Type II collagen-positive progenitors are important stem cells in controlling skeletal development and vascular formation

The ablation of Col2+ cells causes the death of newborn mice with loss of all vertebral bone and most other bones and cartilage

To understand the contribution of Col2+ progenitors to skeletal development, a new mouse model of Col2-cre;DTA+/− was created by breeding mice bearing a DTA transgene downstream of a floxed stop codon (DTA+/+) with Col2-cre mice. Cre- littermates served as controls (wild-type mice, WT mice) (Fig. S1A). On embryonic day 17.5 (E17.5), the Col2-cre;DTA+/− embryos survived and followed Mendelian law (19/80). Interestingly, we found that 70%–75% of mice died between E17.5 and the newborn stage. Approximately 25% of the newborn (16/72) Col2-cre;DTA+/− mice survived for minutes or a few hours after birth and then died due to oxygen insufficiency resulting from a lack of rib cages and other developmental defects.

To test the effect of Cre-mediated DTA ablation, we generated Col2-cre;tdTomato and Col2-cre;DTA+/−;tdTomato mice. Lineage tracing detected abundant levels of tdTomato+ cells in the spine, limbs, ribs, meninges and skull bone of Col2-cre;tdTomato mice (Fig. 1a), but few tdTomato+ cells were detected in the mutant mice compared with the WT mice. Notably, we found that many cells in the WT skull bone, particularly in the cartilaginous nasal capsule, were tdTomato+ (Fig. 1b), which confirmed that Col2+ cells contribute to skull bone and nasal capsule formation. Consistently, the cartilaginous nasal capsule was completely lost in the Col2-cre;DTA+/−;tdTomato newborns, and the skulls of these newborns were smaller +/− than those of the control newborns. Most cells in the lower extremities and spine were labeled, although articular chondrocytes exhibited a weak signal (Fig. 1c, d). In contrast, no tdTomato+ cells could be detected in the spine region and lower extremities of Col2-cre;DTA+/−;tdTomato newborns, which confirmed the effectiveness of cell ablation.

Fig. 1figure 1

Spatial expression of Col2+ lineage progenitors in newborns and removal of the majority of Col2+ cells by DTA. a Fluorescence images showing the pattern of Col2+ lineage progenitors in middle sections of Col2-cre;tdTomato and Col2-cre;DTA+/−;tdTomato newborn mice. b Fluorescence images showing the pattern of Col2+ lineage progenitors in transected skulls of Col2-cre;tdTomato and Col2-cre;DTA+/−;tdTomato mice. White arrow, defective nasal capsule in a Col2-cre;DTA+/−;tdTomato mouse. c Fluorescence images showing the pattern of Col2+ lineage progenitors in the lower extremities of Col2-cre;tdTomato and Col2-cre;DTA+/−;tdTomato newborn mice. Note the complete loss of tdTomato+ cells in the long bones of Col2-cre;DTA+/−;tdTomato mice compared with wild-type newborn mice. d Fluorescence images showing the pattern of Col2+ lineage progenitors in middle sagittal sections of spines of Col2-cre;tdTomato and Col2-cre;DTA+/−;tdTomato newborn mice. n = 6 mice per condition; three independent experiments

The gross appearance of the Col2-cre;DTA+/− newborn mice showed severe dwarfism with extremely shortened limbs, tail and nose, intact but pale skin, cleft palate, and abnormal skull morphology22 (Fig. 2a, Fig. S1B, C).

Fig. 2figure 2

Ablation of Col2+ cells causes lethality in newborn mice due to the absence of endochondral bone and cartilage. a Gross appearance of Col2-cre and Col2-cre;DTA+/− mice at P0. The mutant mouse is small and has an extremely short four-limb pattern. b X-ray of P0 wild-type and mutant embryos. c Skeletons of Col2-cre and Col2-cre;DTA+/− mice at E17.5 and the newborn stage. Embryos and newborns were double stained with Alizarin red/Alcian blue. d Alizarin red/Alcian blue staining of skulls of Col2-cre and Col2-cre;DTA+/− newborns. Red arrow, complete loss of cartilage in Col2-cre;DTA+/− mice. e Alizarin red/Alcian blue staining of the mandibles of Col2-cre and Col2-cre;DTA+/− newborn mice. f Interior view showing the lack of mandibles in the skulls of Col2-cre and Col2-cre;DTA+/− newborn mice. Red arrow, bone loss in Col2-cre;DTA+/− mice. g Hindlimbs of Col2-cre and Col2-cre;DTA+/− newborn mice. Red arrow, bone loss in Col2-cre;DTA+/− mice. h Ribs of Col2-cre and Col2-cre;DTA+/− newborn mice. Red arrow, bone loss in Col2-cre;DTA+/− mice. i Clavicles of Col2-cre and Col2-cre;DTA+/− newborn mice. Red arrow, partial clavicle loss in Col2-cre;DTA+/− mice. n = 6 mice per condition; three independent experiments

A skeletal radiograph examination of Col2-cre;DTA+/− newborns revealed that their vertebral bone was completely lost, and only parts of the skull, mandible, clavicle bone, and tiny bones of the hindlimbs and ribs were observed (Fig. 2b). Consistent with the X-ray results, Alizarin red/Alcian blue staining of mutant embryos at E17.5 revealed the absence of cartilage and bone throughout the body skeleton and only limited Alizarin red staining in the craniofacial bone (Fig. 2c), including the frontal, parietal, temporal, maxillary, interparietal and supraoccipital bones in the skull (Fig. 2d, f) and mandible (Fig. 2e). Interestingly, very small pieces of bone in the hindlimb (Fig. 2G), small parts of the ribs (Fig. 2h), and the clavicle bone were calcified with positive Alizarin red staining (Fig. 2i).

Ablation of Col2+ cells disrupted bone and cartilage development

To shed more light on the function of Col2+ progenitors in mice, we performed sagittal sectioning of Col2-cre control and Col2-cre;DTA+/− newborns. In the Col2-cre newborns, all the bone, cartilage, brain, spinal cord, and organs were well organized and clearly identified (Fig. 3a). However, the entire vertebrae and intervertebral disc, most of the long bone and other bones and all the cartilage were lost in the Col2-cre;DTA+/− mice (Fig. 3a). Most interestingly, despite the severe bone loss in the skull and complete absence of the vertebrae and intervertebral discs observed in the mutant mice, their brain and spinal cord were located in a relatively narrow skull cavity and a “spinal canal”, respectively. However, the morphology of the spinal cord presented a spindle-like structure, which suggested that the spine or vertebral bone may be needed to guide the formation of normal spinal cord morphology.

Fig. 3figure 3

Ablation of Col2+ cells results in absence of the spine and disruption of endochondral skeletal development. a Total view of middle sagittal sections of Col2-cre and Col2-cre;DTA+/− mice at P0. b H&E staining of the middle sagittal sections of Col2-cre and Col2-cre;DTA+/− mice at P0. c Von Kossa staining of the middle sagittal sections of Col2-cre and Col2-cre;DTA+/− mice at P0. d Von Kossa staining of the skull with transection. Yellow arrow, severe bone loss in the back of the skull of Col2-cre;DTA+/− mice. Yellow star, nasal cavity area in Col2-cre and Col2-cre;DTA+/− mice. e Von Kossa staining of craniofacial bone showing loss of the nasal cavity in mutant mice. f Higher magnification of Von Kossa staining showing similar sponge-like bone structures in both wild-type and mutant mice. g Von Kossa staining of bone in the hindlimbs of wild-type and mutant mice. h Higher magnification of Von Kossa staining of the hindlimbs of wild-type and mutant mice. Note that a small piece of well-calcified sponge-like bone structure can be detected in mice with ablation of Col2+ cells. i Von Kossa staining of the middle sagittal section of the spine of wild-type and mutant mice. Red arrow, severe vertebral bone loss in Col2-cre;DTA+/− mice. n = 6 mice per condition; three independent experiments

To gain further insights into the bone changes, histological sections of the skeletons from newborns were examined by H&E (Fig. 3b, Fig. S1D–F) and Von Kossa staining (Fig. 3c). Consistently, we found that the ablation of Col2+ cells resulted in the absence of most parts of the skeleton, including complete loss of the vertebrae and intervertebral disc and all body bones with the exception of parts of the skull and a tiny hindlimb bone, in the Col2-cre;DTA+/− mice. To determine the defects in finer detail, we further analyzed sections of the skull, hindlimbs and spine. The results showed that only the front skull bone could be detected in the Col2-cre;DTA+/− mice, and the nasal cavity and back skull bone were almost absent in these mice compared with the Cre control mice (Fig. 3d–f), which indicated that Col2+ cells also play an important role in skull development. Most interestingly, a small piece of calcified sponge-like bone structure could be detected in the hindlimbs of the Col2+ cell-ablated mice (Fig. 3g, h), which indicated that Col2+ cells dominantly contribute to long bone development but that Col2-negative (Col2−) cells also participate in long bone development. The Von Kossa staining results showed that vertebral bone mineralization was completely absent in Col2-cre;DTA+/− mice, which suggested that Col2+ cells are the major progenitors contributing to vertebral bone development (Fig. 3i). Unexpectedly, even though almost all bones and cartilage were lost in Col2-cre;DTA+/− newborns, the toe and finger digits and intact patterns were normal in these mice, which suggested that limb pattern development is independent of Col2+ cells and bone development in mice (Fig. S1B).

The postnatal deletion of Col2+ cells resulted in mouse growth retardation and a type II collagenopathy phenotype

To assess the contribution of Col2+ cells to postnatal skeletal development in mice, we genetically ablated these cells by inducing the expression of DTA postnatally in Col2+ cells. Specifically, we applied tamoxifen (TM) to either TM-inducible type II collagen Cre (Col2-creERT) or Col2-creERT;DTA+/+ mice on postnatal day 3 (P3) and harvested the mice at four weeks of age. All Col2-creERT;DTA+/+ mice developed postnatal dwarfism with shorter limbs and body lengths (Fig. 4a–e). Skeletal radiographs and whole-mount Alizarin red staining of the skeleton confirmed this observation and revealed skeletal defects, including epiphyseal dysplasia in the long bone, abnormalities of the capital femoral epiphyses and underdevelopment of the femoral head (Fig. 4d, h). Representative micro-CT images showed epiphyseal dysplasia and substantially decreased bone mass in the long bones of Col2-creERT;DTA+/+ mice (Fig. 4f, g). Quantitative analysis revealed that the percentage of bone volume to total bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) of the Col2-creERT;DTA+/+ mice were reduced to approximately 0.62-, 0.53-, and 0.5-fold and that the trabecular spacing (Tb.Sp) was increased 1.5-fold compared with those of the Col2-creERT controls (Fig. 4f). Furthermore, Alizarin red/Alcian blue staining showed loss of bone and cartilage in the femur secondary ossification center in 4-week-old Col2-creERT;DTA+/+ mice (Fig. 4h). Quantitative analysis demonstrated that the lengths of the femur and tibia bones of the mutant group were significantly shortened to 0.55 cm and 0.78 cm, respectively, compared with the lengths of 1.22 cm and 1.4 cm found in the WT mice (Fig. 4i). Moreover, the postnatal ablation of Col2+ cells also impaired skull and long bone and cartilage development (Fig. 4j, k).

Fig. 4figure 4

Postnatal deletion of Col2+ cells causes mouse growth retardation and a type II collagenopathy phenotype. a, b Quantitative analysis of the body length and tail length of Col2-creERT and Col2-creERT;DTA+/+ mice from P3 to four weeks of age (n = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. c Macroscopic image of 4-week-old littermates. The mutant (right) mouse is small and exhibits dwarfism. d Total view of Col2-creERT and Col2-creERT;DTA+/+ mice double stained with Alizarin red and Alcian blue at four weeks of age. e X-ray of 4-week-old Col2-creERT and Col2-creERT;DTA+/+ mice. f Representative picture of the microCT 3D structure of the femur head of 4-week-old Col2-creERT and Col2-creERT;DTA+/+ mice. g Quantitative analysis of the BV/TV, Both, Tb.N, and Tb.Sp of the femurs of 4-week-old Col2-creERT (CreERT) and Col2-creERT;DTA+/+ (DTA) mice (n = 5 mice per group). The data are presented as the means ± s.ds. h Total view of the lower extremities of Col2-creERT and Col2-creERT;DTA+/+ mice double stained with Alizarin red and Alcian blue at four weeks of age. Yellow arrow, delayed secondary ossification development in Col2-cre;DTA+/− mice. i Quantitative analysis of the femur length and tibia length of 4-week-old Col2-creERT (CreERT) and Col2-creERT;DTA+/+ (DTA) mice (n = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. j Total view of the skulls of Col2-creERT and Col2-creERT;DTA+/+ mice stained with Alizarin red and Alcian blue at four weeks of age. k Total view of the forelimb of Col2-creERT and Col2-creERT;DTA+/+ mice stained with Alizarin red and Alcian blue at four weeks of age. l Safranin O/Fast Green staining of coronal sections of the femurs of 4-week-old Col2-creERT and Col2-creERT;DTA+/+ mice. m High-magnification image showing secondary ossification and cartilage in the femurs of 4-week-old Col2-creERT and Col2-creERT;DTA+/+ mice. n High-magnification picture showing the chondrocyte morphology of 4-week-old Col2-creERT and Col2-creERT;DTA+/+ mice. o Immunofluorescence staining of the GP and AC of 4-week-old Col2-creERT and Col2-creERT;DTA+/+ mice for type 2 collagen. p High-magnification image showing type 2 collagen staining in 4-week-old Col2-creERT and Col2-creERT;DTA+/+ mice. q Phalloidin staining showing the cartilage cytoskeleton of 4-week-old Col2-creERT and Col2-creERT;DTA+/+ mice. r Quantitative measurements of the percentage of cells with intact actin fragments relative to the total cells in Col2-creERT (CreERT) and Col2-creERT;DTA+/+ (DTA) mice (n = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. The statistical significance was determined by one-way ANOVA and Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.000 1, NS not statistically significant

Postnatal deletion of Col2+ cells disrupted endochondral ossification and cell alignment

To gain more insight into the skeletal changes, we performed safranin O/fast green staining of tibial sections from 4-week-old Col2-creERT and Col2-creERT;DTA+/+ mice (TM injected at P3). The results further confirmed the significantly decreased bone mass in the tibia and the absence of secondary ossification centers in Col2-creERT;DTA+/+ mice (Fig. 4i). Higher magnification examination of the secondary ossification center and growth plate (GP) in the tibia of Col2-creERT;DTA+/+ mice showed that the GP pattern was substantially disrupted, and the epiphyses were occupied by disorganized hypertrophic chondrocytes22 (Fig. 4m, n).

Immunofluorescence staining showed that the type II collagen matrix was enriched in articular cartilage (AC) and GP chondrocytes in 4-week-old WT mice (Fig. 4o, p) and was substantially reduced in Col2-creERT;DTA+/+ mice, which indicated that Col2+ cells are essential for type II collagen production. Moreover, the F-actin immunostaining results showed that the cell alignment and pattern in GP chondrocytes were markedly disrupted. The quantitative analysis of these results demonstrated that the percentage of cells with intact actin filaments was decreased from 100% in the WT mice to 9.2% in the mutant mice. These findings indicate that the type II collagen matrix and Col2+ cells play important roles in cell patterning and GP organization (Fig. 4q, r).

Spatial distribution of embryonic and postnatal Col2+ cells in the long bones and knee joints of mice

To investigate the contribution of Col2+ progenitors to skeletal development during the embryonic and postnatal stages, we traced the fate of Col2- expressing cells for different time periods in Col2-cre;tdTomato mice, in which Col2+ cells exhibit tomato fluorescence starting from the embryonic stage, and Col2-creERT;tdTomato mice, in which Col2+ cells express tomato fluorescence protein after treatment with TM at the indicated postnatal time. These findings demonstrated that Col2+ cells from both mouse strains and their descendants were permanently marked by the expression of the red fluorescent protein tdTomato.

To examine how Col2+ progenitors contribute to skeletal development in Col2-cre;tdTomato mice during the embryonic stage, we first examined the long bones and knee joints and found that tdTomato+ cells were present in the AC, GP, bone surface, osteocytes, tendon and meniscus (Fig. 5a). Interestingly, the tdTomato+ fluorescence signal was weaker in the GP but stronger in the trabecular and cortical bone and meniscus at P0 and P8. Notably, the tdTomato+ fluorescence was stronger in every compartment of the knee at P14 and P30 than at P0. With aging, tdTomato+ fluorescence in the trabecular and cortical bone gradually decreased; however, tdTomato+ fluorescence in the AC and GP strengthened from P90 to P180 and then decreased at P365.

Fig. 5figure 5

Spatial distribution of embryonic and postnatal Col2+ cells in mouse long bones and knee. a Representative images from the lineage tracing of embryonic Col2+ cells in long bones and knee joints at different time points (P0, P8, P14, P30, P90, P180 and P365). b Representative images from the lineage tracing of postnatal Col2+ cells in long bones and knee joints at different time points (P6, P8, P14, P30, P90, P180 and P365). This analysis was performed by injecting 75 mg·kg−1 tamoxifen into P3 mice. To better present the data, the information for the Col2-creERT;tdTomato mice in each figure is presented as the harvest time (tamoxifen injection date + tracing time period)., e.g., P6 (3 + 3) means that the harvest date of Col2-creERT;tdTomato mice is P6, that tamoxifen was injected at P3 and that tracing was performed for three days. Because all Col2+ cells were activated starting from the embryonic stage in Col2-cre;tdTomato mice, we included only the date for the harvesting of mouse samples. For example, P8 means that the harvest date for the Col2-cre;tdTomato mice was P8. c Representative images from the lineage tracing of postnatal Col2+ cells in long bones and knee joints activated at different time points (P3, P21, P27, P87, and P362). Tamoxifen (75 mg·kg−1) was injected into mice at the indicated time points. d Representative images from the lineage tracing of Col2+ cells in long bones and knee joints at P90 after activation at different time points (embryonic stage, P3, P30, P60 and P87). e Representative images from the lineage tracing of Col2+ cells in the long bones and knee joints of mice at P30 after activation at the indicated time points (embryonic stage, P3, and P60). This analysis was performed by the injection of 75 mg·kg−1 tamoxifen into Col2-creERT;tdTomato mice at different time points. f Representative images of the long bones and knee joint of Col2-creERT;tdTomato mice at P30. This analysis was performed by the injection of 75 mg·kg−1 vehicle into Col2-creERT;tdTomato mice at P3. g Quantitative measurements of the percentage of tdTomato+ cells with respect to the total cells in (c) (n = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. h Quantitative measurements of the percentage of tdTomato+ cells with respect to the total cells in (d) (n = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. i Quantitative measurements of the percentage of tdTomato+ cells with respect to total cells in (e) (n = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. At least 1 000 cells of each sample were measured. The statistical significance was determined by one-way ANOVA and Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.000 1, NS not statistically significant

To further examine the contribution of postnatal Col2+ cells to long bone and knee joint development, Col2-creERT;tdTomato mice were intraperitoneally (i.p.) administered TM at P3 and harvested at P6, P8, P14, P30, P90, P180, and P365. At P6 and P8, tdTomato+ (Col+) cells were predominantly detected in the AC, secondary ossification center, GP, and meniscus (Fig. 5b). Interestingly, at P14 and P30, the pattern of Col2+ cells in the AC, GP and long bones of these mice was similar to that found in Col2-cre;tdTomato mice, and this pattern involved decreased tdTomato+ fluorescence in the AC and GP but increased fluorescence in the cortical and cancellous bones. At P90, tdTomato+ fluorescence was decreased in the cortical and cancellous bones but increased in the GP and AC. However, starting from P180, the numbers of tdTomato+ cells in cortical and cancellous bones as well as the GP and AC significantly decreased with aging in both Col2-cre;tdTomato and Col2-creERT;tdTomato mice (Fig. 5a, b).

The numbers of Col2+ progenitors decreased during aging

To further explore the effect of age on the fate of Col2+ progenitors, Col2-creERT;tdTomato mice were i.p. injected with TM at P3, P21, P27, P87, and P362, and sections were analyzed 3 days after each injection. As shown in Fig. 5c, tdTomato+ cells were detected in articular chondrocytes, the GP and the meniscus after the administration of TM at P3. However, the number of tdTomato+ cells in mice significantly decreased during aging. Approximately 98.1% of GP chondrocytes were tdTomato+ in the mice administered the TM injection at P3, and the injection of TM at P21, P27, P87, and P362 decreased this percentage to 89.9%, 53%, 32.7%, and 23.2%, respectively. Similarly, approximately 97.8% of articular chondrocytes were tdTomato+ in the mice that were injected with TM at P3, and the injection of TM at P21, P27, P87, and P362 decreased these percentages to 86.2%, 26.4%, 17.2%, and 9.1%, respectively (Fig. 5c, g). In the meniscus, 38.1% of cells were tdTomato+ in the mice that were injected with TM at P3, and the injection of TM at P21, P27, and P87 decreased this percentage to 31.1%, 7.8%, and 6.9%, respectively, whereas only a few positive cells were detected after the injection of TM at P362. The results demonstrated that the numbers of Col2+ progenitors decreased during aging.

To determine how age affects the number and differentiation ability of Col2+ cells, we compared the number of tdTomato+ cells in the knee joints at P90 between Col2-cre;tdTomato mice whose Col2+ cells were activated at the embryonic stage and Col2-creERT;tdTomato mice injected with TM to turn on tdTomato protein expression at P3, P30, P60, and P87 (Fig. 5d). The number of tdTomato+ cells gradually decreased from the embryonic stage to P3, P30, P60 and P87. Specifically, at P90, 98.5% of GP chondrocytes were tdTomato+ when Col2+ was activated at the embryonic stage; however, the percentage of these cells decreased to 98.2%, 55.4%, 47.1%, and 37.2% in the Col2-creERT;tdTomato mice injected with TM at P3, P30, P60 and P87, respectively (Fig. 5D, H). Similarly, approximately 98.4% of articular chondrocytes were tdTomato+ when Col2+ was activated at the embryonic stage, and the injection of TM at P3, P30, P60 and P87 decreased this percentage to 97.9%, 38.7%, 20.1%, and 17.2%, respectively. In the meniscus, 98.8% of cells were tdTomato+ when Col2+ was activated at the embryonic stage, and the injection of TM at P3, P30, P60 and P87 decreased this percentage to 8.1%, 10.4%, 8.4%, and 6.9%, respectively. These results suggested that the numbers and differentiation ability of Col2+ cells decreased during the aging process.

To further confirm that the differentiation ability of Col2+ progenitors decreased with increasing age, we compared the tdTomato+ cells in the knee joints over 1 month of tracing among mice whose Col2+ cells were activated at the embryonic stage, P3 and P60 (Fig. 5e). Approximately 98.4% of articular chondrocytes were tdTomato+ when Col2+ was activated at the embryonic stage, and this percentage decreased to 97.9% and 38.7% when TM was injected at P3 and P60, respectively (Fig. 5E, I). Similarly, 98.5% of GP chondrocytes were tdTomato+ when Col2+ was activated at the embryonic stage, and the injection of TM at P3 and P60 decreased this percentage to 98.2% and 55.4%, respectively. In the meniscus, 98.8% of cells were tdTomato+ when Col2+ was activated at the embryonic stage, and the injection of TM at P3 and P60 decreased this percentage to 8.1% and 1.3%, respectively (Fig. 5e, i). These results further confirmed that the differentiation ability of Col2+ progenitors decreased with age. No detectable td-Tomato fluorescence was detected in the control Col2-creERT;R26-tdTomato mice injected with vehicle, which confirmed the specificity of Cre recombinase activity (Fig. 5f).

In addition, comparing type II collagen expression between embryonic and postnatal Col2+ cells revealed that the expression pattern of type II collagen in Col2-cre;tdTomato mice at P6 was similar to the pattern of Col2+ cells at P6 in Col2-creERT;tdTomato mice injected with TM at P3 (Fig. S2). However, the type II collagen expression pattern in Col2-cre;tdTomato mice was completely different from the patterns of Col2+ cells at P30, P180 and P365 in Col2-creERT;tdTomato mice injected i.p. with TM at P27, P177 or P362 (Fig. S2A–D).

To further demonstrate the differentiation ability of Col2+ cells during aging, we performed in vitro primary cell culture. Col2+ chondrocytes were harvested from 5-day-, 1-month- and 5-month-old mice, and the differentiation ability was then tested through chondrogenesis and osteogenesis differentiation assays. The results showed that the differentiation potential of Col2+ cells decreased with increasing age. A quantitative analysis of the Alizarin red and Alcian blue staining results showed that Col2+ chondrocytes from P5 and 1-month-old mice had markedly stronger osteogenic and chondrogenic differentiation potential than Col2+ chondrocytes from 5-month-old mice (Fig. S3). These results further indicate that the differentiation ability of Col2+ progenitors gradually decreased during aging.

CD31+ cells originating from Col2 progenitors expressed Col2 throughout their lifespan until the time points examined

Our results showed that the gross appearance of the Col2-cre;DTA+/− newborn mice exhibited a lack of blood vessel phenotype with pale skin (Fig. 2a, Fig. S1B, C). To further examine whether Col2+ cells contribute to blood vessel formation, we first traced the Col2+ cell locations in the skeleton of Col2-cre;tdTomato mice and found that Col2+ cells (tdTomato+) were present in the GP, AC (chondrocytes) and bone (osteoblasts and osteocytes) of 4-week-old Col2-cre;tdTomato mice (Fig. 6a, Fig. S4). Surprisingly, some CD31+ cells in blood vessels of the long and skull bones were also labeled with tdTomato+ fluorescence (Fig. 6b–f). Flow cytometry results showed that approximately 25.4% of CD31+ cells in the long bones of 4-week-old Col2-cre;tdTomato mice were Col2+ (Fig. 6b, c), whereas almost all CD31+ blood vessel cells in skull bone were Col2+ (Fig. 6d). Additionally, many CD31+ blood vessel cells in the brain, eyeball, heart, and skin were Col2+ (Fig. 6e, Fig. S5A–C), but none of the CD31+ cells found in the kidney vasculature during development were Col2+ (Fig. S5D). Consistently, we found that the ablation of Col2+ cells led to a marked decrease in the numbers of CD31+ blood vessels in the skull bone and eyes of Col2-cre;DTA+/+;tdTomato mice compared with those detected in the control Col2-cre;tdTomato mice (Fig. 6f, Fig. S5E, F). These results indicate that Col2+ cells can contribute to skeletal blood vessel formation.

Fig. 6figure 6

Col2+ progenitors have multilineage differentiation ability, including CD31+ blood vessels. a Representative fluorescent images of the distal femur showing that cartilage cells, osteoblasts and osteocytes are Col2+. Yellow arrow, osteoblasts and osteocytes of 4-week-old Col2-cre;tdTomato mice. b Flow cytometry analysis was performed using dissociated long bone marrow cells collected from 4-week-old Col2-cre;tdTomato mice. Representative dot plots show that some Col2+ bone marrow stromal cells were CD31+ (n = 3 mice per condition; three independent experiments). c Representative fluorescent images of the distal femurs of 4-week-old Col2-cre;tdTomato mice showing that some CD31+ cells in long bones were Col2+ cells. d Representative fluorescent images from the calvarial bone of 4-week-old Col2-cre;tdTomato mice showing that almost all CD31+ cells were Col2+ cells. e Representative fluorescent images from the craniofacial bone of 4-week-old Col2-cre;tdTomato mice showing that almost all CD31+ cells were Col2+ cells. Yellow arrow, overlapping staining of blood vessels. f The gross appearance of the head of Col2-cre and Col2-cre;DTA+/− newborn mice. Note that the mutant mice showed a marked loss of blood vessels in the skull. Yellow arrow, blood vessels in each group. g Representative picture showing that Col2+ cells from the calvaria bone of Col2-cre;tdTomato mice exhibit tube formation capacity. h Representative picture showing that Col2+ cells from the cartilage of Col2-cre;tdTomato mice exhibit tube formation capacity. i Representative picture showing that Col2− cells from the cartilage of Col2-cre;tdTomato mice do not have tube formation capacity. All the data are reported as the means ± s.ds. The statistical significance was determined by one-way ANOVA and Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.000 1. NS not statistically significant

To further investigate whether Col2+ cells could form vascular lumens in vitro, we harvested Col2+ cells from the calvarial bone and cartilage of 1-month-old Col2-cre;tdTomato mice and tested the angiogenic tube formation capacity through angiogenesis assays. Indeed, Col2+ cells from calvarial bone and cartilage formed vascular lumens in vitro (Fig. 6g–i).

Both hematopoietic and blood vessel endothelial cells are differentiated from hemangioblasts of mesodermal cells.1 To determine whether Col2+ cells in the bone marrow contribute to hematopoietic cell-derived osteoclast formation, Bone marrow cells(BMCs) from Col2-cre;tdTomato mice were induced with RANKL/M-CSF. As shown in Fig. S6, osteoclasts did not exhibit tdTomato fluorescence. Moreover, TRAP staining for osteoclastogenesis assays confirmed that Col2+ cells cannot differentiate into osteoclasts.

Col2+ cells in the GP and AC displayed stem cell properties

Our results showed that Col2+ cells were located in the AC, bone marrow and GP and that the ablation of Col2+ cells completely disrupted skeletal development with the exception of craniofacial bone development. To determine whether Col2+ cells exhibit stem cell properties, we first performed a CFU-F activity assay. BMCs, GP chondrocytes and articular chondrocytes were isolated from 4-week-old Col2-cre;tdTomato mice, and the Col2+ cells were then sorted, as shown in Fig. 7a. The quantification of tdTomato+ CFU-F colonies revealed that Col2+ cells from BMCs, GP chondrocytes and articular chondrocytes could all form CFU-F colonies (Fig. 7a).

Fig. 7figure 7

Col2-positive progenitors from different tissues have varied differentiation potential. a CFU-F assay of Col2+ bone marrow stem cells (BMSCs), GP progenitors and AC progenitors showing that every cell type can form CFU colonies. b Adipogenic differentiation of Col2+ BMSCs, GP progenitors and AC progenitors. c Osteogenic differentiation of Col2+ BMSCs, GP progenitors and AC progenitors. d Chondrogenic differentiation of Col2+ BMSCs, GP progenitors and AC progenitors. e Merged pictures showing Col2+ cells cultured from the calvaria of Col2-cre;tdTomato and Col2-cre;DTA+/−;tdTomato newborns. Note that few Col2+ cells were detected in the Col2+ cell ablation group, confirming the effectiveness of the cell ablation technique. f Assay of the trilineage differentiation of cells cultured from the calvarial bone of Col2-cre and Col2-cre;DTA+/− newborns (n = 3 mice per condition; three independent experiments). g Different migration abilities of cells cultured from Col2-cre and Col2-cre;DTA+/− newborns. h Quantitative measurements of migration ability based on the results shown in (g) (n = 3 mice per condition; three independent experiments). All the data are reported as the means ± s.ds. The statistical significance was determined by one-way ANOVA and Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.000 1, NS not statistically significant

To further assess whether Col2+ cells have multiple lineage differentiation capabilities, Col2+ cells from the bone marrow, GP and AC were induced with osteogenic, chondrogenic and adipogenic media for the indicated times (Fig. 7b). Interestingly, Col2+ cells sorted from BMCs could differentiate into osteoblasts, chondrocytes and adipocytes, whereas Col2+ cells from the GP and AC could differentiate only into osteoblasts and chondrocytes but not into adipocytes (Fig. 7b–d). These findings suggested that Col2+ cells from BMCs are likely bone mesenchymal stem cells (BMSCs) at earlier stages, whereas a large population of Col2+ cells from the GP and AC were BMSCs at later stages. Most interestingly, the Alcian blue and Alizarin red staining of the cells showed that Col2+ cells from the GP exhibited markedly higher osteogenic and chondrogenic differentiation potential than Col2+ cells from articular chondrocytes and BMCs (Fig. 7c, d).

Oct4 and Sox2 are stem cell markers.23,24 To determine whether Col2+ cells express stem cell markers, we coimmunostained sections of the GP, bone marrow and AC tissues from newborn and 1-month-old Col2-cre;tdTomato mice to detect the expression of Oct4 and Sox2. Our data showed that Oct4 and Sox2 expression overlapped with Col2+ cells in the bone marrow, GP and AC (Fig. S7A), which confirmed that Col2+ cells from these three sources have stem cell properties. Moreover, our data showed that Oct4- and Sox2-positive cells overlapped with some Col2+ cells in the bone marrow but with markedly fewer Col2+ cells in the GP and AC than in the bone marrow of Col2-cre;tdTomato mice (Fig. S7B, C).

Col2-negative cells from the calvarial bone but not cartilage show high unipotent osteogenic potential

According to the lineage tracing results shown in Fig. 3c, both Col2+ and Col2− cells are present in calvarial bone. To further test whether Col2− cells also have differentiation potential, calvarial bone cells were isolated from the calvaria of newborn Col2-cre;tdTomato (control) and Col2-cre;DTA+/−;tdTomato mice. We found that approximately 50% of cells in the control group were Col2+, but almost no Col2+ cells were detected in the Col2-cre;DTA+/−;tdTomato group, which confirmed the effectiveness of Co2+ cell ablation (Fig. 7e). Moreover, we found that the cells of the control calvarial bone could differentiate into three lineages, namely, osteoblasts, chondrocytes and adipocytes. Interestingly, Col2− cells could not differentiate into chondrocytes and adipocytes but exhibited markedly higher potential to differentiate into osteoblasts than control cells (Fig. 7f). Additionally, the wound healing assay showed that Col2− cells had stronger migration ability than the control cells (Fig. 7g, h).

To investigate whether the differentiation potential differs between Col2+ and Col2− cells, we isolated Col2+ and Col2− cells from the bone marrow of the long bones of 1-month-old Col2-cre;tdTomato mice. We found that both Col2+ and Col2− cells were capable of differentiating into osteoblasts and chondrocytes (Fig. S8). A quantitative analysis of Alcian blue- and Alizarin red-stained cells showed that Col2+ cells from the bone marrow have markedly higher osteogenic and chondrogenic differentiation potential than Col2−cells (Fig. S8).

Col2+ progenitors contributed to chondrocyte differentiation and blood vessel formation during fracture healing

To test whether Col2+ cells contribute to postnatal chondrocyte differentiation and blood vessel formation, we created a closed femoral fracture with an intramedullary nail fixation model in 10-week-old mice as previously described25 to trace Col2+ cells during fracture healing. We first induced tdTomato expression in Col2+ cells of Col2-creERT; tdTomato mice through the injection of TM at P3, induced a fracture in the mice at 10 weeks of age and harvested tissues two weeks after the fracture surgery. In the noninjured mice, the contralateral nonfractured femur exhibited prominent tdTomato+ cells in the AC and metaphysis but few tdTomato+ cells in the bone marrow (Fig. 8a). However, strong tdTomato expression was detected throughout the fractured callus, including both bony and cartilaginous regions (Fig. 8b, c). Immunofluorescent staining for CD31 and Col2α1 showed that 95.5% of the CD31+ cells and 43% of the Col2α1+ cells overlapped with tdTomato+ (Col2+) cells in the callus area of Col2-creERT;tdTomato mice. In contrast, only 25.3% of CD31+ cells and 12.1% of Col2α1+ cells overlapped with tdTomato+ (Col2+) cells in the intact long bones of Col2-creERT;tdTomato mice (Fig. 8b, c). Immunofluorescent staining for blood vessels in the fractured callus showed that the percentage of CD31+ cells decreased markedly to 3.5% in Col2-creERT;DTA+/+;tdTomato mice compared with 12.5% in Col2-creERT;tdTomato mice (Fig. 8d). These results indicate that Col2+ cells play an important role in blood vessel and bone regeneration during fracture healing.

Fig. 8

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