We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.
The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.
During development, endochondral bone formation accounts for over 80% of the skeletal volume. It is widely believed that it begins with the apoptosis of hypertrophic chondrocytes. Subsequently, the cells from the underlying bone marrow invade and initiate angiogenesis, followed by new bone deposition by bone marrow- and periosteum-derived cells1,2. The cell fate of hypertrophic chondrocytes (HCs), however, has been an issue of debate for decades3. Initially, HCs were considered to be the end of the chondrocyte differentiation pathway, and apoptosis was generally thought to be the ultimate fate of HCs. Now, some researchers suggest that at least some HCs could survive and contribute to endochondral bone formation. Although they proposed that growth plate chondrocytes had the ability to transdifferentiate into osteoblasts based on ultrastructure, immunohistochemical staining, and in vitro studies46, none of these methods were definitive in demonstrating chondrocyte contribution to the osteoblast lineage.
The cell lineage tracing technique provides a more rigorous way to study cell fate. Briefly speaking, a recombinase enzyme, which is only expressed in a specific type of cell, stimulates the expression of the reporter gene. In this way, this type of cell and their descendants are permanently labeled7. The Cre-loxP system is commonly used in lineage tracing. Cre (the recombinase enzyme) will excise the STOP sequence between the two loxP sites and activate the reporter in a specific cell line (Figure 1A). In some cases, the investigator can choose a favorable time point to activate Cre by using a drug, such as tamoxifen, causing Cre to fuse to a modified form of the estrogen receptor (CreERT2)8. Fluorescent reporters have become the standard in lineage tracing experiments because they dramatically reduce the complexity and improve the accuracy and efficiency of cell fate tracing8,9. tdTomato is becoming the best choice among fluorescent reporters since it has the brightest fluorescent protein and the strongest epifluorescence, making it easily visualized7 (Figure 1A).
By using the Rosa26tdTomato lineage tracing system, our group and other investigators have shown that HCs can change their phenotype into bone cells during development10-14. To accomplish this, we developed two sets of tracing combinations with Rosa26tdtomato (ubiquitous expression in all cells)/Cre (specific to chondrocytes) mice: 2.3Col1a1-GFP (specific to osteoblasts) and immunofluorescence (specific to bone cells). The data demonstrate that both methods are viable ways to study cell fate in vivo.
All protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Texas A&M University College of Dentistry.
1. Animal Breeding
2. Material Preparation
3. Slide Preparation for Confocal Microscopy (Cell Lineage Tracing Only, No Combination with Immunofluorescence)
4. Immunohistochemical Staining for Runx2 and DMP1
NOTE: The IgG control is necessary for immunohistochemical staining to avoid false-positive signals. The staining for the experimental and control groups needs to be performed simultaneously.
5. Confocal Microscopy
Chondrocytes Directly Transform into Bone Cells (Osteoblasts and Osteocytes) in the Mandibular Condyle and Long Bone.
Aggrecan, a critical gene for chondrogenesis, is mainly expressed in early and mature chondrocytes18. As a result, the injection of tamoxifen at 2 weeks of age in Agg-CreERT2; Rosa26tdTomato mice activated the red-tomato reporter in all chondrocytes and their daughter cells. The 2.3Col1a1-GFP line gave rise to a green-fluorescing color in collagen 1-expressing bone cells, specifically osteoblasts and pre-osteocytes. The yellow color (red combined with green) indicated the presence of chondrocyte-derived bone cells that expressed the collagen 1 gene. In Figure 2A, most of the bone cells in the condylar process beneath the cartilage were red or yellow (a red cell that had begun to secrete green-fluorescing Col1a1, thereby appearing yellow when the two fluorophores were superimposed). 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 (Figure 2B) and the metaphysis (Figure 2C).
To further investigate these results, we crossed Col10al-Cre mice with Rosa26tdTomato; 2.3Col1a1-GFP mice. Col10a1 is a highly specific marker for HCs, expressed only in HCs and their descendants. Moreover, Col10a1-Cre is non-inducible, which reflects cell differentiation from the very beginning of Col10a1 expression (at E14.5). In the trabeculae near the cartilage-bone interface (superior level) of 3-week-old mice, red and some yellow fluorescing cells were predominant, while green fluorescing cells were scarce. In a slightly more inferior area (middle level), the majority of the cells were fluorescing yellow, with slightly fewer fluorescing red. Green fluorescing cells appeared to dominate only in the most inferior area of the condylar process (inferior level) (Figure 3).This trend indicates that condylar growth is mostly contributed to by transformed bone cells from HCs. The distribution of the red and green fluorescing cells in the condylar process is also consistent with the inferior-to-superior direction of condylar growth.
The cell lineage tracing technique clearly demonstrates that apoptosis is not the only fate for HCs. Both the Agg-CreERT2 and Col10a1-Cre lines indicate that chondrocyte-derived bone cells are the major source for bone development in the condylar process and in the epiphysis and metaphysis in long bone.
Co-application of Immunofluorescent Staining and Cell Lineage Tracing Enables the Tracking of Cell Differentiation.
The cell lineage tracing technique can also be combined with fluorescent immunohistochemistry to determine the cell type by the detection of a cell-specific marker. This method has several advantages for the study of cell fate. First, the researcher can still define the cell characteristics by selecting appropriate markers, even without the specific GFP mouse line, which broadens the application for the cell lineage tracing technique. Secondly, it simplifies the generation of compound mice. For instance, we only need to generate Agg-CreERT2; Rosa26tdTomato compound mice to produce the red color after activating Cre (at postnatal day 3), instead of creating Agg-CreERT2; 2.3Col1a1-GFP; Rosa26tdTomato mice, which requires more crosses.
In this study, we chose two antibodies for immunofluorescent staining. One was against Runx2, a critical transcriptional factor during osteoblast maturation (early stage of osteogenic cells); the other was against DMP1, expressed in mature osteocytes (late stage of osteogenic cells). In Figure 4, the green fluorescence represents the Runx2 or DMP1 antibody expression in the 2-week-old condylar process. In Figure 4A, three types of colored cells in the subchondral bone are present. The yellow color (superimposition of red and green fluorescence) in the nucleus represents the chondrocyte-derived bone cells expressing Runx2, indicating that these cells were immature osteoblasts on the bone surface. In addition, there were red-fluorescing bone cells that did not express Runx2 (red fluorescence without superimposed green). These cells represent mature chondrocyte-derived bone cells in the bone matrix. The least common cells were those with only green-fluorescing nuclei, indicating non-chondrocyte-derived bone cells with Runx2 expression or a transformation that occurred before Cre activation. The image suggests that the red bone cells and those with yellow nuclei were the majority cell populations contributing to the condylar subchondral bone formation.
On the other hand, almost every red, chondrocyte-derived bone cell in the bone matrix had DMP1 staining around their cell bodies. Few osteocytes were positive for DMP1 but lacked red cell bodies (Figure 4B). There are also two possible explanations for these cells: either they are non-chondrocyte-derived bone cells, or they are chondrocyte-derived bone cells that transformed prior to the tamoxifen injection. In addition, no red bone cells on the surface of the bone expressed DMP1, indicating that they were not mature osteocytes.
Taken together, the data from the expression patterns for both early- (Runx2) and late-stage markers (DMP1) of osteogenic cells were consistent with the previous outcome using Cre combined with 2.3Col1a1-GFP. These data demonstrate that the combination of immunofluorescence and Rosa26tdTomato tracing is a good way to study cell fate. More importantly, investigators can distinguish the cell type, identify the differentiation stage, and observe the transformed cell numbers simultaneously by using immunofluorescence with different markers, which provides more information than Rosa26tdTomato tracing alone.
Figure 1. The Mechanism for Cell Lineage Tracing and the Generation of the Compound Mice. A)The Cre-loxP system is commonly used in lineage tracing. Cre excises the STOP sequence between the two loxP sites, and the tdTomato protein (fluorescing red) is permanently expressed in the specific cell line. B) Three animal models are mentioned in this paper. We used 2.3Col1a1-GFP; Rosa26tdTomato mice and crossed them with Col10a1-Cre mice. Col10a1-Cre is non-inducible, which reflects the cell differentiation from the very beginning of Col10a1 expression (at E14.5). C) Aggrecan-Cre ERT2(Agg-CreERT2) is another Cre line also crossed with 2.3Col1a1-GFP; Rosa26tdTomato mice. Cre was activated at 2 weeks of age by a tamoxifen injection (Tm: tamoxifen). D) In order to combine the immunofluorescence with cell lineage tracing, we generated Agg-CreERT2; Rosa26tdTomato mice, activated Cre at postnatal day 3, and performed the Runx2 and DMP1 immunofluorescence (Tm: tamoxifen). Please click here to view a larger version of this figure.
Figure 2. The Transformation from Chondrocytes to Bone Cells (Osteoblasts and Osteocytes) in the Mandibular Condyle and Long Bone Using Agg-CreERT2; 2.3Col1a1-GFP; Rosa26tdTomato Compound Mice. A) Cre was activated by tamoxifen on postnatal day 14, and the mice were sacrificed at 4 weeks old. There were three colored cells in the condylar process: pure red (chondrocyte-derived bone cells), yellow (red combined with green, indicating chondrocyte-derived bone cells that expressed the collagen 1 gene), and pure green (non-chondrocyte derived bone cells with the collagen 1 gene). Most of the bone cells in the condylar process beneath the cartilage were yellow or pure red (white arrows), while few bone cells were green (blue arrows). These data provide strong evidence that chondrocytes directly transform into bone cells and contribute to the formation of the condylar process during development (Tm: tamoxifen; C: cartilage; B: bone). B, C) The majority of bone cells in the epiphysis and metaphysis were chondrocyte-derived (white arrow: chondrocyte-transformed bone cells in yellow or pure red; blue arrow: non-chondrocyte-derived bone cells in pure green; AC: articular cartilage; GP: growth plate). Please click here to view a larger version of this figure.
Figure 3. The Transformation from Chondrocytes to Bone Cells in the Mandibular Condyle Using Col10al-Cre; 2.3Col1a1-GFP; Rosa26tdTomato Compound Mice. A) In the condylar process from 3-week-old mice, green bone cells were a distinct minority in the trabeculae near the cartilage-bone interface (superior level, a1), whereas red and some yellow cells were predominant. In the middle level (a2), yellow cells were in the majority, with slightly fewer red cells. Green cells appeared to be in the majority only in the most inferior area (a3). Please click here to view a larger version of this figure.
Figure 4. The Co-localization of two Markers for Osteogenic Cells with the Lineage-tracing Background in the Condylar Process Using 2-week-old Agg-CreERT2; Rosa26tdTomato Compound Mice. A) The yellow color in the nuclei represents chondrocyte-derived bone cells expressing Runx2 (white arrows) on the surface of the trabecular bone beneath the condyle cartilage. The pure red bone cells without Runx2 expression represent mature chondrocyte-derived bone cells in the bone matrix (yellow arrows). The fewest cells were those carrying only green nuclei, representing either non-chondrocyte-derived bone cells or transformed bone cells from chondrocytes before Cre activation (Tm: tamoxifen). B) In the trabecular bone beneath the condyle cartilage, almost every red chondrocyte-derived bone cell in the bone matrix carried DMP1 staining around their cell bodies (white arrows). Only a few of the osteocytes were positive for DMP1 but lacked red color in their cell bodies (yellow arrows). There are two possibilities for these cells: non-chondrocyte-derived bone cells or chondrocyte-derived bone cells arising before tamoxifen injection. Please click here to view a larger version of this figure.
Due to technological limitations, it is always difficult to investigate the behavior of cells in vivo. However, the cell lineage tracing technique is proving to be a powerful tool for studying cell biology7-9. In this study, we further improve this protocol by combining it with immunofluorescence. In this way, cell fate can be defined by multiple related markers, which broadens the application of lineage tracing. Moreover, this co-localization of immunofluorescence and tomato signal simultaneously displays the number of progeny of the founder cell, their location, and their differentiation status, providing more information than cell lineage tracing alone. In addition, the use of cell-specific markers can simplify the generation of compound mice, accelerating the investigation.
The specificity of Cre is crucial for the unequivocal attribution of lineage and the accuracy of the results20,21. It is very important to select the appropriate Cre lines to verify the cell fate. In this study, we chose Col10a1, because it is considered a specific marker for hypertrophic chondrocytes10,11, and aggrecan, because it is also a well-recognized marker for chondrocytes18. There are also good Cre models used in other fields. The Scleraxis-Cre (Scx-Cre) line, for example, is useful in tendon and ligament research since scleraxis is a basic helix-loop-helix transcription factor that is a marker for the tendon and ligament cell lineage22. Another Cre called DMP1-Cre is commonly used in skeleton- and tooth-related studies because DMP1 is highly expressed in odontoblasts and osteocytes23,24.
Compared with non-inducible Cre systems, the activation of inducible Cre can be restricted spatially and temporally20. However, some limitations should be considered when designing the experiment and interpreting the results of inducible Cre models. First, the dose of tamoxifen may change the efficiency of Cre activation and the number of labelled cells. Low doses will label the population of interest at a clonal density25; high doses may label the entire progenitor pool7,26. Thus, the dose must be chosen depending on the purpose of the experiment. Second, tamoxifen has potential toxicity, especially in high doses27,28. For this reason, it is better to decrease the dose to avoid late-term abortions when injecting during pregnancy29.
In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo. In the future, investigators can attempt to simultaneously perform immunofluorescence with two different antibodies over the tomato signal background. This method can show the expression pattern for two markers in one section, making it easier for the investigator to compare and analyze the results. Moreover, this co-application can be further improved by in situ immunofluorescence to decrease non-specific staining that may exist through conventional immunofluorescence.
The authors have nothing to disclose.
This study was supported by NIH grant DE025014 to JQF.
Tamoxifen | Sigma | T5648 | activate the Cre event |
Paraformaldehyde | Sigma | P6148 | fix the sample |
Ethylenediaminetetraacetic acid | Alfa Aesar | A10713 | decalcify the hard tissue |
Sucrose | Sigma | S0389 | dehydrate the tissue |
Hyaluronidase from bovine testes | Sigma | H4272 | retrieve antigen for immunochemical staining |
Bovine serum albumin | Sigma | A3059 | blocking solution |
primary antibody for Runx2 | Cell Signal | D1L7F | primary antibody for immunochemical staining |
primary antibody for DMP1 | provided by Dr. Chunlin Qin | primary antibody for immunochemical staining | |
anti-rabbit IgG | Sigma | 18140 | control for immunochemical staining |
secondary antibody | Invitrogen | A11008 | second antibody for immunochemical staining |
OCT | Tissue-Tek | 4583 | embed the sample for frozen section |
Tween 20 | Fisher Scientific | BP337 | PBST |
non-fluorescing antifade mountant | Life technologies | P36934 | mounting slides |
DAPI | Life technologies | P36931 | nuclear staining |
Hydrophobic Barrier Pen | Vector Laboratories | circle the section on the slide for for immunochemical staining | |
Xylazine | AnaSed | anesthetization | |
Ketaset | Zoetis | anesthetization | |
cryosection machine | Leica | CM1860 UV | |
confocal microscope | Leica | DM6000 CFS |