Eya protein phosphatase activity regulates Six1–Dach–Eya transcriptional effects in mammalian organogenesis
XUE LI1, KENNETH A. OGHI1, JIE ZHANG1, ANNA KRONES1, KEVIN T. BUSH2, CHRISTOPHER K. GLASS3, SANJAY K. NIGAM2, ANEEL K. AGGARWAL4, RICHARD MAAS5, DAVID W. ROSE6 & MICHAEL G. ROSENFELD1
1 Howard Hughes Medical Institute, School and Department of Medicine, UCSD, 9500 Gilman Drive, Room 345, La Jolla, California 92093-0648, USA
2 Departments of Medicine and Pediatrics, School and Department of Medicine, UCSD, 9500 Gilman Drive, La Jolla, California 92093-0693, USA
3 Department of Cellular and Molecular Medicine, School and Department of Medicine, UCSD, 9500 Gilman Drive, La Jolla, California 92093-0651, USA
4 Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, New York 10029-6574, USA
5 Department of Medicine/Division of Genetics, Brigham and Women's Hospital/Harvard Medical School, 20 Shattuck Street, Thorn 1010 Boston, Massachusetts 02115, USA
6 Department of Medicine, University of California, San Diego 9500, Gilman Drive, La Jolla, California 92093-0673, USA
Correspondence and requests for materials should be addressed to X.L. (seli@ucsd.edu) or M.G.R. (mrosenfeld@ucsd.edu).
The precise mechanistic relationship between gene activation and repression events is a central question in mammalian organogenesis, as exemplified by the evolutionarily conserved sine oculis (Six), eyes absent (Eya) and dachshund (Dach) network of genetically interacting proteins. Here, we report that Six1 is required for the development of murine kidney, muscle and inner ear, and that it exhibits synergistic genetic interactions with Eya factors. We demonstrate that the Eya family has a protein phosphatase function, and that its enzymatic activity is required for regulating genes encoding growth control and signalling molecules, modulating precursor cell proliferation. The phosphatase function of Eya switches the function of Six1–Dach from repression to activation, causing transcriptional activation through recruitment of co-activators. The gene-specific recruitment of a co-activator with intrinsic phosphatase activity provides a molecular mechanism for activation of specific gene targets, including those regulating precursor cell proliferation and survival in mammalian organogenesis.
Transcriptional repression and activation of genes control essential cellular functions including cell proliferation, differentiation and cell death during organogenesis. Genetic studies in Drosophila, for instance, have identified a synergistic nuclear complex consisting of sine oculis (so) DNA-binding homeodomain factor1 and eyes absent (eya)2 and dachshund (dac)3 nuclear cofactors, mutations of which lead to the failure of eye formation, and ectopic expression of which leads to additional eye formation4, 5. The highly conserved vertebrate homologues Six1–6 (ref. 6), Eya1–4 (ref. 7) and Dach1–2/Ski/Sno (ref. 8) respectively, are co-expressed in multiple organs, including eye, inner ear, pituitary gland, muscle and kidney, and thereby provide an excellent model system to study transcriptional regulatory mechanisms during organogenesis. Indeed, Dach2, Eya2 and Six1 synergistically regulate myogenesis in chicken somite culture9.
Both Eya and Dach have been proposed to function as cofactors for Six, and genetic studies in Drosophila have demonstrated synergistic interactions between so, eya and dac during eye development10, 11. Eya has no apparent DNA-binding activity and is translocated from the cytoplasm to the nucleus by Six proteins, and serves as a co-activator of Six in the regulation of downstream genes12. In contrast, Dach is closely related to Ski/Sno8, 13, which are known transcription co-repressors that form strong repression complexes with N-CoR, Sin3A and members of histone deacetylases in order to functionally repress downstream gene expression14.
Six6, together with Dach, represses the cyclin-dependent protein kinase inhibitor p27kip1, thus controlling retinal and pituitary precursor cell proliferation14. Six3, which is closely related to Six6, also functions as a transcriptional repressor, interacting with the Groucho/Tle co-repressor families15, and it is essential for eye and other rostral structure development in mice by means of direct repression of Wnt genes16. Thus, through interactions with specific co-repressors, Six3 and Six6 function as evolutionarily conserved tissue-specific transcriptional repressors required for precursor cell proliferation and differentiation.
Here we demonstrate genetic interactions between Six and Eya factors that regulate precursor cell proliferation and survival during mammalian organogenesis. Our data provide initial evidence for gene-specific recruitment of a co-activator (Eya) with essential intrinsic protein phosphatase activity that is required for regulation of specific gene targets controlling precursor cell proliferation and survival during mammalian organogenesis.
Six1 regulates precursor cell proliferation and survival
On the basis of the actions of Six6 and Six3, we investigated the potential role of Six1 in the target tissues in which it is highly expressed, including kidney, otic placode, skeletal muscle, pituitary and developing nasal structures. This was achieved by generating mice null for the Six1 genomic locus using homologous recombination in embryonic stem cells by replacing the functionally conserved Six domain (SD) and DNA-binding homeodomain (HD) with the IRES-LacZ and PGK-Neo sequences14. This results in loss of Six1 transcripts and expression of LacZ, which recapitulates the endogenous Six1 expression pattern (Fig. 1a; see also Supplementary Fig. 1). Profound effects were noted in virtually all organs in which Six1 is expressed, including a failure of renal organogenesis with variable penetrance; that is, from virtual absence of kidneys to a marked, often asymmetrical reduction of kidney size (Fig. 1b). Immunohistochemical analyses of the rudimentary kidneys from Six1-/- mutants revealed no obvious aberrancy of the branching process17 (data not shown). Muscle development was also profoundly affected, exemplified by a marked reduction in 5-bromo-4-chloro-3-indolyl--D-galactoside (X-gal) staining of forelimb bud (Fig. 1c). Indeed, most migratory hypaxial muscles, including those of forelimbs, the diaphragm and the tongue were missing in mutant embryos (Figs 1d and 2a). Hindlimb muscles were less affected, especially at the proximal region (see below, and data not shown). All early markers of muscle development (see below) appeared normally, in accordance with an independent analysis18, but the number of potential Six1-expressing cells (LacZ+) was markedly decreased (Figs 1c, 2a and 3d). There was also a defect of nasal development, but despite strong expression of Six1, the anterior pituitary showed only a minimal (<10%) decrease in size at low penetrance in Six1-/- mice (see below, and data not shown). In addition, there was invariably severe rib-cage deformation, often with the fusing of distal rib cartilage (Fig. 1e, panels 1–4) and virtual loss of the inner ear structures (Fig. 1e, panels 5–8).
Figure 1 Six1 is required for the development of multiple organs. Full legend
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Figure 2 Molecular analyses of the developmental muscle and kidney defects. Full legend
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Figure 3 Decreased cell proliferation, increased cell death and diminished c-Myc gene expression. Full legend
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On the basis of these data it became important to define the molecular basis of Six1-induced alterations in target tissue development. We therefore analysed expression of key markers, including Pax3 (ref. 19), Lbx1 (refs 20–22), c-Met and Hgf (ref. 23), and the results demonstrated normal cellular expression of all of these markers but severe reduction of muscle precursor cells, consistent with the X-gal staining pattern. Notably, the expression level of Gdnf24 and Six2 (ref. 25) per cell was reduced compared with a heterozygous littermate (Fig. 2a). Marker genes, including Eya1 (ref. 25), Pax2 (ref. 26), c-Ret27 and Wt1 (ref. 28), as well as Wnt genes29, displayed normal cellular expression during early kidney organogenesis at embryonic day (E)10.5 and E11.0 (ref. 30) (Fig. 2b, c), but the expression level of Gdnf and Six2 per cell was reduced, as seen in limb muscle, and Lbx2 was diminished. Together, our results suggest a proliferation/survival defect during both muscle and kidney development in Six1-/- mutant mice. In order to investigate these possibilities, we evaluated the rate of cell proliferation by 5-bromodeoxyuridine (BrdU) labelling in vivo at E9.5, E10.5 and E11.5, finding that, in affected tissues, there was a profound decrease (60.5% compared with 21.5%) in cells exhibiting BrdU incorporation (Fig. 3a and data not shown). A TdT-mediated dUTP nick end labelling (TUNEL) assay revealed an increased level of apoptosis in Six1-/- mutant mice in regions of hypaxial muscle precursors (fourfold) and developing kidney (3.5-fold) (Fig. 3a), consistent with results for the eye imaginal disc of Drosophila with the so mutation1.
To examine further whether these Six1-dependent proliferation events were cell autonomous, we used a single-cell nuclear microinjection assay. Nuclear microinjection of purified IgG against Six1 or Eya3 into C2C12 cells—a murine myoblast cell line that expresses several members of the Six/Eya/Dach family, including Six1 and Eya3—inhibited BrdU incorporation, indicating that both factors were required for effective cell-autonomous proliferation of C2C12 cells (Figs 3b and 4). To confirm these findings, we designed and tested small interfering (si)RNAs against specific murine sequences of Six1 and Eya3. We found that microinjecting the optimal siRNAs directly into the nucleus resulted in consistently efficacious knockdown of RNA transcripts within 24 h of siRNA injection. The targeted transcripts were undetectable by polymerase chain reaction with reverse transcription (RT–PCR) using RNA from cells injected with specific siRNAs, but not cells injected with control siRNAs (Supplementary Fig. 2a). The specificity of the effect was confirmed by 'rescue' with microinjection of purified bacterially expressed holoproteins (see below, Fig. 4i, and data not shown). Both Six1 and Eya3 siRNAs inhibited proliferation of C2C12 cells (Fig. 3c). In contrast, using AIF-1 staining as a marker of apoptosis, anti-Six1 or anti-Eya3 IgGs or specific siRNAs did not cause detectable apoptosis of C2C12 cells (data not shown), indicating a major cell-autonomous role of Six1 and Eya3 in proliferation regulation in these cells.
Figure 4 Eya has intrinsic phosphatase activity and this is required for Six1-mediated gene activation and cell proliferation. Full legend
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Because of the limited material available for the study of early organogenesis, our initial efforts to determine potential Six1 gene targets used C2C12 cells after transient overexpression of either VP16–Six1 or engrailed repressor domain–Six1 fusion proteins. Transiently transfected cells were sorted based on the co-expression of enhanced green fluorescent protein (EGFP) marker. Gene expression profiles of these cell populations were analysed using Affymetrix Genechips (murine 74Av2). This analysis provided a number of potential targets, of which we evaluated 50 of the most statistically significant candidates, including Gdnf, Six2 and c-Myc, by in situ hybridization of E11.0 and E13.5 Six1-/-mutant and wild-type littermate controls. Expression of c-Myc in the mutant was virtually absent in both limb-muscle precursor cells and developing kidney precursor cells (E11.0, E13.5) (Fig. 3d). Expression of Pax3 and Pax2 in adjacent sections provided confirmation of the expression of both transcripts in the same muscle and kidney precursor cells that failed to express c-Myc (Fig. 3d), consistent with the functions of the Myc and Gdnf genes during kidney organogenesis24, 31, 32.
To investigate whether c-Myc and Gdnf could be direct Six1 target genes, we first analysed the promoter sequences of both genes, finding that the c-Myc promoter harbours a Six consensus site (ATCCTGA) immediately 3' to the E2F site (Fig. 3e). Similarly, three perfect consensus Six1 sites were identified in the first intron of the Gdnf gene. To evaluate the potential roles of the Six1 gene, we used the single-cell nuclear microinjection assay33-35, finding that the expression of both c-Myc and Gdnf reporters was dependent on the expression of Six1 (Fig. 3e, and data not shown), as injection of anti-Six1 IgG decreased the basal reporter gene expression in C2C12 cells. Furthermore, chromatin immunoprecipitation (ChIP) in C2C12 cells demonstrated that both Six1 and Eya1/3 were present on c-Myc and Gdnf regulatory sequences (Fig. 3f).
Functions of Eya phosphatase activity
Because So and Eya synergistically induce eye formation in Drosophila, where genetic linkage of so–eya–dac is well established10, 11, we wished to determine the roles of Eya and Dach in Six1-mediated proliferation events. Co-transfection analysis using the well-established Six1-responsive myogenin promoter18 showed that Six1 actively repressed reporter gene expression in heterologous 293 cells, whereas expression of Eya3 reversed repression and displayed some degree of activation above baseline levels (Fig. 4a), suggesting a functional interaction between Six1 and Eya3 for downstream gene activation.
In order to study the mechanism of the effects of Eya3, we first analysed the primary amino acid sequence of all known members of the Eya family. These sequences contained a consensus of two sequence motifs corresponding to the haloacid dehalogenase (HAD) family of phosphohydrolases, which is composed of phosphatases, P-type ATPases, L-2-HADs and epoxide hydrolase, among others36, 37 (Fig. 4b). The HAD family is characterized by a DXXX(T/V) (where represents a hydrophobic residue) motif near the amino terminus, with the first aspartate acting as a phosphoryl acceptor during substrate dephosphorylation (Fig. 4b). The fourth residue in this motif is commonly an aspartate in phosphatases (DXDX(T/V)), a threonine in P-type ATPases (DKTGT) and a tyrosine in L-2-HADs37. In addition, phosphatase and ATPases contain a conserved GDGXXD motif near the carboxy terminus, whereas HADs contain a different SSXXXD sequence. The motifs at the N and C termini of an analogous Eya conserved domain (WDLDETI and GDGVEE) suggest a phosphatase function, akin to that of phosphatase members of this family, such as phosphoserine phosphatase (PSP), FCP1 and small CTD phosphatases (SCPs)37-39.
PSP catalyses the dephosphorylation of L-phosphoserine in the biosynthetic pathway for L-serine37, whereas SCPs and FCP1 dephosphorylate the RNA polymerase II (RNAP II) C-terminal domain (CTD) during transcriptional regulation38-40. The crystal structure of PSP reveals that the first aspartate (indicated in bold font) in the N-terminal motif (DXDX(T/V)) is responsible for nucleophilic attack on the phosphate, whereas the second aspartate (DXDX(T/V)) stabilizes the leaving group during the dephosphorylation reaction41. The aspartates in the C-terminal motif (GDGXXD) facilitate the coordination of the Mg2+ ion in the active site. Correspondingly, mutation of one or more of these conserved aspartates in Eya is expected to cause a loss of phosphatase activity, as in the case of PSP and FCP1 (refs 37, 38–39).
To test experimentally this putative enzymatic activity of Eya, we first used the artificial phosphatase substrate p-nitrophenylphosphate (pNPP) (Fig. 4c), and showed that the purified, bacterially expressed Eya1 and Eya3 holoproteins indeed exhibit phosphatase activity, and that a single point mutation of the first aspartate to alanine (Eya3(mut)) in the DXDX(T/V) catalytic motif abolishes this activity (Fig. 4c, e, and data not shown). Furthermore, Eya3 phosphatase displayed dual specificity in vitro, using phosphotheronine/serine and phosphotyrosine peptide substrates (Fig. 4d). Similar to SCPs and FCP1, Eya1 and Eya3 could dephosphorylate purified RNAP II CTD polypeptide labelled by phosphorylation in vitro with ERK 1/2 (Fig. 4e, and data not shown). Kinetic parameters of Eya3 were assessed by steady-state measurements of the rate of pNP production as a function of pNPP concentration (7.5–70 mM) (Fig. 4f). A double reciprocal plot of initial 1/velocity versus 1/[pNPP] fitted well to a straight line (R2 = 0.9975) and yielded Km (Michaelis constant), Kcat (catalytic rate constant) and Vmax (velocity of enzyme-catalysed reaction at infinite concentration of substrate) values of 25 mM, 0.074 s-1 and 119 mM s-1, respectively. Compared with known CTD phosphatases, the turnover number is approximately 37-fold higher than that reported for pNPP hydrolysis by Saccharomyces cerevisiae FCP1 (Kcat of 0.002 s-1) and about 27-fold lower than that for Schizosaccharomyces pombe FCP1 (Kcat of 2 s-1). The Km for pNPP binding is comparable to that of S. pombe FCP1 (Km of 19 mM) and about 2.5-fold lower than that of S. cerevisiae FCP1 (Km of 60 mM). In all, the catalytic efficiency of Eya1 (Kcat/Km) on pNPP substrate is in the range of the related members of this specific family of phosphatases, including that of S. cerevisiae and S. pombe FCP1 (refs 38, 40).
To determine whether this enzymatic activity might be functionally required for the effects of Eya3 on gene transcription, we evaluated the ability of Eya3 to overcome the inhibitory actions of a Gal4–Six1 fusion protein on a UAS-dependent reporter, using the single-cell nuclear microinjection assay. Notably, wild-type Eya3 protein fully reversed the inhibitory actions of Gal4–Six1, whereas Eya3(mut) failed to do so (Fig. 4g). Similarly, Six1 exerted a repressive effect on a reporter driven by Six1-responsive elements, and this effect was fully reversed by the actions of Eya3, but not by Eya3(mut) (Fig. 4h). Immunoprecipitation and nuclear translocation analyses provided evidence that the point mutation (Eya3(mut)) did not disrupt Six1–Eya3 or Eya3–Dach1 interactions (Fig. 5h, i, and data not shown).
Figure 5 Eya3 phosphatase activity is required to recruit CBP and relieve Dach-mediated repression. Full legend
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These data raised the possibility that Eya phosphatase activity is required to regulate cell proliferation. Using C2C12 cells as a model, we evaluated the effect of the phosphatase activity using anti-Eya3 IgG or Eya3 siRNAs. Both treatments inhibited C2C12 cell proliferation as assessed by BrdU incorporation, and this was entirely reversed by co-injection of Eya3 wild-type protein; however, the Eya3(mut) protein failed to rescue the block to proliferation (Fig. 4i). These data suggest that Eya phosphatase activity is essential for regulating precursor cell proliferation.
A mammalian two-hybrid assay using the myogenin Six1-regulated reporter revealed that although Dach1 co-expression enhanced Six1-dependent repression, VP16–Dach1 acted as a strong activator, confirming a functional interaction between Six1 and Dach1 (Fig. 5a). To investigate the potential role of Eya in Six–Dach-mediated repression, we used single-cell nuclear microinjection experiments, finding that a Gal4–Dach fusion protein, together with Six1, strongly repressed UAS-tk reporter gene expression. Co-expression of wild-type Eya3 protein reversed this effect (Fig. 5b). In contrast, Eya3(mut) failed to prevent the repression activity (Fig. 5b), suggesting that Eya phosphatase function is required to reverse Dach–Six-mediated repression.
We therefore sought to evaluate a potential functional role of Dach1 in C2C12 cells. Nuclear microinjection of anti-Dach1 IgG or Dach1 siRNA revealed that Dach1 was required for effective serum-stimulated proliferation (Fig. 5c, see also Supplementary Fig. 2a and data not shown). This raised the possibility that both Dach and Eya are required for Six1-mediated gene activation. Therefore, we first used ChIP assays to examine whether both Dach and Eya were present on the Six target genes in C2C12 cells. Using IgGs that recognized both Eya1 and Eya3 or Dach1 and Dach2, we found that Six1 along with Eya1/3 and Dach1/2 were present on both c-Myc and Gdnf regulatory regions (Fig. 5d, and data not shown). To ascertain co-recruitment, a sequential ChIP analysis was performed35, revealing co-occupancy of Six1 with Dach1/2 and Eya3, as well as co-occupancy of Eya1/3 and Dach1/2, on both c-Myc and Gdnf regulatory regions (Fig. 5e, and data not shown). We then tested whether Dach has a role in gene activation using single-cell nuclear microinjection assays in C2C12 cells. We found that a specific Dach1 siRNA inhibited reporter gene expression driven by Six1-responsive elements comparable to that observed with anti-Six1 IgG (Fig. 5f), indicating that Dach1 is required for activation of Six1 target genes. These results are consistent with the finding that Dach and Eya can interact with CREB-binding protein (CBP) and synergistically enhance Gal4–Six5 function on a UAS reporter in a transient transfection assay42.
The requirement of the Dach1 co-repressor to activate transcription prompted the question of whether Eya phosphatase activity is required to modulate the recruitment of other co-repressors or co-activators to Six target genes. To begin to investigate this issue, we transfected a reporter that was dependent on a Six1-response element into 293 cells, along with expression vectors encoding Six1, Dach1 and either wild-type Eya3 or Eya3(mut) (Supplementary Fig. 2b), and used ChIP to evaluate the presence of co-repressors and co-activators. In cells overexpressing wild-type Eya3, the CBP co-activator was recruited along with gene activation, as indicated by recruitment of polymerase II (Fig. 5g). In contrast, Eya3(mut), although expressed at a comparable level, failed to permit recruitment of CBP and polymerase II (Fig. 5g, see also Supplementary Fig. 2b). Therefore, the phosphatase activity of Eya is required to switch Six1 function from repression to activation, permitting recruitment of co-activators, including CBP.
Six1 and Eya1 synergistically regulate organogenesis
We wished to assess further the Eya–Six interactions in a genetic model. Because Eya1-/- mice represent the best-characterized mutant model30, the mutant phenotype of which clearly resembles that of Six1-/- mice in renal and otic development, we elected to cross Six1+/- and Eya1+/- mice. Eya1+/- Six1+/- double heterozygous mice were crossed to obtain Eya1-/- Six1-/- mice. We found that the double heterozygous mice have a defect in kidney development (Fig. 6b), which is not observed in single heterozygotes for either gene deletion alone, suggesting that Six1 and Eya1 act in the same genetic pathway. Notably, there is a complete absence of all hypaxial muscle in Six1-/- Eya-/- double-gene-deleted mice and severe reduction of epaxial muscle (Fig. 6a, and data not shown), a phenotype resembling that of Myog-/-, MyoD-/- Myf5-/- and Pax3-/- Myf5-/- mutants43-46. Intriguingly, although mutation of Six1 or Eya1 has minimal or no effect on pituitary development, mice with both genes deleted have a pituitary that is approximately 5–10-fold smaller by volume than the wild-type gland (Fig. 6a), an indication that Six1 and Eya1 also function in distinct pathways. Together these data provide genetic evidence of Six–Eya interactions during vertebrate organogenesis.
Figure 6 Genetic interaction between Six1 and Eya1 in regulating muscle, pituitary and kidney development. Full legend
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Discussion
In addition to the large repertoire of tissue-specific, DNA-binding transcription factors, a large, intricately regulated network of co-activator and co-repressor complexes combinatorially modulate transcription in a tissue- and promoter-specific fashion47-49. The well-defined genetic relationship between Six–Eya–Dach has been exploited here, revealing a novel aspect of this co-regulatory network. We have demonstrated that the ability of Six1 to act both as a repressor or activator is based, at least in part, on the recruitment of opposing cofactors; that is, the Dach co-repressors and the Eya co-activators. Eya, however, can act to reverse Dach co-repressor function on the basis of its intrinsic phosphatase activity, allowing it to function as a required co-activator and to permit the recruitment of other co-activators, including CBP. The Six–Eya–Dach regulatory network thereby defines a molecular mechanism by which a recruited co-activator with phosphatase function can serve to derepress target genes (Fig. 6c). Thus, as well as the large number of cellular processes regulated by diverse protein phosphatases50, a phosphatase can also serve as a promoter-specific transcriptional co-activator. Our results provide a conceptual framework for understanding the Six–Eya–Dach genetic interactions that are likely to be prototypic of other co-activator/co-repressor strategies in mammalian organogenesis.
Methods
Generation of Six1-/- mice The procedure for generating Six1-/- mice was essentially the same as described previously14. Briefly, targeting construct was generated by replacing partial Six domain and homeodomain with IRES-LacZ/PGK-neo sequence of Six1 genomic locus from a 129sv genetic background. Five independent R1 embryonic stem clones (a gift of J. D. Marth), of which three were used for generating knockout mice, were identified by Southern blot, screening with both 5' and 3' outside probes. All three lines showed identical phenotypes.
Antibodies and experimental procedures Immunohistochemistry, in situ hybridization, skeletal preparation and paint filling were carried out essentially as described14. The anti-Eya1, anti-Six1 (Santa Cruz), and anti-Eya3 (generated by immunizing rabbits with a synthetic peptide (KDADDQARKNMTVKNRGK)) antibodies were specific on western blot analysis. All antisera were purified for both single-cell nuclear microinjection assay and ChIP assay.
BrdU and TUNEL assays BrdU and TUNEL assays were performed as described previously14. Briefly, pregnant female mice were injected with 0.1 mg g-1 body weight of BrdU/PBS. Embryos were isolated and fixed in 10% neutral buffered formalin overnight. Ten-micrometre cryostat sections were used for both BrdU staining and TUNEL assays. All sections were counter stained with 4,6-diamidino-2-phenylindole (DAPI) for cell counting or photography.
Single-cell nuclear microinjection and ChIP assays The single-cell nuclear microinjection and ChIP assays were performed as described with >300 cells injected for each point14. Six1, Eya3 and Dach1 siRNAs correspond to DNA sequences 5'-AAGAACGAGAGCGTGCTCAAG-3', 5'-AACAGTGATGCTGAGACCACA-3' and 5'-AAAGTGGCTTCCTTTACGGTG-3', respectively. Nonspecific siRNA is from Dharmacon. PCR primers for c-Myc promoter and control primers corresponding to the p27Kip1 coding region were as described previously14. Primers used for Six1-dependent reporters were 5'-CTTTATGTTTTTGGCGTCTTCCAT-3' and 5'-AATGTATCTTATGGTACTGTAAC-3'. The negative control primers corresponded to 2 kilobases downstream of the Six1 elements, and the sequences were 5'-AGCCATACCACATTTGTAGAGG-3' and 5'-GACGATAGTCATGCCCCGCG-3'. Gdnf primers were 5'-TTGTGACTCTTGAGAAGGGTG-3' and 5'-TAGCCAGAGCAATTCGACAAC-3'. Proliferation assay after single-cell injection was done by measuring BrdU immunoactivity as described previously34.
In vitro phosphatase assays In vitro phosphatase assays were the same as described previously39. The Eya3 phosphatase kinetic parameters were estimated using pNPP substrate ranging from 7.5 to 70 mM40. Briefly, the reaction mixture containing 2.5 µg purified Eya3 protein in 200 µl phosphatase buffer (50 mM Tris-HCl, pH 5.3, 10 mM MgCl2, 10% glycerol, 0.5 mM dithiothreitol) and pNPP substrate was incubated for 60 min at 37 °C. The reaction was stopped by adding 800 µl of 0.2 M NaOH and 50 mM EDTA, and the pNP production was determined by measuring A410 and standard. Phosphate release from the phosphothreonine (pT; KRpTIRR), phosphoserine (pS; RRApSVA), or phosphotyrosine peptides (pY1; RRLIEDAEpYAARG, pY2; TSTEPQpYQPGENL; Upstate Biotech) was assayed as described39 according to the manufacturer's suggestion.
Supplementary information accompanies this paper.
