A Subset of Chondrogenic Cells Provides Early Mesenchymal Progenitors in Growing Bones

The hallmark of endochondral bone development is the presence of cartilaginous templates, in which osteoblasts and stromal cells are generated to form mineralized matrix and support bone marrow hematopoiesis. However, the ultimate source of these mesenchymal cells and the relationship between bone progenitors in fetal life and those in later life are unknown. Fate-mapping studies revealed that cells expressing cre-recombinases driven by the collagen II (Col2) promoter/enhancer and their descendants contributed to, in addition to chondrocytes, early perichondrial precursors prior to Runx2 expression and, subsequently, to a majority of osteoblasts, Cxcl12 (chemokine (C-X-C motif) ligand 12)-abundant stromal cells and bone marrow stromal/mesenchymal progenitor cells in postnatal life. Lineage-tracing experiments using a tamoxifen-inducible creER system further revealed that early postnatal cells marked by Col2-creER, as well as Sox9-creER and aggrecan (Acan)-creER, progressively contributed to multiple mesenchymal lineages and continued to provide descendants for over a year. These cells are distinct from adult mesenchymal progenitors and thus provide opportunities for regulating the explosive growth that occurs uniquely in growing mammals

Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow

Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed “Bone CLARITY,” a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato+ osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato+ cells in their native three-dimensional environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells. We demonstrate the value of the clearing-imaging pipeline by quantifying changes in the population of Sox9-tdTomato–labeled osteoprogenitor cells after sclerostin antibody treatment. Bone tissue clearing is able to provide fast and comprehensive visualization of biological processes in intact bone tissue

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone–related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment

Hif-1α regulates differentiation of limb bud mesenchyme and joint development

Recent evidence suggests that low oxygen tension (hypoxia) may control fetal development and differentiation. A crucial mediator of the adaptive response of cells to hypoxia is the transcription factor Hif-1α. In this study, we provide evidence that mesenchymal condensations that give origin to endochondral bones are hypoxic during fetal development, and we demonstrate that Hif-1α is expressed and transcriptionally active in limb bud mesenchyme and in mesenchymal condensations. To investigate the role of Hif-1α in mesenchymal condensations and in early chondrogenesis, we conditionally inactivated Hif-1α in limb bud mesenchyme using a Prx1 promoter-driven Cre transgenic mouse. Conditional knockout of Hif-1α in limb bud mesenchyme does not impair mesenchyme condensation, but alters the formation of the cartilaginous primordia. Late hypertrophic differentiation is also affected as a result of the delay in early chondrogenesis. In addition, mutant mice show a striking impairment of joint development. Our study demonstrates a crucial, and previously unrecognized, role of Hif-1α in early chondrogenesis and joint formation

T Lymphocytes Amplify the Anabolic Activity of Parathyroid Hormone through Wnt10b Signaling

SummaryIntermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism

Disruption of BMP Signaling in Osteoblasts Through Type IA Receptor (BMPRIA) Increases Bone Mass*

Bone morphogenetic proteins (BMPs) are known as ectopic bone inducers. The FDA approved BMPs (BMP2 and BMP7) for clinical use. However, direct effects of BMPs on endogenous bone metabolism are not yet well known. We conditionally disrupted BMP receptor type IA (BMPRIA) in osteoblasts during weanling and adult stages to show the impact of BMP signaling on endogenous bone modeling and remodeling. Cre recombination was detected in immature osteoblasts in the periosteum, osteoblasts, and osteocytes but not in chondrocytes and osteoclasts after tamoxifen administration. Bmpr1a conditional knockout mice (cKO) showed increased bone mass primarily in trabecular bone at P21 and 22 wk as determined by H&E staining. Vertebrae, tails, and ribs showed increased radiodensity at 22 wk, consistent with a significant increase in BMD. Both μCT and histomorphometry showed an increase in trabecular BV/TV and thickness of cKO adult bones, whereas osteoclast number, bone formation rate, and mineral apposition rate were decreased. Expression levels of bone formation markers (Runx2 and Bsp), resorption markers (Mmp9, Ctsk, and Tracp), and Rankl were decreased, and Opg was increased in adult bones, resulting in a reduction in the ratio of Rankl to osteoprotegerin (Opg). The reduction in osteoclastogenesis through the RANKL–OPG pathway was also observed in weanling stages and reproduced in newborn calvaria culture. These results suggest that Bmpr1a cKO increased endogenous bone mass primarily in trabecular bone with decreased osteoclastogenesis through the RANKL–OPG pathway. We conclude that BMPRIA signaling in osteoblasts affects both bone formation and resorption to reduce endogenous bone mass in vivo

Adenomatous polyposis coli-mediated control of β-catenin is essential for both chondrogenic and osteogenic differentiation of skeletal precursors

Background: During skeletogenesis, protein levels of β-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of β-catenin turnover by down-regulating intracellular levels of β-catenin. Results: To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of β-catenin was used, our approach resulted in the accumulation of wild type β-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells. Conclusion: Our data indicate that a tight Apc-mediated control of β-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner

Genetic evidence for a role of adiponutrin in the metabolism of apolipoprotein B-containing lipoproteins

Adiponutrin (PNPLA3) is a predominantly liver-expressed transmembrane protein with phospholipase activity that is regulated by fasting and feeding. Recent genome-wide association studies identified PNPLA3 to be associated with hepatic fat content and liver function, thus pointing to a possible involvement in the hepatic lipoprotein metabolism. The aim of this study was to examine the association between two common variants in the adiponutrin gene and parameters of lipoprotein metabolism in 23 274 participants from eight independent West-Eurasian study populations including six population-based studies [Bruneck (n = 800), KORA S3/F3 (n = 1644), KORA S4/F4 (n = 1814), CoLaus (n = 5435), SHIP (n = 4012), Rotterdam (n = 5967)], the SAPHIR Study as a healthy working population (n = 1738) and the Utah Obesity Case-Control Study including a group of 1037 severely obese individuals (average BMI 46 kg/m2) and 827 controls from the same geographical region of Utah. We observed a strong additive association of a common non-synonymous variant within adiponutrin (rs738409) with age-, gender-, and alanine-aminotransferase-adjusted lipoprotein concentrations: each copy of the minor allele decreased levels of total cholesterol on average by 2.43 mg/dl (P = 8.87 × 10−7), non-HDL cholesterol levels by 2.35 mg/dl (P = 2.27 × 10−6) and LDL cholesterol levels by 1.48 mg/dl (P = 7.99 × 10−4). These associations remained significant after correction for multiple testing. We did not observe clear evidence for associations with HDL cholesterol or triglyceride concentrations. In conclusion, our study suggests that adiponutrin is involved in the metabolism of apoB-containing lipoprotein