Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation

Abstract

According to current dogma, chondrocytes and osteoblasts are considered independent lineages derived from a common osteochondroprogenitor. In endochondral bone formation, chondrocytes undergo a series of differentiation steps to form the growth plate, and it generally is accepted that death is the ultimate fate of terminally differentiated hypertrophic chondrocytes (HCs). Osteoblasts, accompanying vascular invasion, lay down endochondral bone to replace cartilage. However, whether an HC can become an osteoblast and contribute to the full osteogenic lineage has been the subject of a century-long debate. Here we use a cell-specific tamoxifen-inducible genetic recombination approach to track the fate of murine HCs and show that they can survive the cartilage-to-bone transition and become osteogenic cells in fetal and postnatal endochondral bones and persist into adulthood. This discovery of a chondrocyte-to-osteoblast lineage continuum revises concepts of the ontogeny of osteoblasts, with implications for the control of bone homeostasis and the interpretation of the underlying pathological bases of bone disorders.

Keywords: bone repair; chondrocyte lineage; osteoblast ontogeny.

Fig. 1.

HCs contribute to osteoblastic lineage in mouse endochondral bone. (A) Current view of chondrocyte and osteoblast lineages. (B) In situ hybridization showing mRNA expression of indicated genes during POC formation in E15.0 and E15.5 tibia. Dotted lines indicate the cartilage and perichondrium border. (Scale bar, 200 μm.) (C) LacZ expression (by X-Gal staining, which is pink under dark field) in HCs at E15.0 and HC-derived cells at E15.5 in C10cre::RLacZ tibia. Dotted lines indicate the chondro-osseous junction. (D) Cre and LacZ mRNA at E15.5 in C10cre::RLacZ tibia. (E) Fluorescent signals in P10 tibia of Col10a1-GFP and C10cre::RYFP mice. (Insets) Vertebra. Whole tibiae (wild type and C10cre::RYFP) are shown in dashed Insets. (Scale bar, 1 mm.) (F) P10 tibia of C10cre::Z/EG mouse stained by GFP antibody (brown). Dotted line represents the chondro-osseous junction. GFP-expressing osteoblasts and osteocytes are denoted by black and blue arrows, respectively. (G) X-Gal staining (blue) of P10 C10cre::RLacZ and control tibiae. (Inset) LacZ⁺ osteocyte and bone surface osteoblast LacZ⁺ cell. Col1a1 in situ hybridization reveals X-Gal, Col1a1 double-positive cells in the endosteum. The locations of the sections are denoted in the cartoon on the right. (Scale bar, 100 µm.) (H) X-Gal staining of 3-mo-old tibia from C10cre::RLacZ and control. CB, cortical bone; DP, diaphysis; LH, zone of late HCs; PO, primary ossification center; PS, primary spongiosa; TB, trabecular region.

Fig. 2.

Generation and characterization of the C10creERt mouse line. (A) Targeted Col10a1::CreERt allele (detailed in Fig. S4). (B) In situ hybridization showing Col10a1 and CreERt mRNA in C10creERt::RLacZ at E14.5 and E16.5. (C) Proximal-to-distal temporal progression of hypertrophy and transition shown in X-Gal–stained E15.5 C10cre::RLacZ limb skeletal elements. (D) X-Gal–stained tibia and humerus of E14.5–E16.5 C10creERt::RLacZ mice after tam injection at E12.5. (E) X-Gal–stained bone sections harvested 24 h post tam injection at E14.5 from the same C10creERt::RLacZ fetus. Arrow indicates LacZ⁺ (pink) cell in the forming POC of femur. (F) Col1a1 in situ hybridization on X-Gal–stained sections of different E16.0 bones from the same C10creERt::RLacZ fetus, harvested 36 h post tam injection at E14.5. Arrows in the enlarged areas of the POC indicate LacZ⁺ and Col1a1 double-positive cells. (Scale bar, 100 μm.) Fe, femur; Fi, fibula; Hu, humerus; Mc, metacarpal; Mt, metatarsal; PO, primary ossification center; Ra, radius; Sc, scapula; Ti, tibia; Ul, ulna. B, D, and E are dark-field images.

Fig. 3.

HCs contribute to the full osteoblast lineage revealed by tam-inducible lineage tracing. (A and B) X-Gal staining of C10creERt::RLacZ tibiae sections at E15.5 and E18.5 and postnatal stages (P5, P1m) after tam injection at E13.5. In B, LacZ⁺ cells were found to express OSX, Col1a1, and SOST. (Scale bar, 200 μm.) (C) The proportion of tagged HCs [HC-LacZ⁺/HCs (%)] in the HZ and tagged Col1a1-expressing cells [Col1a1⁺;LacZ⁺/Col1a1⁺ (%)] in fetal and postnatal ossification centers. Tam was injected at E13.5, and tibiae were X-Gal stained at E16.0, E18.5, and P5 (n = 5 for each stage). Col1a1-expressing cells (by in situ hybridization) in the trabecular bone and endosteum were counted. DP, diaphysis; PO, primary ossification center; TB, trabecular region.

Fig. 4.

Postnatal HCs may become osteoblasts and osteocytes. (A) LacZ activity (dark fields of X-Gal staining) in C10creERt::RLacZ tibia 8, 16, and 24 h following tam injection at P5. (Scale bar, 20 μm.) (B) Col1a1 mRNA expression in red fluorescent protein (RFP)-labeled cells in trabeculae of C10creERt::RtdTomato tibia 24 h after tam injection at P5. (C) LacZ⁺ osteocytes were found in 2.5- and 16-mo-old tibia after tam injection at P9. Insets show SOST-expressing LacZ⁺ osteocyte in P2.5m tibia, and persisting LacZ⁺ HCs in P16m tibia (top) and rib (bottom). (Scale bar, 100 μm.) CB, cortical bone; PO, primary ossification center; TB, trabecular region.

Fig. 5.

HC-derived osteoblasts and osteocytes contribute to bone repair and revised concept of osteoblast ontogeny in endochondral bone. (A–F) Fate of LacZ-tagged HCs in grafts of P10 C10cre::RLacZ hypertrophic cartilage inserted into the injury sites in tibia of P3m adult females. Tibiae were analyzed for indicated markers 2, 5, and 8 dpo. Alcian blue and collagen I immunostaining marks cartilage and bone matrix, respectively. LacZ⁺ (blue) osteoblasts (black arrow) and osteocytes (red arrows) of graft origin were identified in the bone repair site. (G–J) Similar to A–F, graft hypertrophic cartilages from C10cre::RYFP mice were inserted into the bone injury site. YFP⁺ cells expressing Col1a1 and OSX were detected at 5 and 8 dpo. (K) A model for the ontogeny of osteoblasts in endochondral bone. Sources of osteoblasts are direct differentiation from periosteal mesenchymal cells to form cortical bone (CB), perichondrium-derived osteoblast progenitors accompanying vascular invasion of the POC, and HC transition to osteoblast lineages. BC, bone collar; BV, blood vessel; GP, growth plate; OB, osteoblast; OY, osteocyte; PE, periosteum; TB, trabecular bone.