Method of Studying Palatal Fusion using Static Organ Culture

The overall goal of this procedure is to describe a method of palatal dissection and static organ culture. This is accomplished by first collecting the mouse embryos. In the second step, the mandible is removed and the palate is dissected.

In the final step, the palatal shelves are seated in a static organ culture. Ultimately, the effects of specific drugs of interest on palatal fusion can be evaluated by assessing the mean fusion score of the culture. This method can help answer key questions about palate development, such as what factors are involved during The fusion process.

Though this method can provide insight into mammalian mouse palatal fusion. It can also be applied to other model organisms such as chicken palates, Maria Serrano and Israel Ibrahim. My graduate students will be demonstrating this procedure to harvest the mouse embryos.

Begin by cleaning the ventral abdominal surface of a 13.5 days post equus pregnant mouse with 70%ethanol. Then using sharp, blunt operating scissors and micro dissecting forceps, open the abdominal cavity. Next, lift the entire uterus and use light operating scissors to cut at the uterine body and at the tips of the uterine horns to separate the uterus from the body cavity, using ethanol sterilized sharp, blunt operating scissors and micro dissecting forceps.

Carefully make an initial small opening in the yolk sack. Then widen the opening and gently push on the yolk sack tissue to exteriorize the embryos, followed by the immediate transfer of the embryos to a Petri dish containing cold PBS. To dissect the palates one at a time, transfer the embryos into a new Petri dish with fresh PBS under a dissecting microscope.

After removing the head of the first embryo, use forceps to carefully grasp the mouse cranium above the eyes and make two incisions on both sides of the lip line. Remove the mandible and the tongue. Then to isolate the maxillary process region, make a cut at eye level and remove the brain eyes, nasal septum, and adjoining tissue.

Place the palatal shelves nasal side up and remove the nasal septum and surrounding tissue, keeping the primary palate attached to maintain the orientation. Next, using a spoon spatula very carefully transfer the palatal shelves nasal side down onto a prepared filter on the top of a grid placed within a culture dish of media. Adjust the media volume to facilitate a proper air media interface.

Then under the dissecting microscope, cut the triangular membrane where the anterior part of the palate is located. Orient the tissue in the anterior portion and use tweezers to place the palatal shelves close to each other. Finally, culture up to three pallets in the same organ culture dish using BGJB culture medium at 37 degrees Celsius and 5%carbon dioxide for 72 hours, changing the medium every 24 hours.

In this experiment, the teratogenic effective nicotine on palatal fusion in vitro using two different strains of mice, was investigated in the palates isolated from CD one mice. The control treated palatal shelves continued the fusion in a time dependent manner while the nicotine treated palates exhibited a delayed fusion, particularly at the six millimolar nicotine concentration in the C 57 mouse palettes. The palatable shelves in the control group also fused in a time dependent manner at 48 hours, however, the palatal shelves from these mouse palettes treated with only 0.6 millimolar of nicotine exhibited an impaired fusion compared to the pallets from control animals suggesting a higher degree of sensitivity for C 57 mice to the effects of nicotine treatment compared to the CD one animals.

While attending this method, it is very important to remember to manipulate the tissue carefully. After watching this video, you should have a good understanding of how to perform static organ culture.