We thank F. Bouallague and the IBENS animal facility for excellent zebrafish care. Research reported in this publication was supported by the Fondation ARC pour la recherche sur le cancer, grants N&#176; SFI20111203770 and N&#176; PJA 20131200143.

<ol>
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The authors have nothing to disclose.

פרוטוקול זה מציג דרך קלה ללמוד את התפקיד של גן מועמד נדידת תאי in vivo, על ידי שילוב של היצירה עוברת chimeric באמצעות השתלה תאית עם הדמיה לחיות. יצירה עוברת פסיפס לימוד הדינמיקה של תא דורש הדמיה ?...

בשנת יצורים רב-תאיים, נדידת תאים היא חיונית הן לפיתוח העובר שבו היא מבטיחה הארגון של תאים לתוך רקמות ואיברים, ובמשך חייו הבוגרים, בו היא לוקחת חלק הומאוסטזיס רקמות (ריפוי פצעים) וחסינות. בנוסף לפונקציות פיזיולוגיות אלה, נדידת תאים הוא מעורב גם במצבים פתולוגיים מגוונים, כולל, ובמיוחד, גרורות סרטניות. נדידת תאים כבר נתחה במבחנה במשך עשרות שנים, מתן הבנה כוללת של המנגנונים המולקולריים הבטיחו תנועות תא על משטחים שטוחים. בשנת vivo עם זאת, תאים עומדים בפני סביבה מורכבת יותר. זה בבירור הופיע בשנים האחרונות כי הגירה בתוך אורגניזם עשויה להיות מושפעת רמזים חיצוניים כגון תאי מטריקס, תאי שכנים או כמוקינים מופרשים מנחת הגירה, וכי מנגנוני נהיגת נדידת תאים עשויים להשתנות ממה תוארה &lt;em&gt; במבחנה 1,2. המנגנונים הבטיחו נדידת תאי vivo קבלו פחות תשומת לב עד כה, בעיקר בשל הקושי הטכני גדל, לעומת במבחנה. בשנת vivo ניתוח נדידת תאים בפרט דורש גישה אופטית ישירה תאים נודדים, טכניקות לתייג תאים ייחודיים כדי לראות דינמיקה המורפולוגיה שלהם, כמו גם רווח או הפסד של הפונקציה מתקרב לבחון את התפקיד של גנים מועמדים. עד כה, רק כמה מערכות מודל מחסה מאפיינים אלה שימשו לנתח בתא vivo הגירה 3. אנחנו לאחרונה השתמשנו ההגירה של צלחת prechordal הפוטנציאלית עוברות דג זברה מוקדם כמערכת מודל הנוח חדשה להעריך את הפונקציה של גני מועמדים בשליטת נדידת תאי vivo 4,5. צלחת prechordal פרוספקטיבי (הידוע גם בשם mesendoderm הקדמי) היא קבוצה של תאים ויוצרים בתחילת Gastrulation בצד הגבי של העובר. במהלך gastrulation בקבוצה זו נודד קולקטיבי כלפי קוטב החיה של העובר 6-8, כדי ליצור את צלחת prechordal, עיבוי mesendodermal, קדמית notochord: ומתחת הצלחת העצבית. החלק הקדמי של צלחת prechordal יהיה להצמיח בלוטת הבקיעה, בעוד החלק האחורי שלה תורם סביר לעמוד בראש mesoderm 9. הודות לפיתוח החיצוני בהירות אופטית של עובר הדג, נדידת תאים ניתן ישירות ובקלות שנצפתה המבנה הזה. השתלת תאים היא טכניקה חזקה מאוד המאפשרת יצירה המהירה וקלה של עוברת פסיפס 10. הבעת סמני cytoskeletal פלורסנט בתוצאות תאים המושתלים בתוך התיוג של תאים בודדים, המורפולוגיה והדינמיקה של אשר ניתן להבחין בקלות. שילוב זה רווח או הפסד של הפונקציה מתקרב היתרי ניתוח של פונקציות אוטונומיות התא של פחיתגן didate. הפרוטוקול המובא מתאר כיצד אנו הערכנו את הפונקציה של Sin1 רכיב TORC2 בשליטת דינמיקת גירה תקטין תא in vivo 5. אבל, כאמור בתוצאות ודנו עוד יותר, זה יכול לשמש כדי לנתח את המשמעות הפוטנציאל של כל גן מועמד בשליטת נדידת תאי in vivo.

<table><tbody><tr><td>Glass capillaries (outside diameter 1.0 mm, inside diameter 0.58&amp;nbsp;mm)</td><td>Harvard Apparatus</td><td>300085</td><td>standard thickness</td></tr><tr><td>Glass capillaries (outside diameter 1.0 mm, inside diameter 0.78 mm)</td><td>Harvard Apparatus</td><td>300085</td><td>thin-walled</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td>10&amp;nbsp;000&amp;nbsp;units penicillin and 10&amp;nbsp;mg streptomycin per ml</td></tr><tr><td>fine tweezers</td><td>Dumont Fine Science Tools</td><td>11254-20</td><td>5F</td></tr><tr><td>glass bottom dishes</td><td>MatTek</td><td>P35G-0-10-C</td><td></td></tr><tr><td>Air transjector</td><td>Eppendorf</td><td>5246</td><td></td></tr><tr><td>Micro-forge</td><td>Narishige</td><td>MF-900</td><td></td></tr><tr><td>Microgrinder</td><td>Narishige</td><td>EG-44</td><td></td></tr><tr><td>Micromanipulator (for injection)</td><td>Narishige</td><td>MN-151</td><td></td></tr><tr><td>Micromanipulator (for cell transplantation)</td><td>Leica</td><td>Leica Micromanipulator</td><td></td></tr><tr><td>Hammilton Syringe</td><td>Narishige</td><td>IM-9B</td><td></td></tr><tr><td>Micropipette puller</td><td>David Kopf Instruments</td><td>Model 720</td><td></td></tr><tr><td>Transplantation mold</td><td>Adapative Science Tools</td><td>PT-1</td><td></td></tr><tr><td>Needle holder</td><td>Narishige</td><td>HI-7</td><td></td></tr><tr><td>Tube connector</td><td>Narishige</td><td>CI-1</td><td></td></tr><tr><td>PTFE tubing</td><td>Narishige</td><td>CT-1</td><td></td></tr></tbody></table>

analyzing in vivo cell migration using cell transplantations and time-lapse imaging in zebrafish embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

הטכניקה הציגה שמשה לנתח את התפקיד של Sin1, אחד מרכיבי הליבה של הטור 2 המורכב (TORC2), בשליטת נדידת תאי vivo. שימוש השתלת תאים מתיר תיוג של תאים מבודדים וניתוח של תופעות תאים אוטונומיים. סרט S1 מציג את ההגירה של ובתאים צלחת prechordal המושתלים. תיוג יקטין ע...

Watch this Scientific Journal Video about Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos at JoVE.com

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

ניתוח<em&gt; In vivo</em&gt; נדידת תאים באמצעות השתלות תא והדמיה זמן לשגות עוברי דג הזברה

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

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Research

JoVE Journal

Developmental Biology

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

7.6K Views.  IBENS, Institut de Biologie de l'École Normale Supérieure. Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration. 

Video: Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

הדמית מבני subcellular בעובר דג הזברה החייה

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

In vivo הדמיה של ביטוי מהונדס ג&#39;ין הפרט רשתית אבות ב Chimeric דג הזברה עוברים לחקר השפעות Cell Nonautonomous

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

פוטון רב זמן לשגות הדמיה כדי להמחיש את התפתחות בזמן אמת: ויזואליזציה של העברת תאי הרכס העצבי עוברי דג זברה

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

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 ...

Biology

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

הדמיה חיה של תנועתיות התא ואת cytoskeleton אקטין של נוירונים בודדים לבין תאים קרסט עצבית בעוברים דג הזברה

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

שימוש בדגי זברה זחלים למחקרים יעילים בגידולי Vivo

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

מחבר זרקור: קידום טיפולים בסרטן באמצעות השתלת קסנו-גידול בדגי זברה

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

שיטה פשוטה, מהירה ויעילה לניתוח השתלת גידול בעוברים שקופים של דגי זברה

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

Cancer Research

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

ססגוניות זמן לשגות הדמיה של דג הזברה טראנסגנטי: חזותי בתאי גזע רשתית הופעל על ידי אבלציה ממוקד תאים עצביים

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

הדמיה חיה של שחול Cell מן האפידרמיס של דג הזברה פיתוח

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

ניתוח

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ממוקד רקמות מפלצות עובריים: דג הזברה Gastrula השתלת תאים

הדמיה חיה של תנועתיות התא ואת cytoskeleton אקטין של נוירונים בודדים לבין תאים קרסט עצבית בעוברים דג הזברה

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ססגוניות זמן לשגות הדמיה של דג הזברה טראנסגנטי: חזותי בתאי גזע רשתית הופעל על ידי אבלציה ממוקד תאים עצביים

הדמיה חיה של שחול Cell מן האפידרמיס של דג הזברה פיתוח