GATA4 is essential for formation of the proepicardium and regulates cardiogenesis Alistair J. Watt, Michele A. Battle, Jixuan Li, and Stephen A. Duncan* Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53202 Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 16, 2004 (received for review February 2, 2004) The role of GATA4 during the earliest stages of cardiogenesis has Defects in the development of extraembryonic endoderm can be not been defined because Gata4 knockout embryos suffer an early circumvented by producing embryos from ES cells by using developmental arrest caused by deficiencies in extraembryonic tetraploid embryo complementation (14). Fetuses generated by visceral endoderm function. We have used tetraploid embryo this procedure are derived solely from ES cells, whereas the complementation to rescue these defects and generated clonal extraembryonic endoderm is tetraploid embryo-derived (15). .embryonic day 9.5 Gata4؊/؊ embryos directly from embryonic stem We have used this procedure to generate Gata4Ϫ/Ϫ embryos cells. GATA4-null embryos display heart defects characterized by These embryos display defects in cardiogenesis and proepicar- disrupted looping morphogenesis, septation, and a hypoplastic dium development, demonstrating an essential function for ventricular myocardium. We find that myocardial gene expression GATA4 in the patterning of the ventral mesoderm. is relatively normal in GATA4-null hearts including expression of GATA6. Moreover, GATA4 expression in the endocardium is dis- Materials and Methods pensable for trabeculae formation. Remarkably, the proepicardium Generation of Plasmids, Gata4؊/؊ ES Cells, and Embryos. Targeting is absent in GATA4-null embryos, blocking formation of the epi- and Cre excision of the Gata4 locus was performed as described cardium. Therefore, we propose that the observed myocardial (16). A targeting vector pGATA4loxPDT was constructed to defects may be a secondary consequence of loss of the proepicar- contain a neo-tk cassette flanked by two loxP sites from plasmid dium. These findings definitively demonstrate a requirement for pHR-1 that were inserted into the first SmaI site upstream of GATA4 during early cardiac development and identify an essential Gata4 exon 3. A single loxP site was introduced into the BamHI factor for generation of the proepicardium. site between exons 5 and 6. Negative selection was provided by the diptheria toxin gene. Methodologies for the generation of ES heart development ͉ septum transversum mesenchyme ͉ cell-derived embryos by tetraploid embryo complementation proepicardial organ have been described elsewhere (14). Pregnant mare serum gonadotropin (PMSG) used in superovulation was obtained n the mouse, the cardiac lineage is specified from cells of the from A. F. Parlow at the National Hormone and Peptide ϩ Iprimitive streak at approximately embryonic day (E) 7.0. The Program (Torrance, CA). Gata4loxP/ ;Tie2Cre mice were gener- nascent cardiac mesoderm migrates anterolaterally, where it ated by breeding Gata4loxP/ϩ mice (Gata4tm1Sad) with Tie2-Cre fuses at the ventral aspect of the embryo to form a linear heart mice (17). tube by E8.5. The linear tube subsequently undergoes complex looping and septation to form the characteristic four-chambered Histochemistry, Immunohistochemistry, and Electron Microscopy heart. Considerable effort has been made in defining the mo- (EM). Immunohistochemistry was performed by using antigen lecular networks controlling mammalian heart development, in retrieval as described elsewhere (16) by using antibodies against part because this knowledge is applicable to human disease GATA4 (Santa Cruz Biotechnology), sarcomeric myosin states such as congenital heart disease and cardiac hypertrophy. (MF20, Developmental Studies Hybridoma Bank, Iowa City, Central to these processes, cardiac-selective transcription factors IA), Wilms’ tumor 1 (WT1) (Cell Marque, Hot Springs, AR), such as MEF2c, NKX2–5, TBX5, and HAND1 have been shown and platelet endothelial cell adhesion molecule (CD31, BD to activate cardiac gene expression and are essential for the Pharmingen). For EM, tissues were fixed in 2% gluteraldehyde proper patterning of the developing heart (1, 2). in cacodylate buffer and embedded in epoxy resin. Semithin The zinc finger transcription factor GATA4 represents an- (2-␮m) sections were stained with toluidine blue, and 80- to other such factor. It is expressed in the lateral plate mesoderm 100-nm sections were contrasted with uranyl acetate and lead at the earliest stages of cardiac differentiation and subsequently citrate for EM. maintained in the cardiac lineage throughout development (3). GATA4 regulates cardiac specific gene expression in vitro (4–6) RT-PCR. RT–PCR was carried out as described (14). Primer and modulates cardiogenesis in Xenopus and in embryonic stem sequences are provided in Supporting Text, which is published as (ES) cell embryoid bodies (7, 8). In mice, an engineered point supporting information on the PNAS web site. mutation in GATA4, predicted to disrupt the interaction with the cofactor friend-of-GATA 2 (FOG2), results in septation and Results and Discussion coronary vasculature defects evident by E12.5 (9), a phenotype We used a two-step Cre-loxP strategy to delete exons 3–5 of the similar to two pedigrees of human congenital heart defects Gata4 gene in ES cells (Fig. 1A). These exons contain both zinc BIOLOGY linked to mutations in GATA4 (10). finger DNA-binding domains and the nuclear localization signal DEVELOPMENTAL Despite these studies, the targeted disruption of GATA4 has that are essential for GATA4 function (12). Both genomic copies failed to resolve a function for this factor at the earliest stages of of Gata4 were targeted sequentially to produce Gata4Ϫ/Ϫ ES heart development. GATA4-null embryos arrest at approxi- mately E8.0 because of defects in ventral morphogenesis, in- cluding a failure of the cardiac mesoderm to form a linear heart This paper was submitted directly (Track II) to the PNAS office. tube (11, 12). However, these defects could be rescued by Abbreviations: En, embryonic day n; ES, embryonic stem; WT1, Wilms’ tumor 1; STM, providing GATA4-null embryos with wild-type visceral septum transversum mesenchyme. endoderm, demonstrating that this phenotype is caused by a *To whom correspondence should be addressed. E-mail: [email protected]. defect in extraembryonic tissue and not the heart, per se (13). © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400752101 PNAS ͉ August 24, 2004 ͉ vol. 101 ͉ no. 34 ͉ 12573–12578 Downloaded by guest on September 30, 2021 they are functionally null for GATA4 (Fig. 6, which is published as supporting information on the PNAS web site ). To address the role of GATA4 in cardiogenesis, we generated embryos from Gata4ϩ/Ϫ and Gata4Ϫ/Ϫ ES cells by aggregating them with wild-type tetraploid embryos. As has been shown (13), Gata4Ϫ/Ϫ embryos complemented with wild-type visceral endoderm sur- vive to E9.5, rescuing the developmental defects, including cardia bifida, associated with conventionally produced Gata4 knockout embryos (Fig. 1 C and D). Southern blot analyses demonstrated that these embryos were genetically identical to the Gata4Ϫ/Ϫ ES cells (Fig. 7, which is published as supporting information on the PNAS web site), and immunostaining ex- periments confirmed that they contained no functional GATA4 protein (Fig. 1 E and F). Control Gata4ϩ/Ϫ ES cell-derived embryos were indistinguishable from wild-type embryos and were viable until at least E12. By comparison, Gata4Ϫ/Ϫ ES cell-derived embryos arrested between E9.5 and E10 (data not shown). It is worth noting that embryos harboring a mutation affecting the interaction of GATA4 with friend-of-GATA 2 (FOG2) survived until E12.5 (9). The earlier embryonic lethality of GATA4-null embryos, therefore, demonstrates a FOG2- independent function for GATA4 during development. To determine the cause of the developmental arrest, we compared somite-matched Gata4ϩ/Ϫ and Gata4Ϫ/Ϫ embryos ranging from 18 to 22 somites. The mutant phenotype reported below was confirmed in embryos from two independent Gata4Ϫ/Ϫ ES cell lines. The most striking anatomical abnormal- ity associated with Gata4Ϫ/Ϫ embryos was a failure of the heart tube to undergo correct looping morphogenesis, characterized by the atrial͞inflow tract region failing to migrate rostrally (Fig. 2 A–D). In addition, the atrioventricular canal and bulboven- tricular groove, structures representing early events in chamber formation, are absent (Fig. 2 A–D). These defects are evident on sections of Gata4Ϫ/Ϫ embryos as a dilated heart tube that has failed to form distinct chambers (Fig. 2 E–H). Histology also reveals that the inflow tract region, which bifurcates at the level of the liver bud into the left and right sinus venosus in Gata4ϩ/Ϫ embryos, is present as a single vessel in Gata4Ϫ/Ϫ embryos (Fig. 2 I and J). Interestingly, vitamin A-deficient (VAD) quail embryos display a similar fused inflow tract (19). However, unlike the VAD model, GATA4-null embryos do not appear to have a blind-ended heart tube (data not shown). The failure of GATA4-null hearts to initiate chamber formation is particularly provocative given the recent finding that septation defects are associated with GATA4 mutations in human congenital heart Ϫ/Ϫ disease (10). Fig. 1. Generation of Gata4 embryos by tetraploid embryo complemen- Ϫ/Ϫ