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The Wilms' tumor gene Wt1 is required for normal development of the retina

2002; Springer Nature; Volume: 21; Issue: 6 Linguagem: Inglês

10.1093/emboj/21.6.1398

1460-2075

Kay‐Dietrich Wagner, Nicole Wagner, Valérie Vidal, Gunnar Schley, Dagmar Wilhelm, Andreas Schedl, Christoph Englert, Holger Scholz,

Pluripotent Stem Cells Research

Article15 March 2002free access The Wilms' tumor gene Wt1 is required for normal development of the retina Kay-Dietrich Wagner Kay-Dietrich Wagner Johannes-Müller-Institut für Physiologie, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany Search for more papers by this author Nicole Wagner Nicole Wagner Medizinische Klinik I, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany Search for more papers by this author Valerie P.I. Vidal Valerie P.I. Vidal Developmental Genetics Group, Max-Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Germany Present address: University of Newcastle, Human Molecular Genetics Unit, Newcastle upon Tyne, NE1 7RU UK Search for more papers by this author Gunnar Schley Gunnar Schley Johannes-Müller-Institut für Physiologie, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany Search for more papers by this author Dagmar Wilhelm Dagmar Wilhelm Forschungszentrum Karlsruhe, Institut für Toxikologie und Genetik, Karlsruhe, Germany Search for more papers by this author Andreas Schedl Andreas Schedl Developmental Genetics Group, Max-Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Germany Present address: University of Newcastle, Human Molecular Genetics Unit, Newcastle upon Tyne, NE1 7RU UK Search for more papers by this author Christoph Englert Christoph Englert Forschungszentrum Karlsruhe, Institut für Toxikologie und Genetik, Karlsruhe, Germany Present address: EPIDAUROS Biotechnologie AG, Am Neuland 1, D-82347 Bernried, Germany Search for more papers by this author Holger Scholz Corresponding Author Holger Scholz Johannes-Müller-Institut für Physiologie, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany Search for more papers by this author Kay-Dietrich Wagner Kay-Dietrich Wagner Johannes-Müller-Institut für Physiologie, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany Search for more papers by this author Nicole Wagner Nicole Wagner Medizinische Klinik I, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany Search for more papers by this author Valerie P.I. Vidal Valerie P.I. Vidal Developmental Genetics Group, Max-Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Germany Present address: University of Newcastle, Human Molecular Genetics Unit, Newcastle upon Tyne, NE1 7RU UK Search for more papers by this author Gunnar Schley Gunnar Schley Johannes-Müller-Institut für Physiologie, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany Search for more papers by this author Dagmar Wilhelm Dagmar Wilhelm Forschungszentrum Karlsruhe, Institut für Toxikologie und Genetik, Karlsruhe, Germany Search for more papers by this author Andreas Schedl Andreas Schedl Developmental Genetics Group, Max-Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Germany Present address: University of Newcastle, Human Molecular Genetics Unit, Newcastle upon Tyne, NE1 7RU UK Search for more papers by this author Christoph Englert Christoph Englert Forschungszentrum Karlsruhe, Institut für Toxikologie und Genetik, Karlsruhe, Germany Present address: EPIDAUROS Biotechnologie AG, Am Neuland 1, D-82347 Bernried, Germany Search for more papers by this author Holger Scholz Corresponding Author Holger Scholz Johannes-Müller-Institut für Physiologie, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany Search for more papers by this author Author Information Kay-Dietrich Wagner1, Nicole Wagner2, Valerie P.I. Vidal3,4, Gunnar Schley1, Dagmar Wilhelm5, Andreas Schedl3,4, Christoph Englert5,6 and Holger Scholz 1 1Johannes-Müller-Institut für Physiologie, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany 2Medizinische Klinik I, Medizinische Fakultät Charité, Humboldt-Universität, Berlin, Germany 3Developmental Genetics Group, Max-Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Germany 4Present address: University of Newcastle, Human Molecular Genetics Unit, Newcastle upon Tyne, NE1 7RU UK 5Forschungszentrum Karlsruhe, Institut für Toxikologie und Genetik, Karlsruhe, Germany 6Present address: EPIDAUROS Biotechnologie AG, Am Neuland 1, D-82347 Bernried, Germany ‡K.-D.Wagner and N.Wagner contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1398-1405https://doi.org/10.1093/emboj/21.6.1398 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Wilms' tumor gene Wt1 is known for its important functions during genitourinary and mesothelial formation. Here we show that Wt1 is necessary for neuronal development in the vertebrate retina. Mouse embryos with targeted disruption of Wt1 exhibit remarkably thinner retinas than age-matched wild-type animals. A large fraction of retinal ganglion cells is lost by apoptosis, and the growth of optic nerve fibers is severely disturbed. Strikingly, expression of the class IV POU-domain transcription factor Pou4f2 (formerly Brn-3b), which is critical for the survival of most retinal ganglion cells, is lost in Wt1−/− retinas. Forced expression of Wt1 in cultured cells causes an up-regulation of Pou4f2 mRNA. Moreover, the Wt1(−KTS) splice variant can activate a reporter construct carrying 5′-regulatory sequences of the human POU4F2. The lack of Pou4f2 and the ocular defects in Wt1−/− embryos are rescued by transgenic expression of a 280 kb yeast artificial chromosome carrying the human WT1 gene. Taken together, our findings demonstrate a continuous requirement for Wt1 in normal retina formation with a critical role in Pou4f2-dependent ganglion cell differentiation. Introduction The Wilms‘ tumor gene WT1 (Wt1 in mice) was originally identified on the basis of its mutational inactivation in 10–15% of all Wilms’ tumors (Hastie, 1994 and references therein). Wilms' tumor of the kidney (nephroblastoma) is the most common solid childhood malignancy, affecting around 1 in 10 000 children (Matsunaga, 1981). Nephroblastoma is a good example of malignant cell growth caused by a failure of the embryonic renal precursor, the metanephric blastema, to differentiate normally (Bennington and Beckwith, 1975). WT1 encodes a zinc finger protein of the C2H2 type, which shares a high degree of structural homology with the early growth response (egr) family of transcription factors (reviewed in Rauscher, 1993). At least 24 different isoforms of WT1 protein exist, which are generated by a combination of alternative splicing, RNA editing and the use of alternative translation start sites (for a recent review see Lee and Haber, 2001). Experimental evidence suggests that the WT1 splice variant which has three amino acids, lysine–threonine–serine (commonly referred as KTS), inserted between zinc fingers three and four of the molecule, plays a role in RNA processing (Englert et al., 1995b; Larsson et al., 1995; Davies et al., 1998; Landomery et al., 1999). The WT1(−KTS) isoform binds with high affinity to GC- and TC-rich DNA consensus sequences and functions as a transcriptional regulator (reviewed in Lee and Haber, 2001). Candidate target genes that are activated by WT1(−KTS) include the cyclin-dependent kinase inhibitor p21Cip1 (Englert et al., 1997), Amphiregulin (Lee et al., 1999), Vitamin D receptor (Maurer et al., 2001; Wagner et al., 2001) and Podocalyxin (Palmer et al., 2001), amongst others. By generating mouse strains in which the Wt1(−KTS) and (+KTS) variants have been specifically removed, it was demonstrated recently that these two isoforms exert distinct functions during sex determination and nephron formation (Hammes et al., 2001). Besides its tumor suppressor function, Wt1 also exerts important roles in embryonic development. Thus, targeted inactivation of Wt1 in mice caused a lack of formation of the kidneys, gonads, spleen and adrenal glands, in addition to mesothelial defects (Kreidberg et al., 1993; Herzer et al., 1999; Moore et al., 1999). Wt1−/− embryos die in utero, presumably from heart failure due to disturbed cardiac growth (Kreidberg et al., 1993; Moore et al., 1999). Based on its spatio-temporal expression pattern (Pritchard-Jones et al., 1990; Pelletier et al., 1991) and the characteristic phenotype of the Wt1−/− embryos (Kreidberg et al., 1993), Wt1 was proposed to exert its unique role in development by allowing cells to flip back and forward between a mesenchymal and epithelial state (Moore et al., 1999). It is noteworthy that Wt1 was also detected in populations of neurons in the brainstem and spinal cord (Armstrong et al., 1993; Rackley et al., 1993). These latter observations raise the interesting possibility that Wt1 is involved in the differentiation of ectodermally derived neuronal tissues. However, experimental evidence to support a role for Wt1 in neuron development has not yet been provided. Notably, patients with Wilms‘ tumor are at an increased risk for developing ocular disorders including aniridia and, less frequently, optic nerve hypoplasia (Nelson et al., 1984; Bickmore and Hastie, 1989). These eye abnormalities in Wilms’ tumor patients are thought to result from concomitant disruption of the aniridia gene PAX6, which lies telomeric of WT1 on human chromosome 11p13 (Ton et al., 1991). A preliminary report on the expression of Wt1 in the developing eye is remarkable as it points towards a possible role for Wt1 in ocular development (Armstrong et al., 1993). In this study, we addressed the question of whether the Wilms' tumor gene Wt1 is required for the differentiation of neurons in the vertebrate retina. Indeed, our findings demonstrate that Wt1 is a critical gene for normal retina formation with impact on the proliferation of retinal progenitors and ganglion cell development. Results Wt1 mRNA and protein is expressed in the developing retina We examined a total of >80 tissue sections, which were obtained from four different mouse retinas, each at the indicated developmental stages. Representative examples of Wt1 expression in the developing retina identified by in situ mRNA hybridization and immunohistochemistry are shown in Figure 1. Wt1 transcripts were initially detected throughout the neural retina and in the developing lens vesicle of 12-day-old (E12) C57BL/6 (B6) mouse embryos (Figure 1). Between E15 and the day after birth (P1), Wt1 expression became restricted to the presumptive retinal ganglion cell layer and was absent from the retinas of adult mice (Figure 1). Wt1 expression in the developing retinas of mice was confirmed by the detection of WT1 immunoreactivity in the future retinal ganglion cell layer of an autopsied 19-week-gestation human embryo (Figure 1). Figure 1.Representative micrographs of tissue sections of wild-type (Wt1+/+) eyes at different stages of development. Wt1 transcripts are detected by mRNA in situ hybridization in the lens vesicle and retinal neuroblasts of E12 mice (A), as well as in the developing ganglion cell layer of E15 (C) and P1 eyes (D). No Wt1 mRNA is visible in the retinas of adult mice (E) and in retinal tissue of E12 embryos hybridized with digoxigenin-labeled Wt1 sense RNA strand (B). Note, that WT1 protein is detected by immunohistochemistry in the inner portion of the retina obtained at autopsy of a 19-week-gestation human embryo (F). l, lens; nbl, neuroblast layer; gcl, ganglion cell layer. Scale bars, 100 μm. Download figure Download PowerPoint Proliferation of the progenitor cells is impaired in Wt1−/− retinas To examine whether Wt1 is important for normal retinal development, we compared the morphology of wild-type and Wt1−/− eyes at different embryonic stages. Forty retinal sections that were obtained from four different normal and Wt1−/− mutant E12 embryos each [C57BL/6 (B6) mouse strain] were analyzed. Representative tissue sections are shown in Figure 2. Wt1−/− mice at E12 had markedly thinner neuroretinas, which contained significantly fewer cells than those of age-matched Wt1+/+ embryos (Figure 2). Compared with the wild-type animals at E12, immunostaining of proliferating cell nuclear antigen (PCNA) was weaker, and incorporation of 5-bromo-2′-deoxyuridine (BrdU) was decreased in the Wt1−/− retinas (Figure 2). Counting of three retinal sections from BrdU-injected mouse embryos at stage E12 (n = 3 each) revealed that ∼10 times fewer BrdU-positive cells were contained in Wt1−/− than in wild-type retina. Reduced cell numbers were also observed in the E12 retinas of Wt1−/− embryos in the MF1×B6 mouse background (see below). However, the phenotypic changes of the mutant retinas were less severe in MF1×B6 than in C57BL/6 (B6) embryos at stage E12 (data not shown). The retinal morphology of heterozygous (Wt1+/−) embryos at E12 was normal and could not be distinguished from that of wild-type animals (data not shown). Figure 2.Representative (immuno)histology of wild-type (+/+) and Wt1 null mutant (−/−) eyes at E12. HE staining reveals notably thinner retinas in Wt1−/− (E and F) than in wild-type embryos (A and B). Immunofluorescent labeling of PCNA is clearly reduced in the mutant (G) versus Wt1+/+ (C) retina. Incorporation of BrdU as an estimate of DNA synthesis is also decreased in Wt1−/− (H) compared with wild-type (D) eyes. l, lens; nbl, neuroblast layer; rpe, retinal pigment epithelium. Scale bars, 100 μm (A, C–E, G and H) and 50 μm (B and F), respectively. Download figure Download PowerPoint Lack of Wt1 causes defects of retinal ganglion cells and optic nerves Disruption of the Wt1 gene in mice with a C57BL/6 (B6) genetic background caused embryonic lethality around mid-gestation (Kreidberg et al., 1993). To analyze the role of Wt1 in retina formation at later developmental stages, we made use of the MF1×B6 mouse strain, in which a certain percentage of Wt1−/− embryos survive until birth (Herzer et al., 1999). In comparison with wild-type MF1×B6 mice, the eyes of the Wt1−/− mutants were notably reduced in size at E18 (1403 ± 30 μm maximum diameter in Wt1−/− versus 1735 ± 29 μm in Wt1+/+ embryos, n = 5 each, P <0.0001, Wilcoxon test). The eyes of heterozygotes were phenotypically normal and indistinguishable from those of wild-type embryos (data not shown). Ganglion cells are among the first to appear in the embryonic mouse retina, most of them between E13 and E16 (Sidman, 1961; Cepko et al., 1996). To explore whether Wt1 is critical for normal ganglion cell differentiation, we performed serial sections through the eyes of wild-type and Wt1−/− E18 embryos (n = 5 each). Histological examination of hematoxylin–eosin (HE)-stained tissue sections revealed that ∼40% fewer cells were contained in the future ganglion cell layer of the mutant retinas compared with that of Wt1+/+ mice (Figure 3A and D). TUNEL labeling was performed to explore whether the diminished number of ganglion cells was due to premature death in the Wt1−/− retinas. Indeed, the counts of TUNEL-positive cells in the E18 retinas of Wt1−/− embryos were increased ∼4-fold compared with age-matched Wt1+/+ mice (n = 5 each) (Figure 3I). TUNEL labeling of tissue sections indicated that ∼60% of the apoptotic cells were located in the developing ganglion cell layer of the Wt1−/− retinas (Figure 3H). Apoptosis was not enhanced in the Wt1−/− retinas prior to E18 (Figure 3I). Figure 3.Morphology of wild-type (A–C) and Wt1−/− mutant (D–F) retinas at E18. A significant (∼40%) reduction in the number of ganglion cells is detected by HE staining in the E18 retinas of Wt1−/− (D) compared with wild-type (A) embryos. Immunofluorescent labeling of NF200 reveals blind-ending optic nerve fibers exiting from the Wt1−/− eyes (F). NF200 immunoreactivity is absent from cross-sections of optic nerves made at a distance of ∼400 μm beyond the optic disc level (E). For comparison, NF200-positive axon bundles are clearly visible in the optic nerves of wild-type retina (B). Shown are representative results from five different embryos in both groups. gcl, ganglion cell layer; onf, optic nerve fibers. Scale bars, 50 μm (A and D), 100 μm (B and E) and 200 μm (C and F). Representative examples of apoptotic cells identified by TUNEL assay in the E18 retinas of wild-type (G) and Wt1−/− (H) mice. The majority (∼60%) of the TUNEL-positive cells are located in the developing ganglion cell layer of the mutant retina (H). (I) The number of apoptotic cells identified by TUNEL assay in the neural retinas of wild-type (Wt1+/+), null mutant (Wt1−/−) and WT280-YAC transgenic embryos at different stages of development. An ∼4-fold increase in apoptotic cells is counted in mutant versus Wt1+/+ retinas at E18. Note that the average number of apoptotic cells in the developing retina is not significantly different between wild-type and WT280–YAC embryos. Five animals were studied in each group at the indicated time points. Five 10 μm retinal sections were made from each embryo close to the optic disc level. Values shown are means ± SEM. Asterisks indicate statistical significance (P 4-fold by the Wt1(−KTS) variant (Figure 5B). However, the Wt1(+KTS) isoform, which is thought to act at the post-transcriptional level (Englert et al., 1995; Larsson et al., 1995; Davies et al., 1998; Landomery et al., 1999), had no significant effect on reporter gene activity (Figure 5B). These findings show that the Wt1(−KTS) isoform can stimulate gene transcription from the putative POU4F2 promoter, suggesting that POU4F2 is a direct transcriptional downstream target gene of Wt1. Figure 5.(A) Expression of Pou4f2 in HEK293 cells stably transfected either with a Wt1(−KTS) expression construct or with the empty pCB6+ vector. Pou4f2 mRNA levels were quantified by real-time RT–PCR using the light cycler system (Roche Molecular Biochemicals) and normalized for GAPDH transcripts. Shown are the data obtained from four independent clones each of pCB6+ and Wt1(−KTS) transfected cells. Note that stable expression of Wt1(−KTS) increased Pou4f2 mRNA levels in HEK293 cells ∼8-fold. The horizontal bars indicate the mean values in each group. (B) Relative luciferase activities measured in the lysates of U2OS human osteosarcoma cells. U2OS cells were transiently co-transfected with phBrn3bUS3.8, Wt1 expression constructs encoding two different splice variants (+KTS/−KTS isoforms), and a cytomegalovirus-promoter driven β-galactosidase expression vector that was used for normalization of transfection efficiencies. Plasmid phBrn3bUS3.8 contained an ∼3.8 kb EcoRV–XhoI genomic sequence from the 5′-regulatory region of the human Pou4f2 gene (schematic drawing) in the pGL2 basic reporter vector. Values shown are means ± SEM of n = 7 experiments each performed in duplicate. P <0.05 was considered statistically significant (ANOVA). Download figure Download PowerPoint Retinal defects of the Wt1−/− embryos are rescued by transgenic YAC complementation Finally, we investigated whether transgenic delivery of a yeast artificial chromosome (YAC) carrying the human WT1 gene was able to rescue the ocular phenotype of the Wt1 null embryos. We have recently established YAC transgenic mouse lines expressing a LacZ reporter gene under the control of the human WT1 locus (Moore et al., 1998). LacZ expression in these lines recapitulated the endogenous pattern of Wt1 (Moore et al., 1998), including expression in the embryonic retina (A.W.Moore, personal communication). Transgenic lines were generated by pronuclear injection of amplified YAC DNA spanning 280 kb of the human WT1 locus (Moore et al., 1998). Notably, no retinal defects were visible in the WT280 transgenic embryos at E18 (Figure 6). In particular, Pou4f2 expression and optic nerve fiber growth were restored in the eyes of WT280–YAC transgenic embryos (Figure 6). Normal morphology of the WT280–YAC retinas is also reflected in a low number of apoptotic cells, which was not significantly different from that of Wt1+/+ retinas at stage E18 (Figure 3I). Figure 6.Rescue of retinal phenotype by YAC complementation of the human WT1 gene in Wt1−/− E18 embryos. Note that normal histology of the developing retina (A), as well as Pou4f2 immunoreactivity in the ganglion cell layer (B) and optic nerve fiber growth (C), are re-established by transgenic expression of a 280 kb YAC construct carrying the human WT1 gene in Wt1−/− embryos. The eyes of WT280–YAC transgenic embryos are indistinguishable by morphological means from their wild-type counterparts at stage E18. gcl, ganglion cell layer; onf, optic nerve fibers. Scale bars, 50 μm (A), 100 μm (B) and 200 μm (C), respectively. Download figure Download PowerPoint Discussion In this study, we demonstrate for the first time that the Wilms' tumor gene Wt1 encodes a regulator of neuron development. The abnormalities in the Wt1−/− mutant retinas indicate that Wt1 is required for at least two critical functions during retinogensis: (i) proliferation of the progenitor cells; and (ii) development of the retinal ganglion cells. Normal cell proliferation is one major determinant of retinal development. The reduced cell numbers and the weaker PCNA and BrdU stainings of the mutant retinas suggest that Wt1 is important for normal neuroblast division during early retinogenesis. Stimulation of cell proliferation by WT1 was demonstrated previously in hematopoietic cells (Yamagami et al., 1996). Thus, inhibition of endogenous WT1 by treatment with antisense oligonucleotides arrested K562 leukemia cells in the G2/M phase, suggesting that WT1 is critical for normal progression through the cell cycle (Yamagami et al., 1998). On the other hand, inducible expression of Wt1 in osteosarcoma cell lines triggered an initial G1 phase arrest that was followed by apoptosis (Englert et al., 1995a). These findings suggest that the effect of WT1 on cell proliferation and growth may be cell type specific and depend on additional co-factors that are differentially expressed between tissues. It is interesting to note that hypocellularity of the Wt1−/− retinas at E12 was less severe in embryos on a mixed MF1×B6 genetic background than in C57BL/6 (B6) mice (our unpublished observation). This observation is in agreement with our previous study on the role of Wt1 in spleen development, indicating that the penetrance of the Wt1−/− phenotype depends on the existence of one or more modifier gene(s) (Herzer et al., 1999). Severe abnormalities were also found in the Wt1−/− retinas at later stages of development. Consistent with the expression of Wt1 in the inner portion of the Wt1+/+ retina, a significant fraction of cells were lost by apoptosis in the developing retinal ganglion cell layer of mutant E18 embryos. Defects in the remaining ganglion cells in the Wt1−/− retinas are indicated by the failure of these cells to give rise to normally growing optic nerve fiber bundles. This phenotype of the Wt1−/− retinas is reminiscent of the abnormalities seen in mice, which lack the POU-domain transcription factor Pou4f2 (also known as Brn-3b) (Erkman et al., 1996; Gan et al., 1996; Wang et al., 2000). In fact, several important findings of this study suggest that Pou4f2 is among the candidate genes that mediate the effects of Wt1 in retinogenesis. First, Pou4f2 (mRNA and protein) is lost in the Wt1−/− retinas. Secondly, Pou4f2 expression and the retinal defects are rescued by transgenic delivery of the human WT1 gene into Wt1−/− embryos. Thirdly, endogenous Pou4f2 transcripts in human embryonic kidney cells are up-regulated by forced expression of Wt1. And finally, Wt1 can transactivate the promoter of the human POU4F2 gene. Notably, the activity of the POU4F2 promoter was stimulated only by the Wt1(−KTS) isoform, which functions as a transcriptional regulator (reviewed in Lee and Haber, 2001). In contrast, the Wt1(+KTS) isoform, which is thought to be involved in post-transcriptional mRNA processing (Englert et al., 1995b; Larsson et al., 1995; Davies et al., 1998; Landomery et al., 1999), had no significant effect.