Human Molecular Genetics, 2001, Vol. 10, No. 24 2775-2781
© 2001 Oxford University Press
Molecular effects of Eya1 domain mutations causing organ defects in BOR syndrome
McLaughlin Research Institute for Biomedical Sciences, 1520 23rd Street South, Great Falls, MT 59405, USA
Received July 16, 2001; Revised and Accepted September 14, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Eya1 is a critical gene for mammalian organogenesis. Mutations in human EYA1 cause branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by varying combinations of branchial, otic and renal anomalies, whereas deletion of mouse Eya1 results in the absence of multiple organ formation. Eya1 and other Eya gene products share a highly conserved 271 amino acid Eya domain that is required for protein–protein interaction. Recently, several point mutations that result in single amino acid substitutions in the conserved Eya domain region of EYA1 have been identified in BOR patients; however, the molecular and developmental basis of organ defects that occurred in BOR syndrome is unclear. To understand how these point mutations cause disease, we have analyzed the functional importance of these Eya domain missense mutations with respect to protein complex formation and cellular localization. We have demonstrated that these point mutations do not alter protein localization. However, four mutations are crucial for protein–protein interactions in both yeast and mammalian cells. Our results provide insights into the molecular mechanisms of organ defects detected in human syndromes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Eya1 gene product is homologous to that of Drosophila eyes absent (eya) gene, an early regulator for Drosophila eye morphogenesis (1). At present, four mammalian Eya genes have been isolated and these genes regulate mammalian organogenesis (2–8). The Eya gene products contain a divergent N-terminal transactivation domain (9) and a highly conserved 271 amino acid C-terminal Eya domain that participates in protein–protein interactions with So and Dac (10,11), the gene products encoded by the Drosophila sine oculis (so) and dachshund (dac) genes, respectively. When misexpressed, combinations of Eya and So, or Eya and Dac, act synergistically to direct the formation of ectopic eyes in Drosophila (10,11). Genetic analyses in Drosophila indicate that eya is epistatic to so (10).
Mutations in the human EYA1 gene cause branchio-oto-renal (BOR) syndrome and branchio-oto (BO) syndromes, autosomal dominant disorders with incomplete penetrance and variable expressivity characterized by combinations of branchial, otic and renal anomalies (4,12–14). Recently, mutations in the human EYA1 were also detected in patients with congenital cataracts and ocular anterior segment anomalies (15). In humans, mutations are known throughout the protein, including mutations within the conserved Eya domain (4,12,13,15). However, the molecular and developmental basis for organ defects detected in patients is not established.
We have recently generated a null Eya1 allele containing a neo cassette replacing most of the conserved Eya domain region (3). Eya1-null animals lack thymus, ears and kidneys due to defective inductive tissue interactions (3; P.-X.Xu, W.Zheng, R.Maas, H.Peters and X.Xu, manuscript in preparation). In thymic, otic and nephric induction, we demonstrated that the expression of the relevant Six genes, homologous to Drosophila so, depends on Eya1 function (3; P.-X.Xu, W.Zheng, R.Maas, H.Peters and X.Xu, manuscript in preparation). This supports the idea that a Eya–Six regulatory hierarchy similar to that operating in Drosophila eye development has been conserved in mammalian organogenesis. However, it is not clear whether Eya1 physically interacts with the relevant Six gene products during mammalian organ development.
As a first attempt to dissect out the molecular mechanisms of organ defects that occur in human BOR syndrome, we have analyzed the functional importance of specific Eya1 domain missense mutations identified in human syndromes. We have focused on testing whether these mutations affect protein–protein interactions of Eya1 with the relevant Six gene products that are co-expressed with Eya1 during branchial arch system, otic and kidney development. Our studies demonstrate that Eya1 physically interacts with the relevant Six proteins via the conserved Eya domain and four specific Eya domain mutations identified in affected patients disturbed the protein–protein interaction of Eya1 with Six1 or Six2. This for the first time provides insight into the molecular and developmental basis for organ defects detected in BOR syndrome.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Eya1 domain mutations abolish protein–protein interactions of Eya1 with Six proteins in GST pull-down and yeast two-hybrid assays
In order to determine the molecular basis for organ defects that occur in BOR syndrome, we sought to assay the effects of point mutations identified in the conserved Eya1 domain region. In human syndromes, 19 mutations in the EYA1 gene were identified (12,13,15). Among these mutations, five are missense mutations present in the Eya domain region, and all these residues are conserved among the Eya1 gene products of man, mouse and the Drosophila eya gene product (Fig. 1A). In particular, two residues, E330 and L472, are identical in all eya family members isolated so far (Fig. 1A). These suggest that these residues are essential for the structure or function of the Eya1 protein. As a first attempt to dissect the molecular mechanisms of organ defects in BOR patients, we examined whether these five amino acid substitutions identified in affected patients are crucial for Eya1 interaction with Six1 or Six2.
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Six1 and Six2 are members of the murine Six gene family homologous to Drosophila so. Our previous studies have shown that the murine Eya1 is epistatic to Six1 in early branchial arch and otic development, whereas Eya1 is epistatic to Six2 in the metanepharic mesenchyme during early kidney development (3). These suggest that a conserved Eya–Six regulatory pathway is used during mammalian organogenesis. In Drosophila eye imaginal disc, Eya directly interacts with So (10). To test whether similar physical interactions occur between the murine Eya1 and the relevant Six proteins, we first performed GST pull-down interaction assays. The GST–Eya1 fusion protein that contained the Eya domain only (GST–E1D) was tested for its ability to interact with 35S-labeled Six1 or Six2 protein. The GST–E1D fusion protein efficiently pulled down 35S-labeled Six1 and Six2 (Fig. 1B).
Having demonstrated that Eya1 physically interacts with Six1 or Six2 via the conserved Eya domain, we next introduced the five amino acid substitutions found in patients into the murine Eya1 domain and tested their effect on protein–protein interactions by a GST pull-down assay. Interestingly, single amino acid substitutions of E330K, S454P or L472R failed to pull-down 35S-Six1 (Fig. 1B), indicating that these three amino acid residues are crucial for the Eya1–Six1 interaction. In contrast, the amino acid substitutions of G393S and R514G pulled down 35S-Six1 as efficiently as the wild-type GST–Eya1D (Fig. 1B), indicating that these two residues are not essential for direct Eya1–Six1 interaction. Similarly, four amino acid substitutions of E330K, G393S, S454P and L472R also reduced the amount of Six2 pulled down by GST–Eya1D (Fig. 1B). Taken together, these results indicate that at least three amino acid residues, E330, S454 and L472, are specifically crucial for the Eya1 domain to interact with Six1 or Six2, therefore, providing important clues on the molecular basis of the defects detected in the tissues derived from brachial arches, ear and kidney of those BOR patients.
We further examined the specificity of the interactions seen with GST pull-downs using the GAL4 yeast two-hybrid system. We fused the Eya1 domain and its mutants with a GAL4 DNA-binding domain and used this as the ‘bait’. ‘Prey’ was constructed as fusion with the GAL4 transcriptional activation domain. Thus, in vivo interactions between the bait and prey will result in lacZ transcription. Co-transformation of the Eya1 domain bait construct and either the Six1 or Six2 prey construct led to strong lacZ expression (Fig. 1C). However, four amino acid substitutions of E330K, G393S, S454P and L472R failed to activate lacZ expression when co-transformed with either the Six1 or Six2 prey construct. These results further indicate that the four amino acid residues are essential for the association of Eya1 with Six1 or Six2.
The effect of the Eya1 domain mutations on nuclear translocation and complex formation of Eya1 by Six proteins
Since Eya1 possesses a potent transcriptional activation function (9), but cannot bind to DNA as assessed by binding-selection experiments (data not shown), Eya1 could be a multi-functional co-activator conferring a transcriptional activation function upon DNA binding proteins. Previous studies have suggested that Eya can function as a co-activator of Six proteins (16). In order to assay the effect of the Eya domain mutations on the complex formation of Eya1 with Six1 or Six2 in the nucleus, we have analyzed the complex formation of Eya1 with Six proteins in cultured 293 cells. 293 cells, which are derived from human kidney epithelial cells, do not express Eya1, Six1 and Six2 gene products as assayed by western blot and immunohistochemistry. Thus, the cells are suitable for transfection experiments. However, Eya2 was found to be endogenously expressed in the cell line (data not shown).
Next, we investigated the intracellular distribution of Flag–Eya1 full-length protein (Flag–E1F), Flag-Eya1 N-terminal region (Flag–E1N) and Flag–Eya1 domain (Flag–E1D) fusion proteins. The Flag–E1F and Flag–E1D were mainly distributed in the cytoplasm in 293 cells transfected with each of these constructs alone (Fig. 2A and B), Flag–E1N was detected as punctate staining that seemed to be associated with nuclear membrane (Fig. 2A). Consistent with previous observations (16), we have found that Six1 and Six2 are distributed in the nucleus in 293 cells transfected with His–Six1 or His–Six2 (data not shown). Interestingly, when co-transfected with His–Six1, Flag–E1F or Flag–E1D was translocated into the nucleus, whereas Flag–E1N remained as punctate staining (Fig. 2A and B). Similarly, the cytoplasmic distribution of Flag–E1D was not altered when cells were co-transfected with Flag–E1D and His–Six3 (Fig. 2B). Six3 is another member of the Six gene family, but is the homolog of Drosophila optx, not of so (17). These results indicate that Eya1 can be translocated into the nucleus by co-expression of Six1 or Six2, and the nuclear translocation was mediated via the conserved Eya domain.
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To investigate whether the translocated Eya1 protein forms a complex with Six1 or Six2 in the nucleus, we performed an immunoprecipitation analysis. Nuclear extracts from the cells transfected Flag–E1D with His–Six1 or His–Six2 were incubated with anti-Flag antibody, recovered by protein G agarose beads, and then developed by SDS–PAGE followed by western blotting with anti-Six1 or anti-Six2 antibody. Figure 2C shows that the Flag–E1D was co-immunoprecipitated with His–Six1 or His–Six2. Similar results were obtained in Cos7 cells (data not shown). These results indicate that translocated Eya1 protein forms a complex with Six proteins via the conserved Eya domain in the nucleus, strongly supporting the idea that Eya1 may function as a co-activator of Six1 and Six2 during early branchial system, otic and kidney development.
We next investigated whether the missense mutations would alter the protein subcellular localization. We introduced the five mutations in the Flag–E1F and Flag–E1D constructs and each construct was transfected into 293 cells. No significant difference in protein localization was observed between the wild-type and mutant constructs (data not shown), indicating that these mutations do not alter its protein subcellular localization. We then asked whether these mutations affect nuclear translocation of Eya1 by Six1 or Six2. As shown in Figure 3A, intense staining was observed in the nucleus in 293 cells when each of the Flag–E1D mutant constructs were co-transfected with Six1, indicating that all these mutations do not prevent nuclear translocation of the Eya1 protein by Six1. Similar results were observed when each of these mutant constructs was co-transfected with Six2 (data not shown).
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We then asked whether these mutations affected Eya1–Six1 complex formation in the nucleus by co-immunoprecipitation. Nuclear extracts from the cells co-transfected with each of the Flag–Eya1 mutant constructs and His–Six1 were first analyzed for the expression of Eya1 and its mutant forms, and Six1 by western blot (Fig. 3B). The expression level of Eya1 and its mutant forms were similar and Six1 was also expressed at almost similar levels in cells co-transfected either with Flag–Eya1 or its mutant forms. Then the same nuclear extracts were incubated with anti-Flag antibody, recovered by protein G agarose beads, and then developed by SDS–PAGE followed by western blotting with anti-Six1 antibody. Although none of the amino acid substitutions completely abolished the Eya1–Six1 complex formation in the nucleus (Fig. 3B), three amino acid substitutions of E330K, S454P and L472R resulted in decreased nuclear complex formation of Eya1–Six1 (Fig. 3C). This indicates that these mutations influence the formation of Eya1–Six1 complexes in the nucleus. In contrast, when these Flag–Eya1 constructs were co-transfected with His–Six2, no significant reduction was observed by co-immunoprecipitation (Fig. 3B and C). Similar results were obtained when Flag–E1D and its mutants were used for co-immunoprecipitation experiments (data not shown).
Thus, we have demonstrated here that Eya1 can interact with Six1 or Six2 in vitro and in cultured cells via the conserved Eya domain and four Eya domain mutations identified in patients abrogate such interaction in yeast cells. In cultured 293 cells, although none of the five Eya domain mutations either affected nuclear translocation of the Eya1 protein or completely abolished Eya1–Six1 complex formation in the nucleus, three mutations resulted in decreased nuclear complex formation of Eya1–Six1. Similar observations were obtained in other cell lines, including Cos7 cells (data not shown). These observations suggest that there are direct and indirect interactions between Eya and Six proteins. Both direct and indirect interactions can be detected in yeast two-hybrid and cell culture systems, whereas GST pull-down assays can only detect direct interaction. In the GST pull-down assay, the G393R did not show an effect on Eya1–Six interaction. However, it abolished the interaction in the yeast two-hybrid system. This suggests that this mutation destroys the interaction of Eya1 with other unknown protein(s) that probably bridge(s) the association of Eya1 with Six proteins.
Among the missense mutations we tested, S454P (located close to the 
-helix 2) and L472R (within the -helix 2) were identified from typical BOR patients (12), whereas G393S (between -helices 1 and 2) was identified from a patient who had cataracts in addition to renal and otic anomalies (15). In contrast, mutations E330K (close to the -helix 1) and R514G (between -helices 2 and 3) were identified from patients whose clinical defects are limited to the eye (15), indicating that the mutated proteins with an amino acid substitution have a dominant effect especially in the morphogenesis of the eye. These suggest that there may be a genotype–phenotype correlation. The middle part of the Eya1 domain, including the second -helix (Fig. 1A), may be crucial for the interaction of Eya1 with a certain set of factors that are specifically expressed in the brachial region, ear and kidney. In contrast, the N- and C-terminal parts, including the first and third -helices, may be crucial for the interaction of Eya1 with other factors that are specifically expressed in the eye. Since G393S causes cataracts in addition to BOR disease, the region containing this residue may be critical for Eya1 to interact with the factors that are expressed in the eye as well as in the tissues derived from the branchial region, ear and kidney.
The mutation E330K showed an effect on Eya1–Six1 or Eya1–Six2 interaction in our assays; however, none of the assays showed that R514G had any effect on the interaction of Eya1 with Six1 or Six2, indicating that this mutation may have an effect on Eya1–Six1 or Eya1–Six2 via other unknown factor(s) that are not expressed in either yeast or 293 cells. It is also possible that R514G mutation has a tissue-specific effect in the eye. Interestingly, Six5, another member of the Six gene family, has been found to be co-expressed with Eya1 in the developing lens and cornea and mice deficient in Six5 develop cataracts (18,19), suggesting a possible interaction between Eya1 and Six5. Recently, we have also isolated the Six5 gene from our yeast two-hybrid screen and are in the process of testing the effect of R514G on Eya1–Six5 interaction. Nonetheless, our preliminary data support that R514G is a disease-causing mutation, since this mutation abolished the interaction of Eya1 with three other gene products isolated from the yeast two-hybrid screen in yeast cells (W.Zheng, C.Buller, R.Schwanke, X.Xu and P.-X.Xu, unpublished data). Detailed analysis is underway in our laboratory.
Since none of the five Eya domain mutations altered the nuclear translocation of Eya1 by Six, it seems likely that the translocation of Eya1 by Six proteins is not via direct Eya1–Six interaction and is probably regulated by other unknown factor(s), which are endogenously expressed in 293 cells. Several molecules involved in signaling pathways have been isolated in our laboratory, and they can interact simultaneously with Eya1 and Six1 in the yeast two-hybrid system (W.Zheng, C.Buller, R.Schwanke, X.Xu and P.-X.Xu, unpublished data). It is possible that some of these factors bridge Eya1 and Six proteins to translocate Eya1 into the nucleus. Studies to establish these details are underway in our laboratory.
The discrepancy we have observed between the yeast two-hybrid and immunoprecipitation from mammalian cell extracts could be due to the fact that other cofactors which mediate indirect interaction of Eya1–Six are endogenously expressed in 293 cells but not in the yeast cells. We have shown here that the three Eya domain mutations reduced the rate of nuclear complex formation of Eya1–Six1 by immunoprecipitation analysis (Fig. 3). The reduction could have resulted from the elimination of direct Eya1–Six1 interactions by the mutations. However, none of the mutations affected the complex formation of Eya1–Six2, suggesting that the nature of the Eya1–Six complex differs based on the combination of Eya1 with different Six proteins. Eya1 is co-expressed with Six1 in early branchial and otic development, with Six2 during early kidney induction, and with Six5 in developing lens and cornea. It seems likely that the co-activation mechanism varies depending upon the promoters of distinct downstream targets. Eya1 probably associates with various transcriptional factors and integrates their effects into diverse pathways to regulate morphogenesis during distinct organ formation. The Eya domain mutations identified in patients are likely to influence the formation of Eya1–Six complexes, which in turn reduce the expression of downstream targets and cause disease.
Thus, these studies begin to establish biochemical details about the interaction between Eya1, Six or other proteins. Our results provide insights into the molecular mechanisms of organ defects that occurred in human patients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmids and site-directed mutagenesis of Eya1 constructs
Eya1 full-length cDNA or the Eya1 domain region was cloned into either pGEX-2T vector for producing the GST–Eya1 fusion protein, pGBT9 vector for yeast two-hybrid assays or pcDNA3 vector for cell culture analysis.
To introduce the same amino acid substitutions found in BOR patients into the mouse Eya1 domain region, PCR-based mutagenesis was performed using the QuickChangeTM mutagenesis kit (Stratagene). Once mutant colonies were identified, the plasmid DNA was isolated and sequenced through the mutation-containing region. Five mutant cDNAs (E330K, G393S, S454P, L472R and R514G) were constructed in the same manner in either pGEX-2T, pGBT9 or pcDNA3 vector.
In vitro GST pull-down assay
Escherichia coli BL21 transformed with GST or GST–Eya1 fusion vectors were induced with 0.1 mM isopropyl-ß-D-thiogalactopyranoside for 1.5 h at 30°C and GST or GST–Eya1 fusion proteins were purified from glutathione–agarose beads. The full-length 35S-labeled proteins were generated from pBluescript II or pcDNA3 vectors by in vitro translation using TNT T7, T3 or Sp6 coupled transcription/translation system and labeled with 35S-methionine (Promega).
For the pull-down reaction, 2–3 µg of purified individual GST fusion proteins attached to glutathione–agarose beads (20 µl suspension) were incubated with 10 µl of each 35S-Met-labeled protein in a buffer containing 20 mM HEPES (pH 7.7), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 0.05% NP-40, 2 mM DTT and 10% glycerol as described by Takeshita et al. (20). The beads were washed four times with binding buffer, resuspended in 20 µl of SDS–PAGE sample buffer and heated at 95°C for 5 min. Proteins were visualized after SDS–PAGE by autoradiography.
Yeast two-hybrid assays
The MATCHMAKER Gal4 two-hybrid system (Clontech) was used for yeast interaction assays. Eya1 domain region was fused with the GAL4 DNA-binding domain of pGBT9, and Six1 and Six2 were fused with the GAL4 activation domain of pGAD424. Small scale LiAc co-transformations of the plasmid DNAs into SFY526 cells were performed as outlined in the Clontech protocols. ß-Galactosidase colony-lift filter and liquid culture ß-galactosidase assays were performed as described in the Clontech protocols.
Cell culture and transfection assays
293 cells derived from human embryonic kidney fibroblast were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U penicillin/ml and 100 µg streptomycin/ml at 37°C under 6% CO2. Transfections were performed using FuGENETM 6 transfection reagent (Roche) according to the manufacturer’s instructions. Forty-eight hours after the transfection, the cells were used for immunostaining or extract preparation.
Immunostaining of cultured cells
Transfected 293 cells were washed with 1x PBS twice and fixed with 4% paraformaldehyde for 15 min. After fixation, the cells were rinsed three times in 1x PBS for 5 min. Anti-Flag, anti-His, anti-Six1 or anti-Six2 antibody was used as primary antibody. Goat anti-mouse or goat anti-rabbit fluorescent antiserum was used as secondary antibody. The signals were viewed by Confocol microscopy.
Preparation of nuclear and cytoplasmic extracts and western blot
Nuclear and cytoplasmic extracts from the 293 cells were prepared as described previously (21). The protein concentrations of the extracts were determined using a protein assay kit (Bio-Rad).
Proteins were analyzed on 12% SDS–PAGE and transferred to a nitrocellulose membrane. Anti-Flag, anti-His, anti-Six1 or anti-Six2 antibody was used as the primary antibody and HRP-coupled goat anti-mouse or goat anti-rabbit antiserum was used as secondary antibody. An ECL kit (Pierce) was used for detection.
Co-immunoprecipitation
Nuclear extracts were prepared from the 293 cells co-transfected with a Flag–Eya1 expression plasmid, plus either an empty vector, His–Six1/pcDNA3, His–Six2/pcDNA3 or His–Six3/pcDNA3 plasmid. Nuclear extracts (30 µg of proteins) were incubated with anti-Flag antibody or purified mouse IgG (Sigma) as a negative control for co-immunoprecipitation analysis as described by Kawakami et al. (21). Anti-Six1 or anti-Six2 antibody was used for western blot.
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ACKNOWLEDGEMENTS |
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We thank Drs P.Mao and X.Bai for technical assistance, Dr P.Maire for providing the Six1 antibody, and Drs G.Carlson and D.Stephenson for critical reading of the manuscript. This work was supported by NSF 0078246 and NIH grant P20RR 12345-02 (P.-X.X).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 406 454 6033; Fax: +1 406 454 6019; Email: pxu@po.mri.montana.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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 D. Zou, D. Silvius, B. Fritzsch, and P.-X. Xu Eya1 and Six1 are essential for early steps of sensory neurogenesis in mammalian cranial placodes Development, November 15, 2004; 131(22): 5561 - 5572. [Abstract] [Full Text] [PDF]
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 R. Grifone, C. Laclef, F. Spitz, S. Lopez, J. Demignon, J.-E. Guidotti, K. Kawakami, P.-X. Xu, R. Kelly, B. J. Petrof, et al. Six1 and Eya1 Expression Can Reprogram Adult Muscle from the Slow-Twitch Phenotype into the Fast-Twitch Phenotype Mol. Cell. Biol., July 15, 2004; 24(14): 6253 - 6267. [Abstract] [Full Text] [PDF]
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 R. G. Ruf, P.-X. Xu, D. Silvius, E. A. Otto, F. Beekmann, U. T. Muerb, S. Kumar, T. J. Neuhaus, M. J. Kemper, R. M. Raymond Jr., et al. SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1-SIX1-DNA complexes PNAS, May 25, 2004; 101(21): 8090 - 8095. [Abstract] [Full Text] [PDF]
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W. Zheng, L. Huang, Z.-B. Wei, D. Silvius, B. Tang, and P.-X. Xu The role of Six1 in mammalian auditory system development Development, September 1, 2003; 130(17): 3989 - 4000. [Abstract] [Full Text] [PDF]
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P.-X. Xu, W. Zheng, L. Huang, P. Maire, C. Laclef, and D. Silvius Six1 is required for the early organogenesis of mammalian kidney Development, July 15, 2003; 130(14): 3085 - 3094. [Abstract] [Full Text] [PDF]
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C. Laclef, G. Hamard, J. Demignon, E. Souil, C. Houbron, and P. Maire Altered myogenesis in Six1-deficient mice Development, May 15, 2003; 130(10): 2239 - 2252. [Abstract] [Full Text] [PDF]
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