Name: a 3-d tail explant culture to study vertebrate segmentation in zebrafish
Uploaded: 2021-06-30T13:00:00
Description: Watch this Scientific Journal Video about A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish at JoVE.com

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.

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

Research

JoVE Journal

Developmental Biology

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

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 ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

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.

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 ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.