Pitx3 potentiates Nurr1 in dopamine neuron terminal differentiation through release of SMRT-mediated...

531RESEARCH ARTICLE

INTRODUCTIONThe dopaminergic neurons of the meso-diencephalic dopaminergicsystem (mdDA system) have been extensively studied in relation toParkinson’s disease, and pioneering studies have explored thepossibility of using cell replacement therapy with stem cells as afuture treatment (Freed et al., 2001; Chung et al., 2002; Kim et al.,2002; Olanow et al., 2003; Andersson et al., 2006; Hedlund et al.,2008). Ultimately, stem cells could be exploited as an unlimitedsource of transplantable DA neurons. However, in order to engineerstem cells with mdDA characteristics, the need to obtain theappropriate dopaminergic phenotype through correct molecularcoding has been underlined (Chung et al., 2005; Martinat et al.,2006; Smidt et al., 2007). Therefore, much effort has been put intounravelling the multi-step process that produces a genuine mdDAneuronal population in vivo, as this is believed to hold the key to thesuccessful engineering of stem cells in vitro.

One of the key regulators for development of mdDA neurons invivo is the orphan nuclear receptor Nurr1 (Nr4a2), which is crucialfor expression of a set of genes involved in DA metabolism, such astyrosine hydroxylase (Th), vesicular monoamine transporter(Vmat2), dopamine transporter (Dat) and aromatic L-amino aciddecarboxylase (Aadc) (Zetterström et al., 1997; Saucedo-Cardenaset al., 1998; Baffi et al., 1999; Le et al., 1999; Wallén et al., 1999;Smits et al., 2003). Another key factor for the development ofmdDA neurons is the paired-like homeobox gene Pitx3. Because ofits highly exclusive expression in mdDA neurons within the brain,Pitx3 is a selective marker for the generation of transplantable EScells (Zhao et al., 2004; Chung et al., 2005; Hedlund et al., 2008).

Pitx3 serves a unique function in mdDA neurons, highlighted by thedramatic loss of neurons of the substantia nigra in Pitx3-deficientmice (Hwang et al., 2003; Nunes et al., 2003; Van der Munckhof etal., 2003; Smidt et al., 2004), which is preceded by deficient Thexpression during embryonic development (Smidt et al., 2004;Maxwell et al., 2005; Jacobs et al., 2007). Strikingly, although Pitx3is expressed in all mdDA neurons, only mdDA neurons of thesubstantia nigra are affected by the loss of Pitx3, emphasizing thecomplicated role of Pitx3 in mdDA neurons and the diverseproperties of subsets within the mdDA neuronal population.

Despite the fact that both Pitx3 and Nurr1 are crucial for properexpression of Th in mdDA neurons, it has long been assumed thatPitx3 and Nurr1 are components of independent developmentalpathways (Smidt et al., 2000; Chung et al., 2005). However, there isgrowing evidence to suggest that these pathways are interconnectedat a functional level. Studies aimed at constructing functional DAneurons from ES cells, have shown that combined transduction ofNurr1 and Pitx3 promotes the maturation of ES cells into adopaminergic phenotype (Martinat et al., 2006). Importantly, therecent discovery of aldehyde dehydrogenase 2 (Ahd2; Aldh1a1) astranscriptional target of Pitx3 in mdDA neurons has highlighted asignificant similarity between the phenotype of Pitx3- and Nurr1-nullmice (Jacobs et al., 2007). Similar to what was observed in Pitx3–/–

embryos, Ahd2 expression is not detected in the midbrain area ofNurr1-deficient embryos during final differentiation of mdDAneurons (Wallén et al., 1999). These observations strongly suggest afunctional relationship between the homeodomain transcription factorPitx3 and the orphan nuclear receptor Nurr1 in the development ofmdDA neurons. In this study, we deduce the molecular mechanism ofthis functional interplay between Pitx3 and Nurr1 in vivo, withextensive consequences for mdDA development. We establish Pitx3as an essential regulator of Nurr1-mediated transcription in mdDAneurons, which positions Pitx3 as a crucial factor for the specificationof the dopaminergic phenotype both in vivo and in stem cellengineering for future treatment of Parkinson’s disease.

Pitx3 potentiates Nurr1 in dopamine neuron terminaldifferentiation through release of SMRT-mediated repressionFrank M. J. Jacobs, Susan van Erp, Annemarie J. A. van der Linden, Lars von Oerthel, J. Peter H. Burbach andMarten P. Smidt*

In recent years, the meso-diencephalic dopaminergic (mdDA) neurons have been extensively studied for their association withParkinson’s disease. Thus far, specification of the dopaminergic phenotype of mdDA neurons is largely attributed to the orphannuclear receptor Nurr1. In this study, we provide evidence for extensive interplay between Nurr1 and the homeobox transcriptionfactor Pitx3 in vivo. Both Nurr1 and Pitx3 interact with the co-repressor PSF and occupy the promoters of Nurr1 target genes inconcert. Moreover, in vivo expression analysis reveals that Nurr1 alone is not sufficient to drive the dopaminergic phenotype in mdDAneurons but requires Pitx3 for full activation of target gene expression. In the absence of Pitx3, Nurr1 is kept in a repressed statethrough interaction with the co-repressor SMRT. Highly resembling the effect of ligand activation of nuclear receptors, recruitment ofPitx3 modulates the Nurr1 transcriptional complex by decreasing the interaction with SMRT, which acts through HDACs to keeppromoters in a repressed deacetylated state. Indeed, interference with HDAC-mediated repression in Pitx3–/– embryos efficientlyreactivates the expression of Nurr1 target genes, bypassing the necessity for Pitx3. These data position Pitx3 as an essentialpotentiator of Nurr1 in specifying the dopaminergic phenotype, providing novel insights into mechanisms underlying developmentof mdDA neurons in vivo, and the programming of stem cells as a future cell replacement therapy for Parkinson’s disease.

KEY WORDS: DA phenotype, Dat, Dopamine, Parkinson’s disease, VMAT2, mdDA

Development 136, 531-540 (2009) doi:10.1242/dev.029769

Rudolf Magnus Institute of Neuroscience, Department of Neuroscience &Pharmacology, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG,Utrecht, The Netherlands.

*Author for correspondence (e-mail: [email protected])

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MATERIALS AND METHODSCell cultureMN9D-Nurr1Tet On13N (MN9D) cells were cultured and transfected asdescribed previously (Jacobs et al., 2007).

Identification of Pitx3 interacting proteinsMN9D cells were transfected with 0.5 μg Pitx3-pcDNA3.1(–)myc-His oran equivalent molar amount of pcDNA3.1(–)myc-His expression vector andHis-tagged proteins were purified using Ni-NTA magnetic agarose beads(Qiagen) according to manufacturer’s protocol. Purified proteins wereseparated by SDS PAGE and visualized by protein gel silver-staining.Subsequently, the gel was fixed in 50% methanol (2�15 minutes) and 5%methanol (10 minutes), rinsed with water (3�10 seconds), soaked in 10 μMDTT (20 minutes), incubated in 0.1% AgNO3 (20 minutes), rinsed withwater and incubated in developer solution (3% NaCO3, 0.05%formaldehyde) until protein bands were visible. The reaction was stoppedby adding citric acid and the gel was washed in water. Protein bands ofinterest were excised from the gel and subjected to nanoLC-ESI-MS MassSpectrometry analysis (Proteome Factory).

Immunoprecipitation (IP) and western blottingMN9D cells or tissue was homogenized in lysis buffer [50 mM Tris HCl(pH 8), 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 0.2% NP-40, 5%glycerol and 0.5 mM DTT + Complete Protease Inhibitor Cocktail(Roche)], and subjected to IP. Two dishes (10 cm in diameter) of MN9Dcells or five ventral midbrains of E14.5 C57Bl6-Jico embryos were usedfor each IP. Magnetic dynal beads (Invitrogen) were blocked in 0.5%BSA and incubated with antibodies overnight. Lysates were incubatedwith antibody-bound beads for 3 hours at 4°C. Beads were washed in lysisbuffer (five times) and captured using a magnetic separator. Elution wasperformed in elution buffer [50 mM Tris (pH 8.0), 10 mM EDTA, 1%(v/v) SDS and Complete protease inhibitors] at 65°C with frequentvortexing. Proteins were separated on Nupage 4-12% gradient gels(Invitrogen), transferred to Hybond C extra (Amersham) and afterovernight blocking in 5% milk powder in PBS at 4°C the membraneswere incubated with antibodies in PBS-T [PBS + 0.05% (v/v) Tween-20].Antibodies used: anti-Nurr1 [E-20; Santa Cruz (SC)], 1:100; anti-Pitx3(Smidt et al., 2004), 1:10.000; anti-PSF (B92; Sigma), 1:2500; anti-Sin3a(K-20; SC), 1:1000; anti-Ncor (C-20; SC), 1:1000; anti-SMRT (H300;SC), 1:200; anti-RXR (sc-774; SC), 1:500; anti-HDAC1 (sc7872; SC),1:300. Blots were incubated with SuperSignal West Dura ExtendedDuration Substrate (Pierce) and exposed to ECL films (Pierce). Whenapplicable, films were digitized on a FLA5000 multi imaging system(Fuji), and the amount of co-immunoprecipitated proteins was determinedby densitometry. Ratios were calculated as relative increase in amount ofco-immunoprecipitated proteins in Pitx3–/– embryos compared withC57BL6 embryos.

Chip-on-chip analysisChromatin immunoprecipitation was performed according to the protocolsupplied by Nimblegen. For the in vitro ChIP-on-chip analysis, MN9D-Nurr1Tet On13N cells were transfected with 0.5 μg Pitx3-pcDNA3.1(–).Nurr1 expression was induced by 3 μg/ml doxycyclin and cells werecultured for 48 hours. Two 10 cm plates were used for each antibody. For thein vivo ChIP-on-chip analysis, five ventral midbrains from C57Bl6-JicoE14.5 embryos were used for each ChIP. Chromatin was sonicated to anaverage length of 500 bp on a Soniprep 150 sonicator through 15 pulses of10 seconds at one-third of maximum power. ChIP was performed with eitherNurr1 antibodies, Pitx3 antibodies or pre-immune serum (Ctrl). ChIP-DNAwas amplified using a Whole Genome Amplification kit (Sigma), accordingto the manufacturer’s protocol and shipped for ChIP-on-chip analysis(Nimblegen) where Cy5-labelled Pitx3- and Nurr1-ChIP samples werehybridized to Cy3-labelled input chromatin on a mouse promoter array set(MM8). Microarray data was analysed using Signalmap software (v1.9).PCR-based validation was performed on 10 ng of amplified ChIP-DNAusing the following primers: Vip, 5�-AGCGACTGAGTTGGAGATTC-3�and 5�-GTAAGCCATGGCACTAGCAC-3�; Vmat2, 5�-GTGTCTACTGT -CTATCACAG-3� and 5�-GTCAAAGTGTCCATGAAGCC-3�; control, 5�-GACCCGTGTCACTGACCTAC-3� and 5�-GCCTGCTAGGAG CAG C -

CTTG-3�. The amount of amplified DNA was determined by densitometryon a FLA5000 multi imaging system (Fuji) and relative enrichment byPitx3- and Nurr1-ChIP over pre-immune serum (Ctrl)-ChIP was calculated.Statistical analysis was performed using Student’s t-test, comparing therelative values of enrichment of promoters of Vip and Vmat2 to the relativevalues of a non-enriched region (control) in the Vmat2 promoter, asdetermined by ChIP-on-chip.

AnimalsPitx3–/– and Nurr1–/– embryos were obtained as described previously (Smitset al., 2003; Jacobs et al., 2007). Pitx3-deficient Aphakia (Pitx3–/–) orC57Bl6-Jico wild-type mice were crossed with Pitx3gfp/gfp mice to obtainPitx3gfp/+ and Pitx3gfp/– embryos. Pitx3gfp/– embryos contain both theclassical Ak allele and an allele in which green fluorescent protein (GFP) isknocked-in the Pitx3 locus (Zhao et al., 2004; Maxwell et al., 2005) and aretherefore Pitx3 deficient. Pitx3gfp/+embryos are heterozygous for wild-typePitx3 and GFP, and are described to have a normal development of themdDA system (Maxwell et al., 2005).

GenotypingGenotypes were determined by PCR analysis as described previously(Saucedo-Cardenas et al., 1998; Jacobs et al., 2007).

In situ hybridizationIn situ hybridization was performed as described previously (Smits et al.,2003; Smidt et al., 2004). The following digoxigenin (DIG)-labelled probeswere used: Aadc; bp 22-488 of the mouse coding sequence (cds) (Smits etal., 2003), Vmat2; bp 290-799 of mouse cds (Smits et al., 2003), Dat; bp789-1153 of rat cds, Nurr1; 3� region of rat Nurr1, En1; 5� region of mousetranscript, D2R; bp 345-1263 of mouse cds.

Tissue cultureVentral midbrains of Pitx3gfp/+ and Pitx3gfp/- embryos at stage E13.5 weredissected in L15 medium (Gibco) and cultured in Neurobasal Medium(Gibco) supplemented with: 2% (v/v) B-27 supplement (Gibco), 18 mMHEPES-KOH (pH 7.5), 0.5 mM L-glutamine, 26 μM beta-mercapto-ethanoland 100 units/ml penicillin/streptomycin. Midbrains were cultured andtreated with either 0 mM, 0.3 mM or 0.6 mM of sodium butyrate (Sigma)for 48 hours.

FACS sortingCultures were dissociated using a Papain dissociation system (Worthington)and cells were sorted on a Cytopeia Influx Cell sorter. Sort gates were set onforward scatter versus side scatter (life cell gate), on forward scatter versuspulse width (elimination of clumps) and on forward scatter versusfluorescence channel 1 (528/38) filter; GFP fluorescence). Cells were sortedusing a 100 μm nozzle at a pressure of 15 PSI with an average speed of 7000cells/second.

Semi-quantitative RT-PCRRNA was isolated in Trizol (Invitrogen), according to manufacturer’sprotocol. Semi-quantitative RT-PCR was performed on equal amounts ofRNA (0.1 ng) using a one-step RT-PCR kit (Qiagen), according to thesupplied protocol. The following primers were used: Vmat2, 5�-GCA G -TCACACAAGGCTACCA-3� and 5�-TGAATAGCCCCATCCAAGAG-3�;D2R, 5�-GCCGAGTTACTGTCATGATC-3� and 5�-ACGGTGCAGAG -TTTCATGTC-3�; Th, 5�-GCCACGTGGAATACACAAAG-3� and 5�-GAGGCATGACGGATGTACTG-3�; Tbp, 5�-GAGAATAAGAGAGC -CACGGAC-3� and 5�-TCACATCACAGCTCCCCAC-3�. The amount ofamplified DNA was determined by densitometry as described above.Statistical analysis was performed by two-way unpaired Student’s t-test,comparing the relative transcript levels of sodium butyrate-treated cultureswith transcript levels of untreated cultures.

Statistical analysisThe quantified results represent the average values of experimentsperformed in triplicate and presented data indicate means with standarderrors (s.e.m.). Statistical analysis was performed using Student’s t-test (two-way unpaired). P≤0.05 is considered significant and are indicated byasterisks, P<0.01 is indicated with double asterisks.

RESEARCH ARTICLE Development 136 (4)

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RESULTSBoth Pitx3 and Nurr1 form a complex with the co-repressor PSFBased on the discussed similarities between Nurr1- and Pitx3-nullmice, we hypothesised that Pitx3 and Nurr1 are interconnected at thelevel of transcriptional regulation. Notably, functional interactionsbetween homeobox transcription factors and nuclear receptors havebeen proposed previously (Tremblay et al., 1999; Shan et al., 2005).In the pituitary gland, Pitx1 interacts with the orphan nuclearreceptor ‘steroidogenic factor 1’ (SF1; Nr5a1) and multiple co-regulators to activate SF1-mediated transcription (Tremblay et al.,1999; Quirk et al., 2001). For mdDA neurons, the identification ofproteins that physically interact with Pitx3 might provide clues tothe relationship between Nurr1 and Pitx3 at the level of genetranscription.

Through affinity purification of Pitx3-His proteins followed bymass spectrometry, we identified the transcriptional co-repressorsPSF (SFPQ; PTB-associated splicing factor) and Nono [non-POU-domain-containing, octamer binding protein/p54(nrb)] as proteinsthat specifically interact with Pitx3 in dopaminergic MN9D cells(Choi et al., 1991) (Fig. 1A). Interestingly, PSF and Nono arereported to form a heterodimeric complex involved in repression ofnuclear receptor-mediated transcription (Mathur et al., 2001; Shav-Tal and Zipori, 2002; Sewer et al., 2002; Dong et al., 2007). PSF hasbeen most extensively studied in relation to nuclear receptorrepression and therefore we focussed on PSF. The interaction ofPitx3 with endogenous PSF in MN9D cells was confirmed by meansof co-immunoprecipitation (Co-IP) (Fig. 1B). To investigatewhether the interaction of Pitx3 with PSF also exists in vivo, weperformed immunoprecipitation (IP) on E14.5 mdDA neurons.Indeed, endogenous PSF clearly co-immunoprecipitated with Pitx3(Fig. 1C), demonstrating the interaction of Pitx3 and the nuclearreceptor co-repressor PSF in developing mdDA neurons.

Most compelling, PSF has been shown to interact with theretinoic acid receptor RXR (Mathur et al., 2001), a well-describedheterodimerization partner for Nurr1 in mdDA neurons (Perlmannand Jansson, 1995; Wallen-Mackenzie et al., 2003). Therefore, wehypothesised that, like Pitx3, Nurr1 might also interact with PSF.Immunoprecipitation with Nurr1 antibodies was performed in

MN9D cells, and endogenous PSF was indeed detected in Nurr1immunoprecipitates (Fig. 1D). Moreover, PSF was also detected inNurr1 and RXR-immunoprecipitates of E14.5 mdDA neurons,suggesting that besides Pitx3, the nuclear receptors Nurr1 and RXRalso interact with PSF in vivo (Fig. 1E). These observations, togetherwith the previously described involvement of PSF in repression ofnuclear receptors, makes PSF an appealing candidate for bridgingPitx3 to Nurr1 in mdDA neurons.

Pitx3 and Nurr1 target the same promoter regionsin the genomeThe identification of PSF as interacting protein of both Pitx3 andNurr1 provided novel leads to the molecular background of thefunctional interplay between Pitx3 and Nurr1. In their role asdevelopmental regulators, both Pitx3 and Nurr1 are crucial for theinduction of Th in mdDA neurons of the substantia nigra (Smidt etal., 2004; Maxwell et al., 2005; Jacobs et al., 2007). In addition, PSFhas been shown to repress the TH-promoter in human CHP-212 cells(Zhong et al., 2006). Although the involvement of Pitx3 or Nurr1has not been investigated in this context, other studies haveindependently shown that both Nurr1 and Pitx3 can bind andactivate the Th promoter (Cazorla et al., 2000; Iwawaki et al., 2000;Lebel et al., 2001; Kim et al., 2003). The finding that both Pitx3 andNurr1 interact with PSF could indicate that Nurr1 and Pitx3 are partof the same transcriptional complex on the Th promoter and possiblyother target genes as part of a general mechanism for transcriptionregulation of mdDA-associated genes.

In order to investigate the occupancy of Pitx3 and Nurr1 onpromoters of a large and unbiased set of genes, we performed ChIP-on-chip analysis. We chose two different approaches to obtain DAneurons. For the first approach, we expressed Pitx3 and Nurr1 indopaminergic MN9D cells and for the second approach we used invivo mdDA neurons derived from E14.5 ventral midbrain.Chromatin immunoprecipitation (ChIP) was performed with eitherPitx3, Nurr1 or control (pre-immune serum) antibodies, and the IP-efficiency was determined by western blot analysis (Fig. 2A,B).Amplified ChIP-DNA fragments were labelled with Cy3 andhybridized to Cy5-labelled input chromatin on a MM8 mousepromoter array (Nimblegen). The ChIP-on-chip analysis in MN9D

533RESEARCH ARTICLEPitx3 outSMRTs Nurr1 in dopamine neurons

Fig. 1. Both Pitx3 and Nurr1 interact with the co-repressor protein PSF. (A) Pitx3-His interacts with the co-repressor complex PSF/Nono. His-tag based affinity purification of Pitx3-His proteins from MN9D cells, followed by protein gel silverstaining, revealed four protein bands thatspecifically interacted with Pitx3-His. Through mass spectrometry analysis, the protein bands of 100, 95 and 75 kDa were identified as PSF and the55 kDa band was identified as Nono. (B,C) Pitx3 interacts with PSF in MN9D cells and in vivo. Lysates of Pitx3 transfected MN9D cells (B) or E14.5mdDA neurons (C) were subjected to IP for Pitx3 or pre-immune serum, and immunoblotted for Pitx3 and PSF. (D,E) Nurr1 interacts with PSF inMN9D cells and in vivo. Lysates of MN9D cells expressing Nurr1 (D) and E14.5 mdDA neurons (E) were subjected to IP for Nurr1, RXR or pre-immune serum, and immunoblotted for Nurr1 and PSF. I, input; IP, immunoprecipitation; Ctrl, pre-immune serum control. D

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cells revealed that 1652 and 541 promoter regions were enriched byChIP for Nurr1 or Pitx3, respectively (Fig. 2C). In the in vivo ChIP,a smaller number of promoter regions was enriched: 208 by Nurr1and 140 by Pitx3 (Fig. 2D). This lower number may reflect the morestringent conditions of promoter occupancy in the in vivo situationcompared with in a cell line. Strikingly, a substantial amount ofpromoter regions that were enriched by Nurr1, were also enrichedby Pitx3 (see Tables S1 and S2 in the supplementary material). Thiswas observed for ~22% and 28% of the Nurr1-enriched promoterregions in MN9D cells and in vivo, respectively. Of special interestwas the enrichment of promoter regions near genes that havepreviously been described to be regulated by Nurr1. The ChIP-on-chip analysis of MN9D cells revealed that the promoter of the Nurr1target gene Vip (vasoactive intestinal peptide) (Luo et al., 2007) washighly enriched by Nurr1-ChIP (Fig. 2E). Remarkably, the same Vippromoter region was clearly also enriched by Pitx3-ChIP, whichsuggests that Pitx3 and Nurr1 bind to the same promoter region of

the Nurr1 transcriptional target Vip. The enrichment of the Vippromoter was validated by PCR on ChIP-DNA, confirming asignificant enrichment of the Vip promoter region by both Pitx3- andNurr1-ChIP compared with Control-ChIP in MN9D cells [Fig.2K,L; n=3; P<0.01 (Pitx3-ChIP)/P<0.05 (Nurr1-ChIP)].

The promoter of Vmat2, a well described target of Nurr1 in vivo,was clearly also enriched by ChIP for both Nurr1 and Pitx3 (Fig.2F). The enrichment was observed in both MN9D cells and E14.5mdDA neurons, which is in line with the described regulatory effectof Nurr1 on Vmat2 expression in MN9D cells and in vivo(Hermanson et al., 2003; Smits et al., 2003). PCR-based validationconfirmed the significant enrichment of the selected Vmat2promoter region by ChIP for both Pitx3 and Nurr1 (Fig. 2K,L; n=3;P<0.01). Within the tiled regions of the promoters of Ahd2, Dat, Thand D2R, which are established as Nurr1-regulated genes in mdDAneurons (Zetterström et al., 1997; Le et al., 1999; Castillo et al.,1998; Wallén et al., 1999; Smits et al., 2003), we did not observe


Fig. 2. Pitx3 and Nurr1 target thesame regions on promoters of Nurr1target genes.(A,B) Immunoprecipitation of Pitx3 andNurr1 from crosslinked chromatin. ChIP-Eluates from E14.5 mdDA neurons wereimmunoblotted for Nurr1 (A) or Pitx3(B). (C,D) Representation of uniquelyand mutually enriched promoter regionsby ChIP for Nurr1 and Pitx3 in MN9Dcells (C) and E14.5 mdDA neurons (D)[false discovery rate (FDR)<0.01].(E-J) Pitx3 and Nurr1 interact with thesame regions within promoters of a setof Nurr1-regulated genes. Note thathigh confidence binding sites oftranscription factors are identified as apositive signal for multiple neighbouringprobes (visualised as peaks). Relativeenrichment of the promoters of Vip (E),Vmat2 (F), Ahd2 (G), Dat (H), Th (I) andD2R (J) by ChIP for Pitx3 and Nurr1 inMN9D cells and in vivo E14.5 mdDAneurons. Regions enriched by both Pitx3and Nurr1 are indicated as series of redpeaks. (K,L) ChIP-PCR validation.Significant enrichment of the regionsindicated in red in E and F within thepromoters of Vip (Pitx3-ChIP; n=3;P=0.002, Nurr1-ChIP; n=3; P=0.012)and Vmat2 (Pitx3-ChIP; n=3; P=0.006,Nurr1-ChIP; n=3; P=0.009) by ChIP forPitx3 and Nurr1, but not pre-immuneserum. No enrichment was observed fora region (Control) in the Vmat2promoter that was not enriched byChIP-on-chip. Data are represented asmean±s.e.m., *P<0.05, **P<0.01; IP,immunoprecipitation; Ctrl, Pre-immuneserum; TSS, transcription start site; AB,antibody; E14.5, dissected E14.5 mdDAarea.

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mutually enriched regions (Fig. 2G-J). However, because on average5 kb of each promoter is represented on the array, we cannot excludethe possibility that Nurr1 and Pitx3 targeted sites are outside the tiledregions. Altogether, these data demonstrate the concerted occupancyof Pitx3 and Nurr1 on a set of promoters, including those of theNurr1-regulated genes Vip and Vmat2.

Pitx3 regulates a set of genes involved indopamine metabolism, previously described asNurr1 target genesThus far, we have shown that Pitx3 and Nurr1 form a complex with acommon co-repressor, and bind to regions within the promoters ofNurr1 target genes. These observations make it tempting tohypothesize that these physical associations have functionalconsequences in mdDA neurons. Two lines of evidence are inagreement with this hypothesis. First, combined transduction of Pitx3and Nurr1 in ES cells leads to increased transcript levels of DA genes,such as Vmat2, Dat, Th and Aadc (Martinat et al., 2006). Second, thePitx3 transcriptional target gene Ahd2 is not detected in the midbrainarea of Nurr1-deficient embryos at E15.5 (Wallén et al., 1999) orE14.5 (F.M.J.J. and M.P.S., unpublished). In order to elucidate thegene-regulatory function of Pitx3 in relation to Nurr1 in thedevelopment of mdDA neurons, we performed in situ hybridizationon sagittal sections of E14.5 Pitx3–/– embryos, and compared themwith E14.5 Nurr1–/– embryos (Fig. 3A). The normal expression of En1and Nurr1 in Pitx3–/– embryos and the conserved expression of En1and truncated Nurr1 transcripts in Nurr1–/– embryos is indicative of anormal presence of mdDA neurons in medial sagittal sections (Fig.3B,C,H,I). As described previously, the embryonic expression of Datand Vmat2 was completely lost in Nurr1–/– mdDA neurons (Fig.3J,K), whereas the expression of D2R and Aadc was stronglydecreased (Fig. 3L,M). Interestingly, Dat was not detected in Pitx3–/–

mdDA neurons, whereas it was clearly expressed in Pitx3+/+ littermatecontrols (Fig. 3D). Furthermore, the expression of Vmat2, which iscompletely absent in Nurr1–/– mdDA neurons, was drastically reducedin Pitx3–/– mdDA neurons (Fig. 3E). Notably, Vmat2 was abundantlyexpressed in the hindbrain of Nurr1- and Pitx3-deficient embryos,clearly demonstrating a mdDA neuron-specific deficiency of Vmat2expression. Similarly, D2R was expressed at barely detectable levelsin Nurr1–/– mdDA neurons (Fig. 3L) and was strongly reduced inPitx3–/– mdDA neurons compared with littermate controls (Fig. 3F).Whereas Aadc expression was clearly decreased in Nurr1–/– mdDAneurons (Fig. 3M), we observed no difference of Aadc expressionbetween Pitx3–/– and Pitx3+/+ embryos in these medial sections (Fig.3G). Altogether, besides the previously described effects on theexpression of Th and Ahd2, loss of Pitx3 clearly affects the expressionof Vmat2, Dat and D2R in mdDA neurons during development.Interestingly, next to Nurr1 as essential factor for the dopaminergicphenotype, Pitx3 is crucial for canonical expression of genespreviously described as Nurr1 downstream targets, strengthening ourhypothesis that Pitx3 and Nurr1 constitute a physical and functionalcomplex in transcription regulation.

Loss of Pitx3 expression in vivo increases theinteraction of the co-repressor SMRT with Nurr1Our data demonstrate the concurrent involvement of Pitx3 andNurr1 in the regulation of the same set of genes within mdDAneurons. However, the relative severity of the analyzed molecularphenotypes suggests that, whereas Nurr1 can be considered a masterswitch on DA-gene transcription, Pitx3 appears to regulate thetranscriptional activity of Nurr1. Generally, the activity of nuclearreceptors is highly regulated by co-regulating proteins. Upon

activation of nuclear receptors, co-repressors are released and co-activators are recruited (Fowler and Alarid, 2004; Wolf et al., 2008).Possibly, recruitment of Pitx3 affects the composition of the Nurr1transcriptional complex in relation to the interaction with co-repressor proteins such as PSF. As we have demonstrated that theco-repressor PSF interacts with both Pitx3 and Nurr1, the interactionof Nurr1 with PSF might depend on the presence of Pitx3. Anotherplausible candidate in this respect is the co-repressor Sin3a, whichis recruited by PSF to mediate nuclear receptor repression (Mathuret al., 2001). In order to test any alterations of the composition of theNurr1 transcriptional complex upon Pitx3 ablation in vivo, weperformed IP for Nurr1 from E14.5 wild-type and Pitx3–/– mdDA


Fig. 3. Pitx3 is crucial for expression of a set of Nurr1 targetgenes. (A) Overview of sagittal sections of 14.5 embryos (middle panelwas adapted from www.genepaint.org). In the right panel, the locationof the mdDA neurons in the medial part of the mdDA area is indicatedin red, the broken line represents the area shown in B-M. (B-M) In situhybridization using DIG-labelled probes for a set of mdDA expressedgenes in Pitx3+/+/Pitx3–/– (B-G) and Nurr1+/+/Nurr1–/– (H-M) littermateembryos at stage E14.5. Homozygous mutant embryos are indicatedwith an asterisk (*). (B,C,H,I) Normal presence of mdDA neurons inPitx3–/– and Nurr1–/– embryos. (B,H) In situ hybridization for Nurr1. Notethat truncated Nurr1 transcripts were still detected in Nurr1–/– embryos(H*). (C,I) In situ hybridization for En1. (D-F,J-L) Expression of a set ofNurr1-regulated genes was affected in Pitx3–/– embryos. In situhybridization for Dat (D,J), Vmat2 (E,K) and D2R (F,L). Note the mdDA-specific defect of Vmat2 expression in Pitx3–/– (E*) and Nurr1–/– (K*)embryos. The red line indicates the location of the mid-hindbrainborder. (G,M) In situ hybridization for Aadc. h, hindbrain; m, midbrain;f, forebrain.

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neurons. Importantly, in accordance with what was observedby in situ hybridization, similar amounts of Nurr1 wereimmunoprecipitated from wild-type and Pitx3–/– mdDA neurons(Fig. 4E). Besides PSF, the PSF-interacting co-repressor Sin3a alsointeracted with Nurr1 in vivo. However, the level of interaction ofNurr1 with either PSF or Sin3a was not affected by the presence ofPitx3 (Fig. 4A,B; n=3). These data indicate that the physicalinteraction of PSF and Sin3a with Nurr1 is unrelated to the potencyof Nurr1 to activate its target genes. This is in line with other studies,reporting that activation of nuclear receptors by ligand binding doesnot result in dissociation of Sin3a or PSF (Mathur et al., 2001).Although no endogenous ligand has been identified for Nurr1, itsligand-binding domain interacts with the co-repressors Ncor(nuclear receptor co-repressor 1) and SMRT (silencing mediator ofretinoic acid and thyroid hormone receptor) (Codina et al., 2004;Lammi et al., 2008). In general, Ncor and SMRT bind to unligandednuclear receptors, and are believed to keep these complexes in arepressed state (Nishihara et al., 2004). Ligand binding to the nuclearreceptor complex results in dissociation of Ncor and SMRT,allowing activation of gene transcription. As a possible regulatorymechanism of Nurr1-mediated transcription in mdDA neurons, therecruitment of Pitx3 to the Nurr1 transcriptional complex mightmimic the effect of ligand activation of Nurr1, by inducingthe dissociation of SMRT or Ncor. To test this, we examinedwhether the level of interaction between Nurr1 and the co-repressorsNcor and SMRT was altered upon ablation of Pitx3 in mdDAneurons. Although we did not detect an interaction between Ncorand Nurr1 (Fig. 4C), the co-repressor SMRT was clearly co-immunoprecipitated with Nurr1. Strikingly, we observed asignificant increase in SMRT interaction with Nurr1 in the absenceof Pitx3 (Fig. 4D). The SMRT interaction with Nurr1 was increasedapproximately threefold in Pitx3–/– mdDA neurons compared withwild type, as determined by densitometry (Fig. 4F; n=4; P<0.01).This suggests that in the absence of Pitx3, the transcriptional activityof Nurr1 is kept in a repressed state by SMRT, and recruitment ofPitx3 leads to activation of Nurr1, through release of the SMRT-mediated repression.

Release of HDAC-mediated repression in Pitx3-deficient embryos restores Nurr1 target geneexpression in vivoSMRT exerts its repressive actions through recruitment of a set ofclass I and class II histone deacetylases (HDACs) (Huang et al.,2000; Guenther et al., 2001; Fischle et al., 2002). The de-acetylatedstate of histones near the transcription start site is responsible forthe repression of gene transcription. The potential involvement ofHDACs in the regulation of Nurr1 was further strengthened by thedetection of endogenous HDAC1 in Nurr1 immunoprecipitatesfrom E14.5 dissected ventral midbrains (see Fig. S1 in thesupplementary material). Our data suggest that Nurr1 occupies thepromoters of target genes in Pitx3–/– embryos but is kept in arepressed state owing to the interaction with SMRT/HDACcomplexes. Hypothetically, interference with HDAC activity inPitx3–/– embryos would result in re-activation of Nurr1-mediatedtranscription, bypassing the necessity for Pitx3. To test thishypothesis, we focussed on the expression of Vmat2 and D2Rbecause of the reduced transcript level throughout the mdDA areain Pitx3–/– embryos (Fig. 5B,C) and on Th because of the mdDAsubset-specific loss of Th expression in Pitx3–/– embryos (Fig. 5D).In order to isolate mdDA neurons for gene expression analysis bymeans of FACS sorting, we set up a breeding scheme to obtainPitx3-deficient Pitx3gfp/– and heterozygous Pitx3gfp/+ embryos(Zhao et al., 2004; Maxwell et al., 2005). Importantly, theexpression of GFP was highly similar in Pitx3gfp/– and Pitx3gfp/+

embryos at stage E14.5 (Fig. 5E), showing the normal presence ofmdDA neurons in Pitx3-deficient mdDA neurons during embryonicdevelopment. Ventral midbrains of Pitx3gfp/+ and Pitx3gfp/– embryoswere dissected at stage E13.5 and cultured for 2 additional days. Tointerfere with HDAC activity, we treated the ventral midbrains withdifferent concentrations of the a-specific HDAC inhibitor sodiumbutyrate, after which GFP-positive mdDA neurons were selectedby FACS sorting (Fig. 5F-H). Importantly, similar numbers of GFP-positive neurons were collected from all treated and untreatedPitx3gfp/– and Pitx3gfp/+ cultures. Equal amounts of RNA from GFP-positive mdDA neurons were subjected to semi-quantitative RT-PCR to determine the relative transcript levels of Vmat2, D2R andTh. Tbp (TATA-box binding protein) was taken along as control,which showed no difference in transcript levels in mdDA neuronsof both genotypes and upon treatment with sodium butyrate (Fig.5I,M; n=3). In heterozygous Pitx3gfp/+ mdDA neurons, nosignificant increase in relative amounts of Vmat2, D2R and Thtranscripts was observed upon sodium butyrate treatment (Fig. 5I-L; n=3). In agreement with what was observed for Vmat2expression by in situ hybridization, a significant lower level ofVmat2 transcripts was observed in untreated Pitx3gfp/– mdDAneurons, at ~7% compared with the level of Vmat2 in untreatedPitx3gfp/+ mdDA neurons (Fig. 5I,J; n=3). Strikingly, treatment with0.3 mM sodium butyrate resulted in a significant increase in Vmat2transcripts, restoring levels to 56% (n=3; P<0.01). Vmat2 levelswere even restored to 84% in cultures treated with 0.6 mM sodiumbutyrate (n=3; P<0.01). Similarly, D2R levels were restored from53% in untreated Pitx3gfp/– mdDA neurons to 98% in 0.6 mM-treated cultures (Fig. 5I,K; n=4; P<0.01) and levels of Th wereincreased from 60% in untreated Pitx3gfp/– mdDA neurons to 94%in cultures treated with 0.6 mM sodium butyrate (Fig. 5I,L; n=3;P=0.05). These data clearly demonstrate that inhibition of HDAC-mediated repression in Pitx3–/– embryos efficiently restores thetranscriptional activation of a set of Nurr1 target genes, bypassingthe necessity for Pitx3. These observations signify our proposedmechanism that in mdDA neurons, full activation of Nurr1 target


Fig. 4. Pitx3 decreases the interaction of Nurr1 with the nuclearreceptor co-repressor SMRT. (A-E) Co-IP of Nurr1 with a set ofnuclear receptor co-repressors in wild-type and Pitx3–/– E14.5 mdDAneurons. Lysates were subjected to IP for Nurr1 or pre-immune serum,and immunoblotted for PSF (A), Sin3a (B), Ncor (C), SMRT (D) or Nurr1(E). (F) The interaction of Nurr1 with SMRT was increased in theabsence of Pitx3. Relative increase of interaction with Nurr1 in Pitx3–/–

mdDA neurons over wild type was calculated for PSF (n=3; P=0.12),Sin3a (n=3; P=0.35) and SMRT (n=4; P=0.002). Data are represented asmean±s.e.m., *P<0.01; I, input; IP, immunoprecipitation; Ctrl, pre-immune serum; WT, wild type.

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genes depends on the release of the SMRT/HDAC-mediatedrepression of the Nurr1 transcriptional complex through theregulatory action of Pitx3.

DISCUSSIONThe regulatory effect of Pitx3 on the association of Nurr1 withSMRT and the finding that Nurr1-mediated transcription of Vmat2,D2R and Th is repressed in a HDAC-dependent manner in theabsence of Pitx3, enabled us to propose a model in which Pitx3 isa key regulator of Nurr1-mediated transcription (Fig. 6).Accordingly, the recruitment of Pitx3 to PSF within the Nurr1transcriptional complex leads to (full) activation of Nurr1 targetgenes, by inducing the release of SMRT/HDAC-mediatedrepression from Nurr1. Ablation of Nurr1, acting as a masterswitch on gene transcription, results in complete loss of target geneexpression. However, ablation of Pitx3 mainly affects Nurr1transcriptional activity, which would still allow Nurr1-mediatedtranscription at a lower level (Vmat2/D2R), or in a certain mdDAsubpopulation (Th). This model also explains why expression ofthe Nurr1 target genes Dat, Vmat2 and D2R is not detected until

after Pitx3 is expressed at E11.5, although Nurr1 is alreadyexpressed at E10 (Simon et al., 2003; Wallen-Mackenzie et al.,2003; Smits and Smidt, 2006; Alavian et al., 2008).

Interestingly, another member of the paired like homeoboxtranscription factors has been described to functionally andphysically interact with nuclear receptors in the pituitary. Pitx1enhances the transcriptional activity of the orphan nuclear receptorSF1 (Tremblay et al., 1999; Quirk et al., 2001; Melamed et al., 2002),and Pitx1, together with the estrogen receptor, modify the repressiveandrogen receptor/SF1 complex on the LHβ promoter (Jorgensenand Nilson, 2001). Moreover, SF1 has also been shown to interactwith PSF (Sewer et al., 2002). The apparent analogy between theregulation of an orphan nuclear receptor by Pitx1 and our data areindicative of a more general mechanism involving PSF, in whichhomeodomain transcription factors modulate nuclear receptor-mediated transcription. Most likely, more components are involvedin the proposed mechanism. The release of the co-repressor SMRTfrom Nurr1 could be accompanied by the recruitment of activatingproteins, although co-activators of Nurr1 have yet to be identified(Castro et al., 1999; Wang et al., 2003; Volakakis et al., 2006; Lammi


Fig. 5. Interference with HDAC-mediatedrepression in Pitx3–/– embryos, restores Nurr1target gene expression. (A) Overview of coronalsections of an E14.5 embryonic brain. Adapted, withpermission, from Kaufman (Kaufman, 1992). Thelocation of the mdDA neurons is indicated in red, thebroken line represents the area shown in B-D. (B-D) Insitu hybridization on coronal sections of E14.5 Pitx3+/+

and Pitx3–/– littermate embryos. Homozygous mutantembryos are indicated with an asterisk. (B,C) Expressionof Vmat2 and D2R was decreased throughout themdDA area in E14.5 Pitx3–/– embryos. (D) Expression ofTh was deficient in a subpopulation of the mdDAneurons in E14.5 Pitx3–/– embryos. (E) A similar patternof GFP-positive neurons in E14.5 Pitx3gfp/+ (E) andPitx3gfp/– (E*) reveals the apparent normal presence ofmdDA neurons in Pitx3-deficient Pitx3gfp/– embryos.(F-H) The FACS sorting gate was set, using a E14.5C57Bl6-Jico (Bl6) reference sample (F) to select GFP-positive mdDA neurons from Pitx3gfp/+ (G) and Pitx3gfp/–

(H) midbrain cultures with a purity of 98% (data notshown). (I-M) Treatment with the HDAC inhibitorsodium butyrate restores expression of Nurr1 targetgenes in Pitx3gfp/– mdDA neurons. (I) RNA from FACS-sorted mdDA neurons derived from cultures treatedwith 0 mM, 0.3 mM or 0.6 mM of sodium butyrate(n=3) were subjected to semi-quantitative RT-PCR.Relative transcript levels were determined bydensitometry and compared with transcript levels inuntreated Pitx3gfp/+ mdDA neurons. Relative valueswere calculated for Vmat2 (J; Pitx3gfp/– 0.3 mM P=0.006,Pitx3gfp/– 0.6 mM; P=0.007), D2R (K; Pitx3gfp/– 0.3 mM;P=0.0006, Pitx3gfp/– 0.6 mM; P=0.001), Th (L;Pitx3gfp/– 0.3 mM; P=0.19, Pitx3gfp/– 0.6 mM; P=0.05) andTbp (M; Pitx3gfp/– 0.3 mM P=0.5, Pitx3gfp/– 0.6 mM; P=0.5).Data are represented as mean±s.e.m., *P=0.05,**P<0.01.

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et al., 2008). Notably, β-catenin has been reported to affect thetranscriptional activity of both Pitx factors and SF1 (Vadlamudi et al.,2005; Hossain and Saunders., 2003) which makes β-catenin aninteresting potential component of the Nurr1/PSF/Pitx3 complex.

In zebrafish, PSF is involved in neuronal differentiation, and lossof PSF results in developmental defects in the mid- and hindbrain(Lowery et al., 2007). Although PSF is expressed in mdDA neurons(data not shown) and forms a complex with Pitx3 and Nurr1, the roleof PSF in the Nurr1 transcriptional complex is unclear. Theinteraction of Nurr1 with PSF is unchanged in Pitx3–/– embryos,which makes it unlikely that Pitx3 is involved in the recruitment ordissociation of PSF to the Nurr1 transcriptional complex.Importantly, besides PSF, we showed that Nurr1 also interacts withSin3a, which is involved in transcriptional repression by recruitingSMRT and HDACs (Wolffe, 1997). This may indicate that the effectof Pitx3 on dissociation of SMRT from the Nurr1 transcriptionalcomplex also involves PSF and Sin3a.

The shared interaction of Pitx3 and Nurr1 with PSF suggests thatPitx3 and Nurr1 are part of the same transcriptional complex. Theresults from the ChIP-on-chip analysis further strengthen thishypothesis, as we identified a number of promoter regions that wereenriched by ChIP for both Pitx3 and Nurr1. Most intriguingly, bothPitx3 and Nurr1 interact with the promoter of Vmat2, which iscrucial for DA metabolism and is regulated by Nurr1 in vivo (Smitset al., 2003). These observations strongly suggest that Pitx3 andNurr1 are recruited in concert with a number of promoters, includingsome that have been previously associated with Nurr1.

The shared physical interaction of Pitx3 and Nurr1 with thepromoter of Vmat2 is translated to function in vivo, as expressionanalysis of E14.5 Pitx3–/– embryos revealed an essential role forPitx3 for the proper expression of a set of described Nurr1 targetgenes, including Vmat2. These observations shed new light on thecomplicated role of Pitx3 in mdDA neurons, as it has been puzzlingfor a long time that although Pitx3 is expressed in all mdDA neurons,only the subpopulation of the substantia nigra is dependent on Pitx3

for induction of Th and subsequential maintenance into adulthood.This discrepancy indeed holds true for the expression of Th,underlining the existence of mdDA subsets with differential relianceon Pitx3. However, based on analysis of the expression patterns ofVmat2, Dat and D2R, the entire mdDA population is affected byPitx3 deficiency. Our observations clearly indicate that in additionto the previously described subset-specific role for Pitx3 in thesubstantia nigra, Pitx3 is crucial for the correct molecular phenotypeof mdDA neurons in general. This finding could be an explanationfor the aberrant electrophysiological properties of surviving Th-positive neurons in Pitx3-deficient mice, which has remainedunexplained until now (Smits et al., 2005).

Interestingly, in vivo expression analysis revealed a strikingdifference between the relative severity of the Nurr1 target genedeficit between Nurr–/– and Pitx3–/– embryos, which is most evidentfor the expression of Vmat2. It appears that although Nurr1 is crucialfor the expression of its target genes, Pitx3 is essential for the levelof Nurr1-mediated transcription. The increased interaction of Nurr1with the co-repressor SMRT in the absence of Pitx3 is a suitablemolecular explanation for this observation. The regulatory effect ofPitx3 on the physical interaction of Nurr1 with the co-repressorSMRT strongly suggests that Pitx3 has the potency to induce therelease of SMRT-mediated repression from the Nurr1 transcriptionalcomplex. Indeed, interference with the repressive activity ofHDACs, which are recruited to unliganded nuclear receptors bySMRT, efficiently restores the transcript levels of the Nurr1 targetgenes Vmat2, D2R and Th in cultured Pitx3gfp/– midbrains. Thisclearly indicates that, in the absence of Pitx3, the Nurr1transcriptional complex is kept in a repressed state by SMRT/HDAC, which signifies the regulatory role of Pitx3 on theexpression of Nurr1 target genes as we proposed in our model.

Altogether, as part of a proposed general modulatory effect of Pitxfactors on nuclear receptor activity, our data provide evidence forthe existence of a transcriptional complex involving Pitx3 andNurr1, positioning Pitx3 as an essential activator of the potency of


Fig. 6. Pitx3 is a crucial regulator of Nurr1-mediated transcription. In the absence of Pitx3, the Nurr1 transcriptional complex is kept in arepressed state by SMRT through recruitment of HDACs, which keep the target gene promoter in a de-acetylated (repressed) state, resulting indeficiency of Nurr1 target gene expression. Interference with HDAC-mediated repression by sodium butyrate restores Nurr1-mediated transcriptionof Nurr1 target genes in the absence of Pitx3. Recruitment of Pitx3 to Nurr1 target gene promoters strongly resembles the effect of ligandactivation of nuclear receptors by inducing the dissociation of SMRT from the Nurr1 transcriptional complex, which favours (full) activation oftranscription of Nurr1 target genes.

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Nurr1 to drive expression of genes essential for mdDA neurons. Inconclusion, our data significantly contribute to a betterunderstanding of mdDA neuron development, as we have shownthat Nurr1 and Pitx3 act in concert to induce the mdDAcharacteristics of neurons, which has large consequences for stem-cell engineering as a future treatment for Parkinson’s disease.

We thank Thomas Perlmann for the generous gift of the MN9D cells, OrlaConneely for providing us with Nurr1 knockout mice, Meng Li for providing uswith the Pitx3-GFP knock-in mice, Koen Dreijerink for providing us with theHDAC antibodies, Simone Smits for many helpful discussions, Ger Arkesteinfor FACS sorting and excellent technical assistance, Jeroen Pasterkamp forhelpful comments on the manuscript, and Roger Koot for his excellent supportin the data analysis. This work was supported by a HIPO-grant (University ofUtrecht) to M.P.S.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/4/531/DC1

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