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Developmental Biology 233, 425–436 (2001)doi:10.1006/dbio.2001.0220, available online at http://www.idealibrary.com on

Pannier is a Transcriptional Target and Partnerof Tinman during Drosophila Cardiogenesis

Kathleen Gajewski,*,1 Qian Zhang,*,1 Cheol Yong Choi,†,1

Nancy Fossett,* Anh Dang,* Young Ho Kim,†Yongsok Kim,† and Robert A. Schulz*,2

*Department of Biochemistry and Molecular Biology, Graduate Program in Genes &Development, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030;and †Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute,National Institutes of Health, Bethesda, Maryland 20892

During Drosophila embryogenesis, the homeobox gene tinman is expressed in the dorsal mesoderm where it functions inthe specification of precursor cells of the heart, visceral, and dorsal body wall muscles. The GATA factor gene pannier isimilarly expressed in the dorsal-most part of the mesoderm where it is required for the formation of the cardial cell lineage.espite these overlapping expression and functional properties, potential genetic and molecular interactions between the

wo genes remain largely unexplored. Here, we show that pannier is a direct transcriptional target of Tinman in theeart-forming region. The resulting coexpression of the two factors allows them to function combinatorially in theegulation of cardiac gene expression, and a physical interaction of the proteins has been demonstrated in cultured cells. Welso define functional domains of Tinman and Pannier that are required for their synergistic activation of the D-mef2ifferentiation gene in vivo. Together, these results provide important insights into the genetic mechanisms controllingeart formation in the Drosophila model system. © 2001 Academic Press

Key Words: cardiogenic factors; D-MEF2; heart; Pannier; protein interactions; Tinman; transcriptional enhancer.

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INTRODUCTION

The study of heart development in Drosophila has beenof significant value, not only to our fundamental under-standing of organogenesis in this model system, but alsothrough the application of genetic insights to the investi-gation of cardiogenesis and disease in evolutionarily ad-vanced organisms. A prime example can be found in thehomeobox gene tinman (tin) and its vertebrate NK-2 classounterparts. tin expression in the dorsal mesoderm isegulated by intercellular signaling mediated by the growthactor Decapentaplegic (Dpp) produced by adjacent ectoder-

al cells (Frasch, 1995; Xu et al., 1998). tin function isequired in the dorsal mesoderm for the formation ofrogenitors of the heart, visceral, and dorsal somaticuscles, and the mutation of this transcription factor

esults in an absence of these mesodermal derivatives

1 K.G., Q.Z., and C.Y.C. contributed equally to this work.2 To whom correspondence should be addressed. Fax: (713) 790-

c0329. E-mail: [email protected].

0012-1606/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

Azpiazu and Frasch, 1993; Bodmer, 1993). Based on theseroperties, tin-related genes were cloned and characterizedn vertebrate species, with several shown to be expressed inhe cardiac lineage (reviewed in Harvey, 1996). Comparableo the role of Dpp in regulating tin and promoting cardio-enesis, bone morphogenetic protein signaling induceskx-2.5 expression and cardiac myogenesis in the chick

mbryo (Schultheiss et al., 1997). Likewise, genetic studiesn the mouse and Xenopus systems have demonstratedssential functions for the Nkx-2.3 and Nkx-2.5 genes inertebrate heart development (Lyons et al., 1995; Fu et al.,998; Grow and Krieg, 1998; Tanaka et al., 1999). Ofedical importance, certain patients with congenital heart

isease have been shown to carry dominant mutations inhe human NKX2–5 gene, resulting in diverse cardiac de-elopmental abnormalities (Schott et al., 1998; Benson etl., 1999).tin is expressed in the dorsal mesoderm in a broad pattern

hat exceeds the limits of the heart-forming region (Yin andrasch, 1998; Gajewski et al., 1999). This observation,
oupled with the inability of pan-mesodermal tin expres-
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426 Gajewski et al.

sion to produce ectopic heart cells (Yin and Frasch, 1998),implicated the involvement of additional factors in thegenesis of cardiac cells within the dorsal-most mesoderm.Based on the requirement of a GATA element for thefunction of a heart enhancer of the D-mef2 differentiationgene (Gajewski et al., 1998) and the demonstrated interac-tion of NKX2–5 with GATA4 in the regulation of cardiacgene expression in vertebrates (Durocher et al., 1997; Lee etal., 1998; Sepulveda et al., 1998), a role was predicted for a

ATA factor in fly heart development. Subsequently, theATA gene pannier (pnr) was shown to be expressed in theeart forming region and required for cardial cell formation

Gajewski et al., 1999). The coexpression of Tin and Pnresults in a synergistic activation of cardiac gene expressionnd the induction of an apparent cardioblast fate in bothesodermal and nonmesodermal cell types. Together,

hese results provide evidence that the two cardiogenicactors work together to program cells into the cardiacineage. Additionally, the forced expression of either Pnr or

ouse GATA4 throughout the Drosophila mesoderm re-ults in supernumerary cardial cells, demonstrating anvolutionarily conserved function between the two GATAenes in heart development.A further complexity in the genetic control of Drosophila

ardiogenesis was found in the analysis of the gene encod-ng the multitype zinc finger protein U-shaped (Ush). Thisegulator was previously shown to be an antagonist of Pnrunction in neuronal precursor cell determination, alteringhe properties of the GATA factor from a transcriptionalctivator to a repressor in cells where the two are coex-ressed (Haenlin et al., 1997; Garcia-Garcia et al., 1999).imilarly, in the dorsal mesoderm, Ush controls cardial cellpecification through its modulation of Pnr transcriptionalctivity (Fossett et al., 2000). A loss of ush function resultsn an overproduction of cardial cells, while a gain ofunction leads to a reduction of cardioblasts, phenotypeshat are directly opposite to those seen with pnr. Thetructurally related Friend of GATA-2 (FOG-2) protein isxpressed during heart formation in the mouse and likewiseunctions as a modulator of GATA4 transcriptional activityn cultured cells (Lu et al., 1999; Svensson et al., 1999;

Tevosian et al., 1999). The recent analysis of targeted genemutations have demonstrated a vital function for FOG-2 inheart development as mutants display morphogenetic ab-normalities and an absence of the coronary vasculature(Svensson et al., 2000b; Tevosian et al., 2000). Additionalevidence that Ush and FOG-2 share functional properties isprovided by the depletion of heart cells in Drosophilaembryos expressing FOG-2 in the mesoderm (N. Fossett, K.Gajewski, S. Tevosian, S. Orkin, and R. Schulz, unpub-lished results).

While substantial data exist on the functions of indi-vidual genes in the specification of the cardiac lineage inDrosophila, less is known about genetic and molecularinteractions between these early-acting cardiogenic factors.In this report, we investigate the regulation of the pnr gene
and the functional interactions of the GATA protein with
Copyright © 2001 by Academic Press. All right

Tin. Our results show that pnr is a direct target of Tintranscriptional activation during heart formation. Addition-ally, we identify regions of the two proteins that areessential for their combinatorial regulation of cardiac geneexpression. These findings contribute needed informationto our understanding of the genetic pathways controllingheart development in Drosophila.

MATERIALS AND METHODS

In Vitro DNA Binding Assays

Electrophoretic mobility shift and DNase I protection assayswere performed as described in Lee et al. (1997). To prepare probes,equimolar amounts of single-strand oligonucleotides were an-nealed and end-labeled with T4 kinase. The sequence of theoligonucleotides used for the gel shift assays were as follows.Wild-type probes: pnrA, 59-CCTCGGTTCTCAAGTGCGGAC-CTG-39; pnrB, 59-CAGGTCCGCACTTGAGAACCGAGG-39. Mu-tant probes: pnrAm, 59-CCTCGGTTCTgAAGTcCGGACCTG-39;pnrBm, 59-CAGGTCCGgACTTcAGAACCGAGG-39. The mu-tated nucleotides are in lower case. For the competitive gel-shiftassays, a 100-fold molar excess of unlabeled DNA was used. For thefootprinting assay, the pnr enhancer DNA was digested withHindIII (plus strand labeling) or XbaI (minus strand labeling), andthe linearized DNAs were dephosphorylated with alkaline phos-phatase. These DNAs were then digested with XbaI (plus strand) orHindIII (minus strand), eluted from a gel, and end-labeled with T4kinase. Binding reactions were undertaken with increasingamounts (0.2, 0.4, and 0.6 mg) of GST-NK4 protein and labeled

NA (12.5 fmol) in 20 ml of binding buffer. After incubation for 15min at room temperature, CaCl2 (final 0.5 mM) and DNase I (0.1unit) were added to the binding mixture. The DNase I digestionswere stopped after 1 min incubation by adding stop buffer followedby analysis via denaturing polyacrylamide gel electrophoresis.

Expression Analyses of pnr Enhancer–lacZ FusionGenes

Transformant strains expressing the 457-bp pnr dorsal meso-derm enhancer–lacZ fusion gene have been described (Gajewskiet al., 1999). To monitor enhancer activity in a tin gain offunction background, pnr-lacZ males were crossed to UAS-tinvirgins (Yin and Frasch, 1998), and the resulting heterozygousmales were mated to twi-Gal4 virgins (Baylies and Bate, 1996).

o assay pnr-lacZ expression in tin loss of function embryos,ales were crossed to tin346/TM3, Sb virgins, and animals lack-

ing the balancer chromosome were obtained. Embryos were col-lected from the mating of these flies and immunostained forboth b-galactosidase and Tin, with homozygous embryos iden-ified by the absence of Tin protein. tin346 has been classified as

a null allele of the gene (Azpiazu and Frasch, 1993). Mutationswere introduced into the Tin-binding sites in the pnr enhancerusing the QuickChange Site-Directed Mutagenesis Kit fromStratagene (La Jolla, CA). Primers used were as follows: mutTin1,59-CCTACCGTTCTCCGGTCTgtAGacCGGACCTGCCTCGAT-TTCCC-39 and 59-GGGAAATCGAGGCAGGTCCGgtCTacA-GACCGGAGAACGGTAGG-39; mutTin2, 59-CCTGCCTCGA-TTTCCCGGCTgtAGacCGCGGGACATCATTTTTC-39 and59-GAAAAATGATGTCCCGCGgtCTacAGCCGGGAAATCGA-
GGCAGG-39. Lower case letters correspond to the introduced base
s of reproduction in any form reserved.

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427Functional Interactions of Pannier and Tinman

changes. The processing and antibody staining of whole-mountembryos, and the generation of embryo sections, was as describedin Gajewski et al. (1999).

Colocalization of Tin and Pnr in Cultured Cells

CV-1 cells were cotransfected with GFP- and Myc-tagged Tinand Pnr expression vectors (1 mg per transfection). For the colocal-zation analysis, cells were stained with DAPI, fixed 36 h afterransfection, and subjected to immunohistochemistry using annti-Myc antibody (Invitrogen, San Diego, CA). Signals were de-ected by confocal microscopy and analyzed as described in Kim etl. (1999). For coimmunoprecipitation experiments, nuclear ex-racts were prepared and subjected to immunoprecipitation withnti-Myc antibody followed by Western blot with anti-GFP anti-ody (Clontech, Palo Alto, CA) as described previously (Choi et al.,999a).

Generation of tin and pnr Gene Mutants

For the production of the various UAS-Tin and UAS-Pnr con-structs used in this study, cDNAs encoding either full-length ormutant forms of Tin (Choi et al., 1999b) or Pnr (Winick et al., 1993)were introduced into the P element vector pUAST (Brand andPerrimon, 1993). Transgenic lines harboring the UAS-cDNAs wereestablished by using standard procedures as described in Gajewskiet al. (1997).

To generate Tin(N351Q), a NotI–ApaI DNA fragment from pCMV-NK4 N351Q was inserted into the NotI and ApaI sites of pUAST. Theconstruction of Tin(1–35, 46–416) involved a multistep procedure. A203-bp DNA fragment corresponding to the 59-nontranslated regionthrough amino acid 35 was amplified by PCR with primer NK453 andprimer LEE, 59-CTGGAATTCATCCGCGTTCGTCAGGCT-39. TheDNA was codigested with EcoRI and SalI and subcloned into thecorresponding sites of pKSII-A12, which contains the sequence encod-ing Tin amino acids 46 through 416. The resulting internally deletedDNA was then cloned into the NotI site of pUAST. For the construc-tion of Tin(111–416), Tin(152–416), Tin(192–416), Tin(232–416),Tin(272–416), and Tin(1–109, 192–416), tin cDNAs were generated byPCR amplification with specific primers corresponding to the indi-cated amino acid residues. The primers included NK4–110 3–5, 59-GATCCATGGATGTGGTGGCGCCATCGTC-39 and NK4–1105–3, 59-GATCCATGGTCGTCGTCCCTATCGCCACTC-39; NK4–151, 59-GATCCATGGTCCAGCACTCCGCCTCGGCCTAC-39;NK4–191, 59-GATCCATGGGTGGAGCCACGCCCTCGGCG-39;NK4 –231, 59-GATCCATGGCGACGCAGAAGCTGTGCGT-CAAT-39; NK4–271, 59-GATCCATGGTGACCTCCTCGCGTTC-CGAG-3 9 ; NK4-term, 5 9 -GATTCTAGACTACATGTG-CTGCATCTGTTG-39. The 59-nontranslated region of Tin was alsoamplified with primers NK453NotI, 59-GATGCGGCCGCA-ATTCAAGTGTCGCAA-39 and NK4 ATG, 59-GATCA-CCATGGCTCGCTGCGTGATCACTTG-39. The coding regionDNAs were digested with NcoI and XbaI, while the 59 noncodingDNA was digested with NotI and NcoI, followed by the cloning of theresulting DNAs into the NotI and XbaI sites of the pUAST vector.Immunostaining of embryos expressing the various UAS-tin con-structs with a Tin antibody (Jagla et al., 1997) showed that all mutantproteins were stably expressed and detectable in the transgenic linesused.

To generate the UAS-Pnr constructs, mutant cDNAs were gener-ated using the QuickChange Site-Directed Mutagenesis Kit. Primers
used were as follows: Pnr(E168K), 59-CGAGGGACGCAAGTGCGT-

CAATTG-39 and 59-CAATTGACGCACTTGCGTCCCTCGC-39;Pnr(G184R), 59-GTGGCGACGGGATAGAACCGGACACTATC-39and 59-GATAGTGTCCGGTTCTATCCCGTCG CCAC-39;Pnr(C190S), 59-CACTATCTGTCCAACGCCTGCGGAC-39 and59-GTCCGCAGGCGTTGGACAGATAGTG-39; Pnr(D242S), 59-GG-AGGCGCAACAACAGTGGCGAACCCGTG-3 9 and 5 9 -CACGGGTTCGCCACTGTTGTTGCGCCTCC-39; Pnr(C247S), 59-GGCGAACCCGTGTCCAATGCCTGCG-39 and 59-CGCA-GGCATTGGACACGGGTTCGCC-3 9 ; Pnr (G457stop) ,59-CGGAACCCATTGACACCATGTGACG-39 and 59-GT-CACATGGTGTCAATGGGTTCCG-39. The mutations were veri-fied by DNA sequencing, followed by cloning into the XhoI and XbaIsites of pUAST.

GST Pull-Down ExperimentsGST-Tin fusion proteins and the Pnr in vitro translation (IVT)

product were generated by using methods described previously(Choi et al., 1999b). Fusion proteins were recovered from thesupernatants of sonicated BL21DE3 cells (Invitrogen) and coupledto GSH beads (Amersham Pharmacia, Piscataway, NJ) at roomtemperature. Beads were washed thoroughly to remove unboundprotein and stored at 4°C until use. The Pnr IVT product was eitherused immediately upon synthesis or stored at 270°C for up to 3days. For the pull-down reactions, the relative protein concentra-tion of the GSH-GST-Tin constructs or GSH-GST beads weredetermined by using protein separation by SDS-PAGE and stainingwith Coomassie blue. The volume of beads was adjusted by usingunbound beads so that all preparations had the same relativeamount of protein in a 60-ml volume. As a control, the aliquots ofadjusted bead volumes were assayed again for the same relativeprotein concentration, using SDS-PAGE and Coomassie blue stain-ing. A total of 60 ml of GSH-GST-Tin beads or GSH-GST beads

ere incubated with 10 ml of 35S-methionine-labeled Pnr in bindinguffer (20 mM Tris–HCl, pH 7.6, 2.5 mM MgCl2, 1 mM EDTA, 0.5

mM DTT, 0.15% NP-40, and 25 mg/ml BSA) for 2 h at roomtemperature with nutation. The beads were then washed six timeswith buffer (20 mM Tris–HCl, pH 8.0, and 1% NP-40) to removeunbound IVT product. The GST-Tin/Pnr complexes were elutedfrom the beads by resuspension in 20 ml of SDS reducing buffer andheating for 5 min at 90°C. The entire volume of each reaction wasloaded onto 7.5% SDS-PAGE gels. Gels were run for 40 min at 150V, stained and dried, and exposed to a phosphor screen overnightfollowed by image processing with ImageQuant 5.0.

RESULTS

A pnr Dorsal Mesoderm Enhancer Is Regulated bythe Tin Homeodomain Protein

Around the time of heart precursor cell specification, pnrRNA is detected in the dorsal mesoderm in cells that alsoexpress the tin gene (Gajewski et al., 1999). We havepreviously identified a pnr enhancer that is active in thisheart-forming region and maps to a 457-bp DNA immedi-ately upstream of the gene (Fig. 1A). Because the enhancercontains two putative Tin recognition sites, we investi-gated the binding of the factor to this DNA. A GST-Tinfusion protein was used in a gel-shift assay to test its abilityto bind to the Tin1 sequence TCAAGTG, a known recog-
nition element of the homeodomain protein in mesodermal

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enhancers of the D-mef2, tin, and b3 tubulin genes (Gajew-ki et al., 1997; Lee et al., 1997; Xu et al., 1998; Kremser etl., 1999). Tin can bind specifically to the Tin1 consensusut not to a mutant version of the sequence (Fig. 1B). DNaseprotection assays were also performed with the fusionrotein on the pnr DNA, and two separate footprints werebtained that correspond to the Tin1 and Tin2 sequencesFig. 1C). These in vitro experiments demonstrate that Tinan recognize and bind to two sites within the pnr dorsalesoderm enhancer, suggesting the regulatory DNA might

e a direct target of Tin transcriptional activity.The defined pnr enhancer functions in the dorsal-most

ells of the mesoderm and in cells of the amnioserosa (Figs.A and 2B; Gajewski et al., 1999). To determine whether itsctivity was regulated by Tin around the time of heart cellormation, we monitored the expression of a pnr enhancer–acZ fusion gene in tin gain and loss of function embryos.nitially, we used the Gal4/UAS binary system (Brand and

FIG. 1. Identification of Tin binding sites in a regulatory sequence9-flanking sequence. The location of a 457-bp dorsal mesoderm enites (TCAAGTG) for the protein. (B) Electrophoretic mobility shif

(WT) or mutant (MT) oligonucleotides corresponding to the Tin132P-labeled (2) or (1) strand of the pnr dorsal mesoderm enhancer a1 A, chemical sequencing reaction of the pnr DNA; BSA, reaction wprotected sequences corresponding to the Tin1 and Tin2 sites.

errimon, 1993) to express tin throughout the mesoderm r


nd mesectoderm under the control of the twi-Gal4 driver.e observed an expanded function of the enhancer within

he mesoderm, coupled with ectopic activity in midlineells of the central nervous system (CNS) due to the forcedxpression of Tin (Figs. 2C and 2D). Conversely, in tin nullmbryos, a complete absence of b-galactosidase expressionas observed which demonstrated a requirement of Tin

unction for enhancer activity (Fig. 2H). As a control, weetected a normal pattern of pnr-lacZ transgene expressionn tin heterozygous embryos (Fig. 2G).

The functional importance of the Tin1 and Tin2 sitesithin the pnr control region was assessed by individuallyr simultaneously mutating these sequences and testinghe effects of the alterations on enhancer function. Trans-ormant lines that contained the mutTin1, mutTin2, or

utTin1/2 pnr enhancer-lacZ transgenes showed identicalatterns of a loss of expression from the dorsal mesodermFigs. 2E and 2F). Thus, the presence of both Tin sites is

ream of the pnr gene. (A) Schematic of the pnr gene and its adjacenter is shown on the left; Tin1 and Tin2 indicate consensus bindingy using a Tin fusion protein (GST-NK4) and 32P-labeled wild-typeding site. (C) DNase I protection assay performed with either ancreasing amounts of the Tin fusion protein (GST-NK4). Lanes: Govine serum albumin control protein added. Boxes 1 and 2 indicate

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FIG. 2. Tin is a transcriptional regulator of the pnr dorsal mesoderm enhancer. (A) Ventral view of a whole-mount wild-type embryoexpressing the 457-bp pnr enhancer-lacZ transgene in the dorsal mesoderm. (B) Cross section of an embryo of the same genotype as (A)showing enhancer activity in the dorsal mesoderm. (C) Ventral view of pnr-lacZ expression in a whole-mount embryo of the genotypetwi-Gal4; UAS-tin. b-galactosidase activity is detected in an expanded pattern in the mesoderm (open arrowheads) and de novo along theventral midline (closed arrowhead). (D) Cross section of an embryo of the same genotype as (C) showing ectopic enhancer function in themesoderm (open arrowhead) and derivatives of the mesectoderm (closed arrowhead). (E) Cross section of an embryo expressing the mutTin1pnr-lacZ transgene. b-galactosidase activity is lost from the dorsal mesoderm (open arrowhead) and gained in the dorsal ectoderm. (F) Crosssection of an embryo expressing the mutTin2 pnr-lacZ transgene. The arrowhead points to the absence of enhancer activity. (G) Crosssection of a tin heterozygous embryo double stained for Tin protein (blue) and b-galactosidase activity (brown) produced under the controlof the pnr enhancer. (H) Lack of pnr enhancer function (arrowhead) in a tin homozygous mutant embryo, definitively identified by thebsence of Tin protein. All embryos are at stage 11. Abbreviations: de, dorsal ectoderm; dm, dorsal mesoderm; WT, wild type.IG. 3. Nuclear colocalization and physical interaction of Pnr and Tin in cultured cells. (A) Confocal images showing colocalization of Tin

green) and Pnr (red) within the nucleus (MERGE, yellow). Cells were cotransfected with GFP-Tin and Myc-Pnr expression vectors androteins were detected with GFP signal and anti-Myc antibody. Nuclei were also stained with DAPI (blue). (B) Nuclear extracts fromotransfected cells were subjected to immunoprecipitation followed by Western blot analysis of proteins with the indicated antibodies. Tinnd Pnr are coimmunoprecipitated, indicating the two proteins interact with each other in the nucleus. Abbreviations: IgG, IgG band; IP,
mmunoprecipitation; WB, Western blot.
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.

430 Gajewski et al.


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of these mutagenesis experiments coupled with the tin gainnd loss of function studies demonstrated the pnr dorsalesoderm enhancer is directly regulated by the Tin home-

domain protein. We also note that the transformant em-ryos displayed ectopic b-galactosidase activity in the dor-al ectoderm. The inappropriate ectodermal expression ofhe mutated enhancers may be indicative of the existence ofrepressor protein that interacts with the Tin recognition

equences in these cells (Xu et al., 1998).Since a single GATA sequence was also present in the pnr

nhancer at a position distal to the two required Tin sitesWinick et al., 1993), we monitored the expression of thenr-lacZ transgene in a twi-GAL4;UAS-pnr genetic back-round. No ectopic expression of the reporter sequence wasbserved in these embryos (data not shown), suggesting thatnr enhancer activity is not subject to transcriptionalutoregulation.

Colocalization and Physical Interaction of the Pnrand Tin Proteins in Cultured Cells

We have previously shown that the coexpression of Pnrand Tin in the embryo resulted in a synergistic activation ofcardiac gene expression and the ectopic induction of heart-like cells (Gajewski et al., 1999). To investigate a possiblehysical interaction of the two proteins, we cotransfectedammalian CV-1 cells with Pnr and Tin expression vectors

nd monitored the cellular distribution of the factors byonfocal microscopy and coimmunoprecipitation analysis.s shown in Fig. 3A, cells expressing GFP-tagged Tin andyc-tagged Pnr exhibited colocalization of the proteins in

he nucleus. Furthermore, analysis of nuclear extracts bymmunoprecipitation followed by Western blot revealedhat Tin was coimmunoprecipitated with Pnr (Fig. 3B, lane). Conversely, Pnr was also detected in the Tin immuno-recipitate (Fig. 3B, lane 12). These results indicate that thewo proteins form a physical complex in the cultured cells,onsistent with their ability to work combinatorially in theegulation of cardiac gene expression.

Identification of Tin Protein Domains Essential forIts Functional Interaction with Pnr

The D-mef2 gene is a direct transcriptional target of Tinand Pnr in cardioblasts. A defined heart enhancer for thegene contains a pair of essential Tin binding sites and arequired GATA element located in close proximity to one

FIG. 4. Protein domains needed for transcriptional synergism of Tin wiin the D-mef2 enhancer-lacZ activation assay. The (1) or (2) design

bbreviations: TN, conserved TN domain; HD, homeodomain. (B–G) Sta-mef2-lacZ fusion gene. All embryos are of the genotype twi-Gal4; IIAoint to the de novo expression of D-mef2-lacZ in CNS midline cells whmbryos show a strong overactivation of the fusion gene in the dorsal mes
n the embryos of (C) and (F).

f the Tin recognition sequences (Gajewski et al., 1997,998). Coexpression of the two factors in CNS midline cellsesults in the ectopic activation of the D-mef2 enhancerormally expressed only in cardial cells (Fig. 4B; Gajewskit al., 1999). This result is compatible with the nuclearolocalization and physical interaction of Tin and Pnr inultured cells and provides an embryological assay fordentifying regions of the proteins that are essential forheir functional synergism. Nine deleted or point mutantersions of Tin were tested in the synergism assay (Fig. 4A).in(N351Q) has a single amino acid change in the home-domain and is unable to bind DNA (Choi et al., 1999b).oexpression of this mutant with wild-type Pnr fails to

ctivate the D-mef2 enhancer (Fig. 4C). While a competentomeodomain must be present in Tin for synergism withnr, this region by itself is not sufficient as it fails in theoactivation assay (Fig. 4A; data not shown). The TNomain is a highly conserved 12 amino acid region found inin and most other NK-2 class proteins (Harvey, 1996). A0-amino acid deletion was made within this domain toenerate the Tin(1–35, 46–416) mutant, but this alteredrotein was still able to function combinatorially with PnrFig. 4D). Thus, the TN domain is dispensable in theynergism assay.A transcriptional activation domain has been mapped to

he N-terminal 114 amino acids of Tin by using a cellransfection strategy (Choi et al., 1999b). To determinehether this region was required for functional interactionith Pnr, the Tin(111–416) deletion was generated and

ested. This truncated protein remained competent to syn-rgize with Pnr in the activation of the D-mef2 enhancer,howing that the Tin transactivation domain was notequired (Fig. 4E). However, larger N-terminal deletionsesulted in Tin proteins that were functionally inactive (Fig.A). Specifically, removal of an additional 41 amino acids inin(152–416) identified residues 111 to 151 as essential forin synergism with Pnr (Fig. 4F). The Tin(1–109, 192–416)ariant that contains the transactivation domain and home-domain, but lacks internal sequences including the re-uired 41-amino acid region, is likewise nonfunctional inhe D-mef2 enhancer coactivation assay (Fig. 4G). There-ore, these studies identify two distinct regions of Tineeded for its combinatorial function with Pnr, an internalegment of 41 amino acids adjacent to the transactivationomain and the conserved homeodomain.In support of this in vivo data, we delineated regions ofin that were able to bind to the Pnr protein. Eight GST-Tin

r. (A) Full-length, point mutant, truncated, and deleted forms of Tin usedindicate the ability or inability of a protein to synergize with Pnr.

embryos stained for b-galactosidase activity due to the expression of theD-mef2-lacZ/UAS-tin or UAS-mutant tin; UAS-pnr. Filled arrowheadsen arrowheads indicate the lack of enhancer activation in these cells. All

due to the expression of wild type Pnr. This staining is in focus (arrows)

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fusion proteins containing all or part of the cardiogenicfactor were used in in vitro pull-down assays with 35S-abeled full-length Pnr (Fig. 5). The strongest interactionsere observed with either wild-type Tin or truncated ver-

ions that contained the homeodomain region (Tin A1, A2,23, and A31). Additionally, weaker but discernible bind-

ng of Tin and Pnr was observed with truncated proteinshat contained the functionally defined 111 to 151 regionTin A4, A5, and A34). These molecular results are consis-ent with the requirement of this internal domain for theunctional synergism of Tin with Pnr in the activation ofhe D-mef2 cardiac enhancer in the CNS.

Identification of Pnr Protein Domains Essential forIts Functional Interaction with Tin

We also used the ectopic activation assay to determinethose regions of Pnr that were essential for its functionalsynergism with Tin. Six deleted or point mutant forms weretested for enhancer activation in CNS midline cells (Fig.6A). Pnr(1–457) represents a C-terminal truncation of theGATA factor that maintains zinc fingers 1 and 2, butdeletes two putative amphipathic a helices. ThisC-terminal region has been shown to contain a transcrip-

FIG. 5. Analysis of the Pnr interaction domains of Tin. VariousST-Tin fusion proteins were purified from bacterial cultures,

used to glutathione-Sepharose beads, and subjected to pull-downssays with in vitro-translated 35S-methionine-labeled Pnr protein.

The designations of the Tin proteins are given as A1 to A34, andtheir amino acid compositions are listed in parentheses. Therelative binding of Pnr to the wild-type or mutant forms of Tin wasdetermined from an average of six assays and indicated by (1) or (2)on the right. The inset at the bottom shows the data from one of theexperiments and includes a sample of input 35S-labeled Pnr and a

ST protein control.

tional activation domain (Haenlin et al., 1997), and the t


nability of the truncated protein to synergize with Tinemonstrates an essential requirement of this Pnr sequenceFig. 6B). Pnr(E168K) and Pnr(C190S) contain single aminocid changes in the N-terminal zinc finger that correspondo mutations found in the dominant alleles pnrD4 and pnrD3

(Ramain et al., 1993). These mutations may affect theformation of the first zinc finger and result in proteins thatheterodimerize poorly with the Ush antagonist (Haenlin etal., 1997). However, both Pnr proteins were able to syner-gize with Tin and direct D-mef2 expression in the CNS(Figs. 6C and 6D). In contrast, the mutation of a conservedcysteine residue in zinc finger 2 in Pnr(C247S) inactivatedthe protein in the synergism assay (Fig. 6E). This amino acidchange is likely to influence the formation of theC-terminal zinc finger and identifies this region as anessential functional domain of Pnr in the coactivation ofD-mef2. It is important to note that, although Pnr(1–457)and Pnr(C247S) fail to synergize with Tin, they are compe-tent to bind the homeodomain protein in the GST pull-down assay (data not shown). In combination, these resultssubstantiate that intrinsic functional properties of Pannierare perturbed in the two mutant forms of the GATA factor.

Previous studies have shown that mouse GATA4, but notGATA1, could work with Tin in inducing cardial cellmarkers when coexpressed in the midline cells (Gajewski etal., 1999). One theory for the functional difference betweenthe vertebrate factors in the Drosophila cardiogenic assaywas that certain key amino acids, present in Pnr andGATA4, were not conserved in GATA1. We generated twoPnr proteins that changed amino acids found exclusively inPnr and GATA4 to more closely resemble the GATA1sequence. However, the zinc finger 1 mutant Pnr(G184R)and the zinc finger 2 mutant Pnr(D242S) were both compe-tent in the enhancer coactivation test, demonstrating thatthese conserved residues were not essential for Pnr func-tional interaction with Tin (Figs. 6F and 6G).

DISCUSSION

Results from the current study contribute significantly toour understanding of the genetic control of cardiogenesis inDrosophila. Together with recent work from our lab andothers, we can elaborate a detailed regulatory hierarchyprogramming cardial cell specification and diversification(Fig. 7). The selective induction of the Tin homeodomainprotein in the dorsal mesoderm is mediated by Dpp signalsemanating from the dorsal ectoderm (Frasch, 1995). Thisinvolves the binding of Dpp-activated Smad proteins, andTin itself, to a Dpp-responsive enhancer so as to activate tinexpression (Yin et al., 1997; Xu et al., 1998). The present

ork demonstrates that pnr is a direct transcriptional targetf Tin in cells of the heart forming region. It is noteworthyhat two recent reports have likewise demonstrated theirect activation of cardiac enhancers of the chick andouse gata6 genes by Nkx2.5 (Davis et al., 2000; Molken-

in et al., 2000). These similar findings suggest that impor-


Pws

wi


FIG. 6. Protein domains needed for the transcriptional synergism of Pnr with Tin. (A) Full-length, truncated, and point mutant forms ofnr used in the Dmef2 enhancer-lacZ activation assay. The (1) or (2) designations indicate the ability or inability of a protein to synergizeith Tin. Abbreviations: zf1 and zf2, zinc finger domains 1 and 2; h1 and h2, putative amphipathic a helices 1 and 2. (B–G) Stage 11 embryos

tained for b-galactosidase activity due to the expression of the D-mef2-lacZ fusion gene. All embryos are of the genotype twi-Gal4; IIA237D-mef2-lacZ/UAS-tin; UAS-pnr or UAS-mutant pnr. Filled arrowheads point to de novo expression of D-mef2-lacZ in CNS midline cells,

hile open arrowheads indicate the lack of enhancer activation in these cells. All embryos show a background activation of the fusion gene
n the cephalic mesoderm due to the expression of wild-type Tin. This staining is in focus (arrows) in the embryos of (B) and (E).

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tant regulatory interactions between tin and gata classgenes have been evolutionarily conserved during heartdevelopment. We also show that Pnr and Tin colocalize tothe nucleus of transfected cells, where they form a physicalcomplex. These observations are in accord with the geneticrequirement of pnr and tin for heart precursor formationand the ability of the two factors to work combinatorially inthe activation of cardiac gene expression and the productionof ectopic cardioblast-like cells (Azpiazu and Frasch, 1993;Bodmer, 1993; Gajewski et al., 1999). Their functionalinterplay, along with the negative modulatory effect of Ushon Pnr, culminates in the precise specification of the cardialcell lineage within the dorsal mesoderm (Fossett et al.,000).Subsequent to specification, cardiac progenitors undergo

ymmetric or asymmetric cell divisions to generate non-quivalent populations of cardial cells (Gajewski et al.,000; Ward and Skeath, 2000). These include four pairs of

FIG. 7. Schematic of a genetic regulatory hierarchy controllingardial cell specification and diversification during heart develop-ent in Drosophila. Arrows between genes indicate a direct or

indirect transcriptional activation of a downstream locus. Themerging lines between tinman and pannier represent the combi-natorial function of the two genes in specifying cardioblast precur-sor cells. The blocked lines indicate a negative regulatory effect ofone gene upon another. Boxes highlight progenitor or geneticallydistinct groups of heart cells. The schematic is a simplification ofthe total genetic complexity controlling heart formation in Dro-sophila and does not necessarily list all essential components ofthis developmental process. Abbreviations: 4 cc and 2 cc pairs, fouror two pairs of cardial cells per segment of the dorsal vessel thatexpress the Tin or Svp transcription factors and separable enhanc-ers for the D-mef2 differentiation gene.

ells per segment of the dorsal vessel that express Tin and


wo pairs per segment that express the orphan steroideceptor Seven Up (Svp; Fig. 7). A regulatory interactionxists between these transcription factor genes as onedentified function of Svp in the two cell pairs is to represshe activity of tin (Gajewski et al., 2000). The resultingutually exclusive expression of Tin and Svp leads to the

ctivation of different sets of target genes, including the b3tubulin structural gene in the four cell pair population andindependent enhancers for the D-mef2 differentiation genein either of the cell groups (Kremser et al., 1999; Gajewskiet al., 2000). This presumably confers a needed functionaldiversity to cardial cells within the mature heart.

Our survey of Tin and Pnr protein motifs needed in theD-mef2 coactivation assay reveals both factors requirecompetent DNA-binding domains and at least one addi-tional region for their synergistic interaction. The necessityof the former is not surprising as the D-mef2 control regioncontains clustered Tin and GATA elements that must bepresent for enhancer activity (Gajewski et al., 1997, 1998).Also, several studies have demonstrated that the physicalassociation of NKX2.5 and GATA4 is mediated through thehomeodomain and zinc finger 2 domains (Durocher et al.,1997; Lee et al., 1998; Sepulveda et al., 1998). Given theextensive sequence homology between the fly and mousefactors in these two regions, comparable molecular interac-tions would be predicted for Tin and Pnr.

An unexpected finding of our current work is that, whilethe C-terminal transactivation domain of Pnr is required inthe combinatorial assay, the N-terminal transactivationdomain of Tin is not. One could envision a mechanismwherein the presence of the single domain provided by Pnris sufficient for the activation properties of the het-erodimeric complex. Additionally, it can not be ruled outthat a second transactivation domain exists in Tin that wasnot revealed previously in cell transfection studies (Ranga-nayakulu et al., 1998; Choi et al., 1999b). Also of note is thenonrequirement of a proposed cardiogenic domain of Tinthat maps to the N-terminus of the protein (Park et al.,1998; Ranganayakulu et al., 1998). Specifically, Tin(111–416) is competent to work with Pnr in the cooperativeactivation of the D-mef2 heart enhancer, despite the ab-sence of residues 1 through 42.

Instead, an internal 41-amino acid region between theTin transactivation domain and homeodomain has emergedas a vital sequence for functional interaction with Pnr. Choiet al. (1999b) has recently assigned a repressor activity ofTin to residues 111 through 188, and it is plausible that,based on the biological assay being used, multiple func-tional characteristics may be uncovered within this region.In the context of Tin’s synergistic interaction with Pnr inregulating a defined cardiac enhancer, association of thetwo through this domain may prevent Pnr from interactingwith other proteins such as Ush. At the same time, becauseTin has the potential to act as a transcriptional repressorthat recruits Groucho via this domain (Choi et al., 1999b),the interaction of Tin and Pnr through the essential 111 to
151 subregion may be beneficial to Tin in its role as a

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transcriptional activator by eliminating its possible associa-tion with inhibitory cofactors. Preliminary results suggestthe molecular interaction of Tin and Pnr may be due in partto the presence of this domain.

Relative to the functions of Tin and Pnr in programmingthe cardial cell lineage, it will be critical to decipher thecomplex relationship of these two cooperative factors withthat of the Ush antagonist. How the association of thismultitype zinc finger protein with Pnr changes the GATAfactor from a transcriptional activator to a repressor re-mains a provocative question. Perhaps Ush recruits a re-pressor protein and histone deacetylase to assemble a core-pressor complex. All FOG class proteins, including Ush,contain a binding motif for the transcriptional corepressorC-terminal Binding Protein (CtBP; Fox et al., 1999). How-ever, the importance of this heterologous protein interac-tion has not been firmly established due to conflictingreports on the requirement of CtBP for FOG repressorfunction (Fox et al., 1999; Deconinck et al., 2000; Svenssonet al., 2000a). Whether Ush interacts with CtBP or anotherDrosophila corepressor protein, the specification of thecardial cell lineage will likely depend on the balance be-tween a promoting Tin-Pnr activation system and an in-hibitory Ush-Pnr repressor complex within cells of thedorsal mesoderm.

ACKNOWLEDGMENTS

We thank M. Frasch for providing the Tin antibody, C. Y. Elliottand T. Vachris for technical help, and R. Grenda for assistance withfigures. DNA sequences were determined by the M.D. AndersonDNA Core Sequencing Facility supported by National Institutes ofHealth Grant CA16672. This research was supported by grantsfrom the American Heart Association, Muscular Dystrophy Asso-ciation, and National Institutes of Health (HL59151) (to R.A.S.) andthe National Heart, Lung, and Blood Institute intramural researchprogram (to Y.K.).

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Submitted for publication November 8, 2000Revised January 25, 2001

Accepted January 25, 2001Published online April 16, 2001


pannier is a transcriptional target and partner of tinman during drosophila cardiogenesis

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