Questo studio è stato supportato da una National Institutes of Health (NIH) Core Grant (5P30EY008098) ei fondi di seguito per XW: NIH Concessione R01EY016099 e della Ricerca per la prevenzione Career Development Award cecità. Ringraziamo Lathrop Kira per l&#39;assistenza nel campo dell&#39;imaging confocale.

Giorno 1. Preparazione 1. Impostare croci pesce Impostare attraversa coppie di pesce zebra in serata. Per evitare accoppiamenti casuali prima che il tempo desiderato, utilizzare divisori per separare i pesci femminili e maschili nei cassetti di accoppiamento. GFP-esprimere i pesci transgenici sono usati come fonte di blastomeri donatore. Il pt104 linea transgenica (Zou et al., 2008), qui utilizzato, esprime GFP ubiquitariamente sotto il controllo del promotore EF1. Espressione GFP permette la visualizzazione di cellule del donatore per immagini dal vivo. Altre linee di pesci transgenici che esprimono proteine ​​fluorescenti adatti possono essere utilizzati per esigenze specifiche. 2. Preparare a forma di cuneo agarosio pozzi per lo svolgimento degli embrioni Versare fuso 1% agarosio, preparata in acqua uovo E3 (Westerfield, 2007), in una piastra di Petri. Float uno stampo di plastica che è a forma di cuneo sporgenze sulla soluzione di agarosio e lasciare che la soluzione di agarosio solidificare completamente. Togliere lo stampo con attenzione e immergere il letto di agarosio con acqua E3. 3. Preparare aghi trapianto Utilizzare un estrattore verticale dell&#39;ago (David KOPF Instruments) alla potenza impostazione 11,5 a fare gli aghi tirando pipette di vetro capillare (Precision Instrument World). Suggerimenti ago buono dovrebbe conici con alberi lunghi. Macinare le punte ago con un angolo di 45 gradi con un Grinder Micro (Marishige) fino aperture smussati di 40-45μm di diametro interno sono ottenuti (Fig. 1A). Per pulire la punta dell&#39;ago, collegare un tubo alla fine smussata dell&#39;ago, collegare il tubo di una siringa, e poi aspirare e rilasciare una soluzione al 10% di acido fluoridrico fino a quando tutti i detriti visibile viene rimosso. Successivamente, lavare le punte con acqua distillata, seguito da 100% di etanolo. Asciugare gli aghi fino a che tutti evapora etanolo. E &#39;importante assicurare che le punte siano completamente asciutti: l&#39;etanolo residuo rovinerà gli aghi durante l&#39;alta temperatura fase di lucidatura. Per ammorbidire e lucidare gli spigoli delle punte ad ago, cuocere le punte ago con il calore generato dal filamento di un falsario Micro (Marishige). Non lasciare che le punte ago toccare il filamento durante la lucidatura. La quantità di elettricità che passa anche se il filamento e la distanza tra il filamento e la punta dell&#39;ago può essere regolata per raggiungere condizioni di riscaldamento desiderata. La temperatura non dovrebbe essere troppo alto: questa si scioglierà e deformare la punta dell&#39;ago. Dopo gli spigoli sono levigati, con attenzione toccare il filamento riscaldato con la punta dell&#39;ago e poi tirare delicatamente il filamento lontano da l&#39;ago per generare una estremità appuntita (fig. 1B). Un ago tagliente, liscia e pulita è fondamentale per l&#39;ottenimento di blastomeri donatore integro e per non danneggiare gli embrioni ospitante. 4. Preparare un embrione-posizionamento della sonda Tirare una pipetta di vetro sulla fiamma del gas per ottenere una sonda sottile vetro con un fine sfumate. Questa sonda sarà utilizzata per posizionare gli embrioni secondo il giusto orientamento prima del trapianto. 2 ° giorno. Blastomero trapianto 1. Raccogliere ed embrioni dechorionate Il giorno dopo, togliere i divisori nei cassetti croce per consentire ai pesci di accoppiarsi. Mettere gli embrioni in una piastra di Petri riempita con acqua E3. Poi, gli embrioni dechorionate manualmente strappare la chorions dagli embrioni con un paio di pinze fine. Utilizzare una pipetta di vetro con albero piegato per trasferire gli embrioni attentamente dechorionated in agarosio pozzi. Evitare il contatto degli embrioni con l&#39;aria, che causano gli embrioni per scoppiare. Lasciate che gli embrioni si sviluppano a 28,5 ° C nei pozzi agarosio fino a 3 ore dopo la fecondazione, o HPF. 2. Istituire un apparato di trapianto Mentre gli embrioni progettare, organizzare un apparato trapianto collegando l&#39;ago trapianto al bagno d&#39;olio minerale micro-sistema di pompaggio. Assicurarsi che il sistema non perde e non bolle d&#39;aria intrappolate nel sistema. Riempire di nuovo l&#39;ago intero trapianto con olio minerale, ad eccezione del ,5-1 l di spazio in apertura punta. 3. Trapianto di blastomeri Per trapianto di blastomeri, utilizzare una sonda di posizionamento di vetro per orientare il lato blastomero del 3-4-HPF embrioni verso l&#39;alto. Poi, inserire delicatamente l&#39;ago in un trapianto di embrione donatore e succhiare lentamente in 5-20 blastomeri l&#39;ago. Inserire il blastomero pieno ago in un embrione di accoglienza e rilasciare i blastomeri lentamente. Il sito in cui vengono rilasciati i blastomeri influenzerà in cui la loro progenie localizzare nelle fasi successive dello sviluppo. Per analizzare lo sviluppo della retina e del cervello, le cellule rilasciano al polo animale. Durante il trapianto, assicurarsi che la punta dell&#39;ago non ha contatto con l&#39;aria. Per ridurre al minimo i danni meccanici, gli embrioni ospitante deve essere lasciato nei pozzetti agarosio a 28,5 ° C fino al giorno successivo. Poi, transfer gli embrioni mosaico di piastre di pre-ricoperto con 0,5 ml di 1% agarosio 24-bene, con un embrione in ciascun pozzetto. 1 ml di acqua uovo E3 si aggiunge a ciascun pozzetto. Per prevenire le infezioni batteriche, aggiungere 10 ml di penicillina e streptomicina 100X all&#39;acqua uovo E3. 3 ° giorno. Incorporare e immagini dal vivo degli embrioni mosaico 1. Incorporare gli embrioni Preparare 1,5% di agarosio a basso punto di fusione (PML) in acqua uovo E3 e mantenere la soluzione in un bagno d&#39;acqua 30 ° C. Trasferimento di un embrione mosaico di un piatto FluoroDish coltura di tessuti con un fondo di vetro di 0,17 millimetri di spessore (Strumenti di precisione World). Eliminare l&#39;acqua in eccesso E3 di lasciare l&#39;embrione appena coperto da una minima quantità di E3. Aggiungere 30-50 microlitri LMP fuso 1,5% per l&#39;embrione. Rapidamente posizione l&#39;occhio o il cervello più vicino possibile al fondo di vetro il più possibile prima che la soluzione di agarosio solidifica. Dopo che la soluzione si raffredda, aggiungere ulteriori soluzione di agarosio per proteggere ulteriormente gli embrioni in posizione. Poi, immergere il blocco solidificato agarosio con 2-3 ml di acqua uovo E3. 2. Confocale Imaging Per la microscopia confocale, un acqua-immersion obiettivo invertito (Olympus, Planapo, 40 X/0.90 WLSM, Giappone) con la profondità attenzione spesso è utilizzato. Una goccia d&#39;acqua si aggiunge sul obiettivo e poi inserire un FluoroDish piatto di coltura tissutale sull&#39;obiettivo. Allo sviluppo dell&#39;immagine, eseguire time-lapse imaging di scattare foto degli embrioni ogni 5 minuti. A seconda della natura degli esperimenti, gli intervalli di tempo tra uno scatto e deve essere regolato. Rappresentante risultati La figura 2 illustra un rappresentante di 24 HPF zebrafish mosaico. Espressione GFP evidenzia le cellule del donatore in verde. Le cellule della retina neuroepiteliale coprono l&#39;intero spessore della parete della retina. Il filmato mostra i movimenti delle cellule del donatore nel cervello ospite a 24 HPF. Cellule preavviso morfologie cambiare nel tempo. <img src="/files/ftp_upload/1924/1924fig1.jpg" alt="Figura 1" /> Figura 1. Forme di punte ad ago del trapianto. A. L&#39;apertura smussato della punta di un ago era libero di detriti dopo ripetuti lavaggi acido fluoridrico. B. Una estremità appuntita è stata generata sulla punta dell&#39;ago. <img src="/files/ftp_upload/1924/1924fig2.jpg" alt="Figura 2" /> Figura 2. Visualizzazione di GFP che esprimono cellule del donatore in embrioni di zebrafish chimerico a 24 HPF. Il pannello di sinistra mostra i segnali GFP nel cervello e della retina. Il pannello centrale è una foto in campo chiaro dell&#39;embrione. Il pannello di destra mostra l&#39;immagine fusa. Movie 1. Live imaging di cellule del donatore GFP positive dimostrato la grandi cambiamenti nella morfologia delle cellule cerebrali neuroepiteliale durante lo sviluppo. La singoli fotogrammi del film sono stati raccolti ogni 5 minuti tra le 24 e 25 HPF. <a href="http://www.jove.com/files/ftp_upload/1924/1924_Wei_Movie1.wmv">Clicca qui per vedere il filmato</a> .

<ol>
	<li>Zou, J., Lathrop, K., Sun, M., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+RPE+maintained+by+Nok+is+essential+for+retinal+epithelial+polarity+and+cellular+patterning+in+zebrafish.">Intact RPE maintained by Nok is essential for retinal epithelial polarity and cellular patterning in zebrafish.</a> Journal of Neuroscience. 28 (50), 13684-13695 (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">A guide for the laboratory use of zebrafish (Danio rerio).</a> THE ZEBRAFISH BOOK. , (2007).</li><li>Ho, R. K., Kane, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+action+of+zebrafish+spt-1+mutation+in+specific+mesodermal+precursors.">Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors.</a> Nature. 348, 728-730 (1990).</li><li>Catalano, A., Raymond, P., Goldman, D., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+dou+yan+Mutation+Causes+Patterning+Defects+and+Extensive+Cell+Death+in+the+Retina.">Zebrafish dou yan Mutation Causes Patterning Defects and Extensive Cell Death in the Retina.</a> Developmental Dynamics. 236 (5), 1295-1236 (2007).</li></ol>

In questo video, si dimostra un metodo per blastomero trapianto di analizzare lo sviluppo della retina e del cervello in zebrafish. Questo protocollo può essere utilizzato anche per analizzare lo sviluppo di altri organi e tessuti, semplicemente rilasciando il blastomeri donatore di posizioni corrispondenti negli embrioni di accoglienza. L&#39;etichettatura con le molecole GFP produce uno sfondo più pulito rispetto etichettatura con colorante fluorescente coniugato Destrano (Ho e Kane 1990). Questo perché i residui d...

E3 uovo acqua: <ul><li> 5 mM NaCl </li><li> 0,17 mM KCl </li><li> 0,33 mM CaCl 2 </li><li> 0,33 mM MgSO 4 </li><li> Blu di metilene 0,1% </li></ul>

transplantation of gfp-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos

Cellule cambiare ampiamente nelle loro posizioni e proprietà durante l&#39;embriogenesi. Questi cambiamenti sono regolati dalle interazioni tra le cellule e il loro ambiente. Embrioni chimerici, che sono composti da cellule di diverso background genetico, sono grandi strumenti per studiare le interazioni cellula-cellula mediata da geni di interesse. La trasparenza embrionale del pesce zebra in primi stadi di sviluppo permette la visualizzazione diretta della morfogenesi dei tessuti e organi a livello cellulare. Qui, dimostriamo un protocollo per generare retine chimerico e cervelli in embrioni di zebrafish e di eseguire l&#39;imaging dal vivo delle cellule del donatore. Il protocollo riguarda la preparazione di aghi trapianto, il trapianto di GFP che esprimono blastomeri donatore per GFP-negativi padroni di casa, e l&#39;esame del comportamento delle cellule del donatore vivo sotto microscopia confocale. Con lievi modifiche, questo protocollo può essere usato anche per studiare lo sviluppo embrionale di altri tessuti e organi in zebrafish. I vantaggi di usare buone pratiche agricole per le cellule del donatore etichetta vengono anche discussi.

Watch this Scientific Journal Video about Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos at JoVE.com

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

Dimostriamo un protocollo per generare embrioni di zebrafish chimerico per vivere comportamento imaging cellulare durante l&#39;embriogenesi.

Il trapianto di GFP che esprimono blastomeri per dell&#39;immagine in tempo reale dello sviluppo della retina e del cervello in embrioni di zebrafish Chimerico

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

transplantation-gfp-expressing-blastomeres-for-live-imaging-retinal

Research

JoVE Journal

Biology

10.6K Views.  University of Pittsburgh. Dimostriamo un protocollo per generare embrioni di zebrafish chimerico per vivere comportamento imaging cellulare durante l'embriogenesi.

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

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Due fotoni assotomia e time-lapse confocale in embrioni di zebrafish vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Ricerca

Biologia

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivere Imaging of the Brain Zebrafish embrionali da microscopia confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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.

Generare embrioni di zebrafish Chimerico da trapianto

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 ...

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.

Immagini dal vivo della motilità cellulare e citoscheletro dei singoli neuroni e cellule della cresta neurale in embrioni di zebrafish

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

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

Sviluppo di imaging automatizzati e analisi chimiche per gli schermi zebrafish.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Due-Photon-Based fotoattivazione in Live embrioni di zebrafish

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Dechorionation di embrioni Medaka e trapianto di cellule per la generazione di chimere

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glicani Imaging in embrioni di zebrafish con marcatura metabolica e Chimica Clicca Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.