Lrrc10 is required for early heart development and function in zebrafish

Leucine-rich Repeat Containing protein 10 (LRRC10) has recently been identified as a cardiac-specific factor in mice. However, the function of this factor remains to be elucidated. In this study, we investigated the developmental roles of Lrrc10 using zebrafish as an animal model. Knockdown of Lrrc10 in zebrafish embryos (morphants) using morpholinos caused severe cardiac morphogenic defects including a cardiac looping failure accompanied by a large pericardial edema, and embryonic lethality between day 6 and 7 post fertilization. The Lrrc10 morphants exhibited cardiac functional defects as evidenced by a decrease in ejection fraction and cardiac output. Further investigations into the underlying mechanisms of the cardiac defects revealed that the number of cardiomyocyte was reduced in the morphants. Expression of two cardiac genes was deregulated in the morphants including an increase in atrial natriuretic factor, a hallmark for cardiac hypertrophy and failure, and a decrease in cardiac myosin light chain 2, an essential protein for cardiac contractility in zebrafish. Moreover, a reduced fluorescence intensity from NADH in the morphant heart was observed in live zebrafish embryos as compared to control. Taken together, the present study demonstrates that Lrrc10 is necessary for normal cardiac development and cardiac function in zebrafish embryos, which will enhance our understanding of congenital heart defects and heart disease.     

马兜铃水提液对斑马鱼胚胎的致畸作用和心脏毒性的研究

目的：探索马兜铃水提液对斑马鱼胚胎的致畸作用和心脏毒性.方法：分别用不同浓度的马兜铃水提液和马兜铃酸A（AA）处理斑马鱼胚胎,观察致畸作用和对心脏发育影响.结果：给药组的斑马鱼胚胎出现畸形和死亡;当水提液中AA含量为0.5 μg/mL时,胚胎心率明显减慢;AA含量为5μg/mL时,胚胎在24～48 hpf之间全部死亡;水提液的LC50为1.43 μg/mL.结论：与AA相比,马兜铃水提液对斑马鱼胚胎有着更强的致畸和心脏毒性,且毒性作用具有时间和浓度依赖性.     

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.     

Smarcd3b and Gata5 promote a cardiac progenitor fate in the zebrafish embryo

Development of the heart requires recruitment of cardiovascular progenitor cells (CPCs) to the future heart-forming region. CPCs are the building blocks of the heart, and have the potential to form all the major cardiac lineages. However, little is known regarding what regulates CPC fate and behavior. Activity of GATA4, SMARCD3 and TBX5 - the `cardiac BAF' (cBAF) complex, can promote myocardial differentiation in embryonic mouse mesoderm. Here, we exploit the advantages of the zebrafish embryo to gain mechanistic understanding of cBAF activity. Overexpression of smarcd3b and gata5 in zebrafish results in an enlarged heart, whereas combinatorial loss of cBAF components inhibits cardiac differentiation. In transplantation experiments, cBAF acts cell autonomously to promote cardiac fate. Remarkably, cells overexpressing cBAF migrate to the developing heart and differentiate as cardiomyocytes, endocardium and smooth muscle. This is observed even in host embryos that lack endoderm or cardiac mesoderm. Our results reveal an evolutionarily conserved role for cBAF activity in cardiac differentiation. Importantly, they demonstrate that Smarcd3b and Gata5 can induce a primitive, CPC-like state.     

Whole mount nuclear fluorescent imaging: Convenient documentation of embryo morphology

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. genesis 2012. © 2012 Wiley Periodicals, Inc.     

Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ～6 d.     

The zebrafish/tumor xenograft angiogenesis assay

Here we describe a method to study tumor angiogenesis in zebrafish (Danio rerio) based on the injection of proangiogenic mammalian tumor cells into the perivitelline space of zebrafish embryos at 48 h post-fertilization. Within 24-48 h, proangiogenic tumor grafts induce a neovascular response originating from the developing subintestinal vessels. This can be observed at macroscopic and microscopic levels after whole-mount alkaline phosphatase staining of wild-type zebrafish embryos, or by fluorescence microscopy in transgenic VEGFR2:G-RCFP embryos in which endothelial cells express the green fluorescent protein under the control of the VEGFR2/KDR promoter. Angiogenesis inhibitors added to the injected cell suspension or to the fish water prevent tumor-induced neovascularization. The assay is rapid and inexpensive, representing a novel tool for investigating tumor angiogenesis and for antiangiogenic drug discovery. Also, gene inactivation by antisense morpholino oligonucleotides injection in zebrafish embryos may allow the identification of genes involved in tumor angiogenesis.     

Apoptosis in zebrafish development

Apoptosis (programmed cell death) is important in normal biological processes and in pathogenesis in vertebrates. This review focuses on some of the prominent features of apoptosis during fish development. Caspases and other apoptosis-regulating genes have been cloned from zebrafish (Danio rerio) and other fish species. Elucidation of in vivo functions of apoptosis is focused on development, morphogenesis and sex differentiation. In an attempt to elucidate cause and effect relationships between caspase and development, transgenic zebrafish overexpressing procaspase-3 were generated. Stress-induced apoptosis in zebrafish embryos can be monitored by whole mount TUNEL staining and caspase assay. Thus, zebrafish is a useful experimental model animal for investigation of apoptosis in vivo.     

构建斑马鱼心脏损伤-再生模型的手术方法

斑马鱼心脏再生是近年来心血管再生医学研究的新热点之一, 也是以斑马鱼为模式进行脊椎动物遗传发育研究的一个新的重要方向。通过了解斑马鱼成体心脏再生的过程和研究其分子和细胞机制有可能为诱导哺乳动物成体心脏再生、治疗心肌梗塞等人类心脏疾病提供理论依据。文章主要介绍通过简单的手术切除成体斑马鱼约20%心室造成成体心脏损伤、诱导心脏再生的操作方法与经验。其基本流程主要包括麻醉成鱼、在体视镜下用尖镊撕开斑马鱼心脏腹面的皮肤和心包膜以暴露心脏、用剪刀切除心尖区域的部分心室。这种方法的手术成功率可达90%以上, 操作简便且重复性好, 是目前研究斑马鱼成体心脏损伤-再生的最常用的方法。     

Rapid adaptation of molecular resources from zebrafish and medaka to develop an estuarine/marine model

Many estuary and coastal waters are highly threatened by heavy anthropogenic pollutants. Oryzias melastigma, also called O. dancena, a marine medaka that showed sensitive response to hypoxia and estrogenic endocrine disruptors in previous studies, is becoming a sentinel species for marine ecotoxicology studies. However, the lack of strong molecular foundation and knowledge of early developmental stages hampers its practical applications. Combining our research strength on zebrafish embryos, this study revealed both morphological and molecular (at mRNA and protein levels) development of embryos of this emergent model. Whole mount immunostaining technique specific for O. melastigma was successfully developed based on zebrafish standard protocols. We demonstrated that 17 out of 61 primary antibodies, which were previously tested in zebrafish, showed specific immunoreactivity with O. melastigma. These antibodies clearly illustrated the embryonic development of target tissues (principally neurons) in this medaka. Additionally, partial cDNA fragments of 11 organ-specific marker genes were isolated according to genomic resources of zebrafish, Japanese medaka and other fishes. Of the 11 genes, 8 are widely used as organ markers and their expression patterns were remarkably similar to their homologues in zebrafish and Japanese medaka. The expression profiles of the remaining 3 genes in fish are reported for the first time. These molecular markers (17 antibodies and 11 mRNA probes) can be used as responsive indicators in environmental toxicity evaluation. Moreover, this study brought forward and demonstrated the advantage of transferring techniques and resources from one model to another to hasten the research of interest.     

miR-22心肌特异转基因斑马鱼的构建及心肌肥厚的评估

目的:构建miR-22心肌特异转基因斑马鱼系,在体评估miR-22对于心肌肥厚的作用。方法:构建pTol2-CMLC2-miR-22-IRES-EGFP表达载体。通过显微注射的方法将tol2重组质粒于一细胞期注射入斑马鱼受精卵胚胎中,荧光筛选获得心肌特异表达绿色荧光的斑马鱼胚胎,并稳定表达传代。然后对稳定传代的成年斑马鱼心脏进行心肌肥厚及心功能的检测。结果:成功建立了miR-22心肌特异转基因斑马鱼系,通过定量PCR确定心肌中miR-22表达升高,荧光显微镜观察发现斑马鱼心肌出现绿色荧光。miR-22心脏特异过表达的转基因鱼系的成年鱼与野生对照组相比,出现了心肌肥厚的现象,心肌肥厚分子标志物nppa、myh7明显升高。斑马鱼心脏病理切片结果同样显示出miR-22心肌特异转基因斑马鱼出现了心肌肥厚的现象。结论:成功构建了miR-22心肌特异转基因斑马鱼,为研究心肌中miR-22的生物学功能提供了重要的工具,并证明miR-22心脏特异过表达会引起斑马鱼心肌肥厚。     

Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein.

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.     

Maternal Transfer and Protective Role of the Alternative Complement Components in Zebrafish Danio rerio

Embryos of most fish develop externally and are exposed to an aquatic environment full of potential pathogens, whereas they have little or only limited ability to mount an efficient and protective response. How fish embryos survive pathogenic attacks remains poorly defined. Here we demonstrate that the maternal immunization of female zebrafish with formalin-killed Aeromonas hydrophila causes a significant increase in C3 and Bf contents in the mother, a corresponding rise in the offspring, and induces a remarkable increase in the hemolytic activities in both the mother and offspring. In addition, the embryos derived from the immunized mother are significantly more tolerant to A. hydrophila challenge than those from the unimmunized fish, and blocking C3 and Bf activities by injection of the antibodies against C3 and Bf into the embryos render them more susceptible to A. hydrophila. These results clearly show that the protection of zebrafish embryos against A. hydrophila can be achieved by the maternally-transferred immunity of the complement system operating via the alternative pathway. This appears to be the first report providing in vivo evidences for the protective role of the alternative complement components in the early embryos of zebrafish, paving the way for insights into the in vivo function of other maternally-transferred factors in fish.     

The proepicardium delivers hemangioblasts but not lymphangioblasts to the developing heart

The mass of the myocardium and endocardium of the vertebrate heart derive from the heart-forming fields of the lateral plate mesoderm. Further components of the mature heart such as the epicardium, cardiac interstitium and coronary blood vessels originate from a primarily extracardiac progenitor cell population: the proepicardium (PE). The coronary blood vessels are accompanied by lymph vessels, suggesting a common origin of the two vessel types. However, the origin of cardiac lymphatics has not been studied yet. We have grafted PE of HH-stage 17 (day 3) quail embryos hetero- and homotopically into chick embryos, which were re-incubated until day 15. Double staining with the quail endothelial cell (EC) marker QH1 and the lymphendothelial marker Prox1 shows that the PE of avian embryos delivers hemangioblasts but not lymphangioblasts. We have never observed quail ECs in lymphatics of the chick host. However, one exception was a large lymphatic trunk at the base of the chick heart, indicating a lympho-venous anastomosis and a 'homing' mechanism of venous ECs into the lymphatic trunk. Cardiac lymphatics grow from the base toward the apex of the heart. In murine embryos, we observed a basal to apical gradient of scattered Lyve-1+/CD31+/CD45+ cells in the subepicardium at embryonic day 12.5, indicating a contribution of immigrating lymphangioblasts to the cardiac lymphatic system. Our studies show that coronary blood and lymph vessels are derived from different sources, but grow in close association with each other.     

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter ¹. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury. Open in a separate windowClick here to view.^{(76M, flv)}     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

Cardiac Hypertrophy Involves Both Myocyte Hypertrophy and Hyperplasia in Anemic Zebrafish

Background

An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart.

Methodology/Principal Findings

Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ).

Conclusions/Significance

The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized.