Transgenic mice expressing small interfering RNA against Gata4 point to a crucial role of Gata4 in the heart and gonads Boris Thurisch, Shermi Y Liang 1 , Nanette Sarioglu 2 , Lutz Schomburg 3 , Jo ¨ rg Bungert 1 * and Christof Dame* Department of Neonatology, Charite ´ -Universita ¨ tsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany 1 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA 2 Institute of Pathology, Charite ´ -Universita ¨ tsmedizin Berlin, D-10117 Berlin, Germany 3 Institute for Experimental Endocrinology, Charite ´ -Universita ¨ tsmedizin Berlin, D-13353 Berlin, Germany (Correspondence should be addressed to C Dame; Email: christof.dame@charite.de) *(C Dame and J Bungert designed the research work and contributed equally to this work) Abstract Homozygous deficiency of the transcription factor Gata4 in mice causes lethality due to defects in ventral morphogenesis and heart tube formation. There is increasing evidence demonstrating that GATA4 function is also relevant for normal developed organ systems, including the heart and endocrinum. To analyze the implication of Gata4 beyond development, we generated transgenic mice expressing inducible small interfering RNA against Gata4. In longitudinal analysis, efficient suppression of Gata4 mRNA (down to 80% of wild-type levels) and protein expression in the heart was detected 38 days after induction of Gata4 short hairpin RNA. Decreased Gata4 expression was associated with reduction in the expression of known cardiac target genes, but the function of the heart remained unperturbed at 20–30% of normal Gata4 levels. Interestingly, Gata4 expression was almost abolished in the ovary and testis. This was accompanied in the testis by a significant reduction of GATA4 downstream target genes, such as the genes encoding Mullerian inhibiting substance (MIS) and steroidogenic acute regulatory (StAR) protein. By contrast, expression levels of Mis and Star were only slightly modified in the ovary, and concentrations of circulating FSH and LH were normal in female transgenic mice after induction of Gata4 short hairpin RNA. However, inhibition of Gata4 expression led to the formation of ovarian teratoma in 10% of females. Histology of the teratomas showed predominantly ectodermal and mesodermal structures. Our data demonstrate that Gata4 is critically involved in the function and integrity of the gonads in vivo. Journal of Molecular Endocrinology (2009) 43, 157–169 Introduction The transcription factor GATA4 belongs to a family of six highly conserved zinc finger proteins, which bind to regulatory DNA fragments containing a ‘T/A GATA G/A’ core sequence. GATA factors are critically involved in the development and function of various organs (for review (Lowry & Atchley 2000, Molkentin 2000)). Based on the phenotypes of Gata-deficient transgenic mice and subsequent analysis of downstream target genes, GATA4/5/6 were subgrouped as primary endodermal transcription factors (Molkentin 2000). Each factor exhibits a distinct developmental-stage and tissue-specific expression pattern. Cumulative data show that GATA4 is a critical regulator of cardiac gene expression, modulating cardiomyocyte differen- tiation and adaptive responses of the adult heart. GATA4 is also expressed in various endocrine organs. In the male and female gonads, GATA4 contributes to transcription of multiple hormone-encoding genes (Viger et al. 2008). In mice, homozygous Gata4 deficiency (Gata4 K/K ) results in embryonic lethality between 8 . 5 and 10 . 5 dpc due to defects in yolk sac vasculogenesis, ventral morphogenesis, and heart tube formation (Kuo et al. 1997, Molkentin et al. 1997). Mutant mice with cardiac- specific deletion of Gata4 show hypoplasia of the right ventricle during embryonic development; at later stages this heart fails to react by hypertrophy in conse- quence of hypertension and shows reduced myocyte viability (Zeisberg et al. 2005, Oka et al. 2006). However, transgenic mice with heterozygous Gata4 deficiency (Gata4 C/K ) do not suffer from cardiac failure (Kuo et al. 1997, Pu et al. 2004). Other data from transgenic mice indicate that a critical Gata4 expression level is required for normal cardiac development and function (Pu et al. 2004, Zeisberg et al. 2005, Bisping et al. 2006, Oka et al. 2006, Jay et al. 2007, Rajagopal et al. 2007). 157 Journal of Molecular Endocrinology (2009) 43, 157–169 DOI: 10.1677/JME-09-0030 0952–5041/09/043–157 q 2009 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org