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 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-1α) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2′-deoxyuridine (EdU) incorporation into the DNA. HIF-1α 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.
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-1α) 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 "natural hypoxia" 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-1α 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′-deoxyuridine (EdU) to the embryo medium and measuring its incorporation into the DNA of newly proliferating cells.
This protocol follows the animal care guidelines of the University of Cambridge.
1. Animal Maintenance
2. Induction of Hypoxia In Vivo
3. Control of Successful Hypoxia Induction by Monitoring HIF-1α Levels
4. Monitoring Cell Proliferation in Hypoxia
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 incubation medium using a fiber-optic oxygen sensor (2.1.9) at different time points during the experiment. These data indicate that we have induced stable hypoxia (6-6.5% dissolved oxygen) throughout our experiments (Table 1).
First, we assessed ubiquitous as well as retinal levels of HIF-1α in normoxia and in hypoxia as induced in our hypoxic chamber. HIF-1α is a protein that is stabilized under low oxygen concentrations. We measured HIF-1α levels in whole frog embryo lysates kept under normal oxygen concentration or subjected to 5% hypoxia, and in lysates of their isolated retinas. As shown in Figure 3, HIF-1α is stabilized under hypoxia in both whole embryos and in retinas. The stabilization of HIF-1α is achieved in different wells of the mesh-bottom incubation plate (Figure S1).
As we have previously shown, hypoxia affects proliferation of retinal stem cell progenitors in the Ciliary Marginal Zone (CMZ) of the retina13. Thus, monitoring the proliferation in the CMZ by means of EdU incorporation is a good marker for successful induction of hypoxia. We incubated embryos in a hypoxic chamber maintained at 5% oxygen and assessed retinal proliferation as described Step 4.2. As expected, normoxic control retinas showed intense EdU staining in the CMZ, where proliferating progenitors reside in both frog (Figures 4B and 4D) and zebrafish embryos (Figures 5A and 5C). After oxygen deprivation in the hypoxic chamber, a strong decrease in CMZ progenitor proliferation was observed in both frog (Figures 4C-4D) and zebrafish embryos (Figures 5B, 5C). To determine the extent of the effect, we performed a time-course, incubating the embryos in hypoxic chamber for longer periods of up to 24 h, monitoring proliferation by EdU incorporation as described in Step 4.3. We could show that the decrease in retinal progenitor proliferation was acute and greatest within 2 h, and persisted for many hours (Figure 4D), while embryos developed normally according to their developmental stage. This result suggests that our system can efficiently induce hypoxia in a target tissue of interest, upon short incubation times as well as sustain these conditions for longer periods while supporting normal embryo development.
Figure 1: Schematic Representation of the Experimental Gas Chamber Setup. (A) breeding aquatic tank (22 (length) x 10.5 (width) x 10.5 cm (height)) (B) mesh-bottom 24-well plate (12.8 x 8.6 x1.7 x 1.5 cm well diameter) (C) nylon filter with a mesh diameter of 0.1-0.2 mm (D) tunnels of 10 (length) x 5 mm (diameter) (E) Teflon or PVC rods of appropriate diameter and 30 mm length (F) silicone tubing (G) gas tank with the desired oxygen/CO2 mixture (H) distributor valve (I) gas valve (J) ceramic disc diffuser (K) tank lid. Please click here to view a larger version of this figure.
Figure 2: An Image of the Experimental Gas Chamber Setup. Please click here to view a larger version of this figure.
Table 1: Measurement of Oxygen Concentration upon Different Incubation Times in the Gas Chamber. Please click here to view a larger version of this figure.
Figure 3: HIF-1α Levels Increase upon Hypoxia Induction in Xenopus Embryos and their Retinas. Western blots of protein lysates from whole embryos (A) or from isolated embryonic retinas (B) kept under normal oxygen concentrations or in hypoxia. Blots were probed for HIF-1α subunit and α-tubulin. At least 5 embryos or 22 retinas were taken for each condition (n = 5) (C, D) Quantification of the Western blot performed in (A, B), respectively. Protein levels of HIF-1α were normalized to the protein levels of α-tubulin. Error bars represent standard deviations between experiments. * p-value <0.05, *** p-value <0.001; n = 5. Please click here to view a larger version of this figure.
Figure 4: Hypoxia Decreases Proliferation of Progenitors in the Retinal Stem Cell Niche of Frog Embryos. (A) Schematic representation of a cross section through a 38-stage Xenopus retina, indicating the position of the ciliary marginal zone (CMZ) (B, C) EdU incorporation measured after a 2 h EdU pulse in DAPI-stained retinas from 38 stage frog embryos kept under normoxia (B) or in the hypoxic chamber (C). Magenta = DAPI stain; Grey = EdU stain. Scale bars = 50 µm (D) Quantification of the experiment performed in B and C. Error bars represent standard deviations. *** p-value <0.001; n = 7. For each condition 10-15 retinas were quantified (G) Quantification of EdU-positive cells after an 1 h EdU incorporation in retinas from animals after various times in hypoxia (time in minutes indicated on the x-axis). Error bars represent standard deviations of two independent experiments (n = 2). For each condition and time point, a minimum of 10 retinas was quantified. Please click here to view a larger version of this figure.
Figure 5: Hypoxia Decreases Proliferation of Progenitors in the Retinal Stem Cell Niche of 4 dpf Zebrafish Embryos. EdU incorporation measured after a 2 h EdU pulse in DAPI-stained retinas from 4 dpf old WTe zebrafish embryos kept under normoxia (A) or in the hypoxic chamber (B). Magenta: DAPI stain; Grey: EdU stain. Scale bars = 50 µm (C) Quantification of the experiment performed in B and C. Error bars represent standard deviations. *** p-value <0.001; n = 7. For each condition 10-15 retinas were quantified. Please click here to view a larger version of this figure.
Supplemental Figure 1: HIF-1α Levels Increase upon Hypoxia Induction Consistently in Different Wells of the Plate and between Different Experiments. Western blots of protein lysates from whole Xenopus embryos kept under normal oxygen concentrations or in two different wells of the hypoxic chamber under induction of hypoxia for 2 hours in experiment 1 (A) or in experiment 2 (B). Blots were probed for HIF-1α subunit and α-tubulin. 5 frog embryos of stage 38 were taken for each condition. Experiment 1 and 2 are independent biological replicates (C, D) Quantification of the Western blots performed in (A, B), respectively. Protein levels of HIF-1α were normalized to the protein levels of α-tubulin. Please click here to view a larger version of this figure.
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 – here, retinas. To determine the effectiveness of hypoxia induction, we have monitored the levels of HIF-1α protein in whole embryo and retinal lysates. According to our results, hypoxia induction can be viewed as successful, if an increase of HIF-1α levels can be observed upon 1 h in the hypoxic chamber compared to normoxic control and normalized to global protein levels, e.g., using α-tubulin levels as control. We confirmed the activity of hypoxia in tissue by monitoring stem cell proliferation in retinas by EdU incorporation, and could show, in accordance with our previously published results13, that hypoxia induced using the setup presented here leads to a decrease of retinal stem cell proliferation in both frog and zebrafish embryos.
One of the critical steps of this protocol is to ensure that the hypoxic chamber is properly sealed. It is accomplished by using an appropriate seal on the tank lid, which was silicone grease in our case. In addition, placement inside an incubator will help avoid leakage of atmospheric oxygen into the chamber, by providing an additional barrier. A measurement of oxygen concentration with an appropriate probe or electrode also ensures that hypoxic oxygen levels are kept stable and without fluctuations. Additionally, care should be taken to avoid chamber opening for long time periods while exchanging solutions or samples in between experiments.
Given fast equilibration times of 10 – 15 min for hypoxia establishment, only a little amount of the gas mixture and a low gas flow rate are needed to sustain the system even for longer time periods than described in the representative results (24 h). The solutions used in the experiment should be pre-equilibrated to achieve hypoxia for 15 min prior to incubation in the chamber (as described in 4.2.1). We have used the hypoxic chamber over the period of 48 h of constant hypoxia without any errors as seen from our oxygen concentration measurements (Table 1). It should be noted that correct gas diffusion must be controlled during longer incubation times. Finally, care should be taken while placing the embryos into the wells (Step 2.2.1.) to avoid the mixing up of embryos from different mutant animals/ experimental conditions. This is quite simple to achieve given the relatively high walls of the single wells of the plate.
Despite being a simple but effective and robust system for hypoxia induction, the use of the chamber described here has one drawback for experiments that require use of expensive incubation solutions and media. The minimal volume of media in the chamber tank should not fall below 400 mL. However, adjustments can be made to suit lower volumes: We have succeeded to adjust the length of the rods (Figure 1E) attached to the plate (2.1.4 and 4.2.1) so that we would require only 50 mL of media/ solution. While not ideal for longer incubation times, this setup can fit the same gas tubing and can be appropriately sealed to ensure sufficient hypoxia for up to 8 h.
Taken together, our gas chamber setup can be employed for use with any aquatic model organism and is set apart from other similar commercially available systems by its simplicity, cost-effectiveness and robustness. Once mastered, it can be used for direct in vivo experiments, any given time period and under any desired gas atmosphere including hypoxia, hyperoxia or other gas mixtures.
The authors have nothing to disclose.
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.
Sodium chloride | Sigma | S7653 | NaCl / 0.1X MBS, Embryo medium, 10X TBST |
Potassium chloride | Sigma | P9333 | KCl / 0.1X MBS, Embryo mediu, |
Sodium bicarbonate | Sigma | S5761 | NaHCO3 / 0.1X MBS |
HEPES | Sigma | H3375 | 0.1X MBS |
Magnesium sulfate | Sigma | M7506 | MgSO4 / 0.1X MBS, Embryo medium |
Calcium nitrate | Sigma | 202967 | Ca (NO3)2 / 0.1X MBS |
Calcium chloride | Sigma | C1016 | CaCl2 / 0.1X MBS, Embryo medium |
Methylene blue | Sigma | M9140 | Embryo medium |
Pregnant mare serum gonadotropin | Sigma | CG10 | frog fertilization |
Zebrafish breeding tank | Carolina | 161937 | gas chamber construction |
24-well plate | Thermo Scientific | 142475 | Nunclon Delta Surface, for gas chamber construction |
Epoxy resin | RS Components UK | Kit 199-1468 | |
Gas distributor valve | WPI Luer Valves | Kit 14011 | aquatic tank attachment (Schema 1, H) |
High precision gas valve | BOC | 200 bar HiQ C106X/2B | gas tank attachment (Schema 1, I) |
5% oxygen and 95% N2 gas tank | BOC | 226686-L | hypoxic gas mixture |
ceramic disc diffuser | CO2 Art | Glass CO2 Nano Aquarium Diffuser, DG005DG005 | Schema 1, J |
silicone grease | Scientific Laboratory Supplies | VAC1100 | Schema 1, K |
oxymeter | Oxford Optronix | Oxylite, CP/022/001 | hypoxic chamber setup |
fibre-optic dissolved oxygen sensor | Oxford Optronix | HL_BF/OT/E | hypoxic chamber setup |
plastic pasteur pipette | Sterilin | STS3855604D | for embryo transfer |
MS222 | Sigma Aldrich | E10521-50G | embryo anesthetic |
RIPA buffer | Sigma | R0278-50ML | tissue homogenization |
Protease inhibitor | Sigma | P8340 | tissue homogenization |
Tris | Sigma | 77-86-1 | 4X Laemmli loading buffer, 10X TBST |
Glycerol | Sigma | G5516 | 4X Laemmli loading buffer |
Sodium Dodecyl Sulfate | Sigma | L3771 | SDS, 4X Laemmli loading buffer, 5X Running buffer |
beta-Mercaptoethanol | Sigma | M6250 | 4X Laemmli loading buffer |
Bromophenol Blue | Sigma-Aldrich | B0126 | 4X Laemmli loading buffer |
Trizma base | Sigma | 77-86-1 | 5X Running buffer, Transfer buffer |
Glycine | Sigma | G8898 | 5X Running buffer, Transfer buffer |
Methanol | Sigma | 34860 | Transfer buffer |
Tween 20 | Sigma | P2287-500ML | 10X TBST |
skim milk powder | Sigma | 70166 | Blocking Solution |
Eppendorf microcentrifuge tube | Sigma | T9661 | |
tissue homogenizer | Pellet Pestle Motor Kontes | Z359971 | tissue homogenization |
pellet pestles | Sigma | Z359947-100EA | tissue homogenization |
precast 12% gel | Biorad | Mini-ProteinTGX, 456-1043 | Western Blot |
protein ladder | Amersham | Full-Range Rainbow ladder, RPN800E | Western Blot |
nitrocellulose membrane (0.45 µm) | Biorad | 162-0115 | Western Blot |
anti-HIF-1α antibody | Abcam | ab2185 | Western Blot |
anti-α-tubulin antibody | Sigma | T6074 | Western Blot |
goat anti-rabbit antibody | Abcam | ab6789 | Western Blot |
goat anti-mouse antibody | Abcam | ab97080 | Western Blot |
Pierce ECL 2 reagent | Thermo Scientific | 80196 | Western Blot |
ECL films Hyperfilm | GE Healthcare Amersham | 28906837 | Western Blot |
5-Ethynyl-2′-deoxyuridine | santa cruz | CAS 61135-33-9 | EdU, EdU incorporation |
Phosphate-buffered Saline | Oxoid | BR0014G | 1X PBS |
Formaldehyde | Thermo Scientific | 28908 | Fixation solution |
Sucrose | Fluka | S/8600/60 | Solution solution |
Triton X-100 | Sigma | T9284-500ML | PBST |
Heat-inactivated Goat Serum | Sigma | G6767-100ml | HIGS, Blocking solution (EdU incorporation) |
4',6-diamidino-2-phenylindole | ThermoFisher Scientific | D1306 | DAPI, EdU incorporation |
Dimethyl sulfoxide | Molecular Probes | C10338 | DMSO, EdU incorporation |
glass vial | VWR | 98178853 | EdU incorporation analysis |
Tissue-Plus optimal cutting temperature compound | Scigen | 4563 | embedding medium, EdU incorporation analysis |
cryostat Jung Fridgocut 2800E | Leica | CM3035S | EdU incorporation analysis |
microscope slides Super-Frost plus Menzel glass | Thermo Scientific | J1800AMNZ | EdU incorporation analysis |
EdU Click-iT chemistry kit | Molecular Probes | C10338 | EdU incorporation analysis |
FluorSave | Calbiochem | D00170200 | mounting medium, EdU incorporation analysis |
coverslips | VWR | ECN631-1575 | EdU incorporation analysis |
fluorescent microscope | Nikon | Eclipse 80i | EdU incorporation analysis |
confocal scanning microscope | Olympus | Fluoview FV1000 | EdU incorporation analysis |
Volocity software | PerkinElmer | Volocity 6.3 | EdU incorporation analysis |