Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors.

Vascular development begins when mesodermal cells differentiate into endothelial cells, which then form primitive vessels. It has been hypothesized that endothelial-specific gene expression may be regulated combinatorially, but the transcriptional mechanisms governing specificity in vascular gene expression remain incompletely understood. Here, we identify a 44 bp transcriptional enhancer that is sufficient to direct expression specifically and exclusively to the developing vascular endothelium. This enhancer is regulated by a composite cis-acting element, the FOX:ETS motif, which is bound and synergistically activated by Forkhead and Ets transcription factors. We demonstrate that coexpression of the Forkhead protein FoxC2 and the Ets protein Etv2 induces ectopic expression of vascular genes in Xenopus embryos, and that combinatorial knockdown of the orthologous genes in zebrafish embryos disrupts vascular development. Finally, we show that FOX:ETS motifs are present in many known endothelial-specific enhancers and that this motif is an efficient predictor of endothelial enhancers in the human genome.

Apelin-APJ signaling is a critical regulator of endothelial MEF2 activation in cardiovascular development.

RATIONALE: The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. OBJECTIVE: This study aimed to characterize the embryonic lethal phenotype of the Apj-/- mice and to define the involved downstream signaling targets. METHODS AND RESULTS: We report the first characterization of the embryonic lethality of the Apj-/- mice. More than half of the expected Apj-/- embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj-/- embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj-/- mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj-/- embryos and endothelial cells. CONCLUSIONS: Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

The MADS box transcription factor MEF2C regulates melanocyte development and is a direct transcriptional target and partner of SOX10.

Waardenburg syndromes are characterized by pigmentation and autosensory hearing defects, and mutations in genes encoding transcription factors that control neural crest specification and differentiation are often associated with Waardenburg and related disorders. For example, mutations in SOX10 result in a severe form of Waardenburg syndrome, Type IV, also known as Waardenburg-Hirschsprung disease, characterized by pigmentation and other neural crest defects, including defective innervation of the gut. SOX10 controls neural crest development through interactions with other transcription factors. The MADS box transcription factor MEF2C is an important regulator of brain, skeleton, lymphocyte and cardiovascular development and is required in the neural crest for craniofacial development. Here, we establish a novel role for MEF2C in melanocyte development. Inactivation of Mef2c in the neural crest of mice results in reduced expression of melanocyte genes during development and a significant loss of pigmentation at birth due to defective differentiation and reduced abundance of melanocytes. We identify a transcriptional enhancer of Mef2c that directs expression to the neural crest and its derivatives, including melanocytes, in transgenic mouse embryos. This novel Mef2c neural crest enhancer contains three functional SOX binding sites and a single essential MEF2 site. We demonstrate that Mef2c is a direct transcriptional target of SOX10 and MEF2 via this evolutionarily conserved enhancer. Furthermore, we show that SOX10 and MEF2C physically interact and function cooperatively to activate the Mef2c gene in a feed-forward transcriptional circuit, suggesting that MEF2C might serve as a potentiator of the transcriptional pathways affected in Waardenburg syndromes.

Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis.

Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function and a vasodepressor function in the systemic circulation. The apelin-APJ pathway appears to have opposing physiological roles to the renin-angiotensin system. Here we investigated whether the apelin-APJ pathway can directly antagonize vascular disease-related Ang II actions. In ApoE-KO mice, exogenous Ang II induced atherosclerosis and abdominal aortic aneurysm formation; we found that coinfusion of apelin abrogated these effects. Similarly, apelin treatment rescued Ang II-mediated increases in neointimal formation and vascular remodeling in a vein graft model. NO has previously been implicated in the vasodepressor function of apelin; we found that apelin treatment increased NO bioavailability in ApoE-KO mice. Furthermore, infusion of an NO synthase inhibitor blocked the apelin-mediated decrease in atherosclerosis and aneurysm formation. In rat primary aortic smooth muscle cells, apelin inhibited Ang II-mediated transcriptional regulation of multiple targets as measured by reporter assays. In addition, we demonstrated by coimmunoprecipitation and fluorescence resonance energy transfer analysis that the Ang II and apelin receptors interacted physically. Taken together, these findings indicate that apelin signaling can block Ang II actions in vascular disease by increasing NO production and inhibiting Ang II cellular signaling.

HRC is a direct transcriptional target of MEF2 during cardiac, skeletal, and arterial smooth muscle development in vivo.

The HRC gene encodes the histidine-rich calcium-binding protein, which is found in the lumen of the junctional sarcoplasmic reticulum (SR) of cardiac and skeletal muscle and within calciosomes of arterial smooth muscle. The expression of HRC in cardiac, skeletal, and smooth muscle raises the possibility of a common transcriptional mechanism governing its expression in all three muscle cell types. In this study, we identified a transcriptional enhancer from the HRC gene that is sufficient to direct the expression of lacZ in the expression pattern of endogenous HRC in transgenic mice. The HRC enhancer contains a small, highly conserved sequence that is required for expression in all three muscle lineages. Within this conserved region is a consensus site for myocyte enhancer factor 2 (MEF2) proteins that we show is bound efficiently by MEF2 and is required for transgene expression in all three muscle lineages in vivo. Furthermore, the entire HRC enhancer sequence lacks any discernible CArG motifs, the binding site for serum response factor (SRF), and we show that the enhancer is not activated by SRF. Thus, these studies identify the HRC enhancer as the first MEF2-dependent, CArG-independent transcriptional target in smooth muscle and represent the first analysis of the transcriptional regulation of an SR gene in vivo.

Mef2c is a direct transcriptional target of ISL1 and GATA factors in the anterior heart field during mouse embryonic development.

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 Mef2c locus 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 activated directly by Ets transcription factors through an evolutionarily conserved endothelial cell-specific enhancer.

Members of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors play key roles in the development and differentiation of numerous cell types during mammalian development, including the vascular endothelium. Mef2c is expressed very early in the development of the endothelium, and genetic studies in mice have demonstrated that mef2c is required for vascular development. However, the transcriptional pathways involving MEF2C during endothelial cell development have not been defined. As a first step towards identifying the transcriptional factors upstream of MEF2C in the vascular endothelium, we screened for transcriptional enhancers from the mouse mef2c gene that regulate vascular expression in vivo. In this study, we identified a transcriptional enhancer from the mouse mef2c gene sufficient to direct expression to the vascular endothelium in transgenic embryos. This enhancer is active in endothelial cells within the developing vascular system from very early stages in vasculogenesis, and the enhancer remains robustly active in the vascular endothelium during embryogenesis and in adulthood. This mef2c endothelial cell enhancer contains four perfect consensus Ets transcription factor binding sites that are efficiently bound by Ets-1 protein in vitro and are required for enhancer function in transgenic embryos. Thus, these studies identify mef2c as a direct transcriptional target of Ets factors via an evolutionarily conserved transcriptional enhancer and establish a direct link between these two early regulators of vascular gene expression during endothelial cell development in vivo.

N-twist, an evolutionarily conserved bHLH protein expressed in the developing CNS, functions as a transcriptional inhibitor.

Members of the basic helix-loop-helix (bHLH) transcription factor family play an essential role in multiple developmental processes. During neurogenesis, positive and negative regulation by bHLH proteins is essential for proper development. Here we report the identification and initial characterization of the bHLH gene, Neuronal twist (N-twist), named for its neural expression pattern and high sequence homology and physical linkage to the mesodermal inhibitor, M-twist. N-twist is expressed in the developing mouse central nervous system in the midbrain, hindbrain, and neural tube. This neural expression is conserved in invertebrates, as expression of the Drosophila ortholog of N-twist is also restricted to the central nervous system. Like other bHLH family members, N-Twist heterodimerizes with E protein and binds DNA at a consensus bHLH-binding site, the E box. We show that N-Twist inhibits MASH1-dependent transcriptional activation by sequestering E protein in a dominant negative fashion. Thus, these studies support the notion that N-Twist represents a novel negative regulator of neurogenesis.