To establish the embryo's body plan during development, cells are thought to decode signaling intensities, dynamics, and combinations. We're using optogenetics to manipulate these signaling features in zebrafish embryos. This approach will help us understand how embryonic cells decode signaling to make some of the earliest cell fate decisions.
The optogenetic approaches discussed here use light as a remote control to activate BMP, or nodal signaling, rapidly and reversibly. Because light exposure is easy to direct, these approaches can offer improved experimental control over signaling. Our protocol introduces using these light-sensitive tools in zebrafish embryos.
With this improved temporal and spatial control, it should now be possible to perform precise signaling modifications during zebrafish developments. We expect this to deepen our understanding of how cells decode signaling to make fate decisions, which will help reveal how the embryonic body plan develops. To begin, use an incubator and an LED microplate illuminator to create a light box for controlling light exposure and temperature.
Then, position a six-well plate on the upper shelf of the incubator and check whether the light beam evenly encompasses it. Use a sheet of paper to visualize the distribution of light coverage. Use a light meter to measure irradiance and spatial uniformity.
At least one day before injections, make injection dishes and agarose-coated six-well plates. Rinse an injection dish mold with embryo medium and place gently onto molten agarose. Once the agarose has solidified, delicately use the tab to remove the mold.
Next, prepare flamed-glass pipettes for immunofluorescence experiments on dechorionated embryos. Place the end of a glass pipette into a Bunsen burner flame and rotate continuously until the edges become smooth. Prepare an injection mix using the mRNAs shown here.
For the phenotyping assay, introduce the needle through the chorion directly into the center of the cell at the one-cell stage and inject 1 nanoliter of the injection mix. If conducting an immunofluorescence assay, target the center of the cell of dechorionated embryos at the one-cell stage. For both assays, prepare enough embryos for the unexposed non-injected, unexposed injected, exposed non-injected, and exposed injected embryos.
Select at least 30 embryos per condition. For the immunofluorescence assay, prepare an additional dish containing non-injected embryos as a proxy to assess the progression of developmental stages. After injection, incubate the embryos and Petri dishes at 28 degrees Celsius.
To begin, incubate the injected zebrafish embryos at 28 degrees Celsius. Around 1.5 HPF, at the 4 to 16 cell stage, remove unfertilized and unhealthy embryos. For the phenotyping assay, wrap the unexposed control plate with non-injected and injected embryos in aluminum foil.
Position the foil-covered plate on the lower shelf and the exposed plate on the upper shelf of the 28-degree Celsius light box. After confirming that the dish cover is in place, activate the blue light and shut the light box door to prevent unintended room light exposure. For the immunofluorescence assay around 1.5 HPF, individually wrap the exposed and unexposed dishes and aluminum foil while keeping the proxy dish unwrapped.
Put all the dishes in the 28-degree Celsius light box without activating the LED. Close the light box door to prevent inadvertent exposure to room light. At approximately 5 HPF, use a dissecting scope to evaluate the developmental stage of the proxy embryos.
Also, remove the wrapped dishes to ensure uniform temperature exposure across all dishes. Once the proxy embryos reach 40%epiboly, uncover the exposed dish and place it on the upper shelf of the light box. Keep the unexposed dish covered on the lower shelf.
Immediately activate the blue light, close the door, and set a timer for 20 minutes. To prepare for fixation, eliminate as much room light as possible. Just before fixation, take tubes containing 4%formaldehyde from 4 degrees Celsius and position them beside the light box.
After 20 minutes, open the door of the light box and remove the dish that wasn't exposed to light. Use a flamed-glass pipette tip to remove the light-sensitive embryos from the dish. Then, to immerse the embryos in 4%formaldehyde, submerge the flamed-glass pipette tip in the formaldehyde and allow the embryos to sink into the liquid.
Store fixed embryos at 4 degrees Celsius overnight. For the phenotyping assay, perform the light exposure experiment on bOpto-BMP and bOpto-Nodal expressing zebrafish embryos, and remove them from the light box at one day post-fertilization between 24 to 32 HPF. Using a dissecting scope, evaluate phenotypes and establish a scoring rubric.
Assess embryos while they remain within the chorion for convenience. Utilize a pipette to probe, to reposition embryos for viewing from different angles. For the immunofluorescence assay, after incubating embryos in 4%formaldehyde at 4 degrees Celsius overnight, remove the formaldehyde and rinse them three to five times with PBST.
Discard the PBST and add 100%methanol. Seal the tubes and gently invert them to mix residual PBST and methanol. After washing the embryos two more times with methanol, store them at minus 20 degrees Celsius for at least two hours up to several years.
Using a microscope equipped for optical sectioning, capture images of immunostained embryos. Immunofluorescent staining experiments with appropriate mRNA amounts and light exposure conditions showed that the pSmad levels were similar in non-injected and unexposed embryos, whereas injected light-exposed embryos exhibited higher levels of Smad phosphorylation.
