Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

All animal procedures were performed with the approval of the Vanderbilt University Medical Center Institutional Animal Care and Use Committee.

1. Mouse embryo collection and dissection

1. Mate 8-10 week old female MLC-2v-tdTomato^+/- mice with 8-10 week old male MLC-2v-tdTomato^+/- mice to obtain MLC-2v-tdTomato^+/+, MLC-2v-tdTomato^+/- and MLC-2v-tdTomato^-/- embryos.
2. Check the dams for vaginal plugs every morning. Noon on the day of vaginal plug detection is considered as E0.5.
   NOTE: Vaginal examination for detecting a vaginal plug should be performed in the morning (within 8-24 h after sexual activity), since a vaginal plug can be lost throughout the day.
3. Euthanize the pregnant dams at different days post coitum (e.g., E8.5, E10.5, and E12.5) using CO₂ inhalation followed by cervical dislocation.
4. Lay the mice supine and spray 70% ethanol on the abdomen of mice to avoid mouse hair contamination during dissection.
5. Open the abdominal cavity by incision of both the skin and abdominal wall using sharp surgical scissors and a forcep.
6. Locate bilateral uterine horns in the dorsal part of the abdominal cavity.
7. Separate the entire uterus by carefully cutting above the oviducts on both sides using sharp surgical scissors and a forcep.
8. Place the entire dissected uterus in a 10 cm Petri dish with ice-cold PBS and carefully separate each amniotic sac along the uterine horn using sharp surgical scissors and a forcep.
9. Transfer each embryo into individual wells of 6 well plate filled with ice-cold PBS using a transfer pipette.
10. Under a dissecting microscope, open up an amniotic sac and expose each embryo by cutting off the umbilical cord using sharp surgical scissors and a forcep in an individual well of a 6 well plate with ice-cold PBS.
11. Trim out extra-embryonic tissues as much as possible without damaging the embryo using sharp surgical scissors and a forcep.
    NOTE: Whole mount epifluorescent imaging of whole mouse embryo is usually performed before dissecting the developing heart as described below.
12. Cut the embryo head using sharp surgical scissors and a forcep and transfer to a 1.5 mL tube with 100 µL of buffer A (25 mM NaOH and 0.2 mM EDTA) for genotyping to correlate with the results of epifluorescent imaging.
13. Open the chest of the embryo using fine forceps, remove the heart away from the lungs and vasculature using sharp surgical scissors and forceps, and transfer the dissected embryonic heart into a well of a 12 well plate with PBS using a transfer pipette. All dissection procedures are completed under a dissecting microscope with a fiber optic microscope illuminator.
    NOTE: It was technically difficult to dissect out mouse embryonic hearts at E8.0 or E8.5, because of their extremely small size and fragile structure. Early embryonic hearts (i.e. E8.0 and E8.5) can be examined within the whole mount embryo without dissection.

2. Whole-mount epifluorescence imaging

1. Place the 12 well plate with mouse embryonic hearts under an epifluorescent dissecting microscope.
2. Under an epifluorescent dissecting microscope using fine forceps, position the embryonic heart such that developing ventricles are located close to the examiner.
3. Adjust focusing of the image using a 0.63x objective (zoom range between 3.15x and 18.9x) in bright field mode.
4. Take bright field exposures and capture multiple images. The images were usually obtained by one second exposure. However, exposure times may vary depending on illumination and camera specificationss and need to be optimized for each setup.
5. Turn off a fiber optic microscope illuminator, and set the filter for red fluorescence (Ex545 nm/Em 605 nm) to visualize tdTomato expression.
6. Re-adjust focusing of the image if necessary.
7. Adjust brightness and contrast, take red fluorescent exposures, and capture multiple images.
   NOTE: The following image setting was usually used: 1 s exposure time, 2x gain, 1.0 saturation, and 1.0 gamma correction. The optimal setting needs to be optimized for each experiment. Once the optimal setting is determined, the same setting needs to be used for an entire experiment.

3. Genotyping

1. Boil the samples from step 1.12 for 1 h at 100 °C.
2. Centrifuge for 2 min at 11,360 x g, transfer 20 µL of supernatant into a new 1.5 mL tube with 20 µL of buffer B (40 mM Tris HCl, pH 5.5) and mix them.
3. Take 4.5 µL of the mixed supernatant from step 3.2 as a DNA template, combine it with 0.5 µL of each of the specific forward and reverse primers (10 µM), 10 µL of pre-mixed polymerase and reaction buffer (2x) (see Table of Materials), and then add water to a total volume of 20 µL. Primer sequences are as below.
   F1: 5’-TACCCACGGAGAAGAGAAGGACT-3’
   R1: 5’-TGGACTTCTTGGAACTGACTCTGT-3’
   F2: 5’-ACGGCACGCTGATCTACAAGGT-3’
   R2: 5’-TTTGCGCACAGCCCTGGGAT-3’
4. Run a polymerase chain reaction (PCR) with the following PCR program (Table 1 and Table 2).
5. Run PCR samples and DNA ladder on a 1% agarose gel at 140 V in 1x TAE (Tris-acetate-EDTA) buffer (40 mM Tris-acetate and 1 mM EDTA) for 25 min. Use a 100 bp DNA ladder to estimate the size of the PCR bands.
6. Place the DNA gel on a UV transilluminator to identify the DNA bands and turn on the UV light.