Induction of Hypoxia in Living Frog and Zebrafish Embryos

The overall goal of this invention is to study the effects of hypoxia or hyperoxia on the embryonic tissues of any aquatic organism of interest. This method can help answer key questions in the field of developmental and cell biology, such as embryonic growth restriction, fetal hypoxia, high-altitude adaptation, obesity and solid tumor development. The main advantages of this technique are simplicity, cost-effectiveness and robustness.

It allows induction and sustainment of hypoxi in vivo for long time periods. After collecting and raising xenopus and zebrafish embryos, prepare a mesh-bottom 24-well plate according to the text protocol. Attach plastic rods to the mesh-bottom 24-well plate and place it into the aquatic tank of choice, then using silicone tubing, attach a gas tank with the desired oxygen nitrogen mixture, or another gas mixture of choice, to a distributor valve using an appropriate gas regulator.

Next connect the silicone tubing and the distributor valve to a ceramic disk diffuser inside the aquatic tank. Depending on the type of embryos used in the experiment, fill the hypoxic chamber tank with the appropriate embryo medium, then close the aquatic tank and use silicone grease to coat the lid and carefully seal it. Start diffusing the gas mixture through the ceramic disk diffuser.

Allow the system to equilibrate for 10 to 15 minutes. Adjust the oxygen flow rate in the chamber medium to 0.0017 to 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.

The oxygen flow rate depends on the internal pressure of the gas tank and the outlet pressure of the gas regulator. Both these parameters should be adjusted according to the monitor of the gas regulator. After equilibration, use an oximeter or oxygen probe to measure the oxygen concentration in the water.

If a particular non-room temperature is required, place the hypoxic chamber into a laboratory incubator of the required temperature. Carefully select the embryos for the experiment using whole and live embryos for hypoxia incubation. Place the embryo dishes into the incubator containing the gas incubation chamber, and let them equilibrate to the temperature of the incubator.

Then use a plastic pipette to carefully transfer the embryos into the wells of the meshed 24-well plate of the gas incubation chamber. Carefully label the wells with different genotypes. Ensure minimal reoxygenation of the chamber while placing the embryos into the wells.

Transfer the sample swiftly, and quickly reseal the chamber. Maintain the gas incubation chamber under a constant infusion with the gas mixture of choice for five hours, or for a longer time that suits the experiment. A mixture of 5%oxygen and 95%nitrogen is used here.

Carefully transfer the embryos from the gas incubation chamber back to the normoxic medium corresponding to the embryo type. After preparing buffers and media, and anesthetizing the embryos according to the text protocol, use a tissue homogenizer or a sonicator to homogenize the embryo tissue. Then centrifuge the homogenates to remove debris.

Collect the supernatant and denature the sample at 85 degrees Celsius for five minutes, then add Laemmli gel loading buffer to one X volume by volume. Use the supernatant for western blot analysis, or freeze the samples at negative 20 degrees Celsius. To incorporate EdU into the embryos, fill the gas incubation chamber with 400 to 500 milliliters of five millimolar EuD solution supplemented with 1%volume by volume DMSO.

Connect the gas tubing to the gas cylinder containing a gas mixture of 5%oxygen and 95%nitrogen. Incubate the solution for 15 minutes. Pre-equilibrate the solution with the same gas mixture used in the experiment for 15 minutes prior to the experiment.

Transfer the embryos to the hypoxic chamber, distributing them evenly between the wells, and incubate the embryos for two hours. Finally, analyze the embryos for EdU incorporation according to the text protocol. As shown in this table, desired oxygen concentrations in the incubation medium were induced at different time points throughout the experiments, as monitored by a fiber optic oxygen sensor.

In this experiment HIF-1 alpha which is stabilized under low oxygen concentrations, was measured in whole frog embryo lysates kept under normal oxygen concentrations, or subjected to 5%hypoxia and in lysates of their isolated retinas. In both samples HIF-1 alpha was stabilized under hypoxia. Here frog and zebrafish embryos were incubated in a hypoxic chamber maintained at 5%oxygen, and retinal proliferation was assessed by monitoring EdU incorporation.

As expected normoxic control retinas of frog and zebrafish embryos showed intensive staining in the CMZ, where proliferating progenitors reside. Following the oxygen incubation in the hypoxic chamber a strong decrease in the CMZ progenitor proliferation was observed in both frog and zebrafish embryos. By carrying out a time course it could be shown that the decrease in retinal progenitor proliferation was acute, and greatest within two hours, and persisted for many hours while embryos developed normally according to their developmental stage.

Once mastered, this technique can be done in 30 minutes if it is performed properly. While attempting this procedure it's important to remember to ensure correct gas flow rate, and to avoid unnecessary and excessively long reopening of the chamber. Following this procedure other methods like QPCR can be performed in order to answer additional questions like activation, or down-regulation of certain target genes of HIF-1 alpha and hypoxia, or hyperoxia respectively.

After its development this technique paved the way for the researchers in the field of developmental biology to explore retinal cell proliferation and hypoxia and restricted nutrition in frog and zebrafish embryos. After watching this video you should have a good understanding of how to induce hypoxia or hyperoxia in any aquatic organism, and how to study its effects on the tissue and phenomenon of interest, such as retinal cell proliferation. Don't forget that working with oxygen gas mixtures can be extremely dangerous, and precautions such as ensuring correct use and attachment of gas regulators should always be taken while performing this procedure.