Developmental Biology 233, 425–436 (2001) doi:10.1006/dbio.2001.0220, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE Pannier is a Transcriptional Target and Partner provided by Elsevier - Publisher Connector of 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 in the specification of precursor cells of the heart, visceral, and dorsal body wall muscles. The GATA factor gene pannier is similarly expressed in the dorsal-most part of the mesoderm where it is required for the formation of the cardial cell lineage. Despite these overlapping expression and functional properties, potential genetic and molecular interactions between the two genes remain largely unexplored. Here, we show that pannier is a direct transcriptional target of Tinman in the heart-forming region. The resulting coexpression of the two factors allows them to function combinatorially in the regulation of cardiac gene expression, and a physical interaction of the proteins has been demonstrated in cultured cells. We also define functional domains of Tinman and Pannier that are required for their synergistic activation of the D-mef2 differentiation gene in vivo. Together, these results provide important insights into the genetic mechanisms controlling heart formation in the Drosophila model system. © 2001 Academic Press Key Words: cardiogenic factors; D-MEF2; heart; Pannier; protein interactions; Tinman; transcriptional enhancer. INTRODUCTION (Azpiazu and Frasch, 1993; Bodmer, 1993). Based on these properties, tin-related genes were cloned and characterized The study of heart development in Drosophila has been in vertebrate species, with several shown to be expressed in of significant value, not only to our fundamental under- the cardiac lineage (reviewed in Harvey, 1996). Comparable standing of organogenesis in this model system, but also to the role of Dpp in regulating tin and promoting cardio- through the application of genetic insights to the investi- genesis, bone morphogenetic protein signaling induces gation of cardiogenesis and disease in evolutionarily ad- Nkx-2.5 expression and cardiac myogenesis in the chick vanced organisms. A prime example can be found in the embryo (Schultheiss et al., 1997). Likewise, genetic studies homeobox gene tinman (tin) and its vertebrate NK-2 class in the mouse and Xenopus systems have demonstrated counterparts. tin expression in the dorsal mesoderm is essential functions for the Nkx-2.3 and Nkx-2.5 genes in regulated by intercellular signaling mediated by the growth vertebrate heart development (Lyons et al., 1995; Fu et al., factor Decapentaplegic (Dpp) produced by adjacent ectoder- 1998; Grow and Krieg, 1998; Tanaka et al., 1999). Of mal cells (Frasch, 1995; Xu et al., 1998). tin function is medical importance, certain patients with congenital heart required in the dorsal mesoderm for the formation of disease have been shown to carry dominant mutations in progenitors of the heart, visceral, and dorsal somatic the human NKX2–5 gene, resulting in diverse cardiac de- muscles, and the mutation of this transcription factor velopmental abnormalities (Schott et al., 1998; Benson et results in an absence of these mesodermal derivatives al., 1999). tin is expressed in the dorsal mesoderm in a broad pattern 1 K.G., Q.Z., and C.Y.C. contributed equally to this work. that exceeds the limits of the heart-forming region (Yin and 2 To whom correspondence should be addressed. Fax: (713) 790- Frasch, 1998; Gajewski et al., 1999). This observation, 0329. E-mail: [email protected]. coupled with the inability of pan-mesodermal tin expres- 0012-1606/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 425 426 Gajewski et al. sion to produce ectopic heart cells (Yin and Frasch, 1998), Tin. Our results show that pnr is a direct target of Tin implicated the involvement of additional factors in the transcriptional activation during heart formation. Addition- genesis of cardiac cells within the dorsal-most mesoderm. ally, we identify regions of the two proteins that are Based on the requirement of a GATA element for the essential for their combinatorial regulation of cardiac gene function of a heart enhancer of the D-mef2 differentiation expression. These findings contribute needed information gene (Gajewski et al., 1998) and the demonstrated interac- to our understanding of the genetic pathways controlling tion of NKX2–5 with GATA4 in the regulation of cardiac heart development in Drosophila. gene expression in vertebrates (Durocher et al., 1997; Lee et al., 1998; Sepulveda et al., 1998), a role was predicted for a GATA factor in fly heart development. Subsequently, the MATERIALS AND METHODS GATA gene pannier (pnr) was shown to be expressed in the heart forming region and required for cardial cell formation In Vitro DNA Binding Assays (Gajewski et al., 1999). The coexpression of Tin and Pnr Electrophoretic mobility shift and DNase I protection assays results in a synergistic activation of cardiac gene expression were performed as described in Lee et al. (1997). To prepare probes, and the induction of an apparent cardioblast fate in both equimolar amounts of single-strand oligonucleotides were an- mesodermal and nonmesodermal cell types. Together, nealed and end-labeled with T4 kinase. The sequence of the these results provide evidence that the two cardiogenic oligonucleotides used for the gel shift assays were as follows. Ј factors work together to program cells into the cardiac Wild-type probes: pnrA, 5 -CCTCGGTTCTCAAGTGCGGAC- Ј Ј Ј lineage. Additionally, the forced expression of either Pnr or CTG-3 ; pnrB, 5 -CAGGTCCGCACTTGAGAACCGAGG-3 . Mu- tant probes: pnrAm, 5Ј-CCTCGGTTCTgAAGTcCGGACCTG-3Ј; mouse GATA4 throughout the Drosophila mesoderm re- pnrBm, 5Ј-CAGGTCCGgACTTcAGAACCGAGG-3Ј. The mu- sults in supernumerary cardial cells, demonstrating an tated nucleotides are in lower case. For the competitive gel-shift evolutionarily conserved function between the two GATA assays, a 100-fold molar excess of unlabeled DNA was used. For the genes in heart development. footprinting assay, the pnr enhancer DNA was digested with A further complexity in the genetic control of Drosophila HindIII (plus strand labeling) or XbaI (minus strand labeling), and cardiogenesis was found in the analysis of the gene encod- the linearized DNAs were dephosphorylated with alkaline phos- ing the multitype zinc finger protein U-shaped (Ush). This phatase. These DNAs were then digested with XbaI (plus strand) or regulator was previously shown to be an antagonist of Pnr HindIII (minus strand), eluted from a gel, and end-labeled with T4 function in neuronal precursor cell determination, altering kinase. Binding reactions were undertaken with increasing ␮ the properties of the GATA factor from a transcriptional amounts (0.2, 0.4, and 0.6 g) of GST-NK4 protein and labeled DNA (12.5 fmol) in 20 ␮l of binding buffer. After incubation for 15 activator to a repressor in cells where the two are coex- min at room temperature, CaCl2 (final 0.5 mM) and DNase I (0.1 pressed (Haenlin et al., 1997; Garcia-Garcia et al., 1999). unit) were added to the binding mixture. The DNase I digestions Similarly, in the dorsal mesoderm, Ush controls cardial cell were stopped after 1 min incubation by adding stop buffer followed specification through its modulation of Pnr transcriptional by analysis via denaturing polyacrylamide gel electrophoresis. activity (Fossett et al., 2000). A loss of ush function results in an overproduction of cardial cells, while a gain of Expression Analyses of pnr Enhancer–lacZ Fusion function leads to a reduction of cardioblasts, phenotypes Genes that are directly opposite to those seen with pnr. The structurally related Friend of GATA-2 (FOG-2) protein is Transformant strains expressing the 457-bp pnr dorsal meso- expressed during heart formation in the mouse and likewise derm enhancer–lacZ fusion gene have been described (Gajewski functions as a modulator of GATA4 transcriptional activity et al., 1999). To monitor enhancer activity in a tin gain of in cultured cells (Lu et al., 1999; Svensson et al., 1999; function background, pnr-lacZ males were crossed to UAS-tin virgins (Yin and Frasch, 1998), and the resulting heterozygous Tevosian et al., 1999). The recent analysis of targeted gene males were mated to twi-Gal4 virgins (Baylies and Bate, 1996). mutations have demonstrated a vital function for FOG-2 in To assay pnr-lacZ expression in tin loss of function embryos, heart development as mutants display morphogenetic ab- males were crossed to tin346/TM3, Sb virgins, and animals lack- normalities and an absence of the coronary vasculature ing the balancer chromosome were obtained. Embryos were col- (Svensson et al., 2000b; Tevosian et al., 2000). Additional lected from the mating of these flies and immunostained for evidence that Ush and FOG-2 share functional properties is both ␤-galactosidase and Tin, with homozygous