining factor ,see also V. Scharnhorst et al. / Gene 273 .2001) 141±161 151

Fig. 3. WT1 in gonadal development. WT1 and SF-1 are essential in the development of the bi-potent, undifferentiated gonads. WT1, Dax-1, SF-1, MIS and Sry determine development into the differentiated gonads. The regulatory circuit shown in the lower box supposedly leads to male gonadogenesis. Upregulation of MIS by the transcription factor SF-1 is synergized by WT1 2 KTS and antagonized by Dax-1. WT1 2 KTS by itself stimulates Dax-1 transcription. Both functions of WT1 are inhibited by mutant WT1 proteins as found in the DDS. In addition, WT1 2 KTS forms have now been shown also to activate transcription of the Sry gene, indicating a role for WT1 even further upstream in the cascade of events that determine gonadogenesis. A detailed explanation is given in Section 6.2. review by McElreavey and Fellous, 1999). Mutations in al., 1991c; Rackley et al., 1993; Sharma et al., 1992). The both the Sry and the WT1 gene lead to sex reversal, which latter site of expression is re¯ected in the phenotype of WT1 ®ts very well with the observation that WT1 2 KTS forms knock-out mice, which lack correct mesenchyme-to-epithe- activate transcription from the Sry promoter. Both lium transition of the mesothelium ,Kreidberg et al., 1993). WT1 1 KTS forms and DDS mutant WT1 proteins fail to The development of the diaphragm, which separates the activate this promoter. However, no dominant-negative thoracic and abdominal cavities, is incomplete in WT1 effect of the DDS-mutated WT1 forms was observed, lead- knock-out mice, resulting in an incomplete separation of ing to the hypothesis that in DDS it is mainly haplo-insuf®- the thoracic and the abdominal cavities. The embryos ciency that causes the aberrant gonadal development. display malformed hearts, pericardial bleeding and massive In conclusion, the molecular pathways which account for edema ,Kreidberg et al., 1993). As renal agenesis is not the distinct phenotypes of individuals with different WT1 usually lethal until the immediate postnatal period ,Kleineb- mutations are now being unraveled, but functionally linking recht et al., 1982), the heart abnormalities combined with all different phenotypes to speci®c WT1 mutations will still the edema are the most likely causes for the observed take a long time. embryonic lethality. More recent studies revealed new sites of WT1 expression 6.3. Other sites of WT1 expression by generation of transgenic mouse strains carrying yeast arti®cial chromosomes ,YACs) with the human WT1 Apart from its expression in the developing kidney and promoter directing expression of a b-galactosidase reporter gonads, WT1 is detected in several other sites in the gene ,Moore et al., 1998, 1999). With this approach, WT1 mammalian embryo. WT1 is expressed in the uterus, the expression was found in the proliferating coelomic epithe- spleen, the liver, the thymus, certain areas of the brain and lium, the developing diaphragm and limb, the septum trans- the spinal cord and in the abdominal wall musculature as versum, the early proepicardium, the epicardium and the well as in the mesothelial linings of organs in the thoracic subepicardial mesenchyme. Moore and co-workers used and abdominal cavities ,Armstrong et al., 1992; Pelletier et YAC transgenic constructs spanning the WT1 locus to 152 V. Scharnhorst et al. / Gene 273 .2001) 141±161 complement WT1 knock-out mice. With this approach they lar mechanisms underlying the transition from growth to were able to rescue the heart defects in WT1 knock-out mice differentiation in the LLC-PK1 juvenile pig kidney cell substantiating the relevance of WT1 in heart development line. During cell culture, LLC-PK1 cells undergo several ,Moore et al., 1999). rounds of cell divisions until at con¯uence they differentiate The original study describing the phenotype of WT1 from a rounded cell type to a fully polarized epithelium. knock-out mice was carried out in a C57BL/6 genetic back- Proliferation of these cells is stimulated by Egr-1-dependent ground ,Kreidberg et al., 1993). Herzer et al. ,1999) have transcriptional activation of the G protein a i-2 ,Ga i-2) gene, crossed these mice into different mouse backgrounds, which encoding the a subunit of the heterotrimeric guanine delayed embryonic lethality until birth ,Herzer et al., 1999). nucleotide binding protein Gi-2 ,Kinane et al., 1994). In Therefore, they could assess the requirement of WT1 for the same study, it was shown that Egr-1 binds to a GC- organogenesis in later stages of embryogenesis and found rich element in the Ga i-2 promoter during the proliferation that absence of WT1 results in a failure of spleen develop- phase of LLC-PK1 cells, but is replaced by other DNA- ment. The absence of correct spleen development correlated binding proteins during the polarization phase. In a second with enhanced apoptosis in the primordial spleen cells study, the same authors demonstrated that WT1 is one of ,Herzer et al., 1999). Thus, WT1 is required for the devel- these latter proteins and that increased WT1 levels correlate opment of the spleen and the phenotype of WT1 knock-out with the polarization phase of the kidney cells ,Kinane et al., mice depends on ,a) modi®er gene,s). 1996). Furthermore, overexpression of WT1 repressed tran- Since it had been described before that the Hox11 gene is scription from a reporter construct containing parts of the essential for spleen development, a possible interdepen- Ga i-2 promoter and inhibited proliferation of LLC-PK1 dence of WT1 and Hox11 expression was investigated. cells. In the developing kidney, the Egr-1 gene is expressed Herzer et al. ,1999) found by in situ RNA hybridization in the metanephric mesenchyme, and is downregulated on whole-mount embryos an independent regulation of during renal development ,Rackley et al., 1995), while Hox11 and WT1 expression. In contrast, a more recent expression of WT1 is upregulated during the mesenchyme- report shows decreased WT1 mRNA expression in the to-epithelial transition. The polarization of LLC-PK1 cells spleen anlage of Hox11-null mice ,Koehler et al., 2000). may re¯ect the proliferation to differentiation switch during In addition, they showed that Hox11 protein could activate early renal differentiation and, therefore, can be a useful the WT1 promoter in Hox11-null ®broblasts. These results model system to investigate the molecular changes at this would suggest that the WT1 gene is a downstream target of stage of kidney development. the Hox11 transcription factor. 7.1.2. WT1 is involved in differentiation of leukemic cell lines 7. WT1 in cell culture models of differentiation, WT1 is expressed in stem cells of the bone marrow, but apoptosis and tumorigenesis not in normal mature blood cells ,Fraizer et al., 1995; Patmasiriwat et al., 1996), indicating that WT1 plays a Wilms' tumors seem to arise from pluripotent blastema role in early hematopoiesis. It had been demonstrated before cells of the mesenchyme, which do not differentiate prop- that the WT1 gene is expressed in the leukemic cell lines erly, but instead continue to proliferate ,Hastie, 1994). K562 and HL-60, and that differentiation of these cells in Furthermore, mutations in WT1 are associated with several culture is accompanied by downregulation of WT1 protein developmental abnormality syndromes and gonadoblas- levels ,Phelan et al., 1994; Sekiya et al., 1994), suggesting toma, indicating that WT1 proteins play an important role that WT1 expression is linked to an immature state of leuke- in the regulation of growth and differentiation. Therefore, mic cells. Although one report indicates that the decrease in several studies have addressed the involvement of WT1 in WT1 expression is not a strict prerequisite for the differen- these processes by utilizing cell culture model systems. tiation of K562 cells ,Svedberg et al., 1999), these ®ndings 7.1. Differentiation models and apoptosis are consistent with several reports showing that ectopic expression of various WT1 isoforms delays the in vitro As described above, WT1 has an important function in differentiation of leukemic cell lines, e.g. HL-60, U937 early embryonic organogenesis and its expression in the and K562 ,Inoue et al., 1998; Svedberg et al., 1998; Deuel developing kidney is associated with differentiation of the et al., 1999; Carrington and Algar, 2000). Intriguingly, proliferating mesenchyme into epithelial components of the Deuel et al. ,1999) showed that expression of the zinc-®nger nephrons. Therefore, several groups have made use of plur- domain of WT1 is suf®cient to arrest the macrophage differ- ipotent cell lines to assess the expression and function of entiation of HL-60 cells induced by TPA. These data WT1 during differentiation. suggest that WT1 proteins act, at least partly, by displacing other transcription factors from GC-rich promoter elements. 7.1.1. WT1 is induced during polarization of kidney cells Remarkably, the M1 murine myeloblastic cell line and represses proliferation expressing no endogenous WT1 reacts very differently to Kinane et al. ,1994, 1996) have investigated the molecu- ectopic WT1 expression. Constitutive expression of the V. Scharnhorst et al. / Gene 273 .2001) 141±161 153 1KTS forms induces monocytic differentiation indepen- derived cell line with mesodermal characteristics does not dent of an external stimulus, while M1 cells stably expres- express WT1 protein either ,Scharnhorst et al., 1997). sing the 2KTS forms can not be established because they Kudoh et al. ,1996) failed to establish F9 cells ectopically induce G1 arrest and/or apoptosis ,Smith et al., 1998; expressing WT1,1/2), but could establish F9 cells consti- Murata et al., 1997). tutively synthesizing WT1,2/1). It was found that 24 h Today it is recognized that WT1 is expressed in a wide after adding retinoic acid to WT1,2/1)-expressing F9 range of acute leukemic cell lines and acute leukemias ,see cells, the cells started to undergo apoptosis instead of differ- below), with high expression correlating with less-differen- entiating into endodermal cells. These results suggest that tiated phenotypes ,Pritchard-Jones and King-Underwood, an imbalance in expression of the WT1 isoforms may trig- 1997). ger apoptosis and/or that ectopic expression of WT1,2/1) in differentiating F9 cells generates con¯icting signals and may therefore trigger apoptosis. In this respect it is interest- 7.1.3. Regulation of WT1 expression during differentiation ing to mention that we were not able to establish P19 cells of pheochromocytoma cells overexpressing any WT1 isoform, suggesting that WT1 The rat pheochromacytoma cell line PC12 has been induces apoptosis in proliferating P19 cells ,Scharnhorst widely used over 20 years as a cell culture model to study et al., 1997). neuronal differentiation. Liu et al. ,2000) reported that during nerve growth factor ,NGF)-induced differentiation the expression of the EGF receptor ,EGFR) is downregu- 7.1.5. WT1 can induce and prevent apoptosis lated, and that some TCC repeats in the rat EGFR promoter The results described above suggest that the effects of are necessary for this effect. The same group now shows that WT1 on apoptosis are cell line- and differentiation state- WT1 expression levels are decreasing during NGF-induced dependent and are in agreement with several other reports differentiation of PC12 cells and that this decrease appears demonstrating both pro- and anti-apoptotic effects of WT1. to be necessary for the lower EGFR expression after differ- On the one hand, ectopic expression of WT1 can ef®ciently entiation ,Liu et al., 2001). In their model system, WT1 induce apoptosis in a number of cell lines ,Englert et al., proteins are transcriptionally activating the rat EGFR 1995a; Menke et al., 1997; Murata et al., 1997; Scharnhorst promoter through binding to the TCC repeats. et al., 1999). Interestingly, overexpression of the EGFR The downregulation of WT1 during PC12 differentiation ,Englert et al., 1995a; Menke et al., 1997) or the insulin is reminiscent of what happens during the in vitro differen- receptor ,Menke et al., 1997), products of two target genes tiation of most leukemic cell lines. However, the fact that that are repressed by WT1 in the cell lines studied, can these investigators ®nd a transcriptional activation of the partially rescue cells from WT1-induced apoptosis. EGFR gene is in contrast to earlier reports showing that In contrast, inhibition of WT1 expression by WT1 anti- WT1 proteins actually repress transcription of the EGFR sense oligonucleotides in leukemic cell lines ,Algar et al., promoter ,Englert et al., 1995a), once again demonstrating 1996) or an absence of WT1 in WT1 knock-out mice leads to the cell-type dependence of the effects of WT1 proteins on enhanced apoptosis ,Herzer et al., 1999; Kreidberg et al., promoter activity. 1993). Underscoring an anti-apoptotic function of WT1, it has been found that induction of WT1 expression in cell culture inhibits apoptosis induced by several chemical 7.1.4. WT1 is differentially regulated during differentiation agents ,Mayo et al., 1999) and the p53-dependent apoptosis of embryonal carcinoma and embryonic stem cells in response to UV irradiation ,Maheswaran et al., 1995). Embryonal carcinoma cells and embryonic stem cells can Finally, it has recently been described that the WT1-inter- be used to study the mechanisms underlying early develop- acting protein Par4, whose expression is increased during mental processes and several studies have investigated the induction of apoptosis ,Diaz-Meco et al., 1996; Sells et al., role of WT1 during differentiation of these cells. 1994), enhances apoptosis in response to thapsigargin. The Treatment of F9 and P19 embryonal carcinoma cells with thapsigargin-induced apoptosis can be attenuated by ectopic retinoic acid triggers differentiation into endodermal ,F9) or expression of WT1 ,Sells et al., 1997). Simultaneous over- endodermal and ectodermal ,P19) cells and is accompanied expression of Par4 overcomes the anti-apoptotic effect of by induction of endogenous WT1 proteins ,Kudoh et al., WT1 ,Sells et al., 1997). 1996; Scharnhorst et al., 1997). In F9 cells, the induction These data indicate that, depending on the physiological of WT1 occurs transiently during the ®rst 24 h of differen- conditions, WT1 may either function as a `survival factor' tiation ,Kudoh et al., 1996), whereas WT1 levels in embryo- and, as such, prevent apoptosis, or, in contrast, may induce nic stem cells and P19 cells continuously increase apoptosis and thereby prevent proliferation of, for example, ,Scharnhorst et al., 1997). In line with this, a stable differ- transformed cells. entiated epithelial cell line derived from P19 cells expresses high levels of WT1 protein ,Scharnhorst et al., 1997). Inter- 7.2. Tumorigenesis models estingly, differentiation of P19 cells into mesoderm-derived cell types does not induce WT1 expression and a stable P19- The effects of different WT1 isoforms on growth and 154 V. Scharnhorst et al. / Gene 273 .2001) 141±161 tumor formation have now been tested in several different ingly, WT1,2/2) and WT1,2/1) have no effect on cell lines, of both renal and non-renal origin ,Table 4). tumor growth in the similar 7C1T1 AdBRK cell line Different WT1 isoforms exert growth repressive effects in ,Table 4). This prompted us to investigate the molecular various cell lines tested. One common ®nding of almost all differences between 7C3H2 and 7C1T1 cells and we reports in which WT1 acts as a tumor suppressor is that the found that 7C1T1 cells express about ten-fold higher levels ectopic WT1 expression is lost during tumor growth ,Luo et of endogenous Egr-1 protein than 7C3H2 cells, suggesting al., 1995; McMaster et al., 1995; Menke et al., 1996; that Egr-1 abrogates the effects of WT1 on growth regula- Scharnhorst et al., 2000a). The only exception is the tumors tion ,Scharnhorst et al., 2000a). Furthermore, ectopic derived from RM1 kidney cells transfected with the four expression of Egr-1 in 7C3H2 cells increases their tumor different WT1 isoforms, which retain their very low expres- growth rate and co-expression of Egr-1 and WT1,2/1) sion of WT1 in the tumors isolated from nude mice ,Haber abolishes the need for loss of WT1,2/1) expression during et al., 1993). tumor outgrowth and abrogates the tumor suppressive Our own group has investigated the effects of different effects of WT1,2/1). WT1 isoforms on tumor formation of adenovirus-trans- Since WT1 and Egr-1 are highly homologous in their formed baby rat kidney ,AdBRK) cells in nude mice zinc-®nger domains, regulate transcription from reporter ,Menke et al., 1995, 1996; Scharnhorst et al., 2000a). constructs differentially ,Dey et al., 1994; Kinane et al., Expression of WT1,2/2) in 7C3H2 AdBRK cells clearly 1994, 1996; Lee and Kim, 1996) and have opposite effects increases the tumor growth of these AdBRK cells in nude on cell growth ,Kinane et al., 1994, 1996), it is possible that mice and correlates with an increased ability of the cells to the opposing effects of WT1,2/1) and Egr-1 on tumor grow in serum-free medium ,Menke et al., 1996). Combined growth are due to opposite regulation of common target with the report of Algar et al. ,1996), which demonstrates genes. Thus, aberrant expression of Egr-1 may, in some the necessity of WT1 for proliferation of some leukemic cell cases, play an important role in the development of lines, this suggests that WT1 may, in some cells, function as Wilms' tumor. Indeed, we have found that three out of 16 a `survival factor' by enhancing proliferation and inhibiting Wilms' tumor samples studied express elevated levels of apoptosis. In contrast, stable expression of WT1,2/1) Egr-1, which may contribute to continuous growth instead strongly represses tumor formation by 7C3H2 AdBRK of differentiation and, therefore, play an important role in cells and is correlated with a reduced ability of the cells to the development of Wilms' tumor ,Scharnhorst et al., grow in serum-free medium ,Menke et al., 1996). Strik- 2000a).

Table 4 Effects of WT1 on tumorigenicity

Cell line WT1 isoform,s) tested Effects References

Renal cell lines RM1 ,Wilms' tumor cell line with All four isoforms All four isoforms independently or together greatly ,Haber et al., 1993) mutated WT1) repress colony formation and delay tumor formation in nude mice SM2 ,rhabdoid kidney tumor cell line) All four isoforms No effect on colony formation; effects on tumor ,Haber et al., 1993) growth not reported 7C3H2 ,AdBRK cell line) ,2/1) Strong suppression of tumor take and outgrowth of ,Menke et al., 1995) tumors 7C3H2 ,2/2) Increased tumor growth rate ,Menke et al., 1996) ,1/2),1/1) No effect on tumor growth ,2/1) Decreased tumor growth rate 7C1T1 ,AdBRK cell line) ,2/2) No effect on tumor growth ,Scharnhorst et al., 2000a) ,2/1) No effect on tumor growth G401 ,rhabdoid kidney tumor cell line) ,1/2), ,2/1) Increase in the number of abnormally large and ,McMaster et al., 1995) multinucleated cells; slight decrease in colony formation and signi®cant decrease of tumor growth in nude mice; reduction of tumorigenicity by ,2/1) stronger than by ,2/2)

Cell lines of non-renal origin HT1080 ,®brosarcoma cell line) ,1/2), ,2/1) No effect on tumor growth in nude mice ,McMaster et al., 1995) H-ras-transformed NIH3T3 ,®broblasts) ,2/2) Decreased growth rate, complete contact inhibition; ,Luo et al., 1995) decreased growth in soft agar and inhibition of tumor growth in nude mice M1 myeloblastic leukemia cell line ,2/1), ,1/1) Decreased growth in soft agar; inhibition of tumor ,Smith et al., 2000) growth in scid/scid mice V. Scharnhorst et al. / Gene 273 .2001) 141±161 155 8. WT1 in other malignancies than Wilms' tumor 4 of WT1 ,Gerald et al., 1995; Ladanyi and Gerald, 1994). Karnieli et al. ,1996) could show that the EWS/WT1 2 KTS As mentioned previously, WT1 is involved in the devel- fusion protein activates transcription of the IGF-I receptor opment of some gonadoblastomas and leukemias. Further- gene in reporter assays, whereas WT1 2 KTS represses more, mutations in the WT1 gene play a role in the transcription from the same promoter ,Werner et al., 1993, development of desmoplastic small round cell tumor 1994). Since repression of IGF-I receptor mRNA expres- ,DSRCT) and a small minority of mesotheliomas ,Amin sion by WT1 in G401 is associated with a reduction growth et al., 1995; Kumar-Singh et al., 1997; Park et al., 1993a). rate ,Werner et al., 1995), this indicates that the EWS-WT1 fusion protein may play a causal role in DSRCT. Similarly, 8.1. WT1 in leukemia Lee et al. ,1997) demonstrated that EWS/WT1 2 KTS fusion protein induces the expression of endogenous WT1 mRNA is expressed in the bone marrow, but not in PDGF-A gene in a cell line inducibly expressing EWS/ normal mature blood cells ,Fraizer et al., 1995; Patmasir- WT1, while WT1 2 KTS is known to repress transcription iwat et al., 1996), suggesting a function in early hemato- from reporter constructs containing parts of the PDGF-A poietic development. In addition, WT1 is expressed in acute promoter ,Gashler et al., 1992; Wang et al., 1992). leukemias of myeloid and lymphoid origin as well as in Recently, it has been shown that introduction of EWS/ many acute leukemia cell lines, and expression levels are WT1 2 KTS, but not EWS/WT1 1 KTS, into NIH3T3 cells highest in immature leukemias ,Inoue et al., 1994; Pritch- increases formation of foci, anchorage-independent growth ard-Jones and King-Underwood, 1997). Although in appar- and tumor formation in nude mice ,Kim et al., 1998). ent contrast, a mutation in the remaining allele of WT1 was Combined with the ®nding that EWS/WT1 2 KTS aber- found in a leukemia that occurred as a secondary malig- rantly regulates WT1 target genes, it is likely that the onco- nancy in a WAGR syndrome patient ,Pritchard-Jones et genic effects of the EWS/WT1 gene fusion are through al., 1994) and heterozygous WT1 mutations are present in aberrant regulation of ,a subset of) WT1 target genes by about 10±15% of acute leukemias ,King-Underwood and the EWS/WT1 2 KTS isoform. Since EWS/WT1 2 KTS Pritchard-Jones, 1998; King-Underwood et al., 1996). In and Egr-1 proteins both activate transcription, and since retrospect, the ®rst clue that abnormal WT1 expression the DNA-binding domain of Egr-1 is homologous to the could be involved in leukemia was that one of the two last three zinc ®ngers of WT1, it is conceivable that the groups that ®rst cloned the WT1 gene obtained a WT1 oncogenic effects of EWS/WT1 2 KTS in NIH3T3 cells cDNA clone from a pre-B cell line, in which a serine impor- and of Egr-1 in AdBRK cells may be due to activation of tant for DNA binding ,Bickmore et al., 1992) was replaced at least part of the same target genes. by a phenylalanine ,Call et al., 1990). Since WT1 is expressed in many acute leukemias, WT1 mRNA expression levels are now used as a prognostic factor in myelodysplastic syndromes and acute leukemia. Inoue et 9. Concluding remarks al. ,1994) established a clear correlation between the rela- Since the identi®cation of the WT1 gene it has been tive levels of WT1 expression and the prognosis for acute clearly proven that the WT1 proteins play an essential role leukemia, while Tamaki et al. ,1999) demonstrated that in urogenital development. Still, the exact mechanisms by WT1 expression levels increase during progression of which the various WT1 isoforms perform their biological myelodysplastic syndromes to acute myeloid leukemia. In functions remain largely unknown. Much of the physiolo- both reports, patients with relatively low WT1 mRNA gically relevant information has been obtained from studies expression had a better prognosis than patients with high on patients suffering from the WAGR, Denys±Drash and FS WT1 mRNA levels. Thus, in most leukemias the WT1 syndromes, harboring speci®c types of mutations in the WT1 proteins appear to act as survival factors, although the gene. However, also these studies have not yet answered the presence of mutations in the WT1 gene in some cases ,see most pressing question: what are the relevant target genes of above) would suggest a tumor suppressor function. the WT1 proteins that mediate their biological responses? 8.2. The EWS-WT1 fusion protein in DSRCT The fact that the WT1 gene is expressed into at least 24 different protein isoforms has strongly hampered progress DSRCT is a member of the group of neoplasms referred in this ®eld. Transfection experiments of single isoforms, to as small round cell tumors of childhood and early adult- whose activity might be in¯uenced by the choice of expres- hood, which includes rhabdomyosarcoma, Ewing sarcoma, sion vector, into sometimes apparently non-relevant cell neuroblastoma and lymphoma. types has yielded a long list of putative WT1 target genes. DSRCT is an aggressive tumor that is located in the peri- Other types of experiments are now needed to be able to toneal surfaces of the abdomen. It is de®ned by the chro- separate the wheat from the chaff. One approach might be mosomal translocation t,11;22),p13;q12) that results in the the use of mouse models. The most promising candidate expression of a fusion gene encoding the transactivation could well be the chimeric or, if obtainable, heterozygous domain of the EWS1 protein in frame with zinc ®ngers 2± mouse expressing a WT1 gene with a Denys±Drash-like 156 V. Scharnhorst et al. / Gene 273 .2001) 141±161 mutation that shows features of the DDS ,Patek et al., 1999). Niaudet, P., Moreira-Filho, C.A., Cotinot, C., Fellous, M., 1999. The Microarray analysis of genes that are differentially same mutation affecting the splicing of WT1 gene is present on Frasier expressed at early developmental stages of certain tissues syndrome patients with or without Wilms' tumor. Hum. Mutat. 13, 146± 153. in wild-type versus DDS mutant mice might yield important Baudry, D., Hamelin, M., Cabanis, M.-O., Fournet, J.-C., Tournade, M.-F., clues, although the identi®cation of the primary target genes Sarnacki, S., Junien, C., Jeanpierre, C., 2000. WT1 splicing alterations could still be dif®cult. In analogy to the approach to generate in Wilms' tumors. Clin. Cancer Res. 6, 3957±3965. a DDS-like mouse model, one could try to generate mouse Bickmore, W.A., Porteous, D.J., Christie, S., Seawright, A., Fletcher, J.M., models for FS. The results obtained with such a mutant Maule, J.C., Couillin, P., Junien, C., Hastie, N.D., van Heyningen, V., 1989. CpG islands surround a DNA segment located between translo- mouse could settle the argument of whether FS should be cation breakpoints associated with genitourinary dysplasia and aniridia. classi®ed as a atypical subtype of DDS or as a separate Genomics 5, 685±693. syndrome. 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