We thank T. Fritsch, A. Wolf-Asseburg, I. Linde and S.-M. Tokarski for excellent animal care and technical support. We are grateful to all members of the K&#246;ster lab for intense and helpful discussions. We gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (KO 1949/5-1) and the Federal State of Lower Saxony, Nieders&#228;chsisches Vorab (VWZN2889).

<ol>
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The authors have nothing to disclose.

Here, we present two different protocols to culture primary cells from either 2 dpf zebrafish embryos or adult zebrafish brain.
The preparation of primary cell cultures from 2 dpf zebrafish is relatively easy to perform for anyone with experience in basic cell culture techniques. However, to obtain good and reproducible results, a sufficient number of embryos as starting material is crucial (100 is the minimum). During the raising of the embryos, all possible sources of contamination must be a...

The zebrafish (Danio rerio, D. rerio) is a popular model vertebrate for numerous fields of basic and biomedical research1. Zebrafish embryos develop rapidly ex utero, are transparent, and fit under a microscope, thus providing excellent prerequisites for studying vertebrate development in a living organism. Due to the genetic tractability of zebrafish2, many stable transgenic reporter lines with cell type-specific expression of various fluorescent markers have been established allowing for the observation of specific cell populations. The zebrafish community offers a broad variety of so-called Gal4-driver lines which carry a transgene expressing the synthetic Kal4TA4 (or the KalTA3-equivalent GalFF) gene with the Gal4-DNA-binding domain of yeast fused to viral transcriptional activation domains under the control of cell type-specific enhancers. These driver lines are crossed to effector lines which carry transgenes consisting of a defined upstream activating sequence (UAS) fused to a reporter gene. The Kal4TA4 protein binds to the UAS element, thus activating the cell type-selective expression of the reporter gene3,4. This approach allows for highly diverse combinatorial studies of almost all available enhancer and reporter elements in double-transgenic animals.
However, in-depth live imaging with focus on individual cells or their subcellular contents is limited in a whole and constantly changing embryo. To address specific cell biological questions with highest resolution, the use of cell cultures is often preferable. Some cell lines of zebrafish exist, but they are considered as heavily selected5,6,7 and their propagation is often time-consuming. Furthermore, all the available cell lines are fibroblast derived, limiting experiments using cell culture to one type of cells. Therefore, we established both an efficient and easy-to-use protocol to prepare primary cells directly from zebrafish embryos and adult zebrafish brain, together with approaches to increase the longevity of the culture and to broaden the diversity of cultivated cell types. In addition, we present a procedure to transfect embryonic primary cells with expression constructs for fluorescent organelle markers. Thus, cellular morphologies and subcellular structures can be analyzed with high spatial and temporal resolution in distinct cell types which retain their key features.

<table><tbody><tr><td>&lt;u&gt;Fish lines&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>AB (wild-type)</td><td></td><td></td><td>established by Streisinger and colleagues, available from the Zebrafish International Resource Center (ZIRC)</td></tr><tr><td>Tg(&lt;em&gt;ptf1a&lt;/em&gt;:eGFP)jh1</td><td></td><td></td><td>stable transgenic line in which the enhancer of the zebrafish gene&lt;em&gt; ptf1a &lt;/em&gt;drives expression of the fluorescent protein EGFP (Parsons &lt;em&gt;et al&lt;/em&gt;., 2007)</td></tr><tr><td>Tg(XITubb:DsRed)zf148</td><td></td><td></td><td>stable transgenic line in which the&lt;em&gt; Xenopus &lt;/em&gt;neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed&amp;nbsp; (Peri and N&amp;uuml;sslein-Volhard, 2008)</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>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Equipment&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td></td><td>model 5804 R</td></tr><tr><td>ChemiDoc MP imaging system</td><td>BioRad</td><td></td><td>model XRS+, used to acquire black-and-white images of Petri dishes containing 1 da embryos</td></tr><tr><td>confocal laser scanning microscope</td><td>Leica microsystems</td><td></td><td>model SP8, equipped with 28 &amp;deg;C temperature box and a 63X objective</td></tr><tr><td>epifluorescent microscope</td><td>Leica microsystems</td><td></td><td>model DM5500B, equipped with 28 &amp;deg;C temperature box and a 40X objective</td></tr><tr><td>Gene Pulser Xcell with capacitance extender</td><td>BioRad</td><td>1652661</td><td>electroporation device</td></tr><tr><td>Horizontal shaker</td><td>GFL</td><td></td><td>model 3011</td></tr><tr><td>incubator for cell culture (28 &amp;deg;C)</td><td>Memmert</td><td></td><td>model incubator I</td></tr><tr><td>incubator for embryos (28 &amp;deg;C)</td><td>Heraeus</td><td></td><td>type B6120</td></tr><tr><td>light microscope</td><td>Zeiss</td><td></td><td>model TELAVAL 31</td></tr><tr><td>micro pipettes</td><td>Gilson</td><td></td><td></td></tr><tr><td>sterile work bench</td><td>Bio Base</td><td></td><td>with laminar flow and UV light</td></tr><tr><td>tweezers</td><td>Dumont</td><td></td><td>Style 5, Inox</td></tr><tr><td>vertical tube rotator</td><td>Labinco B.V.</td><td></td><td>model LD-79</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>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Software&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Image Lab Software</td><td>BioRad</td><td></td><td>for the ChemiDoc MP imaging system from BioRad</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td>used for counting 1 dpf embryos by applying the Count particles-tool to the respective black-and-white images; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/. (1997-2016).</td></tr><tr><td>LAS X</td><td>Leica Microsystems</td><td></td><td>for both confocal and epifluorescent microscopes from Leica Microsystems</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>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Plasmids&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>pCS-DCX-tdTomato</td><td>K&amp;ouml;ster Lab</td><td># 1599</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.&lt;/em&gt;, 1994)</td></tr><tr><td>pCS-eGFP</td><td>K&amp;ouml;ster Lab</td><td># 7</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-H2B-mseCFP</td><td>K&amp;ouml;ster Lab</td><td># 2379</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-mClover</td><td>K&amp;ouml;ster Lab</td><td># 3865</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.&lt;/em&gt;, 1994)</td></tr><tr><td>pCS-MitoTag-YFP</td><td>K&amp;ouml;ster Lab</td><td># 2199</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-ss-RFP-KDEL</td><td>K&amp;ouml;ster Lab</td><td># 4330</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-VAMP1-mCitrine</td><td>K&amp;ouml;ster Lab</td><td># 2291</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.,&lt;/em&gt; 1994)</td></tr><tr><td>pSK-UAS:mCherry</td><td>K&amp;ouml;ster Lab</td><td># 1062</td><td>based on the pBluescript-backbone of Stratagene</td></tr><tr><td>Plasmid numbers refer to the database entries of the K&amp;ouml;ster lab. Plasmids are available upon request.</td><td></td><td></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>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Plastic and glass ware&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>BD Falcon Cell Strainer (40 &amp;micro;m)</td><td>FALCON</td><td>REF 352340</td><td>distributed by BD Bioscience, used as &amp;ldquo;landing net&amp;rdquo; to dip deyolked embryos into ethanol and to transfer them quickly to fresh cell culture medium</td></tr><tr><td>1.5 mL reaction tubes</td><td>Sarstedt</td><td>72690550</td><td></td></tr><tr><td>24-well plate</td><td>Sarstedt</td><td>83.3922</td><td></td></tr><tr><td>50 mL falconic tube</td><td>Sarstedt</td><td>62.547.004</td><td></td></tr><tr><td>96-well plate</td><td>Sarstedt</td><td>83.3924.005</td><td></td></tr><tr><td>EasyStrainer (40 &amp;micro;m)</td><td>Greiner Bio-One</td><td>542 040</td><td>with venting slots; used to filter cells after collagenase-mediated dissociation</td></tr><tr><td>electroporation cuvette (0.4 cm)</td><td>Kisker</td><td>4905022</td><td></td></tr><tr><td>glass coverslips</td><td>Heinz Herenz Medizinalbedarf GmbH</td><td>1051201</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Fisher Scientific (Menzel Gl&amp;auml;ser)</td><td>631-0845</td><td></td></tr><tr><td>Neubauer chamber</td><td>Henneberg-Sander GmbH</td><td>9020-01</td><td></td></tr><tr><td>Pasteur pipettes (plastic; 3 mL)</td><td>A. Hartenstein</td><td>PP05</td><td></td></tr><tr><td>Petri dishes (plastic; diameter 10 cm)</td><td>Sarstedt</td><td>821473</td><td>for zebrafish embryos</td></tr><tr><td>pipette tips</td><td>Sarstedt</td><td>Blue (1000 &amp;micro;l): 70762; Yellow (200 &amp;micro;l): 70760002; White (10 &amp;micro;L): 701116</td><td></td></tr><tr><td>sterile cell culture dishes (plastic; diameter 3 cm)</td><td>TPP Techno Plastic Products AG</td><td>93040</td><td></td></tr><tr><td>sterile cell culture dishes (plastic; diameter 6 cm)</td><td>Sarstedt</td><td>72690550</td><td></td></tr><tr><td>sterile Petri dishes (plastic; diameter 10 cm)</td><td>Sarstedt</td><td>83.3902</td><td>for brain dissection</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>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Chemicals and Reagents&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Roth</td><td>0601.1</td><td></td></tr><tr><td>4 % paraformaldehyde in 1x PBS</td><td>Sigma-Aldrich</td><td>16005</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>calcium nitrate tetrahydrate</td><td>Sigma-Aldrich</td><td>C1396</td><td></td></tr><tr><td>ethanol p.a. 100%</td><td>Sigma-Aldrich</td><td>46139</td><td></td></tr><tr><td>goat &amp;alpha;-mouse IgG (Fc specific) FITC conjugated</td><td>Thermo Fisher Scientific</td><td>31547</td><td></td></tr><tr><td>HEPES</td><td>Roth</td><td>9105.4</td><td></td></tr><tr><td>high vacuum grease</td><td>DOW CORNING</td><td>3826-50</td><td>silicon grease used for self-made glass bottom dishes</td></tr><tr><td>magnesium sulfate heptahydrate</td><td>Merck</td><td>105886</td><td></td></tr><tr><td>methylene blue</td><td>Serva</td><td>29198.01</td><td></td></tr><tr><td>Monoclonal Anti-Tubulin, Acetylated antibody</td><td>Sigma-Aldrich</td><td>T6793</td><td></td></tr><tr><td>Aqua-Poly/Mount (mounting medium)</td><td>Polyscience</td><td>18606</td><td></td></tr><tr><td>poly-L-lysine</td><td>Biochrom</td><td>L 7240</td><td></td></tr><tr><td>potasssion chloride</td><td>Merck</td><td>104938</td><td></td></tr><tr><td>Skim milk</td><td>Roth</td><td>68514-61-4</td><td></td></tr><tr><td>Texas Red-X Phalloidin</td><td>Thermo Fisher Scientific</td><td>T7471</td><td></td></tr><tr><td>Tricaine</td><td>Sigma-Aldrich</td><td>E10521</td><td>Synonym: Ethyl 3-aminobenzoate methanesulfonate</td></tr><tr><td>Triton X-100</td><td>BioRad</td><td>1610407</td><td></td></tr><tr><td>Trypan Blue</td><td>Gibco by Life Technologies</td><td>15250061</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>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Enzymes&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>collagenase (Type 2)</td><td>Thermo Fisher Scientific</td><td>17101015</td><td>dissolve powder in cell culture medium (8 mg/mL) and sterile-filter the solution, store aliquots at -20 &amp;deg;C</td></tr><tr><td>pronase (from Streptomyces griseus)</td><td>Roche</td><td>11459643001</td><td>distributed by Sigma-Aldrich, dissolve in 30% Danieau (10 mg/mL) and store aliquots at -20 &amp;deg;C</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>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Medium and solutions for cell culture&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>1x PBS (Dulbecco&#039;s Phosphate Buffered Saline)</td><td>Gibco by Life Technologies</td><td>14190-169</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-independent medium</td><td>Gibco by Life Technologies</td><td>18045054</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>filtrated bovine serum (FBS)</td><td>PAN-Biotech</td><td></td><td>individual batch</td></tr><tr><td>glutamine 100x</td><td>Gibco by Life Technologies</td><td>25030081</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>Leibovitz&#039;s L-15 medium</td><td>Gibco by Life Technologies</td><td>11415049</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>PenStrep (10,000 U/mL)</td><td>Gibco by Life Technologies</td><td>15140148</td><td>distributed by Thermo Fisher Scientific</td></tr></tbody></table>

culture and transfection of zebrafish primary cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

Figure 1G shows a transmitted light image of a typical culture derived from wild type embryos with striated myocytes and clusters of neuron-like cells being most abundant. To identify certain cell types more easily, a transgenic line with cell type-specific expression of a fluorescent protein can be used (Figure 1H).
Transfection of a pCS2+-based plasmid<sup class="xref...

Watch this Scientific Journal Video about Culture and Transfection of Zebrafish Primary Cells at JoVE.com

Culture and Transfection of Zebrafish Primary Cells

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

culture-and-transfection-of-zebrafish-primary-cells

Research

JoVE Journal

Developmental Biology

12.4K Views.  Braunschweig University of Technology. We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Video: Culture and Transfection of Zebrafish Primary Cells

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Cancer Research

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a peri-vascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development. 
Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

Here, we present a protocol to establish versatile orthotopic and ectopic zebrafish xenograft models for ocular melanoma to assess the growth kinetics of the primary tumor, dissemination, extravasation and distant, peri-vascular metastasis formation and the effect of chemical inhibition thereon.

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

Environment

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

Robust Zebrafish Cell Transplantation for Regeneration and Development Research

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.

Author Spotlight: Mapping Cellular Connectivity in the Zebrafish Nervous System During Development and Regeneration

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.

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