Heterozygous Tbx5(del/+) mice were generated to study the mechanisms by which TBX5 haploinsufficiency causes cardiac and forelimb abnormalities seen in Holt-Oram syndrome. Tbx5 deficiency in homozygous mice (Tbx5(del/del)) decreased expression of multiple genes and caused severe hypoplasia of posterior domains in the developing heart. Surprisingly, Tbx5 haploinsufficiency also markedly decreased atrial natriuretic factor (ANF) and connexin 40 (cx40) transcription, implicating these as Tbx5 target genes and providing a mechanism by which 50% reduction of T-box transcription factors cause disease. Direct and cooperative transactivation of the ANF and cx40 promoters by Tbx5 and the homeodomain transcription factor Nkx2-5 was also demonstrated. These studies provide one potential explanation for Holt-Oram syndrome conduction system defects, suggest mechanisms for intrafamilial phenotypic variability, and account for related cardiac malformations caused by other transcription factor mutations.

Two members of the GATA family of transcription factors, GATA-4 and GATA-6, are expressed in the developing and postnatal myocardium and are equally potent transactivators of several cardiac promoters. However, several in vitro and in vivo lines of evidence suggest distinct roles for the two factors in the heart. Since identification of the endogenous downstream targets of GATA factors would greatly help to elucidate their exact functions, we have developed an adenovirus-mediated antisense strategy to specifically inhibit GATA-4 and GATA-6 protein production in postnatal cardiomyocytes. Expression of several endogenous cardiac genes was significantly down-regulated in cells lacking GATA-4 or GATA-6, indicating that these factors are required for the maintenance of the cardiac genetic program. Interestingly, transcription of some genes like the alpha- and beta-myosin heavy-chain (alpha- and beta-MHC) genes was preferentially regulated by GATA-4 due, in part, to higher affinity of GATA-4 for their promoter GATA element. However, transcription of several other genes, including the atrial natriuretic factor and B-type natriuretic peptide (ANF and BNP) genes, was similarly down-regulated in cardiomyocytes lacking one or both GATA factors, suggesting that GATA-4 and GATA-6 could act through the same transcriptional pathway. Consistent with this, GATA-4 and GATA-6 were found to colocalize in postnatal cardiomyocytes and to interact functionally and physically to provide cooperative activation of the ANF and BNP promoters. The results identify for the first time bona fide in vivo targets for GATA-4 and GATA-6 in the myocardium. The data also show that GATA factors act in concert to regulate distinct subsets of genes, suggesting that combinatorial interactions among GATA factors may differentially control various cellular processes.

Transcription factors GATA-4, -5, and -6 constitute an evolutionary conserved subfamily of vertebrate zinc finger regulators highly expressed in the developing heart and gut. Genetic evidence suggests that each protein is essential for embryonic development, but their exact functions are not fully elucidated. Moreover, because all three proteins share similar transcriptional properties in vitro, and because transcripts for two or more GATA genes are present in similar tissues, the molecular basis underlying in vivo specificity of GATA factors remains undefined. Knowledge of the exact cell types expressing each protein and identification of downstream targets would greatly help define their function. We have used high-resolution immunohistochemistry to precisely determine the cellular distribution of the GATA-4, -5, and -6 proteins in murine embryogenesis. The results reveal novel sites of expression in mesodermal and ectodermal cells. In particular, GATA-4 and -6 expression was closely associated with yolk sac vasculogenesis and early endoderm-mesoderm signaling. Additionally, GATA-6 was strongly expressed in the embryonic ectoderm, neural tube, and neural crest-derived cells. This pattern of expression closely paralled that of BMP-4, and the BMP-4 gene was identified as a direct downstream target for GATA-4 and -6. These findings offer new insight into the function of GATA-4 and -6 during early stages of embryogenesis and reveal the existence of a positive cross-regulatory loop between BMP-4 and GATA-4. They also raise the possibility that part of the early defects in GATA-4 and/or GATA-6 null embryos may be due to impaired BMP-4 signaling.