We thank the AECOM Zebrafish Core Facility and Cincinnati Children&#39;s Veterinary Services for fish maintenance, the Cincinnati Children&#39;s Imaging Core for technical assistance, Didar Saparov for assistance with video production and Hannah Seawall for editing the manuscript. Research reported in this publication was supported by the National Institute&#160;of General Medical Sciences of the National Institutes of Health under Award Number R35GM140805 to E.M.&#214;. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This protocol involves use of live vertebrate embryos younger than 1 day post-fertilization. All the animal experiments were performed under the ethical guidelines of Cincinnati Children&#39;s Hospital Medical Center; animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (Protocol # 2017-0048).
1. Embryo collection
<ol>
	<li>Set up pairs of zebrafish in crossing tanks the night before the embryo collection day. For precise staging control of embryo de...

<ol>
	<li>Assheton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Growth in length: Embryological Essays. , (1916).</li><li>Gomez, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+segment+number+in+vertebrate+embryos.">Control of segment number in vertebrate embryos.</a> Nature. 454 (7202), 335-339 (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> The Zebrafish Book: a guide for the laboratory use of zebrafish (Danio rerio), 3rd edition. , (1995).</li><li>Icha, J., Weber, M., Waters, J. C., Norden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxicity+in+live+fluorescence+microscopy,+and+how+to+avoid+it.">Phototoxicity in live fluorescence microscopy, and how to avoid it.</a> BioEssays. 39 (1700003), (2017).</li><li>Simsek, M. F., Ozbudak, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+fold+change+of+Fgf+signaling+encodes+positional+information+for+segmental+determination+in+zebrafish.">Spatial fold change of Fgf signaling encodes positional information for segmental determination in zebrafish.</a> Cell Reports. 24 (1), 66-78 (2018).</li><li>Dubrulle, J., Pourqui&#233;, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fgf8+mRNA+decay+establishes+a+gradient+that+couples+axial+elongation+to+patterning+in+the+vertebrate+embryo.">fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo.</a> Nature. 427 (6973), 419-422 (2004).</li><li>Diez del Corral, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+FGF+and+Retinoid+Pathways+Control+Ventral+Neural+Pattern,+Neuronal+Differentiation,+and+Segmentation+during+Body+Axis+Extension.">Opposing FGF and Retinoid Pathways Control Ventral Neural Pattern, Neuronal Differentiation, and Segmentation during Body Axis Extension.</a> Neuron. 40 (1), 65-79 (2003).</li><li>Stern, H. M., Hauschka, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tube+and+notochord+promote+in+vitro+myogenesis+in+single+somite+explants.">Neural tube and notochord promote in vitro myogenesis in single somite explants.</a> Developmental Biology. 167 (1), 87-103 (1995).</li><li>Langenberg, T., Brand, M., Cooper, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+brain+development+and+organogenesis+in+zebrafish+using+immobilized+embryonic+explants.">Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants.</a> Developmental Dynamics. 228 (3), 464-474 (2003).</li><li>Picker, A., Roellig, D., Pourqui&#233;, O., Oates, A. C., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+micromanipulation+in+zebrafish+embryos.">Tissue micromanipulation in zebrafish embryos.</a> Methods in molecular biology. 546 (11), 153-172 (2009).</li><li>Manning, A. J., Kimelman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tbx16+and+Msgn1+are+required+to+establish+directional+cell+migration+of+zebrafish+mesodermal+progenitors.">Tbx16 and Msgn1 are required to establish directional cell migration of zebrafish mesodermal progenitors.</a> Developmental Biology. 406 (2), 172-185 (2015).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203 (3), 253-310 (1995).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139, 3242-3247 (2012).</li></ol>

This article presents a detailed protocol of a tissue culture explant technique we developed and used recently5 for zebrafish embryos. Our technique builds on the previous explant methods in chick8 and zebrafish9,10,11 model organisms. Tail explants prepared with this protocol can survive as long as &gt;12 h in a simple slide chamber, continuing to elongate its major body axis and ...

Metameric segmentation of organisms is widely used in nature. Repeated structures are essential for functionality of lateral organs such as vertebrae, muscles, nerves, vessels, limbs, or leaves in a body plan1. As a result of such physiological and geometric constraints of the axial symmetry, most phyla of Bilateria-such as annelids, arthropods, and chordates-exhibit segmentation of their embryonic tissues (e.g., ectoderm, mesoderm) antero-posteriorly.
Vertebrate embryos sequentially segment their paraxial mesoderm along the major body axis into somites with species-specific intervals, counts, and size distributions. Despite such robustness among individual embryos within a species, somite segmentation is versatile in between vertebrate species. Segmentation happens in a vast regime of time intervals (from 25 min in zebrafish to 5 h in humans), sizes (from ~20 &#181;m in tail somites of zebrafish to ~200 &#181;m in trunk somites of mice) and counts (from 32 in zebrafish to ~300 in corn snakes)2. More interestingly, fish embryos can develop in a wide range of temperatures (from ~20.5 &#176;C up to 34 &#176;C for zebrafish) while keeping their somites intact with proper size distributions by compensating for both segmentation intervals and axial elongation speeds. Beyond such interesting features, zebrafish stays as a useful model organism to study segmentation in vertebrates due to the external, synchronous and transparent development of a plenitude of sibling embryos as well as their accessible genetic tools. Adversely from a microscopy perspective, teleost embryos develop on a bulky spherical yolk, stretching and rounding the gastrulating tissue around it (Figure 1A). In this article, we present a flattened 3-D tissue explant culture for zebrafish tails. This explant system circumvents the spherical constraints of yolk mass, allowing access to high resolution live imaging of fish embryos for somite patterning.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61981/61981fig01.jpg" /> 
Figure 1: Slide Chamber Explant System for Zebrafish Embryos. (A) Zebrafish embryos have advantages for live imaging, such as the transparency of gastrulating embryonic tissue (blue), but the tissue forms around a bulky spherical yolk mass (yellow) which prevents near-objective, high-resolution imaging in intact embryos. Tail explants can be dissected starting with a microsurgical knife (brown) cut from the tissue anterior of somites (red) and continuing at the border with the yolk posteriorly. (B) Dissected tail explants can be placed on a coverslip (light blue) dorsoventrally; keeping neural tissue (light gray) on top and notochord (dark gray) at the bottom. <a href="https://www.jove.com/files/ftp_upload/61981/61981fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Sub-Q Syringe with PrecisionGlide Needle</td>
			<td>Becton, Dickinson and Co.</td>
			<td>REF 309597</td>
			<td>for dechorionating embryos and manipulations</td>
		</tr>
		<tr>
			<td>200 Proof Ethanol, Anhydrous</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>for tissue dissection media</td>
		</tr>
		<tr>
			<td>Calcium Chloride Anhydrous, Powder</td>
			<td>Sigma-Aldrich</td>
			<td>499609</td>
			<td>for tissue dissection media</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Disposable Scalpel, #10 Stainless Steel</td>
			<td>Integra-Miltex</td>
			<td>MIL4-411</td>
			<td>for preparing tape slide wells</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>886-86-2</td>
			<td>(optional) for anesthesizing tissues older than 20 somites stage</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher</td>
			<td>A3160601</td>
			<td>additional for tissue culture media</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG2b, Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>Cat#A-21145; RRID: AB_2535781</td>
			<td>secondary antibody for immunostaining</td>
		</tr>
		<tr>
			<td>L-15 Medium with L-Glutamine w/o Phenol Red</td>
			<td>GIBCO</td>
			<td>21083-027</td>
			<td>for tissue dissection media</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>179337</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Microsurgical Corneal Knife 2.85 mm Angled Tip Double Bevel Blade</td>
			<td>Surgical Specialties</td>
			<td>72-2863</td>
			<td>for tissue dissection</td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-ppERK</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#M8159; RRID:AB_477245</td>
			<td>for ppERK immunostaining</td>
		</tr>
		<tr>
			<td>NucRed Live 647 ReadyProbes Reagent</td>
			<td>Invitrogen</td>
			<td>R37106</td>
			<td>(optional) for live staining of cell nuclei</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Powder, 95%</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td>for fixation of samples for immunostaining</td>
		</tr>
		<tr>
			<td>Rat Tail Collagen Coating Solution</td>
			<td>Sigma-Aldrich</td>
			<td>122-20</td>
			<td>(optional) for chemically activating slide chambers</td>
		</tr>
		<tr>
			<td>Stage Top Incubator</td>
			<td>Tokai Hit</td>
			<td>tokai-hit-stxg</td>
			<td>(optional) for temperature control during live imaging</td>
		</tr>
		<tr>
			<td>Transparent Tape 3/4&#39;&#39;</td>
			<td>Scotch</td>
			<td>S-9782</td>
			<td>for preparing tape slide wells</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Zebrafish: Tg(actb2:2xMCP-NLS-EGFP)</td>
			<td>Campbell et&#160;al., 2015</td>
			<td>ZFIN: ZDB-TGCONSTRCT-150624-4</td>
			<td>transgenic fish with nuclear localized EGFP</td>
		</tr>
		<tr>
			<td>Zebrafish: Tg(Ola.Actb:Hsa.HRAS-EGFP)</td>
			<td>Cooper et&#160;al., 2005</td>
			<td>ZFIN: ZDB-TGCONSTRCT-070117-75</td>
			<td>transgenic fish with cell membrane localized EGFP</td>
		</tr>
	</tbody>
</table>

a 3-d tail explant culture to study vertebrate segmentation in zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

This protocol enables flat geometric culturing of live zebrafish tail explants. Tissue culture presents three major advantages over whole embryos: 1) control of axis elongation speed, 2) control over various signaling (morphogen) sources by simple dissection, and 3) near-objective, high magnification and high NA live imaging.
Chemically untreated slide chambers allow the tail explant to elongate its major axis (Figure 2A) by the skin ectoderm wrapping around the t...

Watch this Scientific Journal Video about A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish at JoVE.com

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...

3-D zebrafish tail explants let's us exploit transparency of zebrafish embryos to its full extent during somite development and vertebrate segmentation. Fill an empty mating tank with fish system water. Clean the strainer with system water.Raise the barriers between mating pairs. Selectively move the fish pair into the fresh water tank. Collect embryos in the strainer through water transfers with an empty tank.Rinse collected embryos with fish system water and flip on a Petri dish. Ethanol-glaze dissection tools. Make two layers of tapes on slides.Cut the horizontal sides of deep chambers. Complete chamber cuts with vertical sides. Peel the tape off from the chamber surface.Pour cell culture media in a tube. Mix calcium chloride 2.8 millimolar final concentration. Add antibiotic solution.Aliquot dissection media in a separate tube. Compliment fetal bovine serum to make tissue culture medium. De-corrinate embryos with needles.Rinse the embryos in a freshwater dish and collect them for explanting. Stabilize the embryo with needle and start de-yoking. Trim the leftover tissues off.Transfer the explants to the coverslip. Flatten the tissue and suck excess media out with pipette. Put the coverslip on growth medium containing slide chamber.Put the explant tissue on a tissue wipe. Apply immersion oil to the objective. Mount the slide chamber on microscope.Adjust laser channels for image acquisition. Check the z-layer limits of the sample. Set-up time-imaging and z-layers.3-D tail explant system keeps cell integration to tail bud and excess elongation intact like whole embryos. It is also possible to stop excess elongation by chemically activating the surface with type one collagen. 3-D zebrafish tail explants system lets us to exploit transparency of zebrafish embryos by letting us to take high-resolution and near-objective microscopy images.Besides that, it lets us to decouple the excess of elongation from morphogen signaling and also control the morphogen signaling sources with simple dissection methods.

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Developmental Biology

4.0K visualizações.  Cincinnati Children’s Hospital Medical Center. Explantas de cauda de zebrafish 3D vamos explorar a transparência dos embriões de zebrafish em toda a sua extensão durante o desenvolvimento de somite e segmentação de vertebrados. Encha um tanque de acasalamento vazio com água do sistema de peixes. Limpe o filtro com água do sistema.Levante as barreiras entre pares de acasalamento. Mova seletivamente o par de peixes para o reservatório de água doce. Colete embriões no coador através de transferências de água com um tanque...