Co-localization of Cell Lineage Markers and the Tomato Signal

The overall goal of this cell lineage tracing technique and subsequent immunofluorescence is to investigate cell biology and cell fate in vivo. This method can help answer key questions in the cell biology and tissue development field regarding the origin of cells that form bone during endochondrogenesis. The main advantage of this technique is to display the number, location and differentiation status of the progeny of the founder cell simultaneously, thus broadening the application of lineage tracing.

Though this method provides insight into the transformation from chondrocytes into osteogenic cells during normal development, it can also be applied to other tissues and disease models such as cell transformation in a tumor. Generally, individuals new to this method will struggle because frozen sectioning is difficult and the section can easily peel off during staining. We first had the idea for this method when we intended to demonstrate the differentiation status of osteogenic cells transformed from chrondocytes in trabecular bone.

In order to trace the lineage of hypertrophic chrondocytes in the mouse, first breed the animals as described in the accompanying text protocol. This allows for cell lineage tracing in conjunction with fluorescent immunohistochemistry to define the chrondocyte's fate. Once the desired mouse has been bred, remove it from its cage.

Then use the left thumb and index finger to grab the skin on the back of the mouse and turn it over exposing the abdomen. With the right hand, pick up a 0.5 milliliter syringe equipped with a 28.5 gauge needle and loaded with Tamoxifen. Place the syringe parallel to the hind legs and insert it into the peritoneum of the mouse.

Inject the solution into the side of the hypogastrium avoiding the liver and bladder in order to activate the Cre system at a favorable time point. After two weeks, prepare the mouse for fixation by properly anesthetizing it and checking the mouse's pedal reflex. Then use tape to fix the four legs of the mouse on a board and expose the abdomen.

Next, saturate the mouse's abdomen with 70%ethanol to sterilize the area and then make an incision from the lower abdomen to the neck along the middle line. Now, pinch and simultaneously pull the skin to the lateral sides to reveal the peritoneal membrane and use dissection scissors to make a longitudinal excision. Cut off and remove the front ribs to expose the heart.

Then in a class I biosafety cabinet, use a syringe loaded with 4%Paraformaldehyde and equipped with a 22 gauge needle to puncture the wall of the heart and enter the left ventricle. While holding the syringe, cut a slot in the right auricle. Then slowly inject the solution into the left ventricle in order to perfuse the cardiovascular system while the blood flushes out of the cut from the right auricle.

After removing the mouse's skin and organs, place the whole body into a 50 milliliter polypropylene centrifuge tube containing 40 milliliters of 4%Paraformaldehyde and fix the animal overnight at 4 degrees Celsius. The next day, transfer the mouse to a Petri dish in the class I biosafety. Use dissection scissors and number 3 and number 5 forceps to carefully remove a mandible and a hind leg from the body.

Next, remove the muscles and tendons on the surface of the hind leg and mandible. To speed the decalcification, cut the mandible into two pieces at the distal region of the third molar. Put the parts that include the condyle and condylar process into 40 milliliters of 10%EDTA to decalcify.

Similarly, cut the femur and tibia in the midshaft to expose the bone marrow cavity in order to accelerate decalcification. Once decalcified, transfer the samples into a 50 milliliter tube containing a 15%sucrose solution to dehydrate the condyle and hind leg. Further dehydrate the samples by transferring them into a fresh 50 milliliter tube containing a 30%sucrose solution.

Then embed the condyle and hind leg in OCT compound along the sagittal plane respectively. Leave the samples in the cryosection machine until the OCT freezes. Next, use a drop of fresh OCT compound to mount the tissue block on the cutting plate.

Wait for approximately 15 minutes before starting to cut the sample in order to ensure that the connection is stable. When ready to begin cutting sections, set the cut depth to 10 microns and section the condyle and hind leg, collecting the sections on slides and storing them at negative 20 degrees Celsius. Just before staining, incubate the slide in a dry 37 degrees Celsius chamber for 10 minutes to remove the water and adhere the section to the slide.

Then wash the slides twice with distilled water for five minutes. Wipe off the water around each section and use a hydrophobic barrier pen to circle the sections. Next, add a drop of DAPI mounting solution into the circle and carefully lay down the coverslip.

For Runx2 and DMP1 immunohistochemistry, prepare the slides as previously shown, washing them twice with distilled water for five minutes. However, instead of adding DAPI mounting solution right away, first treat the sections with hyaluronidase in a humid chamber at 37 degrees Celsius for 30 minutes. Next, wash the sections with PBST three times and incubate them for one hour at room temperature in an appropriate blocking solution.

Then remove the blocking solution and add either rabbit anti-mouse Runx2 or rabbit anti-mouse DMP1 to each slide. Incubate the slides and their primary antibiodies at four degrees Celsius overnight. The next day, wash the sections three times with PBS and then add the secondary antibody solution.

Incubate the slides in the dark at room temperature for two hours. Following incubation, wash the sections three times in PBS and then add a drop of DAPI mounting solution before adding the coverslip. Capture fluorescent cell images using a confocal microscope at wavelengths ranging from 488 micrometers to 561 micrometers.

Take multiple stacked images at 200 hertz using 10X, 20X and 63X lenses. A Tamoxifen injection at two weeks of age activates the red tomato reporter in all chondrocytes and their daughter cells. In addition, all collagen 1 expressing bone cells are green fluorescing.

Yellow cells indicate the presence of chondrocyte-derived bone cells. These results provided strong evidence that these bone cells were derived from chondrocytes. Similar results were also observed during long bone development where the majority of bone cells were derived from chondrocytes in both the epiphysis and the metaphysis.

In the trabeculae, near the cartilage bone interface of three-week-old mice, red and some yellow fluorescing cells were predominant, while green fluorescing cells were scarce. In a slightly more inferior area, the majority of the cells were fluorescing yellow with slightly fewer fluorescing red. Green fluorescing cells appear to dominate only in the most inferior area of the condylar process.

This trend indicates that condylar growth is mostly contributed to transformed bone cells from hypertrophic chondrocytes. The distribution of the red, yellow and green fluorescing cells in the condylar process is also consistent with the inferior to superior direction of condylar growth. In addition to crossing alone, the co-application of immunofluorescent staining and cell lineage tracing enables the tracking of cell differentiation.

Shown here are Runx2 and DMP1 labeled sections showing the presence of immature osteoblasts and mature chondrocyte-derived osteocytes. While attempting this procedure, it is important to remember to select appropriate Cre lines in order to verify cell fate. In the future, investigators can attempt to perform immunofluorescence with two different antibodies over the tomato signal background to obtain more information in one section or use in situ immunofluorescence to decrease nonspecific staining.

After its development, this technique paved the way for researchers in the field of cell biology and tissue development to explore the unknown differentiation capacity of cells. After watching this video, you should have a good understanding of how to combine the cell lineage tracing technique with immunofluorescence to investigate the cell fate and cell biology in vivo.