Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

To maintain the correct cellular organization during organ renewal, tissue approaching the cells move and dividing specific orientations and patterns. It is therefore important to understand the rules and regulations of these dynamic cellular events. The mouse incisor is an emerging model for adult stem cell research, and many genetic tools are available in the system to study gene functions, and to label cells for lineage tracing.

Most studies to date use adult dental samples collected and fixed its specific time points, and this prevents us from examining life cell behavior. In this protocol, we describe a method to track lifestyles in the adult mouse incisor, and this technique will enable future studies that aim to understand how dynamic cell behaviors contribute to the renewal and repairment of dental tissues. To begin, turn the euthanized mouse over so its ventral side faces up and the mandibles are easily accessible.

Secure the mouse's head gently between the thumb and the index finger. Using a razor blade starting from the lower lip toward the neckline, make a mid-sagittal incision on the lower jaw skin. During the incision, use the thumb and index finger to open the cut skin, exposing the underneath muscles and jawbone.

Next, sever the masseter muscles on the buccal side of the lower jaw. Sever the mylohyoid muscles along the inner side of the mandible and remove muscle attachments. Make another incision at the mandibular synthesis, connecting the two hemimandibles, separating them into the left and right halves.

Wedge the razor blade between the mandibular condyle and the temporal mandibular joint to carefully dissect out the hemimandible from the rest of the head. Immediately transfer the dissected mandibles to a Petri dish containing pre-warmed dissection media. Next, under a bright field dissection microscope, use a number 15 surgical blade to remove muscle tissues.

Identify the oval region of the mandible that covers the incisor socket and houses the apical portion of the incisor. Position the mandibles such that the inner or lingual surface is facing upward. While holding the mandible with a pair of serrated forceps, using a number 15 surgical blade, shave off the overlying membrane bone from the condyle towards the molars to generate a window at the oval.

This exposes the soft tissue of the apical incisor on the inner surface. Next, turn the mandible so the outer or buccal surface faces upwards. As demonstrated for the inner mandible, generate a window at the oval region on the outer mandible.

Using a pair of forceps, pick away the remaining bone fragments at the edge, ensuring that the apical end of the tooth is visible from both sides. To isolate the entire tooth, make a clean cut at a plane immediately adjacent to the apical incisor to remove the condylar process. Then make a second cut just posterior to the third molar but dorsal to the incisor, without damaging the tooth, to remove the coronoid process.

Serially cut from the tip of the angular process towards the incisor to gradually remove the ventral mandible. Next, cut away the alveolar bone with molars and any remaining bones that are still attached to the incisor. Transfer the fully dissected incisor to a dish containing warmed dissection media.

Turn on the fluorescent microscope and use the fluorescent signal from tissues to guide the removal of periodontium. Position the inner incisor on its lingual side, and using serrated forceps, hold the tooth in place. Then using a pair of number 5 fine forceps, start tucking on the periodontal tissues covering the apical incisor and the cervical loop region.

Carefully peel off periodontal tissues from the apical bud such that the lateral side of the cervical loop is visible. To begin, add a 500 microliters of warm culture gel to a well in a 24-well plate. Quickly transfer the dissected whole incisors isolated from the mouse to the well.

Swirl the plate a few times to rinse the incisors. Add 400 microliters of warm culture gel to a culture dish and transfer the rinsed incisors to the dish. Orient the incisor to position the cervical loop region at the center of the dish and adjust the tilt of the apical incisor for the desired imaging plane.

Once the gel is set, using a pair of fine forceps, remove any gel on top of the region of interest. Add approximately 150 microliters of warm culture media against the edge of the dish to cover the sample. Place the explant culture at 37 degrees Celsius to allow the tissue to settle.

After one hour, turn on the microscope and the two-photon laser. Secure the culture dish to the stage adapter and place the perfusion adapter ring on top. Connect the stage adapter to a temperature controller to maintain the culture at 37 degrees Celsius.

Connect the inlet and the outlet of the adapter ring to a microperfusion pump. Set the perfusion speed to 20 and initiate the perfusion of the medium over the sample. Place an ACBR on top of the adapter ring and raise the stage so the objective fits through the ACBR to make contact with the culture medium.

Turn the laser wavelength to 920 nanometers to visualize GFP. After locating the sample through an eyepiece, visualize it with a microscope software, then set up Z-step size of four micrometers and a time interval of five minutes for 14 hours. Initiate the time-lapse imaging and save the subsequent vials for downstream data analysis.

In time-lapsed microscopy of the k14-Cre, R26-rtTA, tetO-H2B-GFP cervical loop, the H2B-GFP signals were predominantly observed in the trans-amplifying region of the cervical loop, indicating active cell divisions. Further, the condensation and alignment of chromosomes at the metaphase plate in mitotic cells followed by their segregation during antiphase and into two daughter cells was observed. Most cell divisions were perpendicular or at an oblique angle relative to the basement membrane, with fewer horizontal divisions parallel to the basement membrane.

Cell division events were also apparent in K14-Cre, R26-mT/mG cervical loops where all epithelial cell membranes were labeled green. Mitotic cells were identified by their cell rounding and subsequent cytokinesis. In K14-Cre, R26-mT/mG cervical loops, both horizontal and vertical cell divisions were also observed.