The node and notochordal plate are essential organizers during mouse embryonic development. Today, we will describe two techniques used by developmental biologists to visualize and study these structures. In the first protocol, we will demonstrate how to perform scanning electro microscopy, SEM and in the second, whole mount immunofluorescence.
The node and notochordal plate are transiently present on the surface of the embryo at embryonic day E7.75, emphasis tiny cilia are projecting to the outside, which allow their identification and visualization by SEM. In both protocols, we will demonstrate critical steps in handling the relatively small mouse embryos and focus on how to transfer and route them. To begin, open the abdomen of a euthanized pregnant mouse through the skin and the mesenteries.
Using scissors and fine forceps, remove the uterus. Rinse the uterus briefly in distilled water and place it in a small petri dish containing PBS. Then, use a dissecting microscope and fine forceps to remove the uterine muscles to free the individual decidui.
Under the dissecting microscope, hold each decidua with a pair of forceps and use another pair to make a longitudinal, full thickness incision between the red part and the white part. Make superficial perforations vertically along the white part of the decidua, contiguous with the incision. Then, pull the decidua apart horizontally into two halves and carefully remove the embryo from the white part of the decidua.
After this, transfer the embryos to a new petri dish containing sterile filtered PBS. Remove Reichert's membrane from each embryo, starting at the ectoplacental cone. For future genotyping, remove a small piece of the yolk sac.
Under a chemical hood, transfer the embryos to a microcentrifuge tube containing EM grade fixitive composed of 2.5%glutaraldehyde and sterile filtered PBS. After this, remove the fixitive from the tube without disturbing the embryos. Wash the embryos three times in sterile, filtered PBS for 15 minutes at room temperature.
Dehydrate the embryos in an ethanol series for five minutes each, using 50%70%and 85%ethanol diluted in PBS. Wash three times in 100%ethanol. Store embryos in 100%ethanol at 20 degrees Celsius, or proceed to the next step.
After this, transfer the embroyos to an appropriate vessel and remove any remaining liquid. Add HMDS and ethanol at a ratio of one to one to the embryos for 30 minutes. Then, remove the liquid and add pure HMDS for 30 minutes.
Remove the liquid from the embryos with a pipette and allow them to dry for 30 minutes. Use a small brush and double sided tape to mount the dried embryos on SEM stubs, with the ventral sides up. Insert the stubs into a sputter coating machine for gold particle coating.
One of the most delicate steps in the SEM particle is mounting the embryos in the correct orientation onto the stub. Once the embryo lands on the tape, the orientation cannot be changed anymore. After this, place the coated stubs into an SEM microscope, apply a vacuum, and observe the embryonic nodes and notochordal plate cells with cilia.
After removing the embryos at E7.75, place them in PBS Tween in a petri dish on ice. Under a chemical hood, transfer the embryos to a microcentrifuge tube containing fixative solution. After this, remove the fixative from the tube, taking care not to touch the embryos.
Then, wash the embryos three times in PBS containing 0.2%Triton X-100 for 5 minutes at room temperature on a nutating shaker. Remove the last wash from the embryos and add blocking solution containing PBS Triton, with 10%heat inactivated serum. Block the embryos for at least two hours on a nutator at four degrees Celsius.
Next, remove the blocking and add approximately one milliliter of primary antibody diluted in blocking solution. Incubate the embryos overnight on a nutator at four degrees Celsius. Remove the primary antibody and save it for later use.
Then, rinse the embryos with PBS Triton twice. And wash them three times for 30 minutes on a nutator. Replace the wash with the diluted florescence conjugated secondary antibody against the primary antibody host species.
And incubate overnight on a nutator at four degrees Celsius. Then, remove the secondary antibody and rinse the embryos with PBS Triton twice. Before washing three times for 30 minutes with PBS Triton.
Replace the last wash with PBS Triton containing diluted florescence conjugated phalloidin and diluted DAPI and stain for one hour at room temperature. Then, rinse the embryos twice in PBS Triton. And wash them with PBS Triton for 30 minutes at room temperature.
After this, replace the PBS Triton with PBS and leave the embryos on ice. Then prepare clean, positively charged slides, cover slips and aqueous glycerol based mounting media. Place two pieces of clear tape 15 millimeters apart from each other on the slide.
Then, under the dissecting microscope, use a modified P200 pipette to move an embryo to the slide. Using fine forceps, make two full cuts on the lateral sides of the yolk sac to unfold the embryo. Then, position the embryo with the ventral side up.
Add 50 microliters of mounting media to the embryo. Then, add a dab of mounting media to the cover slip. Place it straddling the two pieces of tape, and lower it slowly onto the embryos using a bent needle.
Use an absorbent wipe to remove excess mounting media, taking care not to move the cover slip. Then, use a generous amount of nail polish to seal the sides of the cover slip. Finally, use a scanning confocal microscope to observe the embryos.
It's critical to not move the cover slip after mounting it over the embryos, because it can distort them for 3D visualization. Using scanning electron microscopy, the formation of the node in wild-type and strip one mutant mouse embryos at E7.75 was observed. Unlike the pit-shaped node in the wild-type, the mutant embryos displayed a flattened and a regular node and nodalcordal plate.
Whole now to immunofluorescence was used to study the node and notochordal plate formation defects in Strip 1 mutant embryos at the cellular level. The data showed that they were abnormal and stunted in the mutant embryos. The node and nodalcortal plate are only transiently present on the surface of the embryo, therefore timing is essential for the success of SEM.
For example, two to four somite embryos are good for SEM analysis of a mature node with long cilia. The purity reagents is also essential for the success of these techniques, especially in probing the ultrastructure by SEM. Tiny impurities that stick to the embryo usually result in huge artifacts.
We believe that these two techniques give complementary information on the structure of the node and nodalcortal plate during normal development and in mutants that show defects in the formation of these structures.
