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...

Vertebrates do not look like jellyfish because the bones of their skeletons are levers that allow movement and protect vital organs. Bones come in an enormous variety of shapes and sizes to accomplish these goals, but, with few exceptions, use one process--endochondral bone formation--to generate the skeleton. The past few years have seen an enormous increase in understanding of the signalling pathways and the transcription factors that control endochondral bone development.

Proper regulation of chondrocyte differentiation is necessary for the morphogenesis of skeletal elements, yet little is known about the molecular regulation of this process. A chicken homolog of Indian hedgehog (Ihh), a member of the conserved Hedgehog family of secreted proteins that is expressed during bone formation, has now been isolated. Ihh has biological properties similar to those of Sonic hedgehog (Shh), including the ability to regulate the conserved targets Patched (Ptc) and Gli. Ihh is expressed in the prehypertrophic chondrocytes of cartilage elements, where it regulates the rate of hypertrophic differentiation. Misexpression of Ihh prevents proliferating chondrocytes from initiating the hypertrophic differentiation process. The direct target of Ihh signaling is the perichondrium, where Gli and Ptc flank the expression domain of Ihh. Ihh induces the expression of a second signal, parathyroid hormone-related protein (PTHrP), in the periarticular perichondrium. Analysis of PTHrP (-/-) mutant mice indicated that the PTHrP protein signals to its receptor in the prehypertrophic chondrocytes, thereby blocking hypertrophic differentiation. In vitro application of Hedgehog or PTHrP protein to normal or PTHrP (-/-) limb explants demonstrated that PTHrP mediates the effects of Ihh through the formation of a negative feedback loop that modulates the rate of chondrocyte differentiation.

SUMMARY During endochondral bone development, the first osteoblasts differentiate in the perichondrium surrounding avascular cartilaginous rudiments; the source of trabecular osteoblasts inside the later bone is, however, unknown. Here, we generated tamoxifen-inducible transgenic mice bred to Rosa26R-LacZ reporter mice to follow the fates of stage-selective subsets of osteoblast lineage cells. Pulse-chase studies showed that osterix-expressing osteoblast precursors, labeled in the perichondrium prior to vascular invasion of the cartilage, give rise to trabecular osteoblasts, osteocytes, and stromal cells inside the developing bone. Throughout the translocation, some precursors were found to intimately associate with invading blood vessels, in pericyte-like fashion. A similar coinvasion occurs during endochondral healing of bone fractures. In contrast, perichondrial mature osteoblasts did not exhibit perivascular localization and remained in the outer cortex of developing bones. These findings reveal the specific involvement of immature osteoblast precursors in the coupled vascular and osteogenic transformation essential to endochondral bone development and repair.

The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (-/-) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (-/-) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.

The parathyroid hormone-related peptide (PTHrP) gene was disrupted in murine embryonic stem cells by homologous recombination, and the null allele was introduced into the mouse germ line. Mice homozygous for the PTHrP null mutation died postnatally, probably from asphyxia, and exhibited widespread abnormalities of endochondral bone development. Histological examination revealed a diminution of chondrocyte proliferation, associated with premature maturation of chondrocytes and accelerated bone formation. Analysis of earlier developmental stages revealed that disturbance in cartilage growth preceded abnormal endochondral bone formation. There were no morphological abnormalities apparent in other tissues. These results provide direct evidence implicating PTHrP in normal skeletal development and serve to emphasize its potential involvement in human osteochondrodysplasias.

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.

We have previously identified a candidate oncogene (PRAD1 or D11S287E) on chromosome 11q13 which is clonally rearranged with the parathyroid hormone locus in a subset of benign parathyroid tumours. We now report that a cloned human placental PRAD1 complementary DNA encodes a protein of 295 amino acids with sequence similarities to the cyclins. Cyclins can form a complex with and activate p34cdc2 protein kinase, thereby regulating progress through the cell cycle. PRAD 1 messenger RNA levels vary dramatically across the cell cycle in HeLa cells. Addition of the PRAD1 protein to interphase clam embryo lysates containing inactive p34cdc2 kinase and lacking endogenous cyclins allows it to be isolated using beads bearing p13suc1, a yeast protein that binds cdc2 and related kinases with high affinity and coprecipitates kinase-associated proteins. Addition of PRAD1 also induces phosphorylation of histone H1, a preferred substrate of cdc2. These data suggest that PRAD1 encodes a novel cyclin whose overexpression may play an important part in the development of various tumours with abnormalities in 11q13.