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.

היתוך Parabiotic כבר כלי רב עוצמה כדי לחקור תפקודים תאיים של גנים מועמדים בדגמים עוברים חומוס בעכברים בוגרים 10-14. לאחרונה, שיטה היתוך blastula תוארה להפקת 15 עוברי דג הזברה סיאמיים. בפרוטוקול הנוכחי, הדרכות וידאו מבוססות משמשות כדי להדגים וטוב יותר לתאר את המתודולו...

יצירת פסיפסים גנטיים (מפלצות) בין wild-type ובעלי החיים מהונדסים גנטיים היא אסטרטגיה ומבוססת וקלסי לחקירת תא-מהותי לעומת פונקציות תא-חיצוני של גני המועמדים 1-6. Blastula השתלת דג הזברה נוצלה נרחב כדי לייצר עובר chimeric ללימודים של תא-אוטונומיה 7-9. בהתאם הרקמה של עניין, לעומת זאת, זה יכול להיות מאתגר למקד תאים תורמים כצפוי לרקמה הרצויה (למשל, דם) 1-3, 7-9. גנטיקאים עכבר זמן מנוצלים שיטות כירורגיות parabiotic ליצור אורגניזמים סיאמיים עם תפוצה משותפת 10-14. בגלל חי parabiotic לשתף דם משותף, אינטראקציות בין תאים שמקורם מן חי אחד עם תאים של בעלי החיים האחרים של רקע גנטי שונה ניתן ללמוד 10-16. לאחרונה, הקבוצה של כרימה Kissa באלגנטיות הפגינו את היכולת creאכלו עוברי דג הזברה סיאמיים ולאחר מכן להשתמש במערכת הזאת כדי ללמוד גזע hematopoietic ו תא אב (HSPC) ההגירה 15. בנוסף, parabiosis דג הזברה שימש לאחרונה לחקור את התפקיד של cadherin אנדותל 5 בתא גזע hematopoietic (HSC) הופעתה והגירה, 16 ו כדי ללמוד את התפקיד של תאי סטרומה בגומחה HSPC במהלך ההתפתחות דג הזברה 17. בניגוד עכברים, עוברי דג הזברה הם שקוף ולאפשר להדמיה ישירה של תאים במהלך ההתפתחות parabiotic, מה שהופך את מערכת רבת עוצמה באופן ייחודי. השירות של parabiosis דג זברה, לעומת זאת, הוא מוגבל על ידי עקום למידה תלולה, וניתוחי parabiotic על העוברים העדינים יכול להיות מאתגר מבחינה טכנית ללא הדרכה מפורטת והדגמה ויזואלית. מטרת פרוטוקול זה היא לספק סט של ברור, צעד-אחר-צעד ללוות הדרכות וידאו מבוסס על איך ליצור עוברי דג הזברה parabiotic ללימודזמני, תא-מהותי, או פונקציות חיצוני תאים של גן מועמד (ים) על ידי היתוך כירורגית לפתח blastulae. שינויים עיקריים והמלצות נוספות להישרדות parabiont הגדלה ויישומים ניסיוניים חדשים כלולים.

<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

parabiosis כירורגי של שתי חיות של רקע גנטי שונה יוצר תרחיש ייחודי ללמוד תא פנימי לעומת תפקידי תא-חיצוני עבור גני מועמדים של עניין, התנהגויות נדידה של תאים, אותות מופרשים הגדרות גנטיות מובהקות. בגלל חי parabiotic לשתף תפוצה משותפת, כל דם או גורם מובל דם חיה אחת יתחלף עם השותף שלה, ולהיפך. לכן, תאים וגורמים מולקולריים מרקע גנטי אחד ניתנים ללמוד בהקשר של רקע גנטי שני. Parabiosis של עכברים בוגרים כבר נעשה שימוש נרחב להזדקנות מחקר, סרטן, סוכרת, השמנת יתר, והתפתחות המוח. לאחרונה, parabiosis של עוברי דג הזברה נעשה שימוש כדי ללמוד את הביולוגיה ההתפתחותית של hematopoiesis. בניגוד לעכברים, האופי השקוף של עוברי דג זברה מתיר הדמיה הישירה של תאים בהקשר parabiotic, מה שהופך אותו שיטה חזקה באופן ייחודי על חקירת fמנגנונים תאיים ומולקולריים undamental. השירות של טכניקה זו, לעומת זאת, הוא מוגבל על ידי עקומת למידה תלולה להפקת עוברי דג הזברה parabiotic. פרוטוקול זה מספק שיטת צעד אחר צעד כיצד בניתוח פתיל blastulae של שני עוברי דג זברה של רקע גנטי שונה לחקור את התפקיד של גני מועמדים מעניינים. בנוסף, עוברי דג הזברה parabiotic סובלניים הלם חום, מה שהופך שליטה הזמני של ביטוי גנים אפשריים. שיטה זו אינה דורשת הגדרה משוכללת בעל יישומים רחבים ללימוד נדידת תאים, מפרט גורל, והבחנת in vivo במהלך התפתחות עוברית.

בקנה אחד עם מחקרים שפורסמו בעבר 15, היתוך parabiotic מוצלח של עוברי דג הזברה תלוי staging והכיוון של שני עוברים ואת הריכוז של methylcellulose. עם רק כמה כלים פשוטים זולים, blastulae בפיתוח התמזגו בניתוח נוצרו שצמחו לעוברי parabiotic בעלי תפוצה משותפת. כלים אלה כללו פיפטה...

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.

הדור של Parabiotic דג הזברה עוברים ידי Fusion כירורגי של פיתוח Blastulae

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 Views.  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.

Video: 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.

Microbead השרשה בעובר דג הזברה

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 ...

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.

יצירת עוברים דג הזברה chimeric על ידי השתלה

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 ...

Biology

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.

ממוקד רקמות מפלצות עובריים: דג הזברה Gastrula השתלת תאים

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.

חיתוך רוחב חוט השדרה בדג הזברה הזחל

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 ...

Neuroscience

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.

השתלה של ה-GFP-לבטא בלסטומרים עבור הדמיה חיה של התפתחות המוח ואת רשתית עוברי דג הזברה chimeric

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.

מכשיר השתלה פשוט ויעיל לעוברי דגי זברה

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.

דור של מסלקי בלסטודרם נאיביים מעוברי דגי זברה

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>השתלת תאי דג זברה חזקה למחקר התחדשות ופיתוח

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>מחבר זרקור: מיפוי קישוריות תאית במערכת העצבים של דגי זברה במהלך התפתחות והתחדשות

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.

השתלת תאים ברזולוציה גבוהה בדגי זברה עובריים וזחלים

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

הדור של Parabiotic דג הזברה עוברים ידי Fusion כירורגי של פיתוח Blastulae

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