Growth plate hypertrophic chondrocytes dedifferentiate into skeletal stem and progenitor cells that give rise to osteoblasts and adipocytes during skeletal development.

Abstract

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, in contrast to periosteal-derived SSPCs, while also contributing to the adipogenic lineage. Additionally, to further understand the mechanism that may control this developmental process as well as to confirm its restriction to early development, we utilized a number of NOTCH signaling genetic tools after observation of active NOTCH signaling at the chondro-osseous junction. Histological and microCT studies of NOTCH signaling gain-of-function in hypertrophic chondrocytes (Col10a1Cre; R26-tdTomatof/+; R26-NICD1f/+) revealed a loss of bone phenotype, embryonically and early postnatally, but was recovered by non-hypertrophic chondrocyte derived osteoblasts by 2-months of age. NOTCH signaling loss-of-function in hypertrophic chondrocytes (Col10a1Cre; R26-tdTomatof/+; RBPjkf/f) exhibited no bone/osteoblast changes in early skeletal development. Our data demonstrates that hypertrophic chondrocyte-derived osteoblasts readily contribute to early skeletal development, but can compensated by non-hypertrophic chondrocyte-derived osteoblasts over time.