We thank Julie R. Perlin for helpful comments on the manuscript. D.I.S. is supported by grants from the American Society of Hematology, the Cooley's Anemia Foundation, and the NIH (K01DK085217 and R03DK100672). E.J.H. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. B.L. is a Howard Hughes Medical Institute Medical Research Fellow. B.W.B. is supported by an Irvington Fellowship from the Cancer Research Institute and a Young Investigator Award from the Conquer Cancer Foundation of ASCO. L.I.Z. is supported by grants from the NIH (R01CA103846, P01HL032262, and R01HL04880), Taub Foundation for MDS Research, Harvard Stem Cell Institute, and is an investigator of the Howard Hughes Medical Institute.

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The authors declare that they have no conflict of interest.

並体結合融合は、大人のマウスモデルおよびニワトリ胚10-14で候補遺伝子の細胞機能を調査するための強力なツールとなっています。より最近では、胞胚融合方法は、結合したゼブラフィッシュ胚15を生成するために記載されています。本プロトコルでは、ビデオベースのチュートリアルを示し、より良い造血開発に関心のある候補遺伝子の、一時的なセル固有の、お...

野生型および遺伝子改変動物の間の遺伝的モザイク（キメラ）の作成 ​​は、細胞固有の候補遺伝子1-6の細胞外部関数に対する調査するための十分に確立し、古典的な戦略です。ゼブラフィッシュにおける胞胚移植は広く、細胞自律7-9の研究のためのキメラ胚を生成するために利用されてきました。目的の組織に応じて、しかし、予想通り、所望の組織（ 例えば、血液）1-3、7-9にドナー細胞を標的化するために挑戦することができます。マウスの遺伝学者は、長い共有循環10-14と結合した生物を生成するために、並体結合外科的方法を利用してきました。並体結合動物が共通の血流を共有しているため、異なる遺伝的背景の他の動物の細胞と一匹の動物由来の細胞間の相互作用は、10-16を研究することができます。最近、カリマ喫茶のグループは、エレガントなCREする能力を示しました移行15（HSPC）結合したゼブラフィッシュ胚を食べ、その後、造血幹細胞および前駆細胞を研究するためにこのシステムを使用しています。また、ゼブラフィッシュ並体結合は最近、造血幹細胞（HSC）の出現と移行、16で内皮カドヘリン5の役割を調査し、ゼブラフィッシュの開発17時のHSPCのニッチで間質細胞の役割を研究するために使用されました。 マウスとは異なり、ゼブラフィッシュの胚は透明であり、システムがユニークでパワフルな作り、並体結合開発中の細胞の直接可視化を可能にします。ゼブラフィッシュにおける並体結合の有用性は、しかし、急な学習曲線によって制限され、かつ繊細な胚上の並体結合手術は、詳細な指示とビジュアルデモせず、技術的に挑戦することができます。このプロトコルの目的は、研究するための並体結合ゼブラフィッシュの胚を生成する方法についてのビデオベースのチュートリアルを伴うために明確な、ステップバイステップの命令のセットを提供することです時間的、細胞固有の、または胞胚を開発するの外科的融合による候補遺伝子（複数可）の細胞外部関数。増加parabiont生存と新しい実験的なアプリケーションのためのキーの変更や追加の推奨事項が含まれています。

<table><tbody><tr><td>Methyl cellulose</td><td>Sigma</td><td>M0387</td><td></td></tr><tr><td>Individual components for E3/HCR:</td><td></td><td>For 1L: 14,61g NaCl, 0,63g KCl, 2,43g CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O, 1,99g MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td></td></tr><tr><td>NaCl (Sodium chloride)</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>KCl (Potassium chloride)</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (Calcium chloride dihydrate)</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (Magnessium sulfate heptahydrate)</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>Hepes (1M ) buffer solution</td><td>ThermoFisher</td><td>15630-080</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td></td></tr><tr><td>&lt;strong&gt;Antibiotics:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pen/Strep</td><td>gibco by Life technologies</td><td>15140-120</td><td></td></tr><tr><td>Ampicilin sodium salt</td><td>Sigma Life Science</td><td>A0166</td><td></td></tr><tr><td>Kanamycin sulfate from Streptomyces kanamyceticus</td><td>Sigma Life Science</td><td>K1377</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td></td></tr><tr><td>50 mg/ml Pronase from Streptomyces griseus</td><td>Roche</td><td>11459643001</td><td></td></tr><tr><td>capillary glass used for needles (Capillary Glass &amp;amp; Filaments)</td><td>Sutter Instrument</td><td>ITEM#: BF 100-50-10</td><td></td></tr><tr><td>Teasing needles with wooden handles</td><td>Fisher Scientific</td><td>S07894</td><td></td></tr><tr><td>Glass Pasteur pipettes</td><td>Fisherbrand</td><td>13-678-20A</td><td></td></tr><tr><td>10 ml pipette pump (green) (Pipette Pump Pipettor)</td><td>Novatech International</td><td>F37898-0000</td><td></td></tr><tr><td>100 mm diameter/ 20 mm deep plastic petri dishes (PETRI DISH, 100/20 MM, PS, CLEAR, WITH VENTS,&lt;br/&gt; HEAVY DESIGN, 15 PCS./BAG )</td><td>Greiner Bio-one</td><td>664102</td><td></td></tr><tr><td>Dextran, Cascade Blue, 10,000 MW, Anionic, Lysine Fixable</td><td>ThermoFisher</td><td>D-1976</td><td></td></tr><tr><td>PTU. working stock is 0.003% (50x is 0.15%). for 500 ml, 0.75 g N-Phenylthiourea</td><td>Sigma-Aldrich</td><td>P7629</td><td></td></tr><tr><td>Tricaine (powder) (Tricaine Methanesulfonato, Tricaine-S)</td><td>Western Chemical Inc</td><td>MS 222</td><td></td></tr><tr><td>LMP agarose (Ultrapure LMP agarose)</td><td>Invitrogen</td><td>16520100</td><td></td></tr><tr><td>plastic transfer pipette (just the wide ended one I think)</td><td>Fisherbrand</td><td>137115AM</td><td></td></tr><tr><td>Glass-bottom 6-well plates used for imaging</td><td>MatTek</td><td>P06G1.5-20-F</td><td></td></tr><tr><td>plastic western gel loading tip fixed on the end of a wood-handled dissecting needle (GELoader tips)</td><td>Eppendorf</td><td>22351656</td><td></td></tr><tr><td>glass cover slips, slides and vacuum grease if mounting for an upright microscope:</td><td></td><td></td><td></td></tr><tr><td>Vaccum grease ( Dow Corning&amp;reg; high-vacuum silicone grease&lt;br/&gt; colorless, weight 5.3 oz (tube) )</td><td>Dow Corning</td><td>Z273554</td><td></td></tr><tr><td>Glass cover slips</td><td>Corning Life Sciences</td><td>2960-244</td><td></td></tr></tbody></table>

generation of parabiotic zebrafish embryos by surgical fusion of developing blastulae

異なる遺伝的背景の2匹の動物の外科並体結合は、異なる遺伝的設定で細胞固有の関心の候補遺伝子のための細胞外因性の役割、細胞の移動行動、および分泌シグナル対を研究するユニークなシナリオを作成します。並体結合動物が共通の循環を共有しているため、一匹の動物からの血液や血液媒介因子は、そのパートナーとその逆と交換されます。したがって、一つの遺伝的背景に由来する細胞および分子因子は、第二の遺伝的背景との関連で研究することができます。成体マウスの並体結合研究の老化、癌、糖尿病、肥満、および脳の発達に広く使用されています。さらに最近では、ゼブラフィッシュ胚の並体結合は、造血の発生生物学を研究するために使用されています。マウスとは対照的に、ゼブラフィッシュ胚の透明な性質は、それFを調査するためのユニークでパワフルな方法作り、並体結合文脈での細胞の直接可視化を可能にしますundamental細胞および分子メカニズム。この技術の有用性は、しかし、並体結合ゼブラフィッシュ胚を生成するための急な学習曲線によって制限されます。このプロトコルは、関心のある候補遺伝子の役割を調べるために、異なる遺伝的背景の2ゼブラフィッシュの胚の胞胚を融合外科的にする方法についてステップバイステップの方法を提供します。また、並体結合ゼブラフィッシュ胚を可能遺伝子発現の時間的制御を行う、熱ショックに対して耐性です。この方法は、高度なセットアップを必要とし、胚発生の間、in vivoでの細胞遊走、運命の仕様、および分化を研究するための幅広い用途を持っていません。

以前に発表された研究15と一致して、ゼブラフィッシュ胚の成功並体結合融合は、ステージングと向き2胚のとメチルセルロースの濃度に依存します。いくつかの簡単な、安価なツールを使用して、外科的に融合した開発胞胚は、共有循環と並体結合胚に成長し、その生成しました。これらのツールは、変更されたパスツールピペット、10ミリリットルのピペッ...

Watch this Scientific Journal Video about Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae at JoVE.com

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

胞胚の開発の外科的融合による並体結合ゼブラフィッシュ胚の生成

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

generation-parabiotic-zebrafish-embryos-surgical-fusion-developing

Research

JoVE Journal

Developmental Biology

11.5K ビュー.  Boston Children’s Hospital. This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

ビデオ: Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

ゼブラフィッシュ胚におけるマイクロビーズの移植

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

発生生物学

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植によるキメラゼブラフィッシュ胚の生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

生物学

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

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.

幼虫のゼブラフィッシュにおける脊髄切断

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 ...

神経科学

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

キメラゼブラフィッシュ胚における網膜と脳の開発のライブイメージングのためのGFP発現割球の移植

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

ゼブラフィッシュ胚の簡易かつ効果的な移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

ゼブラフィッシュ胚からのナイーブ・ブラストデム・エクスプラントの生成

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

<meta charset="utf8"></head><body>再生・開発研究のための堅牢なゼブラフィッシュ細胞移植

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>著者スポットライト:発生および再生中のゼブラフィッシュ神経系における細胞接続性のマッピング

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

ゼブラフィッシュの胚および幼生における高分解能細胞移植

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

胞胚の開発の外科的融合による並体結合ゼブラフィッシュ胚の生成

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