Name: Neural Explant Cultures from Xenopus laevis (Video) | JoVE Journal
Uploaded: 2012-10-15T12:00:00.000+00:00
Duration: 10 min 5 s
Description: Learn neural explant culture from Xenopus laevis embryos for imaging growth cone cytoskeletal dynamics. This method enables high-resolution fluorescence microscopy of protein localization in developing neurons.

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5 minutes

The authors would like to thank Bob Freeman for training and the Kirschner lab for the use of the frog facility, and members of the Van Vactor lab for support. We thank the Nikon Imaging Center at Harvard Medical School for assistance with light microscopy for the images in Figure 1. This work was funded by the following: NRSA NIH fellowship and NIH K99 fellowship to L.A.L, Basic Science Partnership funding (<a href="https://bsp.med.harvard.edu/" target="_blank">https://bsp.med.harvard.edu/</a>) to AEF, and NIH RO1 NS035909 to D.V.V.

Note: We describe the steps in the first two sections only in brief, as excellent protocols with detailed information have been published elsewhere that focus more specifically on these steps (e.g.8-12). Additionally, the general protocol of working with Xenopus spinal neurons in live cell culture has been previously published in a detailed methods article8. We highly recommend reviewing that article as a complement to this video, although here we do provide enough information to successfully perform and troubleshoot the neural tube dissection and plating protocol in step 3.
1. In Vitro Fertilization of Xenopus Eggs
<ol>
 <li>Obtain eggs from female frogs that were injected with chorionic gonadotropin (400 units/frog) 12-18 hr before the egg collection. Collect eggs in 1X Marc's Modified Ringer solution (MMR) (0.1 M NaCl, 2.0 mM KCl, 1.0 mM MgSO4, 2.0 mM CaCl2, 5.0 mM HEPES, pH 7.49).</li>
 <li>Fertilize eggs in vitro with minced testes, as described previously9.</li>
 <li>After at least 20 min, remove embryo jelly coat by incubating embryos in 2% cysteine in 1X MMR (brought to pH 7.8 with NaOH) for 3-5 min. Wash with 0.1X MMR (or 0.1X MBS (Modified Barth's Saline, 1X recipe: 88 mM NaCl, 1 mM KCl, 0.7 mM CaCl2, 1 mM MgSO4, 2.5 mM NaHCO3, 5 mM HEPES, pH 7.8)) 3-5 times, and keep embryos at room temperature until injections, or if desired, place embryos at 14-18 °C to slow development.</li>
</ol>
2. Microinjection of RNA
Note: while we utilize RNA injection here, these techniques are not limited to RNA, and DNA, proteins, or modified nucleic acids for gene manipulation can also be used and have been described previously12.
<ol>
 <li>Prior to experiment, RNAs for labeling cytoskeletal or other structures are transcribed from linearized DNA templates using the mMessage mMachine kit (Ambion). mRNAs require a 5' cap and a 3' polyadenylation tail to promote translation and prevent degradation in embryos. pCS2+ is a commonly-used vector for this purpose, and the transcription kit includes a cap analog during the synthesis reaction. We re-suspend transcribed mRNA in nuclease-free ddH20, at a stock concentration between 500 and 2,000 ng/μl, followed by prompt storage at -80 °C. Prior to injection, dilute RNA in ddH20 to the appropriate concentration for injection. As only a small volume will be injected into embryos, resuspending in buffer is not necessary. Our final injection concentration is typically 50-200 pg/nl, depending on the construct, and we usually will inject up to 4 nl per embryo, although many labs traditionally inject up to 10 nl, which is approximately 1% of the total volume of an embryo. RNA can be toxic at high doses, and the dose per embryo generally ranges from 10 pg to 1 ng, although up to 5 ng can be tolerated depending on the particular gene and purity. However, for imaging protein localization, the lowest possible level should be injected to avoid over-expression artifacts. RNA for EB1-GFP in this protocol is used at final amount of 250 pg per embryo.</li>
 <li>Prepare injection needles by pulling capillary with a needle puller9, to a tip diameter of ~ 0.2 μm. With a Sutter P-87 puller, we use the following settings: heat 644, pull 125, velocity 70, time 250, but these parameters differ depending on machine and must be re-adjusted after changing the filament (we currently use a 2.5 mm square box filament that is 2.5 mm wide). Under a microscope, break needle tip with forceps at an angle to generate a quill-like shape. There are other methods for breaking and filling injection needles (for example11,12 ), but we fill the needle with RNA by placing a small drop (0.5 - 1 μl) to the back end of the needle. For microinjecting into embryos, there are a number of commercially available injection systems, two of the most common being the Pico-injector (Medical Systems), and the Nanoject (Drummond Scientific) (see11 for more information). We use the Medical Systems PLI-100 Pico-Injector, which uses compressed gas for a digitally set period of time in order to deliver consistent nanoliter volumes. Injection volume must be calibrated for each new micropipette using a stage micrometer, and injection time should be adjusted to achieve desired amount of injected RNA.</li>
 <li>Place fertilized embryos at the 1- to 4-cell stage into a plastic dish containing 5% Ficoll in 0.1X MMR. Holding the embryo in place with forceps, or placing in a holding platform (a plastic mesh, with a ~1 mm grid, adhered to the bottom of the dish with plasticine or dental wax), inject desired volume into animal blastomeres (see step 2.1 for details regarding injection volumes). Distribute in several locations throughout embryo, at least one injection into each of the animal blastomeres, to obtain more uniform distribution (for example, for a 4-cell stage embryo, we typically inject 1-2 times in each blastomere, whereas in a 2-cell stage embryo, we inject 2-4 times in each blastomere). A benefit of waiting until the 2-4 cell stage is that you ensure the embryo has begun to cleave normally. However, if time is of the essence, you can start at the 1-cell stage, with little difference in the end result other than more embryos needing to be injected. Older stage embryos can be used as well, and this is particularly useful if specific cell types are targeted for injection, but as we generally prefer more broad expression, we inject in younger embryos to reduce the need for more numerous injections.</li>
 <li>Transfer injected embryos to a plastic dish containing 0.1X MMR and allow them to develop until stage 20-2313. Incubate embryos at 14 to 22 °C depending upon desired speed of development. (For dissecting neural tubes the following day, use ~ 22 °C. For the day after, use ~ 14 °C.)</li>
</ol>
3. Neural Tube Dissection and Plating
<ol>
 <li>Prior to performing the dissections, prepare the culture dishes14 . First, coat coverslips with enough solution of 200 μg/ml poly-lysine in PBS to pool over the surface of the coverslip, of either Mattek or Lab-Tek culture dishes, and incubate for one hour (alternatively, one could use glass coverslips sitting loose in a Petri dish, for later placement on glass slides for imaging and immunocytochemistry). Aspirate and wash with an excess of PBS 3 times, and let dry. Then coat dishes with 10 μg/ml laminin in PBS (we usually use 500 μl per coverslip, enough to pool over the surface) for one hour at 37 degrees. Aspirate laminin solution and wash 3 times with an excess of PBS, being careful not to let laminin-coated surfaces become exposed to the air interface. Replace PBS with culture media (50% Ringer's, 49% L-15 media, 1% Fetal Bovine Serum, plus 50 μg/ml penicillin/streptomycin and gentamycin, pH 7.4 and filter sterilized). (Ringer's Media, 115 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 10 mM Hepes pH 7.4, 0.5 mM EDTA). While our protocol uses this media, it should be noted that this is an enriched media common for older Xenopus retinal ganglion cultures, where neurons have reduced energy reserved. Young spinal cord cultures will survive and grow for more than 24 hr in pure Ringers's Media without additional growth factors, and this is indeed one of the benefits of using this system. Optional: add NT3 and BDNF (to final concentrations of 25 ng/μl each) to culture media to increase axon outgrowth.</li>
 <li>Because of the variability in the expression of injected mRNA, embryos may exhibit a mosaic of fluorescence. Prior to performing dissections, screen embryos for the presence of fluorescence, to identify those embryos which express the fluorescent fusion protein in the neural tube. Place fluorescent embryos at stage 20-23 into an agarose-coated plastic dish filled with Steinberg's media (58 mM NaCl, 0.67 mM KCl, 0.44 mM Ca(NO3)2, 1.3 mM MgSO4, 4.6 mM Tris, pH to 7.8 then autoclaved)5. To make the agarose-coated dish, coat bottom of plastic dish with melted 1% agarose in 0.1X MMR and let harden. This provides a soft surface so that damage does not occur to the fine forceps and tungsten needles used during the subsequent dissection steps. Dissection of embryos beyond stage 23 is possible, but it is more challenging as tissues adhere more closely to each other and thus require longer treatment with collagenase.</li>
 <li>Under a dissecting scope, remove the vitelline membrane with fine forceps, and then isolate the entire dorsal portion of the embryo, by making a series of incisions. While one forceps hold the embryo in place, use the second to make an incision on the side of the embryo to expose the hollow interior. Then, use both forceps to pinch along the tissue between the dorsal and ventral halves of the embryo, thereby cutting the embryo in half to isolate the dorsal portion containing the neural tube.</li>
 <li>Place the dorsal explant in an Eppendorf tube with 2 mg/ml collagenase in Steinberg's media for 15-20 min on a rotator, to loosen tissues, and then pipette dorsal explant into a plastic agarose-coated dish with fresh Steinberg's solution. Alternatively, the explant can be placed in a Petri dish containing the collagenase solution for 15 min, where the entire dissection could then be carried out.</li>
 <li>Using a pair of forceps, gently dissect the neural tube from the dorsal epidermis and the ventral notochord. Slide the tip of the forceps between the epidermis and underlying tissue and slowly pull back the epidermis, revealing the neural tube beneath. Then, use one forceps to hold the tissue and another to slide the tip between the neural tube and notochord. Finally, use forceps to remove the somites on either side of the tube.</li>
 <li>Transfer the neural tube to an agarose-coated dish filled with culture media, as described in step 3.1.</li>
 <li>After collection of several neural tubes, cut them into numerous pieces (approximately 20, if the whole tube is isolated) using sharpened tungsten needles or forceps, and then plate the pieces in culture dishes filled with medium (prepared in step 3.1), spreading out the explants evenly in rows.</li>
 <li>After plating, do not move the dishes because this would disturb the attaching cells. Incubate the plated neural tube explants around 20-22 °C. We typically leave the dish of cells on the bench at room temperature overnight. One of the benefits of Xenopus laevis neurons is that a special incubator for cell culture that regulates CO2 levels or temperature is not needed. Neurites and growth cones can be observed and imaged at room temperature 12-24 hr after plating, although outgrowth can be accelerated with the addition of growth factors. In general, expression of a RNA or plasmid product will result in variable expression between cells, and thus there may be a range of fluorescence expression levels between growth cones. We have observed RNA expression of fluorescent proteins to persist beyond 48 hr after plating, although this depends on the particular construct.</li>
</ol>
4. Representative Results
After following the protocol, which is summarized in Figure 1A, healthy, correctly-dissected and cultured neural explants will send out numerous axons or neurites in every direction on laminin/poly-lysine substrate, shown in Figure 1B. The growth cones at the tips of the axons can be imaged by either DIC optics, as seen in Figure 1C, to image overall motility dynamics, or high-resolution fluorescence microscopy to examine the localization of fluorescently-tagged cytoskeletal proteins, for example, EB1-GFP is shown in Figure 1D.
<img alt="Frog neural regeneration process, mRNA injection, diagram and microscopy images of cell growth." src="/files/ftp_upload/4232/4232fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Summary of experimental protocol and expected outcome. A) Protocol flow-chart. See movie for details regarding dissection on day 3. The entire neural tube should be cut into about 20 equally-sized pieces and spread out evenly in rows on the coverslip. In this cartoon, scissors are depicted to represent cutting the tissue with fine forceps. CG chorionic gonadotropin B) DIC image of axons or neurites growing out of explant (explant at top left corner). C) Higher magnification image of growth cone at tip of a growing axon. D) Fluorescence microscopy image of EB1-GFP in growth cone. Scale bar 10 μm. <a href="https://www.jove.com/files/ftp_upload/4232/4232fig1large.jpg" target="_blank"> Click here to view larger figure</a>.

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<li>Lavery, D. L., Hoppler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gain-of-function+and+loss-of-function+strategies+in+Xenopus%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Gain-of-function and loss-of-function strategies in Xenopus.</a> Methods Mol. Biol. 469, 401-415 (2008).</li>
<li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNieuwkoop%2C+PD+%22Normal+Table+of+Xenopus+Laevis+%28Daudin%29%22">Normal Table of Xenopus Laevis (Daudin)</a>. , Garland Publishing Inc. New York. (1994).</li>
<li>Guirland, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+cAMP+signaling+through+G-protein-coupled+receptors+mediates+growth+cone+attraction+induced+by+pituitary+adenylate+cyclase-activating+polypeptide%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Direct cAMP signaling through G-protein-coupled receptors mediates growth cone attraction induced by pituitary adenylate cyclase-activating polypeptide.</a> J. Neurosci. 23, 2274-2283 (2003).</li>
<li>Spitzer, N. C., Lamborghini, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+the+action+potential+mechanism+of+amphibian+neurons+isolated+in+culture%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">The development of the action potential mechanism of amphibian neurons isolated in culture.</a> Proc. Natl. Acad. Sci. U.S.A. 73, 1641-1645 (1976).</li>
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<li>Weinl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+turning+of+Xenopus+retinal+axons+induced+by+ephrin-A5%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">On the turning of Xenopus retinal axons induced by ephrin-A5.</a> Development. 130, 1635-1643 (2003).</li>
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No conflicts of interest declared.

Xenopus laevis neural explants send out neurites in a very robust manner by 24 hr after plating on the laminin/poly-lysine substrate if conditions are appropriate. With this substrate, growth cones are highly motile and can achieve axon lengths of up to 1 mm, extending in all directions outward from the explant, although typical lengths are 100 &mu;m or more. If neurites do not grow out, there are a limited number of reasons for this to be the case. One possibility is that the neural explants did not properly a...

<table><tbody><tr><td>Chorionic Gonadotropin</td><td>Argent Labs</td><td>C-HCG-ON-10</td><td></td></tr><tr><td>Cysteine</td><td>Sigma-Aldrich</td><td>52-90-4</td><td></td></tr><tr><td>mMessage mMachine kit</td><td>Ambion</td><td>AM1340</td><td></td></tr><tr><td>Capillary Borosil Needles 1.2 mm (OD) x 0.9 mm (ID)</td><td>FHC, Inc.</td><td>30-31-0</td><td></td></tr><tr><td>Ficoll</td><td>Sigma-Aldrich</td><td>F2637</td><td></td></tr><tr><td>Dumont #5 Biologie Inox Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Collagenase</td><td>Sigma-Aldrich</td><td>9001-12-1</td><td></td></tr><tr><td>Mattek dishes</td><td>Mat Tek Corporation</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>L-15</td><td>Invitrogen</td><td>21083-027</td><td></td></tr><tr><td>Poly-l-lysine</td><td>Sigma-Aldrich</td><td>P-1399</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>NT3</td><td>Sigma-Aldrich</td><td>N1905</td><td></td></tr><tr><td>BDNF</td><td>Sigma-Aldrich</td><td>B3795</td><td></td></tr></tbody></table>

neural explant cultures from xenopus laevis

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

The aim of this procedure is to culture Xenopus Lavis growth cones for subsequent high resolution image analysis. This is accomplished by first injecting female frogs with hormone to stimulate egg production. The second step is to collect and fertilize the eggs, and then inject them with mRNA or other constructs of interest.The next day, the embryos are dissected and the neural tubes are isolated, cut up and plated on cover slips. The final step is to image the outgrowing axons and growth cones. Ultimately, high resolution fluorescence microscopy can be used to show changes in protein localization in growth cones over time.Visual demonstration of this method is critical as the neural tube dissections can be difficult to learn from reading a method section without observing the technique firsthand. Obtain eggs from female frogs previously injected with 400 units of chorionic gonadotropin 12 to 14 hours beforehand. Collect the eggs into one X marks modified ringer solution.Then fertilize the collected X in vitro with mince testes as previously described, and add 0.1 XMMR after at least 20 minutes. Remove media and incubate the embryos in 2%cysteine in one XMMR at pH 7.8 for three to five minutes to remove the embryo jelly coat. Then pour the embryos into a beaker.Next, wash the embryos with 0.1 XMMR or 0.1 x modified bath saline three to five times after the final wash. Keep the embryos in 0.1 XMMR at room temperature until injection or if desired. Place the embryos at 14 to 18 degrees Celsius to slow development prior to injection.Dilute previously prepared capped RNA to a final concentration of 50 to 200 picograms banana liter with double distilled water, and then store on ice until micro injection. Next, prepare the injection needle by pulling a bo silicate capillary using a suter puller or similar instrument. Then under a microscope, use forceps to break the needle tip at an angle to generate a quill like shape and backfill the needle with 0.5 to one microliter of the diluted RNA solution.Mount the needle on the micro manipulator A medical system's PLI 100 PICO injector is used here. Now, place the fertilized embryos at the one to four cell stage into a plastic dish containing 5%fol in 0.1 XMMR. Calibrate the injection volume for each new micro pipette using a radical or stage micrometer.Then set the pico injector to deliver the desired amount of RNA for each injection. Here a one nanoliter injection volume is used. Hold the embryos with forceps or if preferred place on a holding platform and inject the desired volume into the animal blasters.Distribute the injections in several locations throughout the embryo to obtain a uniform distribution of RNA. One to two injections are made in each blaster for a four cell stage embryo, whereas two to four injections are used for a two cell stage embryo. After the injections, transfer the injected embryos to a plastic dish containing 0.1 XMMR and allow them to develop until stage 20 to 23.Depending on the desired speed of development, the embryos are incubated at a temperature of 14 to 22 degrees Celsius coat PLL treated culture dishes with 10 micrograms per milliliter laminin in PBS and place the plates at 37 degrees Celsius for one hour. Once the incubation time has elapsed, wash the plates three times with PBS being careful not to let the laminin coated surface become exposed to the air after the final wash. Replace the PBS with culture media.Finally prepare three agros coated dishes by coating the bottom of a cultured dish with 1%aros in 0.1 XMMR and allowing it to harden. Once hardened, fill one dish with steinberg's media variability in the expression of injected mRNA may cause embryos to exhibit a mosaic of fluorescence prior to performing dissections. Screen embryos for the presence of fluorescence and identify those embryos expressing the fluorescent fusion protein in the neural tube.Then transfer the fluorescent embryos at stage 20 to 23 into the agros coated plastic dish containing Steinberg's Media. Place the dish under a dissecting microscope and use fine forceps to remove the vitelline membrane. Then, while one forceps hold the embryo in place, use the second to make an incision on the side of the embryo and expose the hollow interior.Next, use both sets of forceps to pinch along the tissue between the dorsal and ventral halves of the embryo to cut the embryo in half and isolate the dorsal portion containing the neural tube. Place the dorsal X explan in a micro centrifuge tube containing two milligrams per milliliter, collagenase in Steinberg's media and place on a rotator for 15 to 20 minutes. Then transfer the dorsal explan to an agro coated dish filled with fresh steinberg's media.While working under the microscope, dissect the neural tube from the dorsal epidermis and ventral Noor by slipping the tip of the forceps between the epidermis and underlying tissue, and slowly pulling back the epidermis. Then use one forceps to hold the tissue and another to remove tissue surrounding the neural tube. Next, transfer the dissected neural tube to an agros coated dish filled with fresh culture media.Once several neural tubes have been collected, you sharpen tungsten needles or forceps to cut each tube into around 20 pieces. Then plate the pieces on the laminin coated culture dishes, spreading the eggplants evenly in rows. After plating the neural tubes, the dishes should not be moved as this will disturb the attachment of the cells.Incubate the plated neural tube explan at 20 to 22 degrees Celsius. A cell culture incubator is not required for the culture of XUS lavis neurons 12 to 24 hours after plating image neurons and growth cones at room temperature. Bear in mind that variable expression of RNA may result in a range of fluorescence levels between growth cones.This DIC image shows axons and neurites growing outta the X explan at the top left corner of the field of view. The scale bar in this image and the following images represents 10 microns. This higher magnification movie shows the growth cone at the tip of a growing axon.Finally, this fluorescence microscopy movie shows expression of EB one GFP in a growth cone. While here, we provide the example of imaging, the plus tip fusion protein EB one GFP. This neural explant method can be applied to any number of proteins to elucidate their behaviors within the growth cone during neurite outgrowth.

neural-explant-cultures-from-xenopus-laevis

Research

JoVE Journal

Neuroscience

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Facial Transplants in Xenopus laevis Embryos

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

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

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

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

Learn neural explant culture from Xenopus laevis embryos for imaging growth cone cytoskeletal dynamics. This method enables high-resolution fluorescence microscopy of protein localization in developing neurons.

Neural Explant Cultures from Xenopus laevis

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