Analysis of Postembryonic Heart Development and Maturation in Zebrafish, Danio rerio (original) (raw)
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Analysis of postembryonic heart development and maturation in the zebrafish, Danio rerio
Developmental Dynamics, 2012
Background-Cardiac maturation is vital for animal survival and must occur throughout the animal's life. Zebrafish are increasingly used to model cardiac disease; however, little is known about how the cardiovascular system matures. We conducted a systematic analysis of cardiac maturation from larvae though to adulthood and assessed cardiac features influenced by genetic and environmental factors. Results-We identified a novel step in cardiac maturation, termed cardiac rotation, where the larval heart rotates into its final orientation within the thoracic cavity with the atrium placed behind the ventricle. This rotation is followed by linear ventricle growth and an increase in the angle between bulbous arteriosus and the ventricle. The ventricle transitions from a rectangle, to a triangle and ultimately a circle that is significantly enveloped by the atrium. In addition, trabeculae are similarly patterned in the zebrafish and humans with both muscular fingerlike projections and muscle bands that span the cardiac chamber. Interestingly, partial loss of atrial contraction in myosin heavy chain 6 (myh6/wea hu423/+) mutants result in the adult maintaining a larval cardiac form. Conclusions-These findings serve as a foundation for the study of defects in cardiovascular development from both genetic and environmental factors.
Physiological Genomics, 2010
Shin JT, Pomerantsev EV, Mably JD, MacRae CA. High-resolution cardiovascular function confirms functional orthology of myocardial contractility pathways in zebrafish. Phenotype-driven screens in larval zebrafish have transformed our understanding of the molecular basis of cardiovascular development. Screens to define the genetic determinants of physiological phenotypes have been slow to materialize as a result of the limited number of validated in vivo assays with relevant dynamic range. To enable rigorous assessment of cardiovascular physiology in living zebrafish embryos, we developed a suite of software tools for the analysis of high-speed video microscopic images and validated these, using established cardiomyopathy models in zebrafish as well as modulation of the nitric oxide (NO) pathway. Quantitative analysis in wild-type fish exposed to NO or in a zebrafish model of dilated cardiomyopathy demonstrated that these tools detect significant differences in ventricular chamber size, ventricular performance, and aortic flow velocity in zebrafish embryos across a large dynamic range. These methods also were able to establish the effects of the classic pharmacological agents isoproterenol, ouabain, and verapamil on cardiovascular physiology in zebrafish embryos. Sequence conservation between zebrafish and mammals of key amino acids in the pharmacological targets of these agents correlated with the functional orthology of the physiological response. These data provide evidence that the quantitative evaluation of subtle physiological differences in zebrafish can be accomplished at a resolution and with a dynamic range comparable to those achieved in mammals and provides a mechanism for genetic and smallmolecule dissection of functional pathways in this model organism.
Early Myocardial Function Affects Endocardial Cushion Development in Zebrafish
PLoS Biology, 2004
Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih ÿ/ÿ) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk ÿ/ÿ) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk ÿ/ÿ embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfk s11 variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih-and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.
Santa and Valentine Pattern Concentric Growth of Cardiac Myocardium in the Zebrafish
Development, 2006
During embryogenesis, the myocardial layer of the primitive heart tube grows outward from the endocardial-lined lumen, with new cells added to generate concentric thickness to the wall. This is a key evolutionary step,demarcating vertebrates from more primitive chordates, and is essential for normal cardiac function. Zebrafish embryos with the recessive lethal mutations santa (san) and valentine (vtn) do not thicken, but do add the proper number of cells to the myocardium. Consequently, the heart chambers are huge, constituted of a monolayered myocardium lined by endocardium. This phenotype is similar to that of the heart of glass (heg) mutation, which we described previously as a novel endocardial expressed gene. By positional cloning, we here identify san as the zebrafish homolog of human CCM1, and vtn as the homolog of human CCM2. Dominant mutations of either in humans cause vascular anomalies in the brain, known as cerebral cavernous malformations. The synergistic effects of mor...
Regulation in the heart field of zebrafish
Development (Cambridge, England), 1998
In many vertebrates, removal of early embryonic heart precursors can be repaired, leaving the heart and embryo without visible deficit. One possibility is that this 'regulation' involves a cell fate switch whereby cells, perhaps in regions surrounding normal progenitors, are redirected to the heart cell fate. However, the lineage and spatial relationships between cells that are normal heart progenitors and those that can assume that role after injury are not known, nor are their molecular distinctions. We have adapted a laser-activated technique to label single or small patches of cells in the lateral plate mesoderm of the zebrafish and to track their subsequent lineage. We find that the heart precursor cells are clustered in a region adjacent to the prechordal plate, just anterior to the notochord tip. Complete unilateral ablation of all heart precursors with a laser does not disrupt heart development, if performed before the 18-somite stage. By combining extirpation of the...
Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo
Development (Cambridge, England), 1996
As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether,...
Solving an enigma: Arterial pole development in the zebrafish heart
Developmental Biology, 2006
It is a widely held belief that the arterial pole of the zebrafish heart is unusual among models of comparative cardiogenesis. This is based, in part, on the report that the bulbus arteriosus undergoes a striated-to-smooth muscle phenotypic transition during development. An implication of this is that the zebrafish, a model almost ubiquitously accepted in other fields of comparative biology, may be poorly suited to the study of conotruncal abnormalities in human disease. However, while the use of atrioventricular-specific molecular markers has allowed extensive characterization of the development of the atrium and ventricle, the lack of any bulbus-specific markers has meant that this region of the zebrafish heart is poorly characterized and quite possibly misunderstood. We have discovered that the fluorescent nitric oxide indicator 4,5-diaminofluorescein diacetate (DAF-2DA) specifically labels the bulbus arteriosus throughout development from approximately 48 h post-fertilization. Therefore, using DAF-2DA and an immunohistochemical approach, we attempted to further characterize the development of the bulbus. We have concluded that no such phenotypic transition occurs, that contrary to current thinking, aspects of zebrafish arterial pole development are evolutionarily conserved, and that the bulbus should not be considered a chamber, being more akin to the arterial trunk(s) of higher vertebrates.
In Vivo Assessment of Cardiac Morphology and Function in Heart-specific Green Fluorescent Zebrafish
Journal of the Formosan Medical Association, 2007
The zebrafish (Danio rerio) is a new animal model for cardiac research. 1-3 Its heart contains four components (sinus venosus, atrium, ventricle, and bulbus arteriosus). Although it is equipped with a prototypic vertebrate heart, the studies of genetic control for zebrafish development can reveal some hints to solve human problems. 4 In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we have successfully developed a cardiac-specific promoter containing the upstream sequence of the zebrafish cardiac myosin light chain 2 gene (cmlc2) and generated several germ lines of transgenic zebrafish that have green fluorescent protein (GFP)-tagged hearts. 5 However, excess cardiac expression of GFP has been reported to cause dilated cardiomyopathy in mice. 6 Therefore, we performed this study to evaluate the morphology and functioning of zebrafish hearts with different expressional levels of green fluorescence.
Development of the cardiac conduction system in zebrafish
Gene Expression Patterns, 2016
The cardiac conduction system (CCS) propagates and coordinates the electrical excitation that originates from the pacemaker cells, throughout the heart, resulting in rhythmic heartbeat. Its defects result in life-threatening arrhythmias and sudden cardiac death. Understanding of the factors involved in the formation and function of the CCS remains incomplete. By transposon assisted transgenesis, we have developed enhancer trap (ET) lines of zebrafish that express fluorescent protein in the pacemaker cells at the sino-atrial node (SAN) and the atrio-ventricular region (AVR), termed CCS transgenics. This expression pattern begins at the stage when the heart undergoes looping morphogenesis at 36 hours post fertilization (hpf) and is maintained into adulthood. Using the CCS transgenics, we investigated the effects of perturbation of cardiac function, as simulated by either the absence of endothelium or hemodynamic stimulation, on the cardiac conduction cells, which resulted in abnormal compaction of the SAN. To uncover the identity of the gene represented by the EGFP expression in the CCS transgenics, we mapped the transposon integration sites on the zebrafish genome to positions in close proximity to the gene encoding fibroblast growth homologous factor 2a (fhf2a). fhf2a is represented by three transcripts, one of which is expressed in the developing heart. These transgenics are useful tools for studies of development of the CCS and cardiac disease.