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...

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...

The von Hippel Lindau tumor suppressor protein (pVHL) is a component of a ubiquitin ligase that promotes proteolysis of the transcription factor hypoxia-inducible-factor 1α (HIF1α), the key molecule in the hypoxic response. We have used conditional inactivation of murine VHL(Vhlh) in all cartilaginous elements to investigate its role in endochondral bone development. Mice lacking Vhlh in cartilage are viable, but grow slower than control littermates and develop a severe dwarfism. Morphologically, Vhlh null growth plates display a significantly reduced chondrocyte proliferation rate, increased extracellular matrix, and presence of atypical large cells within the resting zone. Furthermore, stabilization of the transcription factor HIF1α leads to increased expression levels of HIF1α target genes in Vhlh null growth plates. Lastly, newborns lacking both Vhlh and Hif1agenes in growth plate chondrocytes display essentially the same phenotype as Hif1a null single mutant mice suggesting that the Vhlh null phenotype could result, at least in part, from increased activity of accumulated HIF1α. This is the first study reporting the novel and intriguing findings that pVHL has a crucial role in endochondral bone development and is necessary for normal chondrocyte proliferation in vivo.