Transcriptional regulation of the murine Connexin40 promoter by cardiac factors Nkx2-5, GATA4 and Tbx5.

OBJECTIVE: Connexin40 (Cx40) is a gap junction protein expressed specifically in developing and mature atrial myocytes and cells of the conduction system. In this report, we identify cis-acting elements within the mouse Cx40 promoter and unravel part of the complex pathways involved in the cardiac expression of this gene. METHODS: To identify the factors involved in the cardiac expression of Cx40, we used transient transfections in mammalian cells coupled with electrophoretic mobility shift assays (EMSA) and RT-PCR. RESULTS: Within the promoter region, we identified the minimal elements required for transcriptional activity within 150 base pairs (bp) upstream of the transcriptional start site. Several putative regulatory sites for transcription factors were predicted within this region by computer analysis, and we demonstrated that the nuclear factors Sp1, Nkx2-5, GATA4 and Tbx5 could interact specifically with elements present in the minimal promoter region of the Cx40. Furthermore, co-transfection experiments showed the ability of Nkx2-5 and GATA4 to transactivate the minimal Cx40 promoter while Tbx5 repressed Nkx2-5/GATA4-mediated activation. Mutagenesis of the Nkx2-5 core site in the Cx40 promoter led to significantly decreased activity in rat smooth muscle cell line A7r5. Consistent with this, mouse embryos lacking Nkx2-5 showed a marked decrease in Cx40 expression. CONCLUSION: In this work, we cloned the promoter region of the Cx40 and demonstrated that the core promoter was modulated by cardiac transcriptional factors Nkx2-5, Tbx5 and GATA4 acting together with ubiquitous Sp1.

A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field.

Establishment of anteroposterior (AP) polarity is one of the earliest decisions in cardiogenesis and plays an important role in the coupling between heart and blood vessels. Recent research implicated retinoic acid (RA) in the communication of AP polarity to the heart. We utilized embryo culture, in situ hybridization, morphometry, fate mapping and treatment with the RA pan-antagonist BMS493 to investigate the relationship between cardiac precursors and RA signalling. We describe two phases of AP signalling by RA, reflected in RALDH2 expression. The first phase (HH4-7) is characterized by increasing proximity between sino-atrial precursors and the lateral mesoderm expressing RALDH2. In this phase, RA signalling is consistent with diffusion of the morphogen from a large field rather than a single hot spot. The second phase (HH7-8) is characterized by progressive encircling of cardiac precursors by a field of RALDH2 originating from a dynamic and evolutionary-conserved caudorostral wave pattern in the lateral mesoderm. At this phase, cardiac AP patterning by RA is consistent with localized action of RA by regulated activation of the Raldh2 gene within an embryonic domain. Systemic treatment with BMS493 altered the cardiac fate map such that ventricular precursors were found in areas normally devoid of them. Topical application of BMS493 inhibited atrial differentiation in left anterior lateral mesoderm. Identification of the caudorostral wave of RALDH2 as the endogenous source of RA establishing cardiac AP fates provides a useful model to approach the mechanisms whereby the vertebrate embryo confers axial information on its organs.