This protocol allows the analysis of primitive endocardium in a planar orientation. We can analyze the distribution of endocardial cells, the anisotropy of other injunctions, and describe the shape of a single endocardial cell during valve development. Compared to the classical tissue sections, the en face imaging of the inner valve regenerative region allows the analysis of planar cell polarity and dynamics of endocardial cells during valve development in intact embryos.
This method can be used to deepen the knowledge of planar cell polarity during cardio development. Begin by placing the uterus excised from euthanized pregnant mice in a Petri dish containing ice-cold PBT. Remove the individual deciduye from the uterus under a dissecting microscope using fine forceps.
Using fine tweezers, make an incision in the white part of the decidua, which is where the embryo is. Remove it gently, avoid pulling, and transfer the embryos to a new Petri dish with fresh PBT using a P-1000 with the tip cut off, leaving a diameter large enough for an embryo to pass through without breaking. For genotyping, take a little piece of the yolk sac approximately 0.5 square millimeters or a piece of the tail from the posterior end, counting five to 10 somites toward the head and digest it in 100 milliliters of the appropriate buffer.
Under the fume hood, transfer the embryos to ice cold 4%PFA in a two-milliliter microcentrifuge tube at four degrees Celsius. Fix the embryos from two hours to overnight at four degree Celsius on a nutator. Under the fume hood, remove the 4%PFA fixative from the microcentrifuge tubes without touching the embryos.
Discard the PFA in an appropriate container. Wash the embryos five times in cold PBT for 10 minutes at room temperature. Using fine forceps, remove the heart and transfer it to a new 1.5-milliliter microcentrifuge tube with fresh PBT.
Replace PBT with PBS Triton and wash the samples thrice, five minutes each, at room temperature on an orbital shaker. Replace PBS Triton with a blocking solution containing PBS Triton with 10%heat-inactivated bovine serum. Incubate from two hours to overnight at four degree Celsius on an orbital shaker.
Replace the blocking solution with blocking solution containing primary antibody at the desired concentration. Incubate overnight on an orbital shaker at four degrees Celsius. After overnight incubation at four degrees Celsius, incubate the samples for 1.5 hours at room temperature on an orbital shaker.
This step is crucial to increase the signal-to-noise ratio. Remove and keep the primary antibody solution at four degrees Celsius, up to three future experiments. Add sodium azide to a final concentration of 0.02%for best conservation.
Then, wash the embryos thrice with PBS Triton for three minutes each. Wash the embryos with PBS Triton thrice for 30 minutes and keep them on an orbital shaker at four degrees Celsius. After the last wash, add one milliliter of blocking solution containing fluorescence-conjugated secondary antibody against the primary antibody host species.
Then add DAPI to the solution and incubate the embryos overnight on an orbital shaker at four degrees Celsius. After the overnight incubation at four degrees Celsius, incubate the embryos for 1.5 hours at room temperature on an orbital shaker. This step is essential to increase the signal-to-noise ratio.
Repeat the PBS Triton wash steps as mentioned in the text manuscript. Put the hearts on a 35-millimeter Petri dish containing PBS. Using a tungsten wire and fine forceps, isolate the atrioventricular canal or the outflow tract and cut them longitudinally.
Prepare cover slips, slides, forceps, a P-200 pipette with the appropriate tips, a paper towel, and any aqueous glycerol-based mounting media for fluorescence without DAPI. Stick two strips of tape on a slide separated by 0.3 to 0.6 centimeters to create a 3D room space that will allow the samples to conserve their original shape without squashing them. Then, using approximately 20 microliters of buffer, carefully drop the samples onto the slide between the tape strips using a cut P-200 pipette tip.
Use a dissecting microscope to increase the accuracy. Using fine forceps, place the samples with the endocardium facing up and the myocardium down on the slide. Remove excess PBS with a paper towel and avoid touching the sample.
Then, dry the samples for one to two minutes at room temperature to make the samples stick to the slide. Add 40 microliters of aqueous-based mounting media to the tissue. Place the cover slip over the two pieces of tape and lower it slowly onto the tissue with a needle or forceps.
Avoid creating air bubbles. Seal the cover slip using one drop of nail polish on each vertex of the cover slip. Once the nail polish is dry, clean the excess mounting media from the sides of the cover slip by using an absorbent paper towel.
Avoid moving the cover slip. Add nail polish along the cover slip edges to fully seal it, preventing mounting media evaporation. Using an upright or an inverted confocal microscope, take images at 10X magnification for general view and at 63X with 3X zoom for detailed images.
The cell shape of the endocardium was analyzed during the formation of the valves at a cellular resolution. Single cells or clones of a few cells expressed GFP in the endocardium. The GFP expression and combination with VE-cadherin subcellular localization was an effective approach to study the relationship between AJ dynamics and filopodia formation and pre-EMT cells, since these cells developed membrane protrusions as AJs dissolve.
Differences in the intensity of VE-cadherin staining in the endocardium indicated the presence of anisotropic contractility in the embryonic endocardium of the atrioventricular canal at pre-valvular stages. The whole heart was stained with VE-cadherin antibody at E8.5 and E9.5. At E8.5, the endocardium of the atrioventricular canal tended to have a stable epithelial organization, and vertices formed by more than four cells were not detected.
At E9.5, vertices of up to six cells were observed forming rosette-like structures, similar to the cellular organization previously described in epithelial undergoing active cell rearrangements, suggesting that the atrioventricular canal endocardium was undergoing dynamic cellular rearrangement during valve development. A learning curve is necessary to become an expert in this technique. Moreover, it is highly recommended to use Sovereign forceps and tungsten wire.
Today, visualization of pre-valvular endocardium was limited to cross-sections. Our approach offers the possibility to study embryonic valvular endocardium behavior from a planar point of view.
