我们感谢ç张鱼照顾。这项工作是由中国国家自然科学基金（31171074，31371099和31571067至GP）和浦江人才计划（09PJ1401900到GP）的支持。

斑马鱼实验由复旦大学机构动物护理和使用委员会的批准。斑马鱼是根据标准程序20所提出和繁殖。 1.准备<ol><li>培养17 DPF和DPF 25斑马鱼幼体<ol><li>转让2男2女成年斑马鱼（健康，3〜6个月的时候，实验室阿布·斯特兰）的交叉罐傍晚道口前一天。分离男性和女性用的屏障。第二天早上，刷新水箱水并去除障碍，让他们交配。收集100个25mm培养皿受精后1至2小时的卵。 </li><li>在早期发育期（4天）保持在100毫米板用40mL胚胎培养基（EM）约40胚胎在28.5℃，一天刷新EM两次。幼虫转移至1L罐（40幼虫至300mL水系统）和5单丝旦后用活轮虫（全供给，一天两次）进料。经过10 DPF饲料活海虾代替轮虫饮食。转移成鱼重新循环水系统在14 DPF 21。 </li><li>提高幼虫斑马鱼在重新循环水系统，直到17单丝旦或25单丝旦。确保光/暗周期是系统水的14/10小时，pH值约7.2。测量17单丝旦（5.5至6.8毫米），25旦（8到11mm）幼虫22的主体长度。 </li></ol></li><li>制备2％琼脂平板上的清扫。 <ol><li> 4克琼脂添加到200mL的无菌水中。加热该混合物在微波炉中，直到它变成透明的。 </li><li>冷却琼脂溶液中约15分钟，然后将其倒入直径60个毫米的培养皿中（大约1/3体积的板）。存储在4℃下将固化的琼脂平板上。 </li></ol></li></ol> 2.方案1:解剖17生殖腺组织和25单丝旦幼虫<ol><li>加入碎冰进入水箱进行麻醉，17 DPF幼虫。转让麻醉幼虫冷却培养皿用30mL冷的林格氏溶液100毫米。保持鱼在冷冻林格氏液培养至少15分钟，以完全麻醉的幼虫。 </li><li>转移幼虫预冷的琼脂平板上用小塑料勺子。淹没整个鱼身在冷却林格氏溶液10毫升，并轻轻摊放在其一侧。 </li><li>做25X立体显微镜的视野下进行以下操作。卡件与镊子稳定鱼树干。从肛门到心脏与其他镊子纵向撕裂腹部。轻轻卸下身体的一侧的皮肤和肌肉，露出内脏。 </li><li>除去内脏腹仔细鳔的质量。避免损坏连接到鳔性腺。 </li><li>切断鱼鳔和前体之间的连接。拉出整个鱼鳔，轻轻性腺组织。 注意:在大多数情况下，S，在17单丝旦的性腺组织含有周围上皮组织和protonephridium。它们不容易相互分离，但在25 DPF它可以很容易地分离。性腺也连接到肛门。与肛门相互连接左右性腺的结将清晰可见。 </li><li>用镊子小心地将性腺组织从鱼鳔分离和清理周围的脂肪组织。 </li><li>立即分离的性腺组织转移到含有200μL林格氏溶液预冷的1.5毫升离心管中。保持管在冰上直至所有性腺组织从幼虫分开。 </li><li> 2.7 - 如步骤2.1中所述的解剖25单丝旦幼虫性腺。 </li></ol> 3.协议2:分析分离的性腺组织的基因表达<ol><li>从幼虫性腺组织提取总RNA。 <ol><li>转让孤立性腺组织到一个新的无RNase 1。5mL的管和除去林格氏溶液。加入100μL裂解液。涡直至组织完全溶解。 </li><li>根据制造商的说明进行总RNA提取操作。 </li><li>添加1/10体积的10倍的DNA酶I缓冲液和DNA酶I的1μL至RNA溶液并轻轻混合。在37℃下孵育20分钟。 </li><li>添加1/10体积的DNA酶失活试剂的RNA溶液。在70℃下孵育10分钟。测量使用分光光度计总RNA的浓度。存储在-20℃。 </li></ol></li><li>执行第一链cDNA合成<ol><li>使用寡（dT）引物 - 接头按照制造商的方案来进行第一链cDNA的合成。加入RNA的1至2微克至总反应体积〜20μL。 </li><li>孵育在45℃下90分钟的反应溶液中。终止在70℃下5分钟，通过加热进行反应。 </li><li>加入1μlRNA酶H至cDNA，以便lution以除去残留的RNA。在〜13000×g下轻轻混匀，离心10秒。在37℃下孵育20分钟。加入20微升不含核酸酶的水。存储在-70℃。 </li></ol></li><li>执行荧光定量PCR <ol><li>一个循环仪上用荧光染料进行定量PCR。使用下面的PCR循环条件:35个循环的95℃15秒，TM-5℃15秒，68℃30秒。使用的引物序列如下:AMH（正向:GTGGATGGCAGCAGTACGAC;启:GCGGAGAGGTGGAAGAGAGAATG），cyp19a1a（正向:GTCCTGTTGTCTCCTACTGTCG;启:CATTTGAGTTGAATATGATGCCCTG），nanos3（正向:GCTCGGTGTACGCCAAATCAACAT;启:CCAAGTGAAAACACAACACCAGTGC）， 滋养 （正向:ATCGCATAGGAAGAACTGGACGCT;启:CCAAGTGAAAACACAACACCAGTGC）和β肌动蛋白 （正向:AGTGCGACGTGGACATCCGTA;启:GCACTTCCTGTGGACGATGGA）19。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

斑马鱼已成为一个强大的模型，并在发展和疾病相关的研究被广泛使用。在成年斑马鱼的器官，如大脑，心脏，性腺和肾脏的分离的方法，很好地证明了23，24，25。由于体积小，在幼虫斑马鱼性腺组织的动态重构，性腺组织的分离是一项艰巨的任务。以前的研究中使用的整体解剖躯干组织或转基因滋养:基于EGFP细胞分选和激光?...

主要女性性决定簇在野生斑马鱼染色体4被丢失或在家养斑马鱼修改（ 即普通实验室菌株）1。相反，他们必须伴随着环境因素，如温度，缺氧，粮食供应和人口密度的多基因遗传性别决定系统。斑马鱼的性发育的具体机制尚不完全清楚。基本问题时斑马鱼的性别决定发生诸如什么初级性别确定信号（s）为/是，并且该基因的调节性腺变换的第一步骤有待回答2,3。 在斑马鱼性发育的过程中，有几个重要阶段已被确认。在开发的早期阶段，从4 HPF（小时受精后）原始生殖细胞（PGC的）经受说明书中，开始迁移生殖脊和增殖。 PGC号和生殖细胞和体细胞之间的相互相互作用对性腺分化的4个重要。在13 DPF（天受精后），性腺是未分化的阶段。 17个DPF，性腺发育成两个未来的男性和女性双重潜能卵巢。从卵巢细胞凋亡相关的过渡睾丸开始在21至25 DPF并可能持续数周。由35个单丝旦，生殖腺的性别已经确定和性别特异性配子生产正在进行在双侧卵巢和睾丸5，6，7。 迄今为止，不同的候选基因和性别决定机制已经被提出。蛋白质组学和转录分析已经分离的许多基因与性二态表达和这些基因已被用来研究性别分化在斑马鱼8， </sup&gt; 9,10。例如，在斑马鱼幼体中，cyp19a1a基因特别是在卵巢中表达，但不在睾丸11,12。此外，AMH基因在卵泡颗粒细胞弱表达，但在强烈的睾丸支持细胞13。与此相反， 滋养基因在女性和男性斑马鱼的生殖细胞中持续表达，使其成为一个合适的性腺标记14,15。 调查性腺基因表达水平关键是要了解的性别决定和分化的分子机制特别是在双电位卵巢阶段3,9。然而，幼虫斑马鱼和相应小性腺的小尺寸复杂gonada的隔离升组织进行进一步的分子分析。用以前的研究解剖鳃和肛门孔16之间的整个主体区域。该制剂，尽管含有性腺，由多个组织和器官的。可替代地，与特定的性腺-GFP表达的转基因动物，如滋养:EGFP的通过荧光激活细胞分选（FACS）和激光捕获显微解剖17,18用于性腺组织分离。但它们的广泛应用受到限制。在这里，我们描述了一个简单的过程在17 DPF和DPF 25从幼虫斑马鱼隔离性腺组织。我们证明性腺的位置相对于其他器官与周围组织分离形态完整的性腺。进一步的研究表明特定性腺基因如滋养和cyp19a1a在隔离性腺中高度表达通过定量PCR躯干组织（相定量PCR）分析。本协议允许鉴定，分离，RNA纯化和从幼虫斑马鱼性腺特异性基因扩增，由此使生殖腺组织19的随后的分子分析。

<table><tbody><tr><td>Cell culture dish 100 mm</td><td>Corning</td><td>430167</td><td>For embryo incubation</td></tr><tr><td>20x EM</td><td></td><td></td><td>For a 1 L needed: add 17.5 g NaCl, 0.75 g KCl and 2.9 g CaCl&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O; then add 0.41 g KH&lt;sub&gt;2&lt;/sub&gt;PO4, 0.412 g Na&lt;sub&gt;2&lt;/sub&gt;HPO4 anhydrous and 4.9 g MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;7H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>1x EM</td><td></td><td></td><td>Dilute 20x EM in distilled water</td></tr><tr><td>AGAROSE G-10</td><td>Gene</td><td>121985</td><td>For preparing the 2% agar plates</td></tr><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596-026</td><td>For RNA isolation</td></tr><tr><td>Meter glass</td><td>Shen Bo</td><td>250 ml</td><td>For preparing the 2% agar plates</td></tr><tr><td>Microwave Oven</td><td>Midea</td><td>M1-211A</td><td>For heating the AGAR</td></tr><tr><td>Tweezer DUMONT#5INOX</td><td>World Precision Instrument</td><td>500341</td><td>For dissection</td></tr><tr><td>Stereomicroscope</td><td>Motic</td><td>SMZ168</td><td>For dissection</td></tr><tr><td>Pure water equipment</td><td>Millipore</td><td></td><td></td></tr><tr><td>Ringer&amp;rsquo;s solution</td><td></td><td></td><td>For a 1 L needed: Add 6.78 g NaCl, 0.22 g KCl, 0.26 g CaCl&lt;sub&gt;2&lt;/sub&gt; and 1.19 g Hepes; then fill to 1 L; Adjust pH to 7.2. Sterilize by filtration and keep in an autoclaved clear polycarbonate container.</td></tr><tr><td>Transfer pipette</td><td>Samco</td><td>202, 204</td><td></td></tr><tr><td>Metal bath</td><td>QiLinbeier</td><td></td><td>Model GL-150</td></tr><tr><td>Microscope</td><td>Leica&amp;nbsp;</td><td>M205 FA</td><td>For photomicrograph</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>Micro Scale RNA Isolation Kit</td><td>Ambion</td><td>AM1931</td><td>For RNA isolation from gonad tissues</td></tr><tr><td>Dnase I</td><td>Sigma</td><td>AMPD1-1KT</td><td>For DNA digestion in the RNA solution</td></tr><tr><td>RevertAid First Strand cDNA Synthesis Kit</td><td>Thermo Scientific</td><td>#K1631</td><td>For first-strand cDNA synthesis</td></tr><tr><td>Rnase H</td><td>Thermo Scientific</td><td>#EN0202</td><td>For digesting the residual RNA in the cDNA solution.</td></tr><tr><td>SYBR Green Realtime PCR Master Mix</td><td>TOYOBO</td><td>QPK-201</td><td>This product is a Taq DNA polymerase-based 2x master mix for real-time PCR and&amp;nbsp; applicable for intercalation assay with SYBR Green I.</td></tr><tr><td>Spectrophotometer</td><td>Ne Drop</td><td>OD-2000+</td><td>Measuring the concentration of the total RNA</td></tr><tr><td>Mastercycler</td><td>Eppendorf</td><td>AG 22331 Hamburg</td><td>gene expression profiling</td></tr></tbody></table>

dissection of larval zebrafish gonadal tissue

虽然野斑马鱼拥有ZZ / ZW性别决定系统，驯化斑马鱼已经失去了性染色体。他们利用一种多基因遗传性别决定系统，其中几个基因分布在整个基因组共同确定个别鱼类的性别身份。目前，参与调节性腺发育的基因以及它们如何工作仍然是难以捉摸的。通常情况下，隔离生殖腺组织是第一步检查性发育过程。在这里，我们提出了一个程序，从17旦（天受精后）和25 DPF斑马鱼隔离性腺组织。分离的性腺组织可以通过形态学和基因表达谱随后检查。

性腺的解剖上阿布·斯特兰幼虫斑马鱼进行。 图1示出在17和DPF 25单丝旦幼虫斑马鱼的典型性腺组织。首先，皮肤和腹部的一侧的肌肉被切断，露出内脏。去除内脏的质量后，鳔与性腺一起留在树干。性腺附接到鱼鳔的腹侧（ 图1B中的箭头&#39;）。在17个DPF，生殖腺是在双重潜能卵巢阶段。经分离的性腺包含在左侧和?...

Watch this Scientific Journal Video about Dissection of Larval Zebrafish Gonadal Tissue at JoVE.com

Dissection of Larval Zebrafish Gonadal Tissue

在这里，我们提出了隔离幼虫斑马鱼，这将有利于斑马鱼的性别分化和维护的调查性腺组织的协议。

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 ...

dissection-of-larval-zebrafish-gonadal-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.2K 次观看.  Fudan University. 在这里，我们提出了隔离幼虫斑马鱼，这将有利于斑马鱼的性别分化和维护的调查性腺组织的协议。 

视频: Dissection of Larval Zebrafish Gonadal Tissue

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

科研

神经科学

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

生物学

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

发育生物学

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

处理斑马鱼幼虫的肠道细胞以进行单细胞 RNA 测序

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

作者聚焦:分离和分析斑马鱼幼虫的肠道细胞以研究胃肠道发育的转录组学方面 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

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 ...

幼虫斑马鱼性腺组织的解剖

摘要

探索更多视频

此视频中的章节

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

剖析器官的成年斑马鱼

在斑马鱼幼虫脊髓横断

晚期胚胎果蝇性腺的解剖和活体成像

斑马鱼胚胎眼组织显微切割的

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼

幼虫斑马鱼性腺组织的解剖

摘要

探索更多视频

此视频中的章节

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

剖析器官的成年斑马鱼

在斑马鱼幼虫脊髓横断

晚期胚胎 果蝇 性腺的解剖和活体成像

斑马鱼胚胎眼组织显微切割的

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼

晚期胚胎果蝇性腺的解剖和活体成像