The procedures for bone and bone marrow section preparation, immunostaining conditions and antibodies are described in Supplementary Methods. The procedure for BrdU pulse labeling, LTR and subsequent detection has been reported 16 . The mice were fed BrdU (0.8 mg ml 21 in water) for 10 days, during which time 40% of LT-HSCs would divide at least once 31 . Seventy days after BrdU labelling, sections were stained with anti-BrdU antibody. N-cadherin 1 cell countFor quantitative analysis of N-cadherin þ cells, the sections were developed with AEC after being incubated with rabbit anti-N-cadherin antibody for 1 h and horseradish peroxidase (HRP)-conjugated goat anti-rabbit second antibody for 1 h. Three people counted the SNO cells in these sections, blind to the source of the sections. X-ray imageHigh-resolution X-rays (Faxitron MX-20) of bone and bone histomorphometry (OsteoMetrics, Inc.) were performed at the University of Missouri-Kansas City School of Dentistry. 1965-1972 (1996). 193-197 (2000). 10. Simmons, P., Gronthos, S. & Zannettino, A. C. Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals 1-3 . To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs) 4 . Here we show that PPRstimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, liganddependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by g-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.Mammalian bone marrow architecture involves haematopoietic stem cells (HSCs) in close proximity to the endosteal surfaces 5,6 , with more differentiated cells arranged in a loosely graduated fashion as the central longitudinal axis of the bone is approached 5,7,8 . This nonrandom organization of the marrow suggests a possible relationship between HSCs and osteoblasts-osteogenic cells lining the endosteal surface. Osteoblasts produce haematopoietic growth factors [9][10][11] and are activated by parathyroid hormone (PTH) or the locally produced PTH-related protein (PTHrP), through the PTH/ PTHrP receptor (PPR). We tested whether osteoblast...

Skeletal development and turnover occur in close spatial and temporal association with angiogenesis. Osteoblasts are ideally situated in bone to sense oxygen tension and respond to hypoxia by activating the hypoxiainducible factor α (HIFα) pathway. Here we provide evidence that HIFα promotes angiogenesis and osteogenesis by elevating VEGF levels in osteoblasts. Mice overexpressing HIFα in osteoblasts through selective deletion of the von Hippel-Lindau gene (Vhl) expressed high levels of Vegf and developed extremely dense, heavily vascularized long bones. By contrast, mice lacking Hif1a in osteoblasts had the reverse skeletal phenotype of that of the Vhl mutants: long bones were significantly thinner and less vascularized than those of controls. Loss of Vhl in osteoblasts increased endothelial sprouting from the embryonic metatarsals in vitro but had little effect on osteoblast function in the absence of blood vessels. Mice lacking both Vhl and Hif1a had a bone phenotype intermediate between those of the single mutants, suggesting overlapping functions of HIFs in bone. These studies suggest that activation of the HIFα pathway in developing bone increases bone modeling events through cell-nonautonomous mechanisms to coordinate the timing, direction, and degree of new blood vessel formation in bone. IntroductionThe development of the mammalian skeleton takes place in distinct phases involving the initial migration of cells to the site of future bone, condensation of mesenchymal cells, and finally the differentiation of progenitors into chondrocytes and osteoblasts. During intramembranous bone formation, which gives rise to the flat bones of the skull, mesenchymal cells differentiate directly into bone-forming osteoblasts. By contrast, in endochondral bone formation, bones are formed through a 2-stage mechanism that begins with the formation of a chondrocyte anlage, onto which osteoblasts then differentiate and deposit bone. Endochondral bone formation occurs in close spatial and temporal association and proximity to capillary invasion, suggesting that angiogenesis and osteogenesis are coupled.The initial signals for blood vessel invasion into bone are unknown, but tissue hypoxia is believed to be critical for commencement of the angiogenic cascade (1). Hypoxia triggers the changes in oxygen-regulated gene expression via the activation of the Per/Arnt/Sim (PAS)

The complementary DNA encoding a 585-amino acid parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor with seven potential membrane-spanning domains was cloned by COS-7 expression using an opossum kidney cell complementary DNA (cDNA) library. The expressed receptor binds PTH and PTHrP with equal affinity, and both ligands equivalently stimulate adenylate cyclase. Striking homology with the calcitonin receptor and lack of homology with other G protein-linked receptors indicate that receptors for these calcium-regulating hormones are related and represent a new family.

Parathyroid hormone (PTH), a major regulator of mineral ion metabolism, and PTH-related peptide (PIHrP)

A single heterozygous nucleotide exchange in exon M2 of the gene encoding the parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor was identified in a patient with Jansen-type metaphyseal chondrodysplasia, which changes a strictly conserved histidine residue at position 223 in the receptor's first intracellular loop to arginine. Constitutive, ligand-independent adenosine 3',5'-monophosphate accumulation was observed in COS-7 cells expressing the mutant PTH-PTHrP receptor but not in cells expressing the wild-type receptor. This finding explains the severe ligand-independent hypercalcemia and hypophosphatemia, and most likely the abnormal formation of endochondral bone, in this rare form of short-limbed dwarfism.

Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.

Mutations in the fibroblast growth factor 23 gene, FGF23, cause autosomal dominant hypophosphatemic rickets (ADHR). The gene product, FGF-23, is produced by tumors from patients with oncogenic osteomalacia (OOM), circulates at increased levels in most patients with X-linked hypophosphatemia (XLH) and is phosphaturic when injected into rats or mice, suggesting involvement in the regulation of phosphate (Pi) homeostasis. To better define the precise role of FGF-23 in maintaining Pi balance and bone mineralization, we generated transgenic mice that express wild-type human FGF-23, under the control of the alpha1(I) collagen promoter, in cells of the osteoblastic lineage. At 8 wk of age, transgenic mice were smaller (body weight = 17.5 +/- 0.57 vs. 24.3 +/- 0.37 g), exhibited decreased serum Pi concentrations (1.91 +/- 0.27 vs. 2.75 +/- 0.22 mmol/liter) and increased urinary Pi excretion when compared with wild-type littermates. The serum concentrations of human FGF-23 (undetectable in wild-type mice) was markedly elevated in transgenic mice (>7800 reference units/ml). Serum PTH levels were increased in transgenic mice (231 +/- 62 vs. 139 +/- 44 pg/ml), whereas differences in calcium and 1,25-dihydroxyvitamin D were not apparent. Expression of Npt2a, the major renal Na(+)/Pi cotransporter, as well as Npt1 and Npt2c mRNAs, was significantly decreased in the kidneys of transgenic mice. Histology of tibiae displayed a disorganized and widened growth plate and peripheral quantitative computerized tomography analysis revealed reduced bone mineral density in transgenic mice. The data indicate that FGF-23 induces phenotypic changes in mice resembling those of patients with ADHR, OOM, and XLH and that FGF-23 is an important determinant of Pi homeostasis and bone mineralization.

IntroductionIn bone remodeling the activities of osteoblasts, the bone-forming cells, and osteoclasts, cells of hematopoietic origin capable of resorbing bone, must be balanced carefully in order to maintain skeletal integrity (1). The importance of understanding the factors controlling these activities is highlighted by metabolic bone disorders such as osteoporosis, in which the imbalance of bone formation and resorption leads to bone loss. Parathyroid hormone (PTH), a major regulator of calcium homeostasis, plays an important role in both bone formation and resorption. While PTH excess in hyperparathyroidism (2) and in continuous administration of PTH (3) is characterized by large numbers of osteoclasts, rapid bone turnover, and low cortical bone mass, it has long been known that intermittent dosing of PTH can lead to increased trabecular bone mass (4,5). This anabolic effect is due to increased bone formation (6, 7). Interestingly, histomorphometric studies in patients with mild hyperparathyroidism also show an increase in cancellous bone (2). Although osteoblasts likely mediate both the anabolic and catabolic actions of the peptide, the molecular mechanisms underlying this dual effect are incompletely understood.The PTH/PTH-related protein (PTH/PTHrP) receptor (PPR), a G protein-coupled receptor, is believed to mediate many of the actions of both PTH and PTHrP in bone, as shown by mutations in mice and humans. Mice in which the PPR has been ablated by homologous recombination have decreased trabecular bone and increased thickness of cortical bone during fetal development (8). These skeletal abnormalities are similar to those observed in patients with Blomstrand lethal chondrodysplasia, a rare autosomal recessive disorder caused by inactivating PPR mutations (9, 10). Consistent with this crucial role of the PPR in cells of the osteoblast lineage, expression of the mRNA encoding this receptor is detectable in relatively mature osteoblasts and in adjacent stromal cells likely to be osteoblast precursors (11).Jansen's metaphyseal chondrodysplasia (JMC) is a rare form of short-limbed dwarfism caused by activating mutations of the PPR leading to ligand-independent cAMP accumulation (12). Histomorphometric analysis of bone from a patient with this disorder shows exaggerated loss of cortical bone and preservation, or even augmentation of trabecular bone, as is seen in mild primary hyperparathyroidism (13). Parathyroid hormone (PTH), an important regulator of calcium homeostasis, targets most of its complex actions in bone to cells of the osteoblast lineage. Furthermore, PTH is known to stimulate osteoclastogenesis indirectly through activation of osteoblastic cells. To assess the role of the PTH/PTHrelated protein receptor (PPR) in mediating the diverse actions of PTH on bone in vivo, we generated mice that express, in cells of the osteoblastic lineage, one of the constitutively active receptors described in Jansen's metaphyseal chondrodysplasia. In these transgenic mice, osteoblastic function was increased in the t...