Signaling pathways controlling multicellular systems are dynamic. To understand the function of these dynamics, it is essential to be able to subtly modulate these dynamics without affecting overall signaling activity. This protocol uses a microfluidic system that allows for functional dissection of signaling oscillations in developing mouse embryos.
Signaling dynamics have been found in many tissues including adult ones. Microfluidics is a versatile tool that can be adapted to suit many model systems including cell, tissue, and organoid cultures. To begin, prepare the required amount of PDMS by mixing the monomer with the catalyst in a nine-to-one ratio to induce polymerization.
Use disposable tools and make sure that mixing is properly achieved. Place the PDMS mixture in a desiccator and apply vacuum for approximately 30 minutes to remove air. Pour a PDMS layer of approximately three to five millimeters into the chip mold and place back into the desiccator for approximately 30 minutes.
Cure the mold by placing it in an oven overnight at 65 degrees Celsius or lower depending on the mold material. Cut the chip out of the mold using a scalpel. Punch inlet and outlet holes with a one millimeter biopsy punch starting from the inside of the microfluidic chamber.
Clean the glass slide with compressed air. To bond the chip to the glass slide, place the chip and glass slide into the plasma oven with the sides to be bonded facing up. Generate plasma using the protocol specific to the used machine, then bond the chip to the glass by placing the activated surfaces onto each other and applying pressure evenly.
To prepare the tubing and chip for the experiment, cut the tubing at a 45 degree angle and attach one needle to each of the tubings. Place the tubing with needles, the chip, and the plugs in a dish and sterilize them by exposing to ultraviolet light for approximately 15 minutes. To coat the chip with fibronectin, place the chip in a beaker containing PBS plus 1%penicillin or streptomycin at room temperature.
Flush the chip with PBS to remove air with a P200 pipette. Load a three milliliter syringe for each chamber of the chip. Attach the needle to the syringe containing fibronectin and connect the syringe to the syringe pump.
Flush tubing manually to remove air. Attach the outlet tubing to the outlet of the chip. Make sure to push the tubing all the way to the bottom and set a low flow rate to coat the chip.
Let the syringe pump run for at least two hours or overnight. When finished, stop the pump and cut off the tubing right after the needle. Prepare syringes filled with the medium for the experiment and degas both the culture medium and chip within PBS.
Try to flush out or suck up most of the air bubbles from the chip, then install syringes in the pumps and attach inlet tubing to the syringes. To load tissue onto the chip, dissect the most posterior tip of the tail. Flush the chip with culture medium with 25 micromolar HEPES to remove the PBS.
Load the tissue into the chip using a P200 pipette. After each tissue loading step, close the corresponding tissue loading inlet using a piece of PDMS-filled tubing. To assemble the microfluidic setup, attach tubing to the microfluidic chip without getting air bubbles inside.
To do so, ensure that there is a drop of medium present at the end of the tubing. Once all tubing is attached to the chip, take it out of the beaker and place it in a dish containing a wet tissue. Put the dish and approximately 1.5 meters of inlet tubing in an incubator for overnight culture.
Ensure a high humidity to prevent the formation of air bubbles during culture. Alternatively, dry the outside of the chip and place it in a microscope holder, then put the holder and approximately 1.5 meters of inlet tubing inside the incubation chamber of an inverted microscope for a live imaging experiment. After at least 20 minutes of constant flow, start the planned pumping for the experiment.
To entrain notch signaling in the segmenting mouse embryo, use a pumping program of 100 minutes medium and 30 minutes drug pulses repeated until the end of the experiment, typically for 24 hours. For real-time imaging, start imaging after at least 30 minutes. To confirm entrainment of signaling oscillations, multiple experiments are aligned using the timing of the drug pulses and visualized with the Cascade Blue dye.
Quantified oscillations can be detrended and displayed as mean and standard deviation or phases of the oscillations can be calculated. The phase relationship between oscillations of independent posterior embryo cultures to each other and to the external drug pulses can be analyzed. To confirm entrainment, one can for instance determine the period of the endogenous signaling oscillations using the Python-based program pyBOAT.
When applying pulses with a period of 130 minutes, notch signaling oscillations also show a period of 130 minutes. It is critical to prevent the evaporation of liquid during the microfluidic experiment. Otherwise, air bubbles will form in the chip that interfere with medium flow.
To prevent this, degas the medium and the chip and make sure that the humidity in the incubator is high enough. Using this method, signalling dynamics can be modulated and their effects can be analyzed by real-time imaging of fluorescent reporters, immunostainings, or in situ hybridization. Furthermore, the tissue can also be extracted from the chip for further analysis.
One can use this method to study the function of signaling oscillations in embryonic development. It was applied to demonstrate the importance of the phase shift between two oscillating signaling pathways for the segmentation of the mouse embryo. More generally, it can now be used to dissect the mechanism of dynamic signaling coding in multicellular systems.
