Role Of Myocyte Specific-enhancer Factor 2A In Zebrafish Cardiac And Somite Development

The most exciting advance in the biological sciences during the past few decades has been the completion of the human genome project and related initiatives. Large scale efforts are now needed to assign function to these newly discovered genes, an area many have designated as functional genomics. These efforts will likely have greatest success when applied in a highly focused manner using model organisms.A loss-of-function mutation in the human MEF2A causes an autosomal dominant form of coronary artery disease. These data have recently been put into question. Deletion of MEF2A in mice results in sudden cardiac death as well as marked right ventricular dilation that is not explained by increased pressure of the pulmonary vasculature. Despite of the sudden cardiac death phenotype, several differences exist between these mice and humans with mutations in MEF2A. More detailed phenotypic analysis of animals with MEF2A mutations will be required to uncover its biological function.The zebrafish, Danio rerio, offers several distinct advantages as a genetic and embryological model system, including the external fertilization, rapid development and optical clarity of its embryos. Being a vertebrate, the zebrafish has a notochord, blood, heart and vasculature, kidney and optical systems that share many features with corresponding human systems. The zebrafish system bridges the gap between fruit fly/worm and mouse/human genetics, making it feasible to address issues of early development, organ formation, integrative physiology, pharmacology and complex disease. In addition, because of their small size, zebrafish embryos are not completely dependent on a functional cardiovascular system. Even in the total absence of blood circulation, they receive enough oxygen by passive diffusion to survive and continue to develop in a relatively normal fashion for several days, thereby allowing a detailed analysis of animals with severe cardiovascular defects. By contrast, avian and mammalian embryos die rapidly in the absence of a functional cardiovascular system. Forward genetics in zebrafish has led to the identification of several mutations affecting cardiac contractility. So far, in the large scale mutagenesis screens of zebrafish, no mutations were linked to MEF2A locus.The neonatal rat cardiomyocyte model enables heart researchers to study and understand the morphological, biochemical and electrophysiological characteristics of the heart. This model is conducive to a broad spectrum of experiments, such as studiesof contraction, ischaemia, hypoxia and the toxicity of various compounds.Bone morphogenetic proteins (BMPs) are growth and differentiation factors of the transforming growth factor-β superfamily involved in embryogenesis and morphogenesis of various tissues and organs. BMPs and downstream BMP signaling effectors are essential for cardiovascular development. BMP-2, a member of this family of proteins, is an important growth and differentiation factor. Deletion of BMP-2 in mice results in lethality at embryonic day 7.0 10.5 due to malformation of the amnion/chorion and cardiac malformations. The early embryonic lethality of the BMP-2 germline null allele hinders further investigation into BMP-2 function at later stages. The mechanism by which BMP-2 regulates cardiac development and cardiac gene expression has remained obscure.Heart failure is a complex disorder in which cardiac contractility is insufficient to adequately supply blood to other organs. Cardiac contractility is regulated tightly as an essential homeostatic mechanism. Some of the control is neuro-humoral, but much appears intrinsic to the heart. The molecular basis of this intrinsic system is less clear. Heart failure is a complex disorder in which cardiac contractility is insufficient to adequately supply blood to the other organs. An important hallmark of heart failure is reduced myocardial contractility. This syndrome is a common complication ensuing from a wide variety of cardiovascular pathologies. Studies in failing human hearts show numerous morphological changes including degenerative alterations, mutation of contractile elements and marked disorganization of sarcomere. These changes suggest that defects of a contractile gene lead not only to decreased elastic properties of the sarcomere but also to disturbances in sarcomerogenesis.Section 1 Requirements of MEF2A in Zebrafish Cardiac ContractilityIn this study, we describe that MEF2A is highly expressed in heart as well as somites during zebrafish embryogenesis. Knock-down of MEF2A in zebrafish impaires the cardiac contractility and results in sarcomere assembly defects. Dysregulation of cardiac genes in MEF2A morphants suggests that sarcomere assembly disturbances account for the cardiac contractile deficiency. Our studies suggested that MEF2A is essential in cardiac contractility.Section 2 BMP-2 Acts Upstream of MEF2A to Control Embryonic CardiacContractilityWe have previously shown that Myocyte-specific Enhancer Factor 2A (MEF2A), a transcription factor involved in cardiac development, plays an important role in zebrafish embryonic cardiac contractility. The signaling mechanism regulating the expression of MEF-2A transcription factor is largely unclear.Here, we find that Bone Morphogenetic Protein-2(BMP-2) mediates embryonic cardiac contractility upstream of Myocyte-specific Enhancer Factor 2A (MEF-2A) employing zebrafish and neonatal rat cardiomyocyte model systems. We demonstrate that BMP-2 and MEF2A are co-expressed in embryonic and neonatal cardiac myocytes. Furthermore, we provide evidence that BMP-2 is required for cardiac contractility in vitro and in vivo. We further display that MEF2A controls embryonic cardiac contractility. Biochemical studies reveal that MEF-2A expression can be activated by BMP-2 signaling in neonatal cardiomyocytes. Finally, we prove that exogenous MEF2A activity is unable to rescue the dorsalized phenotype in zebrafish embryos lacking BMP-2 function, while ventricle contractility defects can be significantly restored with MEF-2A overexpression, suggesting that BMP-2 mediates embryonic cardiac contractility upstream of MEF-2A. In all, these observations indicate that BMP-2 and MEF-2A are key components of a pathway that controls the onset and maintenance of cardiac ventricular contractility. Functional analysis of BMP-2 should further potentiate our understanding of the molecular mechanism of cardiac development. The BMP2-MEF2A pathway may offer new opportunities for heart failure treatments.Section 3 MEF2A Is Essential for Zebrafish Posterior Somite DevelopmentWe previously showed that MEF2A is highly expressed in zebrafish somites during zebrafish embryogenesis and that morpholino modified antisense oligonucleotides could trigger the specific knock-down of MEF2A in zebrafish. To explore the function of MEF2A in zebrafish somitogenesis, we focused on the phenotype of zebrafish injected with MEF2A MO.In vertebrates, somites form sequentially from anterior to posterior along both sides of the embryonic body axis as the embryo extends posteriorly, giving rise to a species-specific number of segments. Numerous genetic studies demonstrate thatsomite development is governed tightly by genetic factors. The anterior somites appear to be patterned differently to the more posterior ones. However, the molecular basis of this difference is less clear. One obvious difference in anterior somitogenesis observed in mice and zebrafish is the more rapid progression of the somite cycle. In zebrafish, the anterior 6 somites form every 20 min, while the 24 posterior somites form every 30 min.In this study, we find that MEF2A knock-down embryos display a downward tail curvature and have U-shaped posterior somites. Furthermore, we demonstrate that MEF2A is required for Hedgehog signaling. MEF2A inhibition results in induction of apoptosis in the posterior somites. We further find that Hedgehog signaling can negatively regulate MEF2A expression in the somites. Microarray studies reveal a number of genes that are differentially expressed in the MEF2A morphants. Our studies suggest that MEF2A is essential for zebrafish posterior somite development.Section 4 Wortmannin Induces Zebrafish Cardia Bifida through a Mechanism Independent of Phosphoinositide 3-kinase and Myosin Light Chain KinaseIn all vertebrates, the formation of the heart initiates soon after gastrulation, and the heart is the first organ to form and function during embryogenesis. The heart tube, which consists of a myocardial and an endocardial layer, is the result of the fusion of the bilateral heart primordial. During somitogenesis, the myocardial precursors appear to converge at the midline in concert with the anterior lateral plate mesoderm, and fuse to form the heart tube. Large scale genetic screens in zebrafish have identified mutations in eight loci that disrupt this process so far, resulting in the formation of two separate hearts, a phenotype known as cardia bifida. The fusion of the primitive myocardial tubes results in the formation of the definitive heart tube. The molecular events underlying this process are not well understood.Wortmannin was originally isolated from Penicillium wortmannii and has been reported to directly inactivate phosphoinositide 3-kinases (PI3 kinases) by covalent modification of the catalytic subunit with a low nanomolar IC₅₀ [1-6]. Wortmannin can also interact directly with the catalytic domain of myosin light chain kinase (MLCK) at micromolar concentrations. It has been widely used as a selective inhibitor of PI3 kinase or MLCK for the study of signal transduction pathways in different systems.We provide the first evidence that wortmannin treatment of zebrafish embryos can induce cardia bifida in a dose-dependent manner and that wortmannin alters cardiac development between 6 and 16 hours post fertilization. In addition, we demonstrate that wortmannin induces zebrafish cardia bifida through a mechanism independent of phosphoinositide 3-kinase and myosin light chain kinase. Our findings may provide new insights into the cardiomyocyte function and disfunction.