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Multiple roles for the Wilms’ tumour suppressor gene, WT1 in genitourinary development
Molecular and Cellular Endocrinology 140 (1998) 65 – 69
Multiple roles for the Wilms’ tumour suppressor gene, WT1 in genitourinary development Andreas Schedl a, Nicholas Hastie b,* b
a Max-Delbruck Centrum fur Molekulare Medizin, Robert Rossle Str 10, 13122 Berlin Buch, Germany MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland, UK
Abstract Wilms’ tumour is a childhood kidney cancer, and a classic example of cancer arising through disrupted development (Armstrong et al., 1992). It is one of the most common solid paediatric malignancies, affecting one in 10000 children. The genetics of Wilms’ tumour is complicated, with several different genes or chromosomal regions being implicated (Armstrong et al., 1992). However, the gene we know most about is the Wilms’ tumour predisposition gene, WT1 (Bickmore et al., 1992; Bruening and Pelletier, 1996). It is now clear that mutations in this gene in humans can lead to abnormalities of the kidneys and gonads, as well as to the eponymous tumour. Also, as discussed below, WT1 is essential for kidney, testis and ovary development, as revealed in knockout mice. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Wilm’s tumour; WAGR syndrome; Genitourinary development
1. Chromosomal deletions pinpointed the WT1 gene and its possible role in genital abnormalities The identification of the WT1 gene came through the rare association of Wilms’ tumour with another developmental childhood abnormality, aniridia (lack of an iris) in children with the so-called WAGR syndrome (Armstrong et al., 1992), which stands for Wilms’ tumour, aniridia, genitourinary abnormalities and mental retardation. These children suffered constitutional heterozygous deletions of chromosome 11, always encompassing band 11p13. It was proposed that a Wilms’ tumour predisposition gene named WT1 mapped within the deleted region. Analysis of these deletions provided the first clue that WT1 is likely to be involved in genital development as well as development of the kidney. A high proportion of boys with these 11p13 deletions suffer mild pseudo-hermaphroditism, having hypospadias, cryptorchidism, or both. Careful analysis of a large series of deletions suggested that the same
* Corresponding author: Tel.: + 44 131 4678401; fax: + 44 131 5397680.
gene leads to Wilms’ tumour and the genital abnormalities (Bruening et al., 1992). Once the WT1 gene was isolated, this hypothesis was shown to be true (Call et al., 1990; Caricasole et al., 1996).
2. WT1, a tumour suppressor gene encoding a protein with four zinc fingers Intensive positional cloning efforts in 1990 (in the laboratories of David Housman and Gail Bruns) led to the isolation of the WT1 gene mapping to chromosome 11p13 (Bickmore et al., 1992; Bruening and Pelletier, 1996). The WT1 gene was shown to be : 50 kb in size, encoding a protein of :52–54 kDa. This protein had several features of note, particularly four zinc fingers similar to those found in a number of known transcription factors; also a proline–glutamine rich domain typically found in regulatory regions of transcription factors. Large screens carried out by a number of groups have shown that WT1 mutations are found in only 10–15% of all Wilms’ tumour cases (Drummond et al.,
0303-7207/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0303-7207(98)00031-8
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A. Schedl, N. Hastie / Molecular and Cellular Endocrinology 140 (1998) 65–69
Fig. 1. Evolution of WT1 structure and complexity.
1992; Coppes et al., 1993; Drummond et al., 1994; Englert et al., 1995a). About half of these mutations in WT1 are constitutional, the remainder being acquired somatically. It is clear that in nearly all cases both copies of the gene must be mutated for the tumour to develop. Hence, in children who inherit a deletion or mutation of one WT1 allele, the second allele is mutated in the tumour. Thus in this regard, WT1 follows Knudson’s rule and can be classified as a tumour suppressor gene.
tional start site, no alternative exon 5 and no RNA editing in Fugu. On the other hand, the alternative splice that inserts KTS is completely conserved throughout the vertebrate kingdom. As discussed below, this is particularly interesting because we have evidence that the +KTS and − KTS forms of WT1 may have different functions in the cell. Now we are starting to carry out trans-species transgenic experiments to address the question whether the increased complexity of the WT1 gene in mammals plays an important role in the increased complexity of the genitourinary system.
3. There are 16 WT1 isoforms in mammals but only two in fish WT1 is an extremely complex gene that encodes 16 different isoforms through a combination of alternative splicing, alternative translational start sites and RNA editing (Fig. 1) (Gessler et al., 1990; Gashler et al., 1992; Gessler et al., 1994). There are two alternative splices, one inserting an extra exon encoding 17 amino acids downstream of the polyproline – glutamine rich region, the other leading to the insertion of three amino acids, lysine, threonine and serine (KTS) between zinc fingers 3 and 4. All 16 isoforms appear to be conserved throughout mammals, therefore, it is highly likely that they all play important and necessary roles in development or tissue homeostasis. It is very interesting to consider the situation in other vertebrates, particularly fish. Fish have a more primitive genitourinary system than mammals. With this in mind, we have isolated the WT1 gene from the puffer fish, Fugu rubripes. Here it is clear that the WT1 gene of Fugu encodes only two different WT1 isoforms and not the 16 found in mammals (Fig. 1). There appears to be only one transla-
4. WT1 is expressed at highest levels in the developing genitourinary system Comprehensive in situ hybridisation and immunohistochemistry studies have shown that WT1 is expressed at highest levels in the developing derivatives of the intermediate mesoderm (Haber et al., 1990, 1991; Harrington et al., 1993; Hastie, 1994). Highest levels are found in the developing kidney, testis/ovary and mesothelium. The kidney develops through a series of reciprocal induction events between the metanephric mesenchyme and the ureteric bud. First, the metanephric mesenchyme condenses around the bud and then it is induced by the bud to differentiate into epithelial cells which will reorganise into the components of the nephron, the proximal and distal tubules, the loop of Henle and the glomerulus. Immediately after induction the condensing mesenchyme is first induced to proliferate but then, as differentiation proceeds, proliferation ceases. Wilms’ tumours arise from this condensed mesenchyme, presumably because loss
A. Schedl, N. Hastie / Molecular and Cellular Endocrinology 140 (1998) 65–69
of function of the WT1 gene prevents cells from exiting the cycle. Given these considerations, the expression pattern of WT1 during kidney development conforms to expectations. The gene is only expressed in the nephric lineage, not in the ureteric bud. Expression is first detected at low levels in the uncondensed mesenchyme but levels increase dramatically as the mesenchyme condenses and induction starts. As nephrogenesis proceeds, WT1 expression is then restricted to the lower part of the S-shaped body that will form the glomerulus. WT1 expression is very high in the newly forming glomerulus, indicating a role for this gene in the development of this structure. In terms of the gonad, WT1 expression can be detected first in the developing genital ridge and expression is quite high in the mesenchymal component. Highest levels of expression are observed in the Sertoli cells of the testis and the granulosa cells of the ovary.
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is the observation that WT1 can dimerise through a domain in the n-terminus and that mutant forms can inactivate transcriptional activity of a wildtype molecule in transient transfection assays (Kreidberg et al., 1993). Some remarkable DDS mutations support the notion of a different function for the + KTS and −KTS forms of WT1. In several children with DDS the phenotype has arisen through a mutation that affects the splice site leading to the insertion of KTS between zinc fingers 3 and 4 (Larsson et al., 1995). This results in a situation when only the − KTS form of WT1 is produced from one allele but both forms are produced from the other. Hence, there is not expected to be a reduction in the overall amount of WT1, just a reduction in the ratio of + KTS to − KTS forms. These findings suggest that the correct isoform ratio is essential for normal genitourinary development and kidney function. Furthermore, the obvious conclusion is that these two forms of WT1 have different functions.
5. Dominant WT1 mutations in humans can lead to gonadal dysgenesis and severe nephropathy Children with a rare congenital syndrome, known as the Dennys Drash syndrome (DDS), suffer from severe nephropathy, mild to severe gonadal dysgenesis and often Wilms’ tumour. The nephropathy is characterised by diffuse mesangial sclerosis which leads to severe hypertension and death unless kidney transplantation is offered. The gonadal dysgenesis at its most severe can be manifest as streak gonads — essentially the complete absence of epithelial structures. Alternatively XY individuals with DDS can have female genitalia or gonads; in other cases derivatives of both the Wolffian and Mullerian duct may be present. It was shown several years ago that DDS arises inevitably through constitutional WT1 mutations that leave most of the protein intact but which affect the zinc fingers so that DNA binding activity is impaired (Caricasole et al., 1996). Remarkably, around half of the mutations are missense mutations that lead to exactly the same change, an arginine to tryptophan substitution, at residue 394 in zinc finger 3. Crystallographic modelling of another zinc finger protein has shown that this arginine is essential for function and directly contacts a guanosine residue in target DNA (Kennedy et al., 1996). Another fascinating aspect is that these mutations are heterozygous, only one WT1 allele being mutated, the other wildtype; yet the phenotype is much more severe than the situation with the WAGR syndrome where one WT1 allele is deleted completely. Hence, these mutant forms of WT1 are either acting dominantly so that the protein is taking on new deleterious functions, or the mutant molecules are acting in the dominant negative mode, i.e. leading to the inactivation of the wildtype molecule. In support of this latter hypothesis
6. Knockout mice show that WT1 is essential for kidney and gonad development Whereas evidence from human genetics supports the idea that normal WT1 function is important in the development of the kidney and gonad, it is necessary to use mice to test the consequences for development of a complete lack of WT1 function. Kreidberg et al. produced Wt1 null mice using gene targeting in ES cells (Little et al., 1992). Homozygous null animals died at mid-gestation, probably through abnormalities of the heart which appeared to have a disrupted mesothelial layer and pericardium. However, the most dramatic and satisfying aspect of the phenotype was complete absence of a metanephric kidney, testis and ovary. Further studies showed that the metanephric mesenchyme forms but that there is no ureteric outgrowth and the mesenchyme then degenerates through apoptosis. These studies show that WT1 is essential for the very earliest stages of kidney and gonad development. As far as the gonad is concerned, the phenotype is very similar to that found with SF1 knockout mice, hence the two genes act early in gonad development and perhaps interact directly (Luo et al., 1994). However, it is likely that WT1 also functions at later stages in both kidney and gonad development, in the function of the glomerulus in the kidney and the formation of the sertoli and granulosa cells of the testes and ovaries, respectively. In fact, our recent transgenic experiments support a role for WT1 in the development of the glomerulus.
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7. WT1 acts as a transcriptional repressor Immediately after the primary structure of WT1 was known, it was proposed that the protein acted as a transcription factor. Subsequently it was shown that the zinc fingers of WT1 bind to G-rich DNA; the −KTS isoform of WT1 binds to a G-rich ninemer through the last three zinc fingers, three nucleotides to each finger (Madden et al., 1991). The + KTS form, however, could not bind to this ninemer but could bind to a 12 nucleotide G-rich sequence with lower affinity (Mundlos et al., 1993; Park et al., 1993). Following these observations, Rauscher’s group showed that WT1 can repress the expression of reporter sequences linked to promoters containing WT1 binding sites in transient transfection assays (Pavletich and Pabo, 1991). By fusing the proline – glutamine-rich region of WT1 to the Gal4 transcription factor they were able to transform this classic activator into a repressor (Pavletich and Pabo, 1991). Whilst WT1 normally appears to behave as a repressor, there are conditions where it can act as an activator, and this activator activity seems to reside in amino acids 180 – 294 (Pelletier et al., 1991a,b). If WT1 normally acts as a transcription activator, it would be reasonable to assume that physiological target genes might be those which help to keep kidney stem cells in cycle. Consistent with this idea, WT1 can repress the expression of a variety of such growth genes including the IGF2 (Pelletier et al., 1991a,b), CSF1 (PritchardJones et al., 1990), PDGFA (Rauscher et al., 1990) and EGF receptor genes (Reddy et al., 1995) in transient transfection assays. It is, of course, difficult to prove direct interaction between any transcription factor and a potential target gene; this is particularly difficult for WT1 as many thousands of genes have G-rich binding sites in their promoter regions and might in principle be considered potential target genes. However, there is compelling evidence to believe that IGF2 and EGFR may be true physiological targets for WT1 repression. There is also evidence that WT1 may repress the expression of the developmental gene PAX2 during kidney development (Ryan et al., 1995). Most of the candidate WT1 target genes have been considered in relation to kidney development. So far little has been done in the context of gonad development. SF1 and SRY could both be considered potential WT1 target genes. Clearly, new approaches are required to allow the identity of physiological target genes for transcription factors such as WT1.
8. WT1 may act to regulate RNA splicing as well as transcription We have analysed cell lines set up from fetal mouse tissues, particularly a kidney cell line, M15, to allow us
to study WT1 function in as close to physiological conditions as possible. To our surprise we found that WT1 protein localised to discrete domains in the nucleus, not in a diffuse pattern expected for transcription factors. We went on to show that these domains corresponded to speckles and coiled bodies in which splice factors are concentrated (Sharma et al., 1994). Furthermore, we were able to show that splice factor antibodies were able to coimmunoprecipitate WT1. The antibodies we used detected all WT1 isoforms, so to observe the subnuclear localisation of individual WT1 forms we turned to transfection experiments in Cos2 cells. These experiments showed quite clearly that in most (but not all) transfected cells, the + KTS forms of WT1 localised with splice factors; on the other hand, the − KTS forms localised chiefly in diffuse domains along with transcription factors. These experiments suggested for the first time that WT1 might play a role in splicing as well as transcription. Furthermore, the two different forms of the protein, with and without a three amino acid insert, might have different functions, in agreement with the evidence from human genetics. Since these first studies, the evidence supporting a role for WT1 in RNA metabolism has increased. Firstly, WT1 was shown to be able to bind RNA through the zinc fingers (Van Heyningen et al., 1990). Secondly, through structural modelling, an RNA recognition motif was localised to the n-terminal region of WT1 (Wang et al., 1993). Thirdly, we have been able to show that WT1 can be found in active splice complexes (unpublished observations). These findings raise the possibility that regulated RNA splicing plays a crucial role in the normal development of the genitourinary system, and that abnormalities in such splicing control can lead to a variety of developmental defects including tumours. We are now trying to identify the RNA targets that might interact with and be regulated by WT1. Furthermore, we are trying to use new approaches to identify transcriptional targets for WT1 in the process of genitourinary development. In the long run, identification of upstream regulators of WT1 and downstream targets should provide insights into the pathways that regulate genitourinary development. These upstream and downstream genes will also be candidates for other human developmental disorders of the genitourinary system.
References Armstrong, J.F., Pritchard-Jones, K., Bickmore, W.A., Hastie, N.D., Bard, J.B.L., 1992. The expression of the Wilms’ tumour gene, WT1, in the developing mammalian embryo. Mech. Dev. 40, 85 – 97.
A. Schedl, N. Hastie / Molecular and Cellular Endocrinology 140 (1998) 65–69 Bickmore, W.A., Oghene, K., Little, M.H., Seawright, A., Van Heyningen, V., Hastie, N.D., 1992. Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript. Science 257, 235–237. Bruening, W., Pelletier, J., 1996. A non-AUG translational initiation event generates novel WT1 isoforms. J. Biol. Chem. 271, 8646 – 8654. Bruening, W., Bardeesy, N., Silverman, B.L., Cohn, R.A., Machin, G.A., et al., 1992. Germline intronic and exonic mutations in the Wilms’ tumour gene (WT1) affecting urogenital development. Nat. Genet. 1, 144 – 148. Call, K.M., Glaser, T., Ito, C.Y., Buckler, A.J., Pelletier, J., Haber, D.A., Rose, E.A., Kral, A., Yeger, H., Lewis, W.H., Jones, C., Housman, D.E., 1990. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms’ tumor locus. Cell 60, 509 – 520. Caricasole, A., Duarte, A., Larsson, S., Hastie, N.D., Little, M., Holmes, G., Todorov, I., Ward, A., 1996. RNA binding by the Wilms’ tumour suppressor (WT1) zinc finger proteins. Proc. Natl. Acad. Sci. USA 93, 7562–7566. Coppes, M.J., Liefers, G.J., Paul, P., Yeger, H., Williams, B.R.G., 1993. Homozygous somatic WT1 point mutations in sporadic unilateral Wilms tumor. Proc. Natl. Acad. Sci. USA 90, 1416 – 1419. Drummond, I.A., Madden, S.L., Rohwer-Nutter, P., Bell, G.I., Sukhatme, V.P., Rauscher, F.J. III, 1992. Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1. Science 257, 674–678. Drummond, I.A., Rupprecth, H.D., Rohwer-Nutter, P., Lopez-Guisa, J., Madden, S.L., et al., 1994. DNA recognition by splicing variants of the Wilms’ tumor suppressor, WT1. Mol. Cell. Biol. 14, 3800 – 3809. Englert, C., Hou, X., Maheswaran, S., Bennett, P., Ngwu, C., Re, G.G., Garvin, A.J., Rosner, M.R., Haber, D.A., 1995a. WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis. EMBO J. 14, 4462–4675. Gashler, A.J., Bonthron, D.T., Madden, S.L., Rauscher, F.J. III, Collins, T., Sukhatme, V.P., 1992. Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1. Proc. Natl. Acad. Sci. USA 89, 10984–10988. Gessler, M., Pustka, A., Cavenee, W., Neve, R.L., Orkin, S.H., Bruns, G.A.P., 1990. Homozygous deletion in Wilms’ tumours of a zinc finger gene identified by chromosome jumping. Nature 343, 774 – 778. Gessler, M., Konig, A., Arden, K., Grundy, P., Orkin, S., et al., 1994. Infrequent mutation of the WT1 gene in 77 Wilms’ tumors. Hum. Mutat. 3, 212 – 222. Haber, D.A., Buckler, A.J., Glaser, T., Call, K.M., Pelletier, J., et al., 1990. An internal deletion within an 11p13 zinc finger gene contributes to the development of Wilms’ tumor. Cell 61, 1257069. Haber, D.A., Sohn, R.L., Buckler, A.J., Pelletier, J., Call, K.M., Housman, D.E., 1991. Alternative splicing and genomic structure of the Wilms’ tumor gene WT1. Proc. Natl. Acad. Sci. USA 88, 9618 – 9622. Harrington, M.A., Konicek, B., Song, A., Xia, X., Fredericks, W.J., Rauscher, F.J. III, 1993. Inhibition of colony-stimulating factor-1 promoter activity by the product of the Wilms’ tumor locus. J. Biol. Chem. 268, 21271 – 21275. Hastie, N.D., 1994. The genetics of Wilms’ tumor—a case of disrupted development. Annu. Rev. Genet. 523–558.
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Kennedy, D., Ramsdale, T., Mattick, J., Little, M., 1996. An RNA recognition motif in WT1 revealed by structural modelling. Nat. Genet. 12 (3), 329 – 332. Kreidberg, J.A., Sariola, H., Loring, J.M, Maeda, M., Pelletier, J., et al., 1993. WT1 is required for early kidney development. Cell 74, 679 – 691. Larsson, S.H., Charlieu, J.-P., Miyagawa, K., Engelkamp, D., Ross, A., Van Heyningen, V., Hastie, N.D., 1995. Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing. Cell 81, 391 – 401. Little, M.H., Prosser, J., Condie, A., Smith, P.J., Van Heyningen, V., Hastie, N.D., 1992. Zinc finger point mutations within the WT1 gene in tumour patients. Proc. Natl. Acad. Sci. 89, 4791–4795. Luo, X., Ikeda, Y., Parker, K.L., 1994. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77, 481 – 490. Madden, S.L., Cook, D.M., Morris, J.F., Gashler, A., Sukhatme, V.P., Rauscher, F.J. III., 1991. Transcriptional repression mediated by the WT1 Wilms tumor gene product. Science 253, 1550–1553. Mundlos, S., Pelletier, J., Darveau, A., Bachmann, M., Winterpacht, A., Zabel, B., 1993. Nuclear localization of the protein encoded by the Wilms’ tumor gene WT1 in embryonic and adult tissues. Development 119, 1329 – 1341. Park, S., Schalling, M., Bernard, A., Maheswaran, S., Shipley, G.C., et al., 1993. The Wilms’ tumour gene WT1 is expressed in murine mesoderm-derived tissues and mutated in a human mosothelioma. Nat. Genet. 4, 415 – 420. Pavletich, N.P., Pabo, C.O., 1991. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1A. Science 252, 809 – 817. Pelletier, J., Bruening, W., Li, F.P., Haber, D.A., Glaser, T., Housman, D.E., 1991a. WT1 mutations contribute to abnormal genital system development and hereditary Wilms’ tumour. Nature 353, 431–434. Pelletier, J., Bruening, W., Kashtan, C.E., Mauer, S.M., Manivel, J.C., et al., 1991b. Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys Drash syndrome. Cell 67, 437 – 447. Pritchard-Jones, K., Fleming, S., Davidson, D., Bickmore, W., Porteous, D.J., et al., 1990. The candidate Wilms’ tumour gene is involved in genitourinary development. Nature 346, 194–197. Rauscher, F.J., Morris, J.F., Tournay, O.E., Cook, D.M., Curran, T., 1990. Binding of the Wilms’ tumor locus zinc finger protein to the EGR-consensus sequence. Science 250, 1259 – 1262. Reddy, J.C, et al., 1995. WT1-mediated transcriptional activation is inherited by dominant negative mutant proteins. J. Biol. Chem. 270, 10878 – 10884. Ryan, G., Steele Perkins, V., Morris, J.F., Rauscher, F.J., Dressler, G.R., 1995. Repression of Pax-2 by WT1 during normal kidney development. Development 121, 867 – 875. Sharma, P.M., Bowman, M., Madden, S.L., Rauscher, F.J., Sukumar, S.L., 1994. RNA editing in the Wilms’ tumor susceptibility gene, WT1. Genes Dev. 8, 720 – 731. Van Heyningen, V., Bickmore, W.A., Seawright, A., Fletcher, J.M., Maule, J., et al., 1990. Role of the Wilms’ tumor gene in genital development? Proc. Natl. Acad. Sci. USA 87, 5383 – 5386. Wang, Z.Y., Qui, Q.Q., Deuel, T.F., 1993. The Wilms’ tumor gene product WT1 activates or suppresses transcription through separate functional domains. J. Biol. Chem. 268 (13), 9172 – 9175.
Multiple roles for the Wilms’ tumour suppressor gene, WT1 in genitourinary development Andreas Schedl a, Nicholas Hastie b,* b
a Max-Delbruck Centrum fur Molekulare Medizin, Robert Rossle Str 10, 13122 Berlin Buch, Germany MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland, UK
Abstract Wilms’ tumour is a childhood kidney cancer, and a classic example of cancer arising through disrupted development (Armstrong et al., 1992). It is one of the most common solid paediatric malignancies, affecting one in 10000 children. The genetics of Wilms’ tumour is complicated, with several different genes or chromosomal regions being implicated (Armstrong et al., 1992). However, the gene we know most about is the Wilms’ tumour predisposition gene, WT1 (Bickmore et al., 1992; Bruening and Pelletier, 1996). It is now clear that mutations in this gene in humans can lead to abnormalities of the kidneys and gonads, as well as to the eponymous tumour. Also, as discussed below, WT1 is essential for kidney, testis and ovary development, as revealed in knockout mice. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Wilm’s tumour; WAGR syndrome; Genitourinary development
1. Chromosomal deletions pinpointed the WT1 gene and its possible role in genital abnormalities The identification of the WT1 gene came through the rare association of Wilms’ tumour with another developmental childhood abnormality, aniridia (lack of an iris) in children with the so-called WAGR syndrome (Armstrong et al., 1992), which stands for Wilms’ tumour, aniridia, genitourinary abnormalities and mental retardation. These children suffered constitutional heterozygous deletions of chromosome 11, always encompassing band 11p13. It was proposed that a Wilms’ tumour predisposition gene named WT1 mapped within the deleted region. Analysis of these deletions provided the first clue that WT1 is likely to be involved in genital development as well as development of the kidney. A high proportion of boys with these 11p13 deletions suffer mild pseudo-hermaphroditism, having hypospadias, cryptorchidism, or both. Careful analysis of a large series of deletions suggested that the same
* Corresponding author: Tel.: + 44 131 4678401; fax: + 44 131 5397680.
gene leads to Wilms’ tumour and the genital abnormalities (Bruening et al., 1992). Once the WT1 gene was isolated, this hypothesis was shown to be true (Call et al., 1990; Caricasole et al., 1996).
2. WT1, a tumour suppressor gene encoding a protein with four zinc fingers Intensive positional cloning efforts in 1990 (in the laboratories of David Housman and Gail Bruns) led to the isolation of the WT1 gene mapping to chromosome 11p13 (Bickmore et al., 1992; Bruening and Pelletier, 1996). The WT1 gene was shown to be : 50 kb in size, encoding a protein of :52–54 kDa. This protein had several features of note, particularly four zinc fingers similar to those found in a number of known transcription factors; also a proline–glutamine rich domain typically found in regulatory regions of transcription factors. Large screens carried out by a number of groups have shown that WT1 mutations are found in only 10–15% of all Wilms’ tumour cases (Drummond et al.,
0303-7207/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0303-7207(98)00031-8
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A. Schedl, N. Hastie / Molecular and Cellular Endocrinology 140 (1998) 65–69
Fig. 1. Evolution of WT1 structure and complexity.
1992; Coppes et al., 1993; Drummond et al., 1994; Englert et al., 1995a). About half of these mutations in WT1 are constitutional, the remainder being acquired somatically. It is clear that in nearly all cases both copies of the gene must be mutated for the tumour to develop. Hence, in children who inherit a deletion or mutation of one WT1 allele, the second allele is mutated in the tumour. Thus in this regard, WT1 follows Knudson’s rule and can be classified as a tumour suppressor gene.
tional start site, no alternative exon 5 and no RNA editing in Fugu. On the other hand, the alternative splice that inserts KTS is completely conserved throughout the vertebrate kingdom. As discussed below, this is particularly interesting because we have evidence that the +KTS and − KTS forms of WT1 may have different functions in the cell. Now we are starting to carry out trans-species transgenic experiments to address the question whether the increased complexity of the WT1 gene in mammals plays an important role in the increased complexity of the genitourinary system.
3. There are 16 WT1 isoforms in mammals but only two in fish WT1 is an extremely complex gene that encodes 16 different isoforms through a combination of alternative splicing, alternative translational start sites and RNA editing (Fig. 1) (Gessler et al., 1990; Gashler et al., 1992; Gessler et al., 1994). There are two alternative splices, one inserting an extra exon encoding 17 amino acids downstream of the polyproline – glutamine rich region, the other leading to the insertion of three amino acids, lysine, threonine and serine (KTS) between zinc fingers 3 and 4. All 16 isoforms appear to be conserved throughout mammals, therefore, it is highly likely that they all play important and necessary roles in development or tissue homeostasis. It is very interesting to consider the situation in other vertebrates, particularly fish. Fish have a more primitive genitourinary system than mammals. With this in mind, we have isolated the WT1 gene from the puffer fish, Fugu rubripes. Here it is clear that the WT1 gene of Fugu encodes only two different WT1 isoforms and not the 16 found in mammals (Fig. 1). There appears to be only one transla-
4. WT1 is expressed at highest levels in the developing genitourinary system Comprehensive in situ hybridisation and immunohistochemistry studies have shown that WT1 is expressed at highest levels in the developing derivatives of the intermediate mesoderm (Haber et al., 1990, 1991; Harrington et al., 1993; Hastie, 1994). Highest levels are found in the developing kidney, testis/ovary and mesothelium. The kidney develops through a series of reciprocal induction events between the metanephric mesenchyme and the ureteric bud. First, the metanephric mesenchyme condenses around the bud and then it is induced by the bud to differentiate into epithelial cells which will reorganise into the components of the nephron, the proximal and distal tubules, the loop of Henle and the glomerulus. Immediately after induction the condensing mesenchyme is first induced to proliferate but then, as differentiation proceeds, proliferation ceases. Wilms’ tumours arise from this condensed mesenchyme, presumably because loss
A. Schedl, N. Hastie / Molecular and Cellular Endocrinology 140 (1998) 65–69
of function of the WT1 gene prevents cells from exiting the cycle. Given these considerations, the expression pattern of WT1 during kidney development conforms to expectations. The gene is only expressed in the nephric lineage, not in the ureteric bud. Expression is first detected at low levels in the uncondensed mesenchyme but levels increase dramatically as the mesenchyme condenses and induction starts. As nephrogenesis proceeds, WT1 expression is then restricted to the lower part of the S-shaped body that will form the glomerulus. WT1 expression is very high in the newly forming glomerulus, indicating a role for this gene in the development of this structure. In terms of the gonad, WT1 expression can be detected first in the developing genital ridge and expression is quite high in the mesenchymal component. Highest levels of expression are observed in the Sertoli cells of the testis and the granulosa cells of the ovary.
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is the observation that WT1 can dimerise through a domain in the n-terminus and that mutant forms can inactivate transcriptional activity of a wildtype molecule in transient transfection assays (Kreidberg et al., 1993). Some remarkable DDS mutations support the notion of a different function for the + KTS and −KTS forms of WT1. In several children with DDS the phenotype has arisen through a mutation that affects the splice site leading to the insertion of KTS between zinc fingers 3 and 4 (Larsson et al., 1995). This results in a situation when only the − KTS form of WT1 is produced from one allele but both forms are produced from the other. Hence, there is not expected to be a reduction in the overall amount of WT1, just a reduction in the ratio of + KTS to − KTS forms. These findings suggest that the correct isoform ratio is essential for normal genitourinary development and kidney function. Furthermore, the obvious conclusion is that these two forms of WT1 have different functions.
5. Dominant WT1 mutations in humans can lead to gonadal dysgenesis and severe nephropathy Children with a rare congenital syndrome, known as the Dennys Drash syndrome (DDS), suffer from severe nephropathy, mild to severe gonadal dysgenesis and often Wilms’ tumour. The nephropathy is characterised by diffuse mesangial sclerosis which leads to severe hypertension and death unless kidney transplantation is offered. The gonadal dysgenesis at its most severe can be manifest as streak gonads — essentially the complete absence of epithelial structures. Alternatively XY individuals with DDS can have female genitalia or gonads; in other cases derivatives of both the Wolffian and Mullerian duct may be present. It was shown several years ago that DDS arises inevitably through constitutional WT1 mutations that leave most of the protein intact but which affect the zinc fingers so that DNA binding activity is impaired (Caricasole et al., 1996). Remarkably, around half of the mutations are missense mutations that lead to exactly the same change, an arginine to tryptophan substitution, at residue 394 in zinc finger 3. Crystallographic modelling of another zinc finger protein has shown that this arginine is essential for function and directly contacts a guanosine residue in target DNA (Kennedy et al., 1996). Another fascinating aspect is that these mutations are heterozygous, only one WT1 allele being mutated, the other wildtype; yet the phenotype is much more severe than the situation with the WAGR syndrome where one WT1 allele is deleted completely. Hence, these mutant forms of WT1 are either acting dominantly so that the protein is taking on new deleterious functions, or the mutant molecules are acting in the dominant negative mode, i.e. leading to the inactivation of the wildtype molecule. In support of this latter hypothesis
6. Knockout mice show that WT1 is essential for kidney and gonad development Whereas evidence from human genetics supports the idea that normal WT1 function is important in the development of the kidney and gonad, it is necessary to use mice to test the consequences for development of a complete lack of WT1 function. Kreidberg et al. produced Wt1 null mice using gene targeting in ES cells (Little et al., 1992). Homozygous null animals died at mid-gestation, probably through abnormalities of the heart which appeared to have a disrupted mesothelial layer and pericardium. However, the most dramatic and satisfying aspect of the phenotype was complete absence of a metanephric kidney, testis and ovary. Further studies showed that the metanephric mesenchyme forms but that there is no ureteric outgrowth and the mesenchyme then degenerates through apoptosis. These studies show that WT1 is essential for the very earliest stages of kidney and gonad development. As far as the gonad is concerned, the phenotype is very similar to that found with SF1 knockout mice, hence the two genes act early in gonad development and perhaps interact directly (Luo et al., 1994). However, it is likely that WT1 also functions at later stages in both kidney and gonad development, in the function of the glomerulus in the kidney and the formation of the sertoli and granulosa cells of the testes and ovaries, respectively. In fact, our recent transgenic experiments support a role for WT1 in the development of the glomerulus.
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7. WT1 acts as a transcriptional repressor Immediately after the primary structure of WT1 was known, it was proposed that the protein acted as a transcription factor. Subsequently it was shown that the zinc fingers of WT1 bind to G-rich DNA; the −KTS isoform of WT1 binds to a G-rich ninemer through the last three zinc fingers, three nucleotides to each finger (Madden et al., 1991). The + KTS form, however, could not bind to this ninemer but could bind to a 12 nucleotide G-rich sequence with lower affinity (Mundlos et al., 1993; Park et al., 1993). Following these observations, Rauscher’s group showed that WT1 can repress the expression of reporter sequences linked to promoters containing WT1 binding sites in transient transfection assays (Pavletich and Pabo, 1991). By fusing the proline – glutamine-rich region of WT1 to the Gal4 transcription factor they were able to transform this classic activator into a repressor (Pavletich and Pabo, 1991). Whilst WT1 normally appears to behave as a repressor, there are conditions where it can act as an activator, and this activator activity seems to reside in amino acids 180 – 294 (Pelletier et al., 1991a,b). If WT1 normally acts as a transcription activator, it would be reasonable to assume that physiological target genes might be those which help to keep kidney stem cells in cycle. Consistent with this idea, WT1 can repress the expression of a variety of such growth genes including the IGF2 (Pelletier et al., 1991a,b), CSF1 (PritchardJones et al., 1990), PDGFA (Rauscher et al., 1990) and EGF receptor genes (Reddy et al., 1995) in transient transfection assays. It is, of course, difficult to prove direct interaction between any transcription factor and a potential target gene; this is particularly difficult for WT1 as many thousands of genes have G-rich binding sites in their promoter regions and might in principle be considered potential target genes. However, there is compelling evidence to believe that IGF2 and EGFR may be true physiological targets for WT1 repression. There is also evidence that WT1 may repress the expression of the developmental gene PAX2 during kidney development (Ryan et al., 1995). Most of the candidate WT1 target genes have been considered in relation to kidney development. So far little has been done in the context of gonad development. SF1 and SRY could both be considered potential WT1 target genes. Clearly, new approaches are required to allow the identity of physiological target genes for transcription factors such as WT1.
8. WT1 may act to regulate RNA splicing as well as transcription We have analysed cell lines set up from fetal mouse tissues, particularly a kidney cell line, M15, to allow us
to study WT1 function in as close to physiological conditions as possible. To our surprise we found that WT1 protein localised to discrete domains in the nucleus, not in a diffuse pattern expected for transcription factors. We went on to show that these domains corresponded to speckles and coiled bodies in which splice factors are concentrated (Sharma et al., 1994). Furthermore, we were able to show that splice factor antibodies were able to coimmunoprecipitate WT1. The antibodies we used detected all WT1 isoforms, so to observe the subnuclear localisation of individual WT1 forms we turned to transfection experiments in Cos2 cells. These experiments showed quite clearly that in most (but not all) transfected cells, the + KTS forms of WT1 localised with splice factors; on the other hand, the − KTS forms localised chiefly in diffuse domains along with transcription factors. These experiments suggested for the first time that WT1 might play a role in splicing as well as transcription. Furthermore, the two different forms of the protein, with and without a three amino acid insert, might have different functions, in agreement with the evidence from human genetics. Since these first studies, the evidence supporting a role for WT1 in RNA metabolism has increased. Firstly, WT1 was shown to be able to bind RNA through the zinc fingers (Van Heyningen et al., 1990). Secondly, through structural modelling, an RNA recognition motif was localised to the n-terminal region of WT1 (Wang et al., 1993). Thirdly, we have been able to show that WT1 can be found in active splice complexes (unpublished observations). These findings raise the possibility that regulated RNA splicing plays a crucial role in the normal development of the genitourinary system, and that abnormalities in such splicing control can lead to a variety of developmental defects including tumours. We are now trying to identify the RNA targets that might interact with and be regulated by WT1. Furthermore, we are trying to use new approaches to identify transcriptional targets for WT1 in the process of genitourinary development. In the long run, identification of upstream regulators of WT1 and downstream targets should provide insights into the pathways that regulate genitourinary development. These upstream and downstream genes will also be candidates for other human developmental disorders of the genitourinary system.
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