This work was supported by the Support from Wellcome Trust SIA Award 100329/Z/12/Z to W.A.H. and the DFG fellowship KH 376/1-1 awarded to H.K.

This protocol follows the animal care guidelines of the University of Cambridge.
1. Animal Maintenance
<ol>
	<li>Frog embryos 
	NOTE: Embryos can be raised and maintained according to the animal and laboratory facility. Here, an example of the animal maintenance is described.
	<ol>
		<li>Prepare 0.1x Modified Barth&#39;s Solution (MBS) solution: 0.88 mM NaCl, 10 &#181;M KCl, 24 &#181;M NaHCO3, 100 &#181;M HEPES, 8.2 &#181;M MgSO4, 3.3 &#181;M Ca(NO3)2, and 4.1 &#181;M CaCl2, pH&#160;7.6.</li>
		<li>Obtain Xenopus laevis embryos by in vitro fertilization.
		<ol>
			<li>Induce ovulation in adult female African clawed frogs (Xenopus laevis) by injection with pregnant mare serum gonadotropin one week (60 IU) and 12 h (500 IU) prior to egg laying.</li>
			<li>Collect oocytes and fertilize them in vitro with homogenized male testis.</li>
			<li>Raise embryos in 0.1x MBS at 16 - 18 &#176;C. Stage the embryos according to the Normal table of Xenopus laevis14. Take care in selecting whole and alive embryos for the experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Zebrafish embryos 
	NOTE: Embryos can be raised and maintained according to the animal and laboratory facility. Here, an example of the animal maintenance is described.
	<ol>
		<li>Prepare embryo medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.00001% methylene blue.</li>
		<li>Maintain and breed WT&#160;AB strain Danio rerio at 26.5 &#176;C.</li>
		<li>Collect the embryos on the morning of fertilization, raise until 4 d post fertilization (dpf) at 28 &#176;C in embryo medium prepared as described above, select and stage as previously described15.</li>
	</ol>
	</li>
</ol>
2. Induction of Hypoxia In Vivo
<ol>
	<li>Construction of the gas incubation chamber
	<ol>
		<li>Use a breeding aquatic tank (Figure 1A) that is normally used in the fish facility for hypoxic chamber construction. This tank should fit a 24-well plate.</li>
		<li>Prepare a mesh-bottom 24-well plate as depicted in Figure 1. Remove the plastic bottom of the 24-well plate (Figure 1B), e.g., by using a milling machine for this purpose.</li>
		<li>Fill in the space between the plastic of the wells and the walls of the plate with epoxy resin. Attach any nylon filter (Figure 1C) with a mesh diameter of 0.1-0.2 mm to the resin-coated plastic and allow the plate to be completely dry.</li>
		<li>Once dry, drill three or four tunnels (Figure 1D) of 10 mm length and 5 mm diameter into the resin- and mesh-coated plate walls. Attach plastic or Teflon rods (Figure 1E) of appropriate diameter and 30 mm length to the tunnels and place the plate into the tank of choice.</li>
		<li>Place the mesh-bottom 24-well plate into the aquatic tank of choice. Using silicone tubing (Figure 1F), attach a gas tank (Figure 1G) with the desired O2/N2 mixture or another gas mixture of choice to a distributor valve (Figure 1H) using an appropriate gas regulator (I) (BOC 200 bar). Here, use gas tanks containing a mixture of 5% oxygen and 95% N2 in the hypoxia experiments presented here.</li>
		<li>Adjust the oxygen flow rate in the chamber medium to 0.0017-0.0019 cubic meters per second. Under this gas flow rate, no disturbance of the medium should be seen in the wells of the plate.
		<ol>
			<li>Use the high purity two-stage gas regulator for this purpose. Set the regulator to reduce the inner pressure of the gas tank (1000 - 2500 PSI) to a delivery pressure of 10 PSI throughout the experiment. Thus, in accordance with the specifications of this gas regulator, a flow rate of 0.00189 cubic meters per second was achieved throughout the experiment.</li>
		</ol>
		</li>
		<li>Connect the silicone tubing and the distributor valve to a ceramic disc diffuser (Figure 1J) inside the aquatic tank. Close the aquatic tank and carefully seal the lid, coating it with silicone grease (Figure 1K) (compare with the Figure 1). If a particular non-room temperature is required, place the hypoxic chamber into a laboratory incubator of required temperature.</li>
		<li>Depending on the type of embryos used in the experiment, fill the hypoxic chamber tank with the appropriate embryo medium and start diffusing the gas mixture through the ceramic disc diffuser. Equilibrate for 10-15 min.</li>
		<li>After equilibration with the desired oxygen mixture, measure oxygen concentration in the water using an oxymeter or oxygen probe. Here, use an oxymeter with the fiber-optic dissolved oxygen and temperature sensor for this purpose.</li>
	</ol>
	</li>
	<li>Induction of hypoxia in embryos
	<ol>
		<li>Carefully select the embryos for the experiment, use whole and alive embryos for the hypoxia incubation. Here, us frog embryos of stage 38 or zebrafish embryos 4 dpf&#160;in the experiments presented here.</li>
		<li>Place the embryo dishes into the incubator (Figure 1K) containing the gas incubation chamber and let them equilibrate to the temperature of the incubator.</li>
		<li>Carefully transfer the embryos into the wells of the meshed 24-well plate (Figure 1B) of the gas incubation chamber, using a plastic pipette. Always use a plastic pipette for embryo transfer throughout the protocol. 
		NOTE: Each well of this plate can contain up to 5 frog embryos of stage 38 or up to 7 zebrafish embryos of 4 dpf. Label the wells carefully if using different genotypes.</li>
		<li>Maintain the gas incubation chamber under a constant infusion with the gas mixture of choice for 5 h or for a longer time that suits the experiment. Use a mixture of 5% O2 and 95% N2 here.</li>
		<li>Carefully transfer the embryos from the gas incubation chamber back to the normoxic medium corresponding to the embryo type and immediately proceed with the Steps 3 or 4.</li>
		<li>If a further normoxic treatment is required, carefully transfer the embryos back to the normoxic medium corresponding to the embryo type.</li>
	</ol>
	</li>
</ol>
3. Control of Successful Hypoxia Induction by Monitoring HIF-1&#945; Levels
<ol>
	<li>Preparations
	<ol>
		<li>Prepare 0.4 mg/mL MS222 solution in 0.1x MBS.</li>
		<li>Prepare homogenization buffer: Purchase Radioimmunoprecipitation Assay buffer (RIPA buffer) for homogenization of tissue and supplement the amount of buffer necessary for your experiment with Protease Inhibitor to a final concentration 1:100 v/v.</li>
		<li>Prepare solutions for Western blot.
		<ol>
			<li>Prepare 4x Laemmli gel loading buffer: 425 mM Tris pH 8.0, 40% v/v Glycerol, 8% v/v SDS, 4% v/v beta-mercaptoethanol, 0.5% w/v Bromophenol Blue, 10 mL total volume ddH2O.</li>
			<li>Prepare 5x running buffer: 15 g Trizma base, 72 g Glycine, 5 g SDS, 1 L total volume ddH2O.</li>
			<li>Prepare Transfer buffer: 2.66 g Trizma base, 14.4 g glycine, 200 mL 100% methanol, 1 L total volume ddH2O.</li>
			<li>Prepare 10x&#160;TBST: 5 mL 1 M Tris pH 8.0, 30 mL 5M NaCl, 2.5 mL Tween 20, 1 L total volume H2O.</li>
			<li>Prepare blocking solution: 4% w/v skim milk powder in 1x TBST.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Collection of tissue
	<ol>
		<li>Anaesthetize the embryos by bathing in 0.4 mg/mL MS222 solution16,17 and transfer them into a plastic reaction tube filled with homogenization buffer using a plastic pipette. Use 20 &#181;L buffer per embryo. Note that at least 5 frog embryos of stage 38 or 7 zebrafish embryos of 4 dpf are required to determine a significant change in HIF1&#945; protein levels.</li>
		<li>Homogenize the tissue using an appropriate tissue homogenizer or a sonicator. Remove the debris by centrifugation (15 min; 13,000 x g; 4 &#176;C).
		<ol>
			<li>Alternatively, dissect the retinas from the anaesthetized frog embryos17 and homogenize as above. Use 20 &#181;L of homogenization buffer per 10 retinas.</li>
		</ol>
		</li>
		<li>Take the supernatant using a pipette, denature at 85 &#176;C for 5 min and supplement with Laemmli gel loading buffer to a final concentration of 1x v/v (e.g., add 5 &#181;L of 4x Laemmli buffer to 15 &#181;L homogenate). Use this supernatant for the Western blot or freeze the samples at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Western Blot
	<ol>
		<li>Load the samples prepared as described in Step 3.2 on a precast 12% gel in an electrophoresis system using 1x v/v Running buffer. Use a protein ladder for protein size determination. Transfer the proteins onto a nitrocellulose membrane (0.45 &#181;m membrane) in Transfer buffer for 1 h packed on ice at 4 &#176;C, as per manufacturer&#39;s protocol.</li>
		<li>Block unspecific binding sites incubating the membrane in Blocking buffer for 60 min. Follow with 3 x 10 min washes in 1x TBST.</li>
		<li>Incubate the membrane in solutions of rabbit anti-HIF-1&#945; antibody and mouse anti-&#945;-tubulin diluted 1:100 v/v and 1:5000 v/v, respectively, in Blocking solution, for 2.5 h&#160;at RT. Follow with 3 x 10 min washes in 1x TBST.</li>
		<li>For detection, incubate in goat anti-rabbit and goat anti-mouse HRP-conjugated antibodies diluted 1:1000 v/v in Blocking solution for 1 h. Follow with 3 x 10 min washes in 1x TBST and visualize the HRP staining using a commercial kit and a developer machine of choice.</li>
		<li>Quantify the amount of HIF1&#945; protein using digital quantification.</li>
	</ol>
	</li>
</ol>
4. Monitoring Cell Proliferation in Hypoxia
<ol>
	<li>Preparations
	<ol>
		<li>Prepare 5 mM EdU solution in the embryo medium of choice. The solution can be used immediately or aliquoted and stored at -20 &#176;C for up to 6 months.</li>
		<li>Prepare Phosphate-Buffered Saline (PBS) or purchase PBS from commercial resources. Autoclave for 10 min at 115 &#176;C.</li>
		<li>Prepare solutions for the detection of EdU incorporation.
		<ol>
			<li>Prepare fixation solution: 4% v/v of 16% Formaldehyde stock solution in PBS.</li>
			<li>Prepare sucrose solution: 30% w/v sucrose in PBS.</li>
			<li>Prepare PBST: 0.1% v/v Triton X-100 in PBS.</li>
			<li>Prepare Blocking solution: 10% v/v Heat-inactivated Goat Serum (HIGS) in PBST.</li>
			<li>Prepare DAPI solution: 1:1000 v/v 4&#39;,6-diamidino-2-phenylindole (DAPI) in PBST.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incubation of embryos in hypoxia for EdU incorporation
	<ol>
		<li>Fill the gas incubation chamber with 400-500 mL of 5 mM EdU solution supplemented with 1% v/v DMSO. Pre-equilibrate the solution with the same gas mixture used in the experiment for 15 min prior to the experiment. Note that the volume of the incubation solution can be minimized by attaching shorter rods (Figure 1E) to the mesh-bottom plate.</li>
		<li>Connect the gas tubing to the gas cylinder containing a gas mixture of 5% O2 and 95% N2. Incubate the solution for 15 min.</li>
		<li>Transfer the embryos to the hypoxic chamber, distributing them evenly between the wells and incubate for 2 h. Each well can fit up to 5 frog embryos of stage 38 or up to 7 zebrafish embryos 4 dpf.</li>
		<li>Analyze the embryos for EdU incorporation.</li>
	</ol>
	</li>
	<li>Time-course of embryo incubation in hypoxia and EdU incorporation
	<ol>
		<li>Fill the gas incubation chamber with 500 mL of embryo medium of choice. Connect the gas tubing to the gas cylinder containing a gas mixture of 5% O2 and 95% N2 and equilibrate the medium with the gas mixture for 15 min.</li>
		<li>Transfer the embryos to the hypoxic chamber, distributing them evenly between the wells and incubate for desired time period up to 48 h. For the last 1 h, substitute the embryo medium with 5 mM EdU solution supplemented with 1% v/v DMSO.</li>
		<li>Transfer the embryos back to the normoxic dish and analyze for EdU incorporation.</li>
	</ol>
	</li>
	<li>EdU incorporation detection and analysis
	<ol>
		<li>Anaesthetize the embryos by bathing in 0.4 mg/mL MS222 solution.</li>
		<li>Transfer the embryos to an appropriate glass vial and fix by incubating in Fixation solution for 2 h at RT or O/N at 4 &#176;C. Follow with 3 x 10 min washes in PBS. Carefully transfer the embryos to the sucrose solution for tissue cryoprotection. Incubate for 4 h or until the embryos sink to the bottom of the glass vial.</li>
		<li>Embed the embryos in embedding medium (optimal cutting temperature compound). Cryosection blocks with embryos immediately or store for up to 14 d&#160;at -80 &#176;C.</li>
		<li>Cryosection the blocks containing embryos using a cryostat. Collect sections of 12 &#181;m (zebrafish embryos) or 16 &#181;m (frog embryos) thickness on microscope slides and let dry. Process cryosections immediately or store at -20 &#176;C for up to 3 months.</li>
		<li>Detect EdU incorporation by immunofluorescent staining using a commercial chemistry kit. Prepare the amount of EdU Reaction Mix necessary for the number of cryosections in the experiment, as described by the manufacturer.
		<ol>
			<li>For 500 &#181;L of EdU Reaction Mix, use 430 &#181;L of 1X EdU reaction buffer, 20 &#181;L of CuSO4, 1.2 &#181;L of Alexa Fluor azide, 50 &#181;L of 1X EdU buffer additive.</li>
		</ol>
		</li>
		<li>Wash the cryosections once with PBS to remove the embedding medium and 3 x 5 min with PBST to permeabilize the tissue. Incubate the cryosections in Blocking solution for 30 min.</li>
		<li>Incubate the cryosections with the EdU Reaction Mix for 1 h and followed by 3 x 10 min washes in PBST. Costain the cryosections with DAPI solution for 15 min, followed by 3 x 10 min washes in PBST. Mount the slides with stained cryosections in mounting medium, cover with coverslips and leave to dry before analyzing under a fluorescent microscope or storing at 4 &#176;C for up to 1 year.</li>
		<li>Analyze the cryosections with anti-EdU staining under an inverted confocal scanning microscope and determine the number of EdU-positive cells using digital quantification.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Here we have presented an easy but robust new method to induce hypoxia that is adjusted for use with frog and zebrafish embryos but can also be suited for other aquatic organisms. The major advantage of this method lies in its simplicity and cost efficiency. Nevertheless, the results achieved with this method are very robust. We have shown that hypoxia can be efficiently induced in the chamber both in whole embryos as well as in specific tissue &#8211; here, retinas. To determine the effectiveness of hypoxia induction, w...

Hypoxia research has numerous applications. These include investigating the pathogenesis and developing treatments for medical conditions characterized by hypoxia1 and acute high altitude illness2. Hypoxic stress causes major metabolic changes in all organisms requiring oxygen. Hypoxic stress also influences fetal growth and development and the pathogenesis of several human diseases, including intrauterine growth restriction3. Hypoxic stress can not only lead to reduced birth weight, fetal and neonatal mortality, but can also result in many complications in adult life, such as cardiovascular disease, type-2 diabetes, obesity, and hypertension4. Hypoxic stress is also often observed during solid tumor development, when the tumor tissue outgrows its blood supply. It is therefore crucial to be able to study the effects of hypoxia in vivo and directly during embryonic development.
Among the most well-known methods that have been employed to study effects of hypoxia during development is the use of cobalt chloride in the growth medium or incubation of the organism in a hypoxic chamber. Cobalt chloride artificially induces a hypoxic response under normal oxygen concentration, due to its role in the stabilization of hypoxia-inducible factor-1 alpha (HIF-1&#945;) by preventing its proteosomal degradation5,6,7. However, being a convenient method8, the use of cobalt chloride as well as other similar chemical hypoxia mimetics can have unspecific deleterious effect on cells and tissues, e.g., apoptosis9. Therefore, hypoxic chambers are a better method for induction of &#34;natural hypoxia&#34; in living organisms through the course of normal development.
We have focused on developing a system for induction of hypoxia in aquatic animal embryos. Both frogs and zebrafish have now become informative vertebrate model organisms for studies of numerous biological processes, as well as models for various human diseases. Frog and zebrafish embryos develop externally, eliminating the complication of maternal compensation. Further, a fast course of development makes it possible to manipulate environmental factors and observe the phenotypic changes in organ formation in real time. In addition, many components of major signal transduction pathways are highly conserved in these model organisms and have been characterized in detail by a large body of literature. The main advantage in using frogs and zebrafish embryos to study the effects of hypoxia on vertebrate development is that all processes can be monitored directly, since oxygen quickly penetrates the embryos. Thus, in frogs and zebrafish, as in contrast to other model organisms such as mouse embryos, the influence of a specific oxygen concentration can be studied in the tissue of interest, without taking into consideration the presence or lack of functional vasculature.
Most commercially available setups for hypoxic incubation have the disadvantage of being comparably large and having correspondingly high running costs. Apart from their high initial cost and gas consumption, equilibration and maintenance of common hypoxia chambers requires sustainment of constant hypoxic atmosphere against the gas gradient that naturally occurs in these chambers because of their larger size and/or organism respiration. This requires employment of gas fans and a cooling system, which increases the amount of additional necessary equipment, impedes the dexterity of the researcher and overall decreases simplicity of experimental procedure. In contrast, the setup we present here is comparably robust but very cost-effective, small, easy to establish and allows fast gas equilibration, stable hypoxic atmosphere and simple exchange of materials and solutions within the chamber. Our system can be employed for use with any aquatic model organism of interest.
We have constructed a hypoxic chamber that is conveniently small and therefore can be placed inside a common laboratory incubator, which easily allows experimental procedures at any specific temperature. Providing convenient control of temperature as well as oxygen concentration in the medium, the advantage of our system against the commercially available hypoxia incubators lies in its small size and cost efficiency. Thus, our setup can be established using general laboratory supplies available for the majority of research labs and does not require any expensive materials. In addition, our setup does not generate heat, unlike the commercially available hypoxia incubators, and allows use at temperatures lower than room temperature being placed in an incubator. The last is especially critical for the work with cold-blooded organisms such as frogs and fish where developmental and metabolic rates are strongly temperature dependent.
Being very cost-effective and easily built, our gas incubation chamber is nevertheless very versatile in establishing various hypoxic or hyperoxic conditions, as well as enabling quick and easy administration of different media and solutions for a vast number of experimental conditions. In addition, employing a 24-well plate instead of commonly used dishes or laboratory tanks10,11,12, our system allows observation and experimental treatment of several mutant conditions at once.
To control for correct induction of hypoxia, we have monitored the levels of the HIF-1&#945; protein by Western blot detection. In addition, the number of proliferating cells before and after incubation in the hypoxic chamber can be used to determine if hypoxia has been induced in the tissue. This method is based on our previously published results13, showing that proliferation in embryonic retinal stem cell niche decreases upon induction of hypoxia. Thus, we have monitored the level of retinal stem cell proliferation by adding 5-ethynyl-2&#8242;-deoxyuridine (EdU) to the embryo medium and measuring its incorporation into the DNA of newly proliferating cells.

<table><tbody><tr><td>Sodium chloride</td><td>Sigma</td><td>S7653</td><td>NaCl/0.1x MBS, Embryo medium, 10x TBST</td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9333</td><td>KCl/0.1x MBS, Embryo mediu,</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;/0.1x MBS</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>0.1x MBS</td></tr><tr><td>Magnesium sulfate</td><td>Sigma</td><td>M7506</td><td>MgSO&lt;sub&gt;4&lt;/sub&gt;/0.1x MBS, Embryo medium</td></tr><tr><td>Calcium nitrate</td><td>Sigma</td><td>202967</td><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;/0.1x MBS</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C1016</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt;/0.1x MBS, Embryo medium</td></tr><tr><td>Methylene blue</td><td>Sigma</td><td>M9140</td><td>Embryo medium</td></tr><tr><td>Pregnant mare serum gonadotropin (PMSG)</td><td>Sigma</td><td>CG10</td><td>Frog fertilization</td></tr><tr><td>Zebrafish breeding tank</td><td>Carolina</td><td>161937</td><td>Gas chamber construction</td></tr><tr><td>24-well plate</td><td>Thermo Scientific</td><td>142475</td><td>Nunclon Delta Surface, for gas chamber construction</td></tr><tr><td>Epoxy resin</td><td>RS Components UK</td><td>Kit 199-1468</td><td></td></tr><tr><td>Gas distributor valve</td><td>WPI Luer Valves</td><td>Kit 14011</td><td>Aquatic tank attachment (Schema 1, &lt;strong&gt;H&lt;/strong&gt;)</td></tr><tr><td>High precision gas valve</td><td>BOC&amp;nbsp;</td><td>200 bar HiQ C106X/2B</td><td>Gas tank attachment (Schema 1, &lt;strong&gt;I&lt;/strong&gt;)</td></tr><tr><td>5% oxygen and 95% N&lt;sub&gt;2&lt;/sub&gt; gas tank</td><td>BOC</td><td>226686-L</td><td>Hypoxic gas mixture</td></tr><tr><td>ceramic disc diffuser</td><td>CO&lt;sub&gt;2&lt;/sub&gt; Art&amp;nbsp;</td><td>Glass CO2 Nano Aquarium Diffuser, DG005DG005</td><td>Schema 1, &lt;strong&gt;J&lt;/strong&gt;</td></tr><tr><td>silicone grease</td><td>Scientific Laboratory Supplies</td><td>VAC1100</td><td>Schema 1, &lt;strong&gt;K&lt;/strong&gt;</td></tr><tr><td>oxymeter</td><td>Oxford Optronix&amp;nbsp;</td><td>Oxylite, CP/022/001</td><td>Hypoxic chamber setup</td></tr><tr><td>fibre-optic dissolved oxygen sensor</td><td>Oxford Optronix</td><td>HL_BF/OT/E</td><td>Hypoxic chamber setup</td></tr><tr><td>plastic pasteur pipette</td><td>Sterilin</td><td>STS3855604D</td><td>For embryo transfer</td></tr><tr><td>MS222&amp;nbsp;</td><td>Sigma Aldrich</td><td>E10521-50G</td><td>Embryo anesthetic</td></tr><tr><td>RIPA buffer&amp;nbsp;</td><td>Sigma</td><td>R0278-50ML</td><td>Tissue homogenization</td></tr><tr><td>Protease inhibitor</td><td>Sigma</td><td>P8340</td><td>Tissue homogenization</td></tr><tr><td>Tris</td><td>Sigma</td><td>77-86-1</td><td>4x Laemmli loading buffer, 10x TBST</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>4x Laemmli loading buffer</td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma</td><td>L3771</td><td>4x Laemmli loading buffer, 5x Running buffer</td></tr><tr><td>beta-Mercaptoethanol&amp;nbsp;</td><td>Sigma</td><td>M6250</td><td>4x Laemmli loading buffer</td></tr><tr><td>Bromophenol Blue</td><td>Sigma-Aldrich</td><td>B0126</td><td>4x Laemmli loading buffer</td></tr><tr><td>Trizma base&amp;nbsp;</td><td>Sigma</td><td>77-86-1</td><td>5x Running buffer, Transfer buffer</td></tr><tr><td>Glycine</td><td>Sigma</td><td>G8898</td><td>5x Running buffer, Transfer buffer</td></tr><tr><td>Methanol</td><td>Sigma</td><td>34860</td><td>Transfer buffer</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P2287-500ML</td><td>10x TBST</td></tr><tr><td>skim milk powder</td><td>Sigma</td><td>70166</td><td>Blocking Solution</td></tr><tr><td>Eppendorf microcentrifuge tube</td><td>Sigma</td><td>T9661</td><td></td></tr><tr><td>tissue homogenizer</td><td>Pellet Pestle Motor Kontes</td><td>Z359971</td><td>Tissue homogenization</td></tr><tr><td>pellet pestles</td><td>Sigma</td><td>Z359947-100EA</td><td>Tissue homogenization</td></tr><tr><td>precast 12% gel</td><td>Biorad</td><td>Mini-ProteinTGX, 456-1043</td><td>Western Blot</td></tr><tr><td>protein ladder</td><td>Amersham</td><td>Full-Range Rainbow ladder, RPN800E</td><td>Western Blot</td></tr><tr><td>nitrocellulose membrane (0.45 &amp;micro;m)</td><td>Biorad</td><td>162-0115</td><td>Western Blot</td></tr><tr><td>anti-HIF-1&amp;alpha; antibody</td><td>Abcam</td><td>ab2185</td><td>Western Blot</td></tr><tr><td>anti-&amp;alpha;-tubulin antibody</td><td>Sigma</td><td>T6074</td><td>Western Blot</td></tr><tr><td>goat anti-rabbit antibody</td><td>Abcam</td><td>ab6789</td><td>Western Blot</td></tr><tr><td>goat anti-mouse antibody</td><td>Abcam</td><td>ab97080</td><td>Western Blot</td></tr><tr><td>Pierce ECL 2 reagent&amp;nbsp;</td><td>Thermo Scientific</td><td>80196</td><td>Western Blot</td></tr><tr><td>ECL films Hyperfilm</td><td>GE Healthcare Amersham</td><td>28906837</td><td>Western Blot</td></tr><tr><td>5-Ethynyl-2&amp;prime;-deoxyuridine &amp;nbsp;</td><td>santa cruz</td><td>CAS&amp;nbsp;61135-33-9</td><td>EdU, EdU incorporation</td></tr><tr><td>Phosphate-buffered Saline (PBS)</td><td>Oxoid</td><td>BR0014G</td><td>1x</td></tr><tr><td>Formaldehyde</td><td>Thermo Scientific</td><td>28908</td><td>Fixation solution</td></tr><tr><td>Sucrose</td><td>Fluka</td><td>S/8600/60</td><td>Solution solution</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284-500ML</td><td>PBST</td></tr><tr><td>Heat-inactivated Goat Serum (HiGS)</td><td>Sigma</td><td>G6767-100ml</td><td>Blocking solution (EdU incorporation)</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>D1306</td><td>DAPI, EdU incorporation</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Molecular Probes</td><td>C10338</td><td>EdU incorporation</td></tr><tr><td>glass vial</td><td>VWR</td><td>98178853</td><td>EdU incorporation analysis</td></tr><tr><td>Tissue-Plus optimal cutting temperature compound&amp;nbsp;</td><td>Scigen</td><td>4563</td><td>Embedding medium, EdU incorporation analysis</td></tr><tr><td>cryostat Jung Fridgocut 2800E</td><td>Leica&amp;nbsp;</td><td>CM3035S</td><td>EdU incorporation analysis</td></tr><tr><td>microscope slides Super-Frost plus Menzel glass</td><td>Thermo Scientific</td><td>J1800AMNZ</td><td>EdU incorporation analysis</td></tr><tr><td>EdU Click-iT chemistry kit</td><td>Molecular Probes</td><td>C10338</td><td>EdU incorporation analysis</td></tr><tr><td>FluorSave</td><td>Calbiochem</td><td>D00170200</td><td>Mounting medium, EdU incorporation analysis</td></tr><tr><td>coverslips</td><td>VWR</td><td>ECN631-1575</td><td>EdU incorporation analysis</td></tr><tr><td>fluorescent microscope</td><td>Nikon</td><td>Eclipse 80i</td><td>EdU incorporation analysis</td></tr><tr><td>confocal scanning microscope</td><td>Olympus</td><td>Fluoview FV1000</td><td>EdU incorporation analysis</td></tr><tr><td>Volocity software</td><td>PerkinElmer</td><td>Volocity 6.3</td><td>EdU incorporation analysis</td></tr></tbody></table>

induction of hypoxia in living frog and zebrafish embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

Employing the hypoxic chamber system that we present here allows the study of the effects of hypoxia individually and in vivo in whole living animals. Hypoxia can be induced by placing entire frog or zebrafish embryos in the hypoxic chamber (Figure 1), and be undertaken on different combinations of conditions. An image of our completed gas chamber setup is shown in Figure 2. We have monitored oxygen concentration in the ...

Watch this Scientific Journal Video about Induction of Hypoxia in Living Frog and Zebrafish Embryos at JoVE.com

Induction of Hypoxia in Living Frog and Zebrafish Embryos

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

induction-of-hypoxia-in-living-frog-and-zebrafish-embryos

Research

JoVE Journal

Developmental Biology

9.7K Views.  University of Cambridge. We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Video: Induction of Hypoxia in Living Frog and Zebrafish Embryos

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

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

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

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

Biology

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Author Spotlight: Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Neuroscience

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Oxidative Stress in Zebrafish Embryos

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

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

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Summary

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Chapters in this video

Microbead Implantation in the Zebrafish Embryo

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Optogenetic Signaling Activation in Zebrafish Embryos

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Metabolic Profile Analysis of Zebrafish Embryos

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

Analysis of Oxidative Stress in Zebrafish Embryos