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Objectives: To better understand the molecular mechanisms of regeneration and explore the potential signalling pathways as therapeutic targets for heart attacks. Results: After treatment with the MEK inhibitor AZD6244 upon cardiac injury, the core members in MAPK/ERK signalling- mek and erk-demonstrate elevated expression, and these proteins are deposited at the injury site in zebrafish. pERK is also induced in non-cardiomyocytes near the injury site. Furthermore, the induced expression of a dominant-negative form of MEK1 inhibits zebrafish cardiac regeneration, characterized by increased cardiac fibrosis (a hallmark of regenerative failure), reduced or delayed production of regenerative myocardium, and migration of FLI1 endothelial cells, without direct inhibition of cardiomyocyte proliferation. Conclusion: Appropriate activation of MAPK/ERK signalling is essential for zebrafish cardiac regeneration. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor A (Vegfa) signaling regulates cardiovascular development. However, the cellular mechanisms of Vegfa signaling in early cardiogenesis remain poorly understood. The present study aimed to understand the differential functions and mechanisms of Vegfa signaling in cardiac development. A loss-of-function approach was utilized to study the effect of Vegfa signaling in cardiogenesis. Both morphants and mutants for vegfaa display defects in cardiac looping and chamber formation, especially the ventricle. Vegfa regulates the heart morphogenesis in a dose-dependent manner. Furthermore, the initial fusion of the bilateral myocardium population is delayed rather than endocardium. The results demonstrate that Vegfa signaling plays a direct impact on myocardium fusion, indicating that it is the initial cause of the heart defects. The heart morphogenesis is regulated by Vegfa in a dose-dependent manner, and later endocardium defects may be secondary to impaired myocardium-endocardium crosstalk. [ABSTRACT FROM AUTHOR]

Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signalling cascades that regulate cilium formation remain incompletely understood. Here we report that prostaglandin signalling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish leakytail (lkt) mutants show ciliogenesis defects, and the lkt locus encodes an ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes to the cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogated by the Lkt/ABCC4T804M mutant. PGE2 synthesis enzyme cyclooxygenase-1 and its receptor, EP4, which localizes to the cilium and activates the cyclic-AMP-mediated signalling cascade, are required for cilium formation and elongation. Importantly, PGE2 signalling increases anterograde but not retrograde velocity of IFT and promotes ciliogenesis in mammalian cells. These findings lead us to propose that Lkt/ABCC4-mediated PGE2 signalling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis. [ABSTRACT FROM AUTHOR]

Embryonic organs attain their final dimensions through the generation of proper cell number and size, but the control mechanisms remain obscure. Here, we establish Gridlock (GrI), a Hairy-related basic helix-loop-helix (bHLH) transcription factor, as a negative regulator of cardiomyocyte proliferative growth in zebrafish embryos. Mutations in gri cause an increase in expression of a group of immediate-early growth genes, myocardial genes, and development of hyperplastic hearts. Conversely, cardiomyocytes with augmented Grl activity have diminished cell volume and fail to divide, resulting in a marked reduction in heart size. Both bHLH domain and carboxyl region are required for Grl negative control of myocardial proliferative growth. These Grl-induced cardiac effects are counterbalanced by the transcriptional activator Gata5 but not Gata4, which promotes cardiomyocyte expansion in the embryo. Biochemical analyses show that Grl forms a complex with Gata5 through the carboxyl region and can repress Gata5-mediated transcription via the bHLH domain. Hence, our studies suggest that Grl regulates embryonic heart growth via opposing Gata5, at least in part through their protein interactions in modulating gene expression. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Cardiac contractility is regulated tightly as an extrinsic and intrinsic homeostatic mechanism to the heart. The molecular basis of the intrinsic system is largely unknown. Here, we test the hypothesis that bone morphogenetic protein-2 (BMP-2) mediates embryonic cardiac contractility upstream of myocyte-specific enhancer factor 2A (MEF2A). Methods: The BMP-2 and MEF2A expression pattern was analyzed by RT-PCR, Western blotting, whole-mount in situ hybridization, and an in vivo transgenic approach. The cardiac phenotype of BMP-2 and MEF2A knock-down zebrafish embryos was analysed. Cardiac contractions were recorded with a video camera. Myofibrillar organization was observed with transmission electron microscopy. Gene expression profiles were performed by quantitative real-time PCR analysis. Results: 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 and that MEF2A expression can be activated by BMP-2 signaling in neonatal cardiomyocytes. BMP-2 is involved in the assembly of the cardiac contractile apparatus. Finally, we find that exogenous MEF2A is sufficient to rescue ventricular contractility defects in the absence of BMP-2 function. Conclusions: In all, these observations indicate that BMP-2 and MEF2A are key components of a pathway that controls the cardiac ventricular contractility and suggest that the BMP2-MEF2A pathway can offer new opportunities for the treatment of heart failure. [Copyright &y& Elsevier]

γ-Secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen-2), nicastrin, and Aph-1, is the final proteolytic step in generating amyloid β protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen-2 in neuronal survival. We found that (i) knockdown of Pen-2 using antisense morpholino led to a reduction of islet-1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen-2 or other γ-secretase components, (iii) neuronal loss in Pen-2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen-2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet-1 or acetylated tubulin positive neurons in Pen-2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen-2 directly induces a p53-dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen-2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Myocyte-specific enhancer factor 2A (MEF2A) regulates a broad range of fundamental cellular processes including cell division, differentiation and death. Here, we tested the hypothesis that MEF2A is required in cardiac contractility employing zebrafish as a model organism. 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. [Copyright &y& Elsevier]

Abstract: Cardia bifida is an anomaly of the embryonic heart in which the bilateral myocardial rudiments fail to travel to the midline, resulting in the formation of two separate hearts in lateral positions. In zebrafish, eight loci responsible for the cardia bifida phenotype were identified in the large-scale genetic screen. Wortmannin has been reported to be a highly selective inhibitor of phosphoinositide 3-kinase and myosin light chain kinase activity. 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 16h 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. [Copyright &y& Elsevier]

Conventional drug discovery approaches require a priori selection of an appropriate molecular target, but it is often not obvious which biological pathways must be targeted to reverse a disease phenotype1'2. Phenotype-based screens offer the potential to identify pathways and potential therapies that influence disease processes. The zebrafish mutation gridlock (grl, affecting the gene hey2) disrupts aortic blood flow in a region and physiological manner akin to aortic coarctation in humans3-5. Here we use a whole-organism, phenotype-based, small-molecule screen to discover a class of compounds that suppress the coarctation phenotype and permit survival to adulthood. These compounds function during the specification and migration of angioblasts. They act to upregulate expression of vascular endothelial growth factor (VEGF), and the activation of the VEGF pathway is sufficient to suppress the gridlock phenotype. Thus, organism-based screens allow the discovery of small molecules that ameliorate complex dysmorphic syndromes even without targeting the affected gene directly. [ABSTRACT FROM AUTHOR]

The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwick[sup m171] (pik[sup m171]), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pik[sup m242], pik[sup m740], pik[sup m186]; ref. 3) and one allele (pik[sup mvO62H]) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscles. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue. [ABSTRACT FROM AUTHOR]

Discusses a study on the gridlock mutation in the zebrafish Danio rerio which selectively perturbs assembly of the artery. Morphological and functional difference between arteries and veins; Methods used in cloning the gridlock mutation; Genomic sequence of gridlock in mutant embryos; Implications of zebrafish mutation for future research.

Pseudoxanthoma elasticum (PXE), caused by ABCC6/MRP6 mutation, is a heritable multisystem disorder in humans. The progressive clinical manifestations of PXE are accompanied by ectopic mineralization in various connective tissues. However, the pathomechanisms underlying the PXE multisystem disorder remains obscure, and effective treatment is currently available. In this study, we generated zebrafish abcc6a mutants using the transcription activator-like effector nuclease (TALEN) technique. In young adult zebrafish, abcc6a is expressed in the eyes, heart, intestine, and other tissues. abcc6a mutants exhibit extensive calcification in the ocular sclera and Bruch's membrane, recapitulating part of the PXE manifestations. Mutations in abcc6a upregulate extracellular matrix (ECM) genes, leading to fibrotic heart with reduced cardiomyocyte number. We found that abcc6a mutation reduced levels of both vitamin K and pyrophosphate (PPi) in the serum and diverse tissues. Vitamin K administration increased the gamma-glutamyl carboxylated form of matrix gla protein (cMGP), alleviating ectopic calcification and fibrosis in vertebrae, eyes, and hearts. Our findings contribute to a comprehensive understanding of PXE pathophysiology from zebrafish models. [ABSTRACT FROM AUTHOR]

Abstract Photoreceptor cells are highly specialized sensory neurons capable of visual phototransduction. The connecting cilia in the retinal photoreceptors link the inner segment to the outer segment and mediate the transport of opsins in vision. Although our previous study demonstrates that Prostaglandin E2 (PGE 2) signaling is required for ciliogenesis in zebrafish, its roles in retinal ciliogenesis and photoreceptor cell development remain unknown. Here, we investigated the function of prostaglandin signaling in retina and photoreceptor cell development. We generated zebrafish mutations in prostaglandin endoperoxide synthase 1 (PTGS1) and prostaglandin endoperoxide synthase 2 (PTGS2), two rate-limiting enzymes responsible for prostaglandin production. We found that ptgs2b knockdown in ptgs1 -/- ptgs2a -/- double mutants significantly reduced the length of connecting cilia and resulted in severe defects in photoreceptor cell differentiation. Furthermore, mutation in PGE 2 transporter Leakytail (Lkt)/ATP-binding cassette transporter 4 (ABCC4) exhibited loss of connecting cilia and outer segment in photoreceptor cells, leading to mislocalization of opsins in the cell bodies of photoreceptors. Together, our findings suggest that PGE 2 production and transport are crucial for connecting cilia formation and photoreceptor cell development. Highlights • The first study to construct the ptgs1 -/- ptgs2a -/- double mutants in zebrafish. • Deficiency in prostaglandin synthesis affects photoreceptor cell development. • Mutation in PGE 2 export exhibits loss of connecting cilia and mislocalization of opsins. [ABSTRACT FROM AUTHOR]

Mutations in Notch2, Jagged1 or homologs of the Hairy-related transcriptional repressor Hey2 cause congenital malformations involving the non-chamber atrioventricular canal (AVC) and inner curvature (IC) regions of the heart, but the underlying mechanisms have not been investigated. By manipulating signaling directly within the developing chick heart, we demonstrated that Notch2, Hey1 and Hey2 initiate a signaling cascade that delimits the non-chamber AVC and IC regions. Specifically, misactivation of Notch2 signaling, or misexpression of either Hey1 or Hey2, repressed Bmp2. Because Jagged (also known as Serrate in non-mammalian species) ligands were found to be present in prospective chamber myocardium, these data support the model that Notch2 and Hey proteins cause the progressive restriction of Bmp2 expression to within the developing AVC and IC, where it is essential for differentiation. Misactivation or inhibition of Notch2 specifically induced or inhibited Hey1, respectively, but these manipulations did not affect Hey2, implicating Hey1 as the direct mediator of Notch2. Bmp2 within the developing AVC and IC has been shown to induce Tbx2, and we found that Tbx2 misexpression inhibited the expression of both Hey1 and Hey2. Tbx2, therefore, is envisaged to constitute a feedback loop that sharpens the border with the developing AVC and IC by delimiting Hey gene expression to within prospective chamber regions. Analysis of the loss-of-function phenotype in mouse embryos homozygous for targeted disruption of Hey2 revealed an expanded AVC domain of Bmp2. Similarly, zebrafish gridlock (Hey2 homolog) mutant embryos showed ectopic expression of Bmp4, which normally marks AVC myocardium in this species. Thus, Hey pathway regulation of cardiac Bmp appears to be an evolutionarily conserved mechanism to delimit AVC and IC fate, and provides a potential mechanistic explanation for cardiac malformations caused by mutations in Serrate/Jagged1 and Notch signaling components. [ABSTRACT FROM AUTHOR]

An abstract of the article "Zebrafish Genetics and Formation of Embryonic Vasculature," by Tao P. Zhong is presented.

P-21 activated kinases, or PAKs, are serine–threonine kinases that play important roles in diverse heart functions include heart development, cardiovascular development and function in a range of models; however, the mechanisms by which PAKs mediate heart regeneration are unknown. Here, we demonstrate that PAK2 and PAK4 expression is induced in cardiomyocytes and vessels, respectively, following zebrafish heart injury. Inhibition of PAK2 and PAK4 using a specific small molecule inhibitor impedes cardiomyocyte proliferation/dedifferentiation and cardiovascular regeneration, respectively. Cdc42 is specifically expressed in the ventricle and may function upstream of PAK2 but not PAK4 under normal conditions and that cardiomyocyte proliferentation during heart regeneration relies on Rac1-mediated activation of Pak2. Our results indicate that PAKs play a key role in heart regeneration. [ABSTRACT FROM AUTHOR]

Summary: We have developed a robust in vivo small-molecule screen that modulates heart size and cardiomyocyte generation in zebrafish. Three structurally related compounds (Cardionogen-1 to Cardionogen-3) identified from our screen enlarge the size of the developing heart via myocardial hyperplasia. Increased cardiomyocyte number in Cardionogen-treated embryos is due to expansion of cardiac progenitor cells. In zebrafish embryos and murine embryonic stem (ES) cells, Cardionogen treatment promotes cardiogenesis during and after gastrulation, whereas it inhibits heart formation before gastrulation. Cardionogen-induced effects can be antagonized by increasing Wnt/β-catenin signaling activity. We demonstrate that Cardionogen inhibits Wnt/β-catenin-dependent transcription in murine ES cells and zebrafish embryos. Cardionogen can rescue Wnt8-induced cardiomyocyte deficiency and heart-specific phenotypes during development. These findings demonstrate that in vivo small-molecule screens targeting heart size can reveal compounds with cardiomyogenic effects and identify underlying target pathways. [ABSTRACT FROM AUTHOR]

Abstract: In vertebrate embryos, the dorsal aorta and the posterior cardinal vein form in the trunk to comprise the original circulatory loop. Previous studies implicate Hedgehog (Hh) signaling in the development of the dorsal aorta. However, the mechanism controlling specification of artery versus vein remains unclear. Here, we investigated the cell-autonomous mechanism of Hh signaling in angioblasts (endothelial progenitor cells) during arterial–venous specification utilizing zebrafish mutations in Smoothened (Smo), a G protein-coupled receptor essential for Hh signaling. smo mutants exhibit an absence of the dorsal aorta accompanied by a reciprocal expansion of the posterior cardinal vein. The increased number of venous cells is equivalent to the loss of arterial cells in embryos with loss of Smo function. Activation of Hh signaling expands the arterial cell population at the expense of venous cell fate. Time-lapse imaging reveals two sequential waves of migrating progenitor cells that contribute to the dorsal aorta and the posterior cardinal vein, respectively. Angioblasts deficient in Hh signaling fail to contribute to the arterial wave; instead, they all migrate medially as a single population to form the venous wave. Cell transplantation analyses demonstrate that Smo plays a cell-autonomous role in specifying angioblasts to become arterial cells, and Hh signaling-depleted angioblasts differentiate into venous cells instead. Collectively, these studies suggest that arterial endothelial cells are specified and formed via repressing venous cell fate at the lateral plate mesoderm by Hh signaling during vasculogenesis. [Copyright &y& Elsevier]

Abstract: Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid and choline. Studies in cultured cells and Drosophila melanogaster have implicated PLD in the regulation of many cellular functions, including intracellular vesicle trafficking, cell proliferation and differentiation. However, the function of PLD in vertebrate development has not been explored. Here we report cloning and characterization of a zebrafish PLD1 (pld1) homolog. Like mammalian PLDs, zebrafish Pld1 contains two conservative HKD motifs. Maternally contributed pld1 transcripts are uniformly distributed in early embryo. Localized expression of pld1 is observed in the notochord during early segmentation, in the somites during later segmentation and in the liver at the larval stages. Studies in intact and cell-free preparations demonstrate evolutionary conservation of regulation. Inhibition of Pld1 expression using antisense morpholino oligonucleotides (MO) interfering with the translation or splicing of pld1 impaired intersegmental vessel (ISV) development. Incubating embryos with 1-butanol, which diverts production of phosphatidic acid to a phosphatidylalcohol, caused similar ISV defects. To determine where Pld1 is required for ISV development we performed transplantation experiments. Analyses of the mosaic Pld1 deficient embryos showed partial suppression of ISV defects in the segments containing transplanted wild-type notochord cells but not in the ones containing wild-type somitic cells. These results provide the first evidence that function of Pld1 in the developing notochord is essential for vascular development in vertebrates. [Copyright &y& Elsevier]

Abstract: NDRG4 is a novel member of the NDRG family (N-myc downstream-regulated gene). The roles of NDRG4 in development have not previously been evaluated. We show that, during zebrafish embryonic development, ndrg4 is expressed exclusively in the embryonic heart, the central nervous system (CNS) and the sensory system. Ndrg4 knockdown in zebrafish embryos causes a marked reduction in proliferative myocytes and results in hypoplastic hearts. This growth defect is associated with cardiac phenotypes in morphogenesis and function, including abnormal heart looping, inefficient circulation and weak contractility. We reveal that ndrg4 is required for restricting the expression of versican and bmp4 to the developing atrioventricular canal. This constellation of ndrg4 cardiac defects phenocopies those seen in mutant hearts of heartstrings (hst), the tbx5 loss-of-function mutants in zebrafish. We further show that ndrg4 expression is significantly decreased in hearts with reduced tbx5 activities. Conversely, increased expression of tbx5 that is due to tbx20 knockdown leads to an increase in ndrg4 expression. Together, our studies reveal an essential role of ndrg4 in regulating proliferation and growth of cardiomyocytes, suggesting that ndrg4 may function downstream of tbx5 during heart development and growth. [Copyright &y& Elsevier]