We would like to thank C. Burns, C. McRae for outlining the basic principle of this purification protocol, and F. Priller for initial implementation of this method in our lab. S.A.-S. is supported by a Heisenberg professorship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

该协议遵循了德国和柏林国家法律的动物护理准则;斑马鱼的处理是由动物保护（LaGeSo，柏林 - 勃兰登堡）地方当局监控。 1.获取斑马鱼胚胎的心脏提取<ol><li>横心脏记者斑马鱼，例如作为Tg（MYL7:EGFP）转基因twu34线9，以便获得胚胎心脏特异性GFP表达。 </li><li>保持水蛋胚胎在28.5°C，直到所需的萌芽阶段10,11。确保所收集的胚胎人口是同质既遗传和发育阶段以限制的变化的基因表达。 </li><li>手动Dechorionate胚胎。 </li></ol> 2.解剖斑马鱼胚胎的心脏注意:保留所有的解决方案上的冰。如果可能的话，做团队:一个人从教统局解剖的心ryos而另一个人在排序的荧光体视显微镜的心。这允许几百胚胎在几个小时的处理。 <ol><li>转约100胚胎成1.5ml的离心管中。麻醉用三卡因的胚胎（0.16毫克/毫升在E3的培养基）。一旦进入胚胎显然麻醉（他们不游泳和沉积物的试管底部，但他们的心还在跳动）。 </li><li>除去三卡因/ E3溶液和1毫升的L-15/10％FBS培养基中，一旦洗涤胚胎并在冰上保持。 注:L-15/10％FBS培养基中是很重要的，整个解剖过程中，以保持器官和细胞存活，直到心已被转移到RNAlater中或Trizol试剂。 </li><li>加入1毫升的L-15/10％FBS培养基中的胚胎和移液器上下用圆形凝胶加载提示5-8次，直到蛋黄被完全破坏。吸取胚胎逐滴到溶液的表面上，从而使压降将会破裂和将胚轻轻破碎。 注意:如何大力和频率胚胎必须吸取上下取决于胚胎的发育阶段， 即多移液需要在后期（ 例如在56 HPF）。 </li><li>对于第一批解剖胚胎，评估胚的完整性并解剖心在立体显微镜下的比例，因为一些心中可保持附着在胚胎如果过轻轻吸入。调整移液的方式为下轮清扫相应。 注:使用低滞留枪头和离心管，以减少心中坚持的塑料。 </li><li>样品应用到100微米的过滤器放置在50毫升离心管中;冲洗1.5 ml管1毫升的L-15/10％FBS培养基中，然后将此到滤波器，以便最小化样品的损失（一些心可能被粘在管的壁）。 1毫升L洗两次过滤器-15 / 10％FBS中。在此步骤中心通过过滤器，并收集在50ml管中。 </li><li>应用流通到30微米的过滤器放置在一个15毫升的离心管中，用1ml的L-15/10％FBS培养基冲洗过滤器一次。在这第二个过滤步骤中，心被保留在过滤器和较小的碎片被洗掉。 </li><li>转动过滤器倒置并通过施加连续洗涤三次用1ml的L-15/10％FBS冲洗心中从过滤器的成在1％琼脂糖包被的培养皿。 </li><li>手动分离GFP阳性的心从非荧光胚胎碎片（ 如眼镜头）与一对钳下，荧光体视显微镜，集中他们的菜心。根据步骤3收集它们。 注:如果胚胎是年龄超过24 HPF，心中应跳动，表明组织仍然生理正常。 </li></ol> 3.从Dissec分离mRNA特德心<ol><li>收集在尽可能小的体积的心（ 例如 10微升），并吸移管到1.5ml试管含有0.75毫升RNAlater中（在冰上）。确认没有心留在枪头通过查看它在荧光体视显微镜。 </li><li>或者，如果一种化学罩是可以在荧光显微镜的附近，在化学罩所收集的心转移到的Trizol（警告）。这就避免心贴在了枪头，也是心灵的离心过程中可能的损失（步骤3.4，3.5和3.7）。汇集来自几轮孤立的心在同一个管的Trizol，并直接转到步骤3.8;最终的体积应不超过原始的Trizol体积的10％。 注意:请阅读的Trizol材料安全数据表（MSDS）后再使用。处理Trizol试剂罩下，并建议穿戴个人防护装备。避免与皮肤，眼睛和clothi接触纳克。去除所有火源。 </li><li>普尔从几轮隔离心中到相同的管中的RNAlater（在冰上），与组织的收集量的RNA分离的效率提高。如果总样品体积超过原始RNAlater中体积的10％，可以使用一个额外的管0.75毫升RNAlater中存储（在冰上）。 </li><li>离心样品在15700 XG为20分钟，在4°C至沉淀的心。 </li><li>下一个荧光立体显微镜，收集仔细尽可能多的上清液尽可能到装有3毫升的L-15/10％FBS的培养基中在1％琼脂糖包被的培养皿，但是不干扰沉淀的心在离心管的底部。由于心的高达15％的可保留在上清液由于RNAlater中的高粘度，如在步骤3.7中所述检索它们。 </li><li>在化学罩，加0.5毫升的Trizol含有沉淀的心管和保持在冰上。 </li><li>根据荧光microscoPE，传送从1％琼脂糖包被培养皿含有的RNAlater / L-15/10％FBS的溶液的心（来自步骤3.5）转换成另一种1％琼脂糖包被培养皿用3ml的L-15/10％FBS稀释出RNAlater中。收集的心在培养皿的中心，下化学罩在小体积转移成沉淀心中的Trizol的管。 </li><li>涡1.5 ml管中含有的Trizol心中破坏心脏和孵化5分钟，在室温下。 </li><li>在化学罩，加100微升氯仿（警告），调匀（使用前以最大速度离心30秒）的Trizol /氯仿溶液转移到1.5 ml的预纺PLG管。孵育3分钟，在室温下进行。 注意:请阅读氯仿MSDS方可使用。处理氯仿试剂罩下，并建议穿戴个人防护装备。避免与皮肤，眼睛和衣服接触。远离不相容，如金属，碱。 </li><li> CentrifuGE将样品在15700×g离心15分钟，在4℃下，将水相转移到一个新的1.5毫升管。 </li><li>添加5-10微克糖原和250μl的预冷（-20℃）异丙醇;拌匀，孵育过夜，在-20℃。 </li><li>离心样品在15700×g离心30分钟，在4℃，弃去上清液。 </li><li>在15700×g离心在4℃下洗用1ml 75％乙醇，离心沉淀15分钟，并弃去上清液。 </li><li>让乙醇蒸发干燥的粒料在室温下，直到它变成白色（ 例如 10-15分钟）。 </li><li>添加20微升不含RNA酶的DDH 2 O的以沉淀孵育10-15分钟，在55℃以溶解的RNA然后保持在冰上。 </li><li>评估RNA的浓度和纯度通过吸光度测定。通过运行在1％琼脂糖凝胶的样品评估RNA的完整性。 </li></ol>

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The authors have nothing to disclose.

该协议允许斑马鱼胚胎心脏组织的快速富集的基因表达分析。心脏特异性RNA样品的数量和质量在很大程度上取决于几个关键步骤:第一，如果能够防止在该协议中的每一步的心损耗的样品的量有很大的提高，因为RNA纯化将仅具有足够的工作起始材料。第二，样品，这完全取决于实验者的纯度，通过分类和收集的心内的培养皿同时严格排除其它组织确定的。第三，样品的质量主要取决于限制可能发生...

斑马鱼（ 斑马鱼 ）被广泛用于发育生物学研究器官体内 ，由于其快速的，透明的和宫外胚胎发育，结合小尺寸和转基因记者线荧光蛋白的组织特异性表达的可用性。这个小脊椎动物特别适于研究心脏发育，因为早期的斑马鱼胚胎的氧合不依赖于心脏的跳动及血液流动;这些特点使大量心血管突变1,2的表征和斑马鱼现在是一个被广泛认可的模式生物来研究心脏疾病3。 到胚胎发育过程中研究基因表达，转录通常通过整个安装原位杂交（WISH）4分析，RT-qPCR的5，微阵列6，或下一代测序（RNA测序）7。而WISH允许基因表达的整个胚胎内的空间 - 时间分析，转录水平通过RT-qPCR的，微阵列或RNA测序方法，通常进行评估。然而，这些方法需要组织富集为特定的基因表达图谱。 因为斑马鱼胚胎心脏表示整个胚胎的一小部分，心脏发育过程中转录研究需要一个协议，用于解剖和心脏的富集。此外，为了得到生理上相关的数据，以维持心脏组织完全功能，直到RNA提取它是很重要的。在这里，我们描述了一种协议，用于快速分离生理正常和跳动心脏从数百斑马鱼胚胎的有效地获得高品质的RNA样品用于进一步分析。本方法是基于报道的烧伤和麦克雷，2006年8所述的协议，对于心脏组织的富集，这两种方法都使用的转基因心肌记者线并利用斑马鱼胚胎心脏与其他组织的鉴别粘附性能优势。简要地说，通过一个狭窄的枪头吹打许多胚胎上下，心脏被同时从胚体释放并随后从胚胎碎片两个快速过滤步骤中分离;荧光标记的心脏，然后手动地从剩余的碎片来分类并收集用于进一步处理。

<table><tbody><tr><td>Equipment for raising fish and collecting eggs&amp;nbsp;</td><td></td><td></td><td>see the Zebrafish Book&lt;sup&gt;11&lt;/sup&gt; for details</td></tr><tr><td>Fluorescence stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Refrigerated Microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>UV-Spectrophotometer</td><td></td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; Thermo Scientific Nanodrop 2000</td></tr><tr><td>Nucleic acid electrophoresis chamber</td><td></td><td></td><td></td></tr><tr><td>Petri dishes 4 cm &amp;Oslash;, coated with 1% agarose in E3 medium</td><td></td><td></td><td></td></tr><tr><td>Micropipettes and tips (P20, P100, P1000)</td><td></td><td></td><td></td></tr><tr><td>1.5 ml centrifugation tubes</td><td></td><td></td><td></td></tr><tr><td>15 ml and 50 ml centrifugation tubes</td><td></td><td></td><td></td></tr><tr><td>Pair of Dumont #5 forceps</td><td></td><td></td><td></td></tr><tr><td>ExactaCruz&amp;trade; Round Gel Loading Tips in Sterile Rack, 1-200&amp;mu;l&amp;nbsp;</td><td>Santa Cruz</td><td>sc-201732</td><td></td></tr><tr><td>100 &amp;mu;m filter (BD Falcon 100 mm Cell Strainer)</td><td>BD Biosciences</td><td>352360</td><td></td></tr><tr><td>30 &amp;mu;m filter (Pre-Separation Filters-30 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-041-407</td><td></td></tr><tr><td>Phase lock gel, heavy, 1.5 ml tubes&amp;nbsp;</td><td>Prime</td><td>2302810</td><td></td></tr><tr><td>Egg water medium</td><td></td><td></td><td>60 &amp;mu;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td></tr><tr><td>E3 medium&amp;nbsp;</td><td></td><td></td><td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Tricaine (3-amino benzoic acidethylester)</td><td>Sigma-Aldrich</td><td>A-5040</td><td>4 mg/ml Tricaine stock solution, pH 7</td></tr><tr><td>1% agarose (in E3 medium)</td><td></td><td></td><td></td></tr><tr><td>1% agarose gel (in TBE buffer)</td><td></td><td></td><td></td></tr><tr><td>Leibovitz&amp;acute;s L-15 medium&amp;nbsp;</td><td>Gibco</td><td>21083-027</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>RNAlater</td><td>Ambion</td><td>AM7020</td><td></td></tr><tr><td>Trizol</td><td>Ambion</td><td>1559606</td><td></td></tr><tr><td>Glycogen&amp;nbsp;</td><td>Invitrogen</td><td>10814-010</td><td>20 &amp;micro;g/&amp;micro;l in RNase-free water</td></tr><tr><td>chloroform</td><td></td><td></td><td></td></tr><tr><td>isopropanol</td><td></td><td></td><td></td></tr><tr><td>75% ethanol (in DEPC-ddH2O)</td><td></td><td></td><td></td></tr><tr><td>Nuclease-free water or sterilized DEPC treated ddH2O</td><td></td><td></td><td></td></tr><tr><td>Nucleic acid loading buffer</td><td></td><td></td><td></td></tr><tr><td>TBE (Tris/Borate/EDTA) buffer for electrophoresis</td><td></td><td></td><td></td></tr></tbody></table>

large-scale zebrafish embryonic heart dissection for transcriptional analysis

斑马鱼胚胎的心脏是由只有几百细胞，占整个胚胎中只有一小部分。因此，为了防止被掩蔽由全局胚胎转录心脏转录，有必要收集足够数量的心作进一步的分析。此外，如斑马鱼心脏发育迅速进行，心脏收集和RNA提取方法需要快，以保证样品的均匀性。在这里，我们提出了一个快速的手动清扫协议收集功能/跳动的心，从斑马鱼胚胎。这是为随后的心脏特异性RNA提取，以确定心肌特异性基因表达水平通过转录组分析，例如定量实时聚合酶链反应（RT-qPCR的）的必要先决条件。该方法是基于在斑马鱼胚胎心脏和其他组织相比的差的粘合性能;这允许迅速的phy从心外心脏组织由流体剪切力的破坏，逐步过滤和人工采集转基因荧光标记心中的组合SICAL分离。

在这里，我们描述了使用斑马鱼的Tg（MYL7:GFP）的有代表性的心脏解剖实验专在心肌（图1）的转基因twu34线9，它表达绿色荧光蛋白（GFP）。我们收集了两个解剖心，从他们得出评估心脏样品的纯度胚胎。简言之，将纯合的Tg（MYL7:GFP）twu34斑马鱼9进行异型杂交用野生型，使得所有的胚胎心脏的GFP标记。大约500胚胎（56 HPF）转移到1.5毫升管（约100?...

Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

到斑马鱼心脏发育过程中分析的心脏基因表达分布，总RNA已被提取从离体心脏。在这里，我们提出了一个协议，用于收集功能/跳动的心迅速人工清扫，从斑马鱼胚胎获得心脏特异性mRNA。

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

large-scale-zebrafish-embryonic-heart-dissection-for-transcriptional

Research

JoVE Journal

Developmental Biology

14.3K 次观看.  Max Delbrück Center for Molecular Medicine. 到斑马鱼心脏发育过程中分析的心脏基因表达分布，总RNA已被提取从离体心脏。在这里，我们提出了一个协议，用于收集功能/跳动的心迅速人工清扫，从斑马鱼胚胎获得心脏特异性mRNA。 

视频: Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

科研

发育生物学

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

心肌机械变形（DIAMOND）的位移分析揭示了胚胎斑马鱼心脏功能的分段异质性

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

生物工程

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

生物学

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

斑马鱼胚胎快速心脏动力学的光片显微镜

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

CRISPR-Cas9介导的斑马鱼心脏中的精确敲入编辑

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...