Hedgehog activation promotes osteogenic fates of growth plate resting zone chondrocytes through transient clonal competency.

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-Indian hedgehog feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and traced the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with patched-1-floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large, concentric, clonally expanded cell populations within the resting zone ("patched roses") and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell descendants migrated away from the growth plate and transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a potentially novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.

Growth plate resting zone chondrocytes acquire transient clonal competency upon Hedgehog activation and efficiently transform into trabecular bone osteoblasts.

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-indian hedgehog (Ihh) feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP + resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we utilized a tamoxifen-inducible PTHrP-creER line with Patched-1 ( Ptch1 ) floxed and tdTomato reporter alleles to specifically activate Hedgehog signaling in PTHrP + resting chondrocytes and trace the fate of their descendants. Hedgehog-activated PTHrP + chondrocytes formed large concentric clonally expanded cell populations within the resting zone (' patched roses ') and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP + cell-descendants migrated away from the growth plate and eventually transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP + skeletal stem cells.

Chondrocytes in the resting zone of the growth plate are maintained in a Wnt-inhibitory environment.

Chondrocytes in the resting zone of the postnatal growth plate are characterized by slow cell cycle progression, and encompass a population of parathyroid hormone-related protein (PTHrP)-expressing skeletal stem cells that contribute to the formation of columnar chondrocytes. However, how these chondrocytes are maintained in the resting zone remains undefined. We undertook a genetic pulse-chase approach to isolate slow cycling, label-retaining chondrocytes (LRCs) using a chondrocyte-specific doxycycline-controllable Tet-Off system regulating expression of histone 2B-linked GFP. Comparative RNA-seq analysis identified significant enrichment of inhibitors and activators for Wnt signaling in LRCs and non-LRCs, respectively. Activation of Wnt/ß-catenin signaling in PTHrP+ resting chondrocytes using Pthlh-creER and Apc-floxed allele impaired their ability to form columnar chondrocytes. Therefore, slow-cycling chondrocytes are maintained in a Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating maintenance and differentiation of PTHrP+ skeletal stem cells of the postnatal growth plate.

A Wnt-mediated transformation of the bone marrow stromal cell identity orchestrates skeletal regeneration.

Bone marrow stromal cells (BMSCs) are versatile mesenchymal cell populations underpinning the major functions of the skeleton, a majority of which adjoin sinusoidal blood vessels and express C-X-C motif chemokine ligand 12 (CXCL12). However, how these cells are activated during regeneration and facilitate osteogenesis remains largely unknown. Cell-lineage analysis using Cxcl12-creER mice reveals that quiescent Cxcl12-creER+ perisinusoidal BMSCs differentiate into cortical bone osteoblasts solely during regeneration. A combined single cell RNA-seq analysis demonstrate that these cells convert their identity into a skeletal stem cell-like state in response to injury, associated with upregulation of osteoblast-signature genes and activation of canonical Wnt signaling components along the single-cell trajectory. ß-catenin deficiency in these cells indeed causes insufficiency in cortical bone regeneration. Therefore, quiescent Cxcl12-creER+ BMSCs transform into osteoblast precursor cells in a manner mediated by canonical Wnt signaling, highlighting a unique mechanism by which dormant stromal cells are enlisted for skeletal regeneration.

Growth Plate Borderline Chondrocytes Behave as Transient Mesenchymal Precursor Cells.

The growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly at the periphery of the growth plate immediately adjacent to the perichondrium are "borderline" chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single-cell RNA-seq analysis revealed subpopulations of Col2a1-creER-marked neonatal chondrocytes and their cell type-specific markers. A tamoxifen pulse to Pthrp-creER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow, and became Col(2.3 kb)-GFP+ osteoblasts and Cxcl12-GFPhigh reticular stromal "CAR" cells. Interestingly, these borderline chondrocyte-derived marrow cells were short-lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage-tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. © 2019 American Society for Bone and Mineral Research.

Autocrine regulation of mesenchymal progenitor cell fates orchestrates tooth eruption.

Formation of functional skeletal tissues requires highly organized steps of mesenchymal progenitor cell differentiation. The dental follicle (DF) surrounding the developing tooth harbors mesenchymal progenitor cells for various differentiated cells constituting the tooth root-bone interface and coordinates tooth eruption in a manner dependent on signaling by parathyroid hormone-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR). However, the identity of mesenchymal progenitor cells in the DF and how they are regulated by PTHrP-PPR signaling remain unknown. Here, we show that the PTHrP-PPR autocrine signal maintains physiological cell fates of DF mesenchymal progenitor cells to establish the functional periodontal attachment apparatus and orchestrates tooth eruption. A single-cell RNA-seq analysis revealed cellular heterogeneity of PTHrP+ cells, wherein PTHrP+ DF subpopulations abundantly express PPR. Cell lineage analysis using tamoxifen-inducible PTHrP-creER mice revealed that PTHrP+ DF cells differentiate into cementoblasts on the acellular cementum, periodontal ligament cells, and alveolar cryptal bone osteoblasts during tooth root formation. PPR deficiency induced a cell fate shift of PTHrP+ DF mesenchymal progenitor cells to nonphysiological cementoblast-like cells precociously forming the cellular cementum on the root surface associated with up-regulation of Mef2c and matrix proteins, resulting in loss of the proper periodontal attachment apparatus and primary failure of tooth eruption, closely resembling human genetic conditions caused by PPR mutations. These findings reveal a unique mechanism whereby proper cell fates of mesenchymal progenitor cells are tightly maintained by an autocrine system mediated by PTHrP-PPR signaling to achieve functional formation of skeletal tissues.

Resting zone of the growth plate houses a unique class of skeletal stem cells.

Skeletal stem cells regulate bone growth and homeostasis by generating diverse cell types, including chondrocytes, osteoblasts and marrow stromal cells. The emerging concept postulates that there exists a distinct type of skeletal stem cell that is closely associated with the growth plate1-4, which is a type of cartilaginous tissue that has critical roles in bone elongation5. The resting zone maintains the growth plate by expressing parathyroid hormone-related protein (PTHrP), which interacts with Indian hedgehog (Ihh) that is released from the hypertrophic zone6-10, and provides a source of other chondrocytes11. However, the identity of skeletal stem cells and how they are maintained in the growth plate are unknown. Here we show, in a mouse model, that skeletal stem cells are formed among PTHrP-positive chondrocytes within the resting zone of the postnatal growth plate. PTHrP-positive chondrocytes expressed a panel of markers for skeletal stem and progenitor cells, and uniquely possessed the properties of skeletal stem cells in cultured conditions. Cell-lineage analysis revealed that PTHrP-positive chondrocytes in the resting zone continued to form columnar chondrocytes in the long term; these chondrocytes underwent hypertrophy, and became osteoblasts and marrow stromal cells beneath the growth plate. Transit-amplifying chondrocytes in the proliferating zone-which was concertedly maintained by a forward signal from undifferentiated cells (PTHrP) and a reverse signal from hypertrophic cells (Ihh)-provided instructive cues to maintain the cell fates of PTHrP-positive chondrocytes in the resting zone. Our findings unravel a type of somatic stem cell that is initially unipotent and acquires multipotency at the post-mitotic stage, underscoring the malleable nature of the skeletal cell lineage. This system provides a model in which functionally dedicated stem cells and their niches are specified postnatally, and maintained throughout tissue growth by a tight feedback regulation system.

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

BACKGROUND: Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice. RESULTS: First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant. CONCLUSIONS: Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Filamin-interacting proteins, Cfm1 and Cfm2, are essential for the formation of cartilaginous skeletal elements.

Mutations of Filamin genes, which encode actin-binding proteins, cause a wide range of congenital developmental malformations in humans, mainly skeletal abnormalities. However, the molecular mechanisms underlying Filamin functions in skeletal system formation remain elusive. In our screen to identify skeletal development molecules, we found that Cfm (Fam101) genes, Cfm1 (Fam101b) and Cfm2 (Fam101a), are predominantly co-expressed in developing cartilage and intervertebral discs (IVDs). To investigate the functional role of Cfm genes in skeletal development, we generated single knockout mice for Cfm1 and Cfm2, as well as Cfm1/Cfm2 double-knockout (Cfm DKO) mice, by targeted gene disruption. Mice with loss of a single Cfm gene displayed no overt phenotype, whereas Cfm DKO mice showed skeletal malformations including spinal curvatures, vertebral fusions and impairment of bone growth, showing that the phenotypes of Cfm DKO mice resemble those of Filamin B (Flnb)-deficient mice. The number of cartilaginous cells in IVDs is remarkably reduced, and chondrocytes are moderately reduced in Cfm DKO mice. We observed increased apoptosis and decreased proliferation in Cfm DKO cartilaginous cells. In addition to direct interaction between Cfm and Filamin proteins in developing chondrocytes, we showed that Cfm is required for the interaction between Flnb and Smad3, which was reported to regulate Runx2 expression. Furthermore, we found that Cfm DKO primary chondrocytes showed decreased cellular size and fewer actin bundles compared with those of wild-type chondrocytes. These results suggest that Cfms are essential partner molecules of Flnb in regulating differentiation and proliferation of chondryocytes and actin dynamics.

OBIF, an osteoblast induction factor, plays an essential role in bone formation in association with osteoblastogenesis.

In vertebrate bone formation, the functional mechanisms of transcription factors in osteoblastic differentiation have been relatively well elucidated; however, the exact roles of cell-extrinsic molecules are less clear. We previously identified human and mouse Obif, an osteoblast induction factor, also known as Tmem119, which encodes a single transmembrane protein. OBIF is predominantly expressed in osteoblasts in mouse. While exogenous Obif expression stimulated osteoblastic differentiation, knockdown of Obif inhibits the osteoblastic differentiation of pre-osteoblastic MC3T3-E1 cells. In order to investigate an in vivo role of OBIF in bone formation, we generated Obif-deficient mice by targeted gene disruption. Analyses of micro-computed tomography (mCT) revealed that Obif(-/-) mice exhibit significantly reduced cortical thickness in the mid-shaft of the femur at postnatal day 14 (P14). Furthermore, progressive bone hypoplasia is observed after 8 weeks. The expression levels of osteoblast marker genes, Collagen 1a1, Osteopontin, Runx2, and Osterix, in the calvaria were decreased in Obif(-/-) mice at P4. These data indicate that Obif plays an essential role in bone formation through regulating osteoblastogenesis.

Isolation and characterization of a novel plasma membrane protein, osteoblast induction factor (obif), associated with osteoblast differentiation.

BACKGROUND: While several cell types are known to contribute to bone formation, the major player is a common bone matrix-secreting cell type, the osteoblast. Chondrocytes, which plays critical roles at several stages of endochondral ossification, and osteoblasts are derived from common precursors, and both intrinsic cues and signals from extrinsic cues play critical roles in the lineage decision of these cell types. Several studies have shown that cell fate commitment within the osteoblast lineage requires sequential, stage-specific signaling to promote osteoblastic differentiation programs. In osteoblastic differentiation, the functional mechanisms of transcriptional regulators have been well elucidated, however the exact roles of extrinsic molecules in osteoblastic differentiation are less clear. RESULTS: We identify a novel gene, obif (osteoblast induction factor), encoding a transmembrane protein that is predominantly expressed in osteoblasts. During mouse development, obif is initially observed in the limb bud in a complementary pattern to Sox9 expression. Later in development, obif is highly expressed in osteoblasts at the stage of endochondral ossification. In cell line models, obif is up-regulated during osteoblastic differentiation. Exogenous obif expression stimulates osteoblastic differentiation and obif knockdown inhibits osteoblastic differentiation in preosteblastic MC3T3-E1 cells. In addition, the extracellular domain of obif protein exhibits functions similar to the full-length obif protein in induction of MC3T3-E1 differentiation. CONCLUSIONS: Our results suggest that obif plays a role in osteoblastic differentiation by acting as a ligand.