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.