Boettger, T.

Grow, M.

Kessel, M.

Krieg, P.

Newman, C.

Ni, K.

Reecy, J.

Schwartz, R.

English

MPG.PuRe

Gene expression patternTransient cardiac expression of the tinman-family homeobox gene,XNkx2-10Craig S. Newmana, James Reecyb,1, Matt W. Growa, Karen Nia, Thomas Boettgerc,Michael Kesselc, Robert J. Schwartzb, Paul A. Kriega,*aDivision of Molecular Cell and Developmental Biology, School of Biological Sciences, University of Texas at Austin, Austin, TX 78712, USAbDepartment of Cell Biology, Baylor College of Medicine, Houston, TX 77035, USAcMax-Planck-Institut fur biophysikalische Chemie, Am Fassberg, D-37077 Gottingen, GermanyReceived 18 May 1999; received in revised form 20 September 1999; accepted 29 October 1999AbstractIn Drosophila, the tinman homeobox gene is absolutely required for heart development. In the vertebrates, a small family of tinman-relatedgenes, the cardiac NK-2 genes, appear to play a similar role in the formation of the vertebrate heart. However, targeted gene ablation of one ofthese genes, Nkx2-5, results in defects in only the late stages of cardiac development suggesting the presence of a rescuing gene function earlyin development. Here, we report the characterization of a novel tinman-related gene, XNkx2-10, which is expressed during early heartdevelopment in Xenopus. Using in vitro assays, we show that XNkx2-10 is capable of transactivating expression from promoters previouslyshown to be activated by other tinman-related genes, including Nkx2-5. Furthermore, Xenopus Nkx2-10 can synergize with the GATA-4 andSRF transcription factors to activate reporter gene expression. q 2000 Elsevier Science Ireland Ltd. All rights reserved.Keywords: Nkx; tinman; Heart; Pharyngeal endoderm1. Results and discussionSomewhat unexpectedly, mice lacking Nkx2-5 functionare able to form beating heart tissue and the majority ofmyocardial differentiation markers are expressed normally(Lyons et al., 1995). This result suggests that additionaltinman-related genes expressed in the developing heartmay partially substitute for Nkx2-5 function in theknock-out mouse (Lyons et al., 1995). Using low strin-gency screening we have identi®ed XNkx2-10, a newmember of the vertebrate tinman-related gene family(Fig. 1A). The XNkx2-10 protein is 263 amino acidslong, shorter than the Xenopus homologues of XNkx2-3(338 amino acids) and XNkx2-5 (299 amino acids) andcloser in length to the zebra®sh nkx2-7 coding region(267 amino acids). The XNkx2-10 homeodomain sequencediffers from all previously described Tinman-relatedproteins (Fig. 1B). Speci®cally, the methionine and gluta-mine residues located at homeodomain positions 11 and15, respectively, are not present in any other Tinmanfamily proteins (Harvey, 1996). Indeed, the XNkx2-10homeodomain differs at ten positions from chickencNkx2-8, at nine positions from XNkx2-5, at six positionsfrom XNkx2-3 and at ®ve positions from zebra®sh Nkx2-7.Outside of the homeodomain, XNkx2-10 contains both aTN domain and an NK-2 Speci®c Domain related to thosein other Tinman-family proteins. The primary sequencetherefore suggests that XNkx2-10 is a novel member ofthe vertebrate tinman gene family.By RNase protection assay (Fig. 1C) XNkx2-10 is ®rsttranscribed in the early neurula stage embryo. This is laterthan the onset of XNkx2-5 expression which occurs at themid-gastrula stage in Xenopus (Tonissen et al., 1994). Bywhole-mount in situ hybridization, early expression ofXNkx2-10 closely resembles that of XNkx2-3 and XNkx2-5.XNkx2-10 transcripts are ®rst visible at the late neurula stagein a position consistent with the location of precardiac tissue(Fig. 2A). By the early tailbud stage, cardiac precursor cellsMechanisms of Development 91 (2000) 369±3730925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved.PII: S0925-4773(99)00291-9www.elsevier.com/locate/modo* Corresponding author. Department of Cell Biology and Anatomy,University of Arizona College of Medicine, 1501 N. Campbell Avenue,P.O. Box 245044, Tucson, AZ 85724, USA. Tel.: 11-520-626-9370;fax: 11-520-626-2097.E-mail address: pkrieg@u.arizona.edu (P.A. Krieg)1 Present address: Department of Animal Science, Iowa State University,Ames, IA 50011, USA.and also the overlying anterior pharyngeal endoderm areXNkx2-10 positive (Fig. 2B,C,F,G). At stage 28, myocardialprecursor cells initiate the expression of cardiac differentia-tion markers (Chambers et al., 1994; Drysdale et al., 1994). Itis at this stage, that XNkx2-10 expression begins to decreasein the forming myocardium (Fig. 2D,H). In contrast, bothXNkx2-3 and XNkx2-5 transcripts remain abundant in thedeveloping heart throughout embryonic development (Fig.2E,K) (Tonissen et al., 1994; Evans et al., 1995; Cleaver etal., 1996). Whereas cardiac expression of XNkx2-10decreases in the mid-tailbud stage embryo, transcripts remainextremely abundant in the pharyngeal endoderm (Fig. 2D,E).XNkx2-10 expression decreases in the pharyngeal endodermand becomes undetectable by the late tadpole stage (stage42). PCR analysis fails to detect Nkx2-10 expression in anyadult tissue tested (heart, spleen, kidney, liver, lung, pancreasand skeletal muscle).We have carried out preliminary experiments to deter-mine the transcription regulating properties of Nkx2-10.First, we tested for transactivating ability by co-transfect-ing variable amounts of a plasmid containing XNkx2-10sequences, together with the 3£ (A20)-TATA-LUCconstruct (Chen and Schwartz, 1996), into the CV-1cultured cell line. In parallel experiments, XNkx2-10sequences were co-transfected with the minimal cardiacactin promoter construction. As shown in Fig. 3A, lowlevels of XNkx2-10 produce little effect, but higher levelscause a signi®cant increase in reporter gene activity overbackground. In the case of 3£ (A20)-TATA-LUC a 14-foldincrease is observed and for Ca SRE1-TATA-LUC thestimulation is 7-fold. The ability of XNkx2-10 to co-oper-ate with other transcription factors was also tested. Asshown in Fig. 3B, transfection of 150 ng of XNkx2-10plasmid alone, results in a less than 2-fold increase in CaSRE1-TATA-LUC reporter gene activity, and transfectionwith the MADS class transcription factor, SRF, results in a6-fold increase in luciferase activity (Fig. 3B). However,co-transfection of both XNkx2-10 and SRF leads to a 17-fold activation of luciferase activity. Similarly, expressionof GATA4 alone results in only a slight increase in luci-ferase activity, while co-transfection of both GATA4 andXNkx2-10 leads to a 4-fold increase (Fig. 3B). Thesestudies indicate that XNkx2-10 functions as a weak tran-scriptional activator and this activity is synergized by co-operation with other cardiac-expressed transcriptionfactors. Overall, Nkx2-10 is a weaker activator thanNkx2-5, but approximately equivalent to Nkx2-8 whentested in identical assays. For comparison, Nkx2-5 co-transfection with GATA-4 increases Ca SRE1-TATA-LUC reporter activity approximately 15-fold while Nkx2-8 plus GATA-4 results in an approximately 2-fold increase(Sepulveda et al., 1998).We have also tested the effects of XNkx2-10 expression inthe Xenopus embryo. Although previous studies indicatethat over-expression of either Nkx2-3 or Nkx2-5 in the Xeno-pus embryo results in enlarged hearts (Chen et al., 1996;Cleaver et al., 1996), we have been unable to replicate thiseffect using Nkx2-10. Although one must be cautious withthe interpretation of negative results, these experimentssuggest that the biological activities of Nkx2-5 and Nkx2-10 are not identical.C.S. Newman et al. / Mechanisms of Development 91 (2000) 369±373370Fig. 1. (A) DNA sequence of the XNkx2-10 gene. The coding region ofXNkx2-10 and the derived protein sequence are shown. Portions of thissequence were previously reported in Newman and Krieg (1998), wherethe gene was called Nkx2-9. The conserved TN, homeo- and NK-2 Speci®cdomains are highlighted by overlines, underlines and dash underlinesrespectively. The GenBank accession number is AF127224. (B) Compar-ison the homeodomain sequences of cardiac NK-2 genes from frog, chickand zebra®sh (Tonissen et al., 1994; Evans et al., 1995; Lee et al., 1996;Boettger et al., 1997; Brand et al., 1997; Reecy et al., 1997). (C) RNaseprotection assay to determine the onset of XNkx2-10 gene expression. RNAsamples from early (stage 10.5) and late (stage 12) gastrula and early (stage14) and late (stage 19) neurula were assayed. The XNkx2-10 probe consistedof the ®nal 310 nt of the coding region. The ubiquitous Xmax gene (Tonis-sen and Krieg, 1994) is used as a loading control.2. Experimental proceduresApproximately 106 plaques of a Xenopus stage 42tadpole cDNA library were screened at low stringencywith the coding region of the chicken cNkx2-8 gene. All®lters were washed in 5£ SSC/0.1% SDS at 378C. RNaseC.S. Newman et al. / Mechanisms of Development 91 (2000) 369±373 371Fig. 2. Expression pattern of XNkx2-10 by wholemount in situ hybridization. (A) Anterior view of a late neurula stage embryo showing XNkx2-10 expression inendodermal and mesodermal cells (arrow) adjacent to the neurual tube. (B) Lateral view of an early tailbud embryo showing XNkx2-10 transcripts in thepharyngeal endoderm and cardiac primordia (arrow). (C) Ventral view of the embryo in (B) showing both cardiac (black arrows) and pharyngeal (whitearrowheads) staining. (D) Mid tailbud embryo (stage 32) stained for both XHex (purple), which marks the developing liver (Newman et al., 1997) and XNkx2-10 (blue). The heart (arrow), directly anterior to the liver (small arrowhead) is negative for XNkx2-10 at this stage. Note the extensive pharyngeal staining,extending anteriorly past the forming thyroid (large arrowhead). (E) Mid tailbud embryo (stage 32) stained for both XHex (purple) and XNkx2-5 (blue).Compare with (D) and note the strong cardiac staining (arrow). (F±H) Sections of embryos stained for XNkx2-10. (I±K) Sections of embryos stained for XNkx2-5. (F and I) Parasagittal section through an early tailbud stage (stage 22) embryo showing expression of both XNkx2-10 and XNkx2-5 in the endoderm and themesoderm. (G and J) Cross-section through a tailbud stage embryo (stage 26) showing transcripts in both the pharyngeal endoderm (large arrowhead) andcardiac mesoderm (small arrowhead). (H and K) Cross-section through a late tailbud stage embryo. Note the lack of XNkx2-10 expression in the folded heart(small arrowhead) and the lack of XNkx2-5 expression in the pharyngeal endoderm (large arrowhead). Arrows highlight expression of both XNkx2-5 andXNkx2-10 in the mesoderm immediately dorsal to the heart. In situ analysis was performed as described by Harland (1991).protection analysis was performed as described by Kriegand Melton (1987) using ten embryo equivalents of totalRNA.For transcription studies, CV-1 ®broblasts were main-tained in Dulbecco's modi®ed Eagle's medium (DMEM)containing 10% fetal bovine serum. Proliferating CV-1®broblasts were transfected using Lipofectamine (Gibco),with a total of 2.5 mg of DNA, consisting of 3£ (A20)-TATA-LUC or Ca SRE1-TATA-LUC (1 mg; Chen andSchwartz, 1995; Chen et al., 1996) in the presence orabsence of pCS2-XNkx2-10 and/or pCGN-SRF (150 ng,gift of Dr. Ron Prywes). All transfections were balancedfor a constant amount of pCGN control vector. After 6 hat 378C, 3 ml of DMEM containing 3% horse serum and 15mg/ml of insulin was added to the transfected cells, whichwere then incubated for an additional 60 h. Cells werewashed with phosphate buffered saline and lysed with 400ml of Reporter Lysis Buffer (Promega). Lysates wereanalyzed for luciferase activity as described previously(Reecy et al., 1997).AcknowledgementsWe wish to thank Mike King for providing the excellentstage 42 embryo cDNA library and other members of thelaboratory for critical comments on the manuscript. Thiswork was supported NIH grants PO1HL49953 to R.J.S.and HL52746 to P.A.K.ReferencesBoettger, T., Stein, S., Kessel, M., 1997. The chicken Nkx2-8 homeoboxgene: a novel member of the NK-2 gene family. Dev. Genes Evol. 207,65±70.Brand, T., Andree, B., Schneider, A., Buchberger, A., Arnold, H.-H., 1997.Chicken NKx2-8, a novel gene expressed during early heart and foregutdevelopment. Mech. Dev. 64, 53±59.Chambers, A.E., Logan, M., Kotecha, S., Towers, N., Sparrow, D., Mohun,T.J., 1994. The RSRF/MEF2 protein SL1 regulates cardiac muscle-speci®c transcription of a myosin light chain gene in Xenopus embryos.Genes Dev. 8, 1324±1334.Chen, C.Y., Schwartz, R.J., 1995. Identi®cation of novel DNA bindingtargets and regulatory domains of a murine tinman homoedomainfactor. Nkx2-5. J. Biol. Chem. 270, 15628±15633.Chen, C.Y., Schwartz, R.J., 1996. Recruitment of the tinman homologNkx2-5 by serum response factor activates cardiac b-actin gene tran-scription. Mol. Cell. Biol. 16, 6372±6384.Cleaver, O.B., Patterson, K.D., Krieg, P.A., 1996. Over-expression of thetinman-related genes XNkx-2.5 and XNkx-2.3 in Xenopus embryosresults in myocardial hyperplasia. Development 122, 3549±3556.Drysdale, T.A., Tonissen, K.F., Patterson, K.D., Crawford, M.J., Krieg,P.A., 1994. Cardiac troponin I is a heart-speci®c marker in the Xenopusembryo: expression during abnormal heart morphogenesis. Dev. Biol.165, 432±441.Evans, S., Yan, W., Murillo, M.P., Ponce, J., Papalopulu, N., 1995. tinman,a Drosophila homeobox gene required for heart and visceral mesodermspeci®cation, may be represented by a family of genes in vertebrates:XNkx-2.3, a second vertebrate homologue of tinman. Development 121,3889±3899.Harland, R., 1991. In situ hybridization: an improved whole-mount methodfor Xenopus embryos. Methods Cell Biol. 36, 685±695.Harvey, R.P., 1996. NK-2 homeobox genes and heart development. Dev.Biol. 178, 203±216.Krieg, P.A., Melton, D.A., 1987. In vitro RNA synthesis with SP6 RNApolymerase. Methods Enzymol. 155, 397±415.Lee, K.H., Xu, Q., Breitbart, R.E., 1996. A new tinman-related gene. nkx2-7, anticipates the expression of nkx2-5 and nkx2-3 in zebra®sh heart andpharyngeal endoderm. Dev. Biol. 180, 722±731.Lyons, I., Parsons, L.M., Hartley, L., Li, R., Andrews, J.E., Robb, L.,Harvey, R.P., 1995. Myogenic and morphogenetic defects in the hearttubes of murine embryos lacking the homeobox gene Nkx-2.5. GenesDev 9, 1654±1666.Newman, C.S., Krieg, P.A., 1998. tinman-related genes expressed duringheart development in Xenopus. Dev. Gen. 22, 230±238.Newman, C.S., Chia, F., Krieg, P.A., 1997. The XHex homeobox gene isexpressed during development of the vascular endothelium: over-expression leads to an increase in vascular endothelial cell number.Mech. Dev. 66, 83±93.Reecy, J.M., Yamada, M., Cummings, K., Sosic, D., Chen, C.-Y., Eichele,G., Olson, E.N., Schwartz, R.J., 1997. Chicken Nkx2-8: novel homeo-C.S. Newman et al. / Mechanisms of Development 91 (2000) 369±373372Fig. 3. Transcription regulation properties of XNkx2-10. (A) Relative lightunits after transfection of cultured cells with varying amounts of XNkx2-10plasmid. The reporter construction was either 3£ (A20)-TATA-LUC (solidbars) or Ca SRE1-TATA-LUC (open bars). Activation is assessed relativeto the control experiment using pCG vector alone (indicated as 0 ng). Themean and standard deviation of two independent experiments are presented.(B) Relative activation of transcription of the Ca SRE1-TATA-LUC repor-ter construction, following transfection with combinations of plasmids. Inall cases, 150 ng of DNA encoding the transcription factor was transfectedinto the cultured CV1 cells. The mean and standard deviation of two inde-pendent experiments are presented.box gene expressed in early heart progenitor cells and pharyngeal pouch-2 and -3 endoderm. Dev. Biol. 188, 295±311.Sepulveda, J.L., Belaguli, N., Nigam, V., Chen, C-Y., Nemer, M.,Schwartz, R.J., 1998. GATA-4 and Nkx-2.5 coactivate Nkx-2 DNAbinding targets: role for regulating early cardiac gene expression.Mol. Cell. Biol. 18, 3405±3415.Tonissen, K.F., Krieg, P.A., 1994. Analysis of a variant Max sequenceexpressed in Xenopus laevis. Oncogene 9, 33±38.Tonissen, K.F., Drysdale, T.A., Lints, T.J., Harvey, R.P., Krieg, P.A., 1994.XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: evidence for aconserved role in cardiac development. Dev. Biol 162, 325±328.C.S. Newman et al. / Mechanisms of Development 91 (2000) 369±373 373

Transient cardiac expression of the tinman-family homeobox gene, XNkx2-10.

Transient cardiac expression of the tinman-family homeobox gene, XNkx2-10.

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