Mef2cis a direct transcriptional target of ISL1 and GATA factors in the anterior heart field during mouse embryonic development

Abstract

The vertebrate heart forms initially as a linear tube derived from a primary heart field in the lateral mesoderm. Recent studies in mouse and chick have demonstrated that the outflow tract and right ventricle originate from a separate source of mesoderm that is anterior to the primary heart field. The discovery of this anterior, or secondary, heart field has led to a greater understanding of the morphogenetic events involved in heart formation;however, many of the underlying molecular events controlling these processes remain to be determined. The MADS domain transcription factor MEF2C is required for proper formation of the cardiac outflow tract and right ventricle, suggesting a key role in anterior heart field development. Therefore, as a first step toward identifying the transcriptional pathways upstream of MEF2C, we introduced a lacZ reporter gene into a bacterial artificial chromosome (BAC) encompassing the murine Mef2clocus and used this recombinant to generate transgenic mice. This BAC transgene was sufficient to recapitulate endogenous Mef2c expression,and comparative sequence analyses revealed multiple regions of significant conservation in the noncoding regions of the BAC. We show that one of these conserved noncoding regions represents a transcriptional enhancer that is sufficient to direct expression of lacZ exclusively to the anterior heart field throughout embryonic development. This conserved enhancer contains two consensus GATA binding sites that are efficiently bound by the zinc finger transcription factor GATA4 and are completely required for enhancer function in vivo. This enhancer also contains two perfect consensus sites for the LIM-homeodomain protein ISL1. We show that these elements are specifically bound by ISL1 and are essential for enhancer function in transgenic embryos. Thus, these findings establish Mef2c as the first direct transcriptional target of ISL1 in the anterior heart field and support a model in which GATA factors and ISL1 serve as the earliest transcriptional regulators controlling outflow tract and right ventricle development.

MEF2C is required for the normal allocation of cells between the ventricular and sinoatrial precursors of the primary heart field

“…The role of MEF2C in AHF differentiation is beginning to be defined through promoter and mutant analyses. This research places mef2c downstream of isl1 and foxh1, and upstream of bop and hand2 for transcriptional regulation within the AHF and its derivatives (Cai et al, 2003;Dodou et al, 2004;von Both et al, 2004;Phan et al, 2005). However, AHF defects alone do not explain the reduced size and transcriptional abnormalities in the LV, hich is formed exclusively from the PHF, nor the absence of a morphologically normal AVC, which is derived from both fields (Cai et al, 2003;.…”

“…The anterior region expresses fgf10 as well as isl1, contributes to the heart solely through the arterial pole, and is known as the anterior heart field (AHF) (Kelly et al, 2001;Mjaatvedt et al, 2001;Waldo et al, 2001;Kelly and Buckingham, 2002;Zaffran et al, 2004). Moreover, the AHF is transcriptionally distinct from the posterior cells of the SHF in that promoter elements of the mef2c and nkx2.5 genes are active in the AHF but not the posterior region (Dodou et al, 2004;Takeuchi et al, 2005). Lineage tracing experiments using the AHF regulatory elements from the mef2c gene show that the outflow tract (OFT), right ventricle (RV), and ventricular septum are AHF derivatives (Verzi et al, 2005).…”

“…Recent reports highlight the importance of MEF2C in formation and transcriptional regulation of AHF-derived structures, leading to the hypothesis that part of the mef2c Ϫ/Ϫ phenotype can be explained as defects in AHF-derived structures (Dodou et al, 2004;von Both et al, 2004). Because the AHF-derived cells in the mef2c Ϫ/Ϫ embryo are capable of incorporating into the heart relatively normally, the dysfunction appears to be in later stages of development (Verzi et al, 2005).…”

“…Moreover, the bop promoter contains essential MEF2 sites, suggesting a genetic route from mef2c to RV formation through the direct activation of the bop promoter and the, possibly indirect, induction of hand2 expression (Phan et al, 2005). A potential upstream genetic pathway within the AHF from isl1 and foxh1 to mef2c has also been uncovered (Dodou et al, 2004;von Both et al, 2004). Within the AHF, isl1 directly regulates transcription of the mef2c gene in combination with GATA factors through an element 5Ј of the transcriptional start site (Dodou et al, 2004).…”

“…A potential upstream genetic pathway within the AHF from isl1 and foxh1 to mef2c has also been uncovered (Dodou et al, 2004;von Both et al, 2004). Within the AHF, isl1 directly regulates transcription of the mef2c gene in combination with GATA factors through an element 5Ј of the transcriptional start site (Dodou et al, 2004). Similarly, foxh1 regulates transcription of mef2c in combination with nkx2.5 through a 3Ј element (von Both et al, 2004).…”