Using this method we can directly exam heart tube formation, looping, and full chamber formation without further experimental manipulations of the mouse embryo. Although, we use MLC-2v-tdTomato mice as an example, this simple method can be widely used with other fluorescent reporter mouse lines. The main advantage of this protocol is that it is very simple and does not require any complex techniques or equipment.
It can be performed at regular lab setting. After mating female with male mice, both MLC-2v-tdTomato 8 to 10 weeks old, check the dams for vaginal plugs every morning. Lay the pregnant dams, euthanized at different days post coitum, in supine position and spray the abdomen with 70%ethanol.
Use sharp surgical scissors and forceps to open the abdominal cavity by incision of both the skin and abdominal wall. After locating bilateral uterine horns in the dorsal part of the abdominal cavity, separate the entire uterus using sharp surgical scissors and forceps to carefully cut above the oviducts on both sides. Place the entire dissected uterus in a 10 centimeter petri dish with ice cold PBS.
Using sharp surgical scissors and forceps, carefully separate each amniotic sac along the uterine horn. Use tweezers to transfer each embryo into a 35 millimeter culture plate filled with ice cold PBS. Using sharp surgical scissors and forceps, open an amniotic sac and expose each embryo by cutting off the umbilical cord and then trim out extra embryonic tissues as much as possible without damaging the embryos.
Under a dissecting microscope with a fiber optic microscope illuminator, cut off the embryo's head and transfer it to a 1.5 milliliter tube with 100 microliters of buffer A for later genotyping to correlate with the results of epifluorescent imaging. Using fine forceps, open the chest of the embryo. With sharp surgical scissors and forceps, remove the heart away from the lungs and vasculature.
Use tweezers to transfer the dissected embryonic heart into a 35 millimeter culture plate with PBS. Place the plate with mouse embryonic hearts under an epifluorescent dissecting microscope. Use fine forceps to position the embryonic hearts such that developing ventricles are facing the examiner.
Adjust focus using a 0.63x objective in bright-field mode. Take bright-field exposures and capture multiple images. To visualize tdTomato expression, turn off a fiber optic microscope illuminator and set the filter for red fluorescence.
Readjust focusing of the image if necessary, adjust brightness and contrast, take red-fluorescent exposures, and capture multiple images. For embryo genotyping, boil previously isolated head samples in buffer A at 100 degrees Celsius for 30 minutes. Centrifuge for two minutes at 11, 360 times G.Transfer 20 microliters of supernatant into a new 1.5 milliliter tube with 20 microliters of buffer B and mix.
Use 4.5 microliters of the mixed supernatant as a DNA template and combine it with 0.5 microliters of each of the 10 micromolar-specific forward and reversed primers, 10 microliters of 2x-premixed polymerase and reaction buffer and then add water to a total volume of 20 microliters. Run a PCR as described in the manuscript. Load the completed reaction in 100 base pair DNA ladder on a 1%agarose gel and run at 140 volts in 1x TAE buffer for 25 minutes.
Place the completed gel on a UV transilluminator and turn on the UV light to identify the DNA bands. MLC-2v is considered to be the earliest marker for ventricular chamber specification during heart development. In MLC-2v-tdTomato reporter knock-in mice, relatively weak expression of tdTomato in the linear heart tube at E8.0 becomes stronger at E8.5 as shown using whole-mount epiflourescent imaging of the embryos.
Using whole-mount epiflourescent imaging of the dissected heart, ventricular chamber formation during mouse heart development can be easily determined. In the dissected heart from a whole mouse embryo at E10.5, MLC-2v-tdTomato reporter expression was shown in the ventricular portion of the heart and not in the inflow tract, the outflow tract, or the future atria. At E12.5 to E13.5, tdTomato reporter is exclusively expressed in the ventricles of the four-chambered heart.
The similar ventricular-specific expression pattern was shown in the dissected mouse embryo at E16.5. After whole imaging, genotype of the embryos was determined using two sets of primers for PCR genotyping, confirming homozygous embryos carrying the tdTomato knock-in allele, heterozygous, and the wild-type embryos. This method is pretty straightforward and simple to perform.
The critical step is dissecting embryonic hearts. Understanding cardiac anatomy during heart development is necessary to successfully isolate heart from the whole embryo.
