Controlling Chondrocyte Differentiation Research Articles

Inflammatory and Metabolic Signaling Interfaces of the Hypertrophic and Senescent Chondrocyte Phenotypes Associated with Osteoarthritis.

Osteoarthritis (OA) is a complex disease of whole joints with progressive cartilage matrix degradation and chondrocyte transformation. The inflammatory features of OA are reflected in increased synovial levels of IL-1β, IL-6 and VEGF, higher levels of TLR-4 binding plasma proteins and increased expression of IL-15, IL-18, IL-10 and Cox2, in cartilage. Chondrocytes in OA undergo hypertrophic and senescent transition; in these states, the expression of Sox-9, Acan and Col2a1 is suppressed, whereas the expression of RunX2, HIF-2α and MMP-13 is significantly increased. NF-kB, which triggers many pro-inflammatory cytokines, works with BMP, Wnt and HIF-2α to link hypertrophy and inflammation. Altered carbohydrate metabolism and the upregulation of GLUT-1 contribute to the formation of end-glycation products that trigger inflammation via the RAGE pathway. In addition, a glycolytic shift, increased rates of oxidative phosphorylation and mitochondrial dysfunction generate reactive oxygen species with deleterious effects. An important surveyor mechanism, the YAP/TAZ signaling system, controls chondrocyte differentiation, inhibits ageing by protecting the nuclear envelope and suppressing NF-kB, MMP-13 and aggrecanases. The inflammatory microenvironment and synthesis of key matrix components are also controlled by SIRT1 and mTORc. Senescent chondrocytes represent the functional end stage of hypertrophic differentiation and characteristically upregulate p16 and p21, but also a variety of inflammatory cytokines, chemokines and metalloproteinases, developing the senescence-associated secretory phenotype. Senolysis with dendrobin, miR29b-5p and other agents has been shown to be efficient under experimental conditions, and appears to be a promising tool for the treatment of OA, as it restores COL2A1 and aggrecan synthesis, suppressing NF-kB and destructive metalloproteinases.

TAp63γ influences mouse cartilage development.

Depletion of tumor protein p63 results in severe epithelial as well as limb defects in mice, suggesting that p63 is also required for endochondral ossification during long bone development. A key stage in endochondral ossification is chondrocyte hypertrophy, which has been associated with elevated levels of the p63 variant TAp63γ. To investigate the role of TAp63γ in chondrocyte differentiation and maturation, we developed stable TAp63γ expressing ATDC5 cells. Compared to control cells, TAp63γ cells showed significant upregulation of Col10a1 after 4 and 7 days in culture. Moreover, alkaline phosphatase, Alizarin red, and Alcian blue staining were stronger in TAp63γ cells, suggesting that TAp63γ promotes chondrocyte proliferation, hypertrophic differentiation, and possibly matrix mineralization. To investigate the in vivo function of TAp63γ during endochondral bone formation, we established transgenic mice that express flag-tagged TAp63γ driven by Col10a1 regulatory elements. Skeletal staining of transgenic mice at postnatal day 1 showed accelerated ossification in long bone, tail, and digit bones compared to wild-type littermates. Furthermore, Sox9 expression was reduced and Runx2 expression was increased in the proliferative and/or hypertrophic zones of these mice. Altogether, these results suggest that TAp63γ promotes endochondral ossification and skeletal development, at least partially via controlling chondrocyte differentiation and maturation.

Hypoxia promotes maintenance of the chondrogenic phenotype in rat growth plate chondrocytes through the HIF-1α/YAP signaling pathway.

The Hippo-yes-associated protein (YAP) signaling pathway was previously identified to serve an important role in controlling chondrocyte differentiation and post-natal growth. Growth plate cartilage tissue is avascular, and hypoxia-inducible factor (HIF)-1α is essential for chondrocytes to maintain their chondrogenic phenotype in a hypoxic environment. In the present study, the role of hypoxia and HIF-1α in the regulation of YAP in chondrocytes was investigated. The data demonstrated that hypoxia promoted the maintenance of the chondrogenic phenotype, HIF-1α expression and YAP activation in chondrocytes in a time-dependent manner. Hypoxia promoted YAP activation in a Hippo-independent manner. Inhibiting the expression of HIF-1α decreased the activation of YAP and downregulated the expression of sex-determining region-box 9 protein (SOX9) under hypoxic conditions, while the upregulation of HIF-1α by cobalt chloride promoted the expression and nuclear translocation of YAP and upregulated the expression of SOX9 and collagen II chain under normoxic conditions. In addition, inhibition of YAP expression under hypoxia did not affect the expression of the HIF-1α signaling pathway, but inhibited the up-regulation of SOX9 expression caused by hypoxia. In addition, reoxygenation following hypoxia inhibited the activation of YAP caused by hypoxia in chondrocytes, whereas the upregulation of SOX9 and collagen II chain also appeared to be inhibited. In conclusion, the results of the present study demonstrated that hypoxia promoted YAP activation via HIF-1α. Therefore, the HIF-1α/YAP signaling axis may serve an important role in controlling growth plate chondrocyte differentiation and the maintenance of the chondrogenic phenotype in growth plate chondrocytes.

A Tumor Suppressor Gene Product, Platelet-Derived Growth Factor Receptor-Like Protein Controls Chondrocyte Proliferation and Differentiation.

The platelet-derived growth factor receptor-like (PDGFRL) gene is regarded as a tumor suppressor gene. However, nothing is known about the molecular function of PDGFRL. In this study, we initially clarified its function in chondrocytes. Among all cell lines examined, the PDGFRL mRNA level was the highest in chondrocytic HCS-2/8 cells. Interestingly, the proliferation of chondrocytic HCS-2/8 cells was promoted by PDGFRL overexpression, whereas that of the breast cancer-derived MDA-MB-231 cells was inhibited. Of note, in PDGFRL-overexpressing HCS-2/8 cells, the expression of chondrocyte differentiation marker genes, SOX9, ACAN, COL2A1, COL10A1, and ALP, was decreased. Moreover, we confirmed the expression of PDGFRL mRNA in normal cartilage tissue and chondrocytes. Eventually, the expression of PDGFRL mRNA in condrocytes except in the case of hypertrophic chondrocytes was demonstrated in vivo and in vitro. These findings suggest that PDGFRL plays the different roles, depending upon cell types. Particularly, in chondrocytes, PDGFRL may play a new and important role which is distinct from the function previously reported. J. Cell. Biochem. 118: 4033-4044, 2017. © 2017 Wiley Periodicals, Inc.

Inactivation of Fam20B in Joint Cartilage Leads to Chondrosarcoma and Postnatal Ossification Defects.

During endochondral ossification, chondrocytes embed themselves in a proteoglycan-rich matrix during the proliferation-maturation transition. Accumulating evidence shows that proteoglycans are essential components for chondrocyte proliferation and differentiation. When we conditionally inactivated FAM20B (Family with sequence similarity 20 member-B), which is a newly identified xylose kinase essential for glycosaminoglycan (GAG) formation on the protein core of proteoglycans, from the dental mesenchyme using Osr2-Cre, which is also strongly expressed in joint cartilage, we found chondrosarcoma in the knee joint and remarkable defects of postnatal ossification in the long bones. Mechanistic analysis revealed that the defects were associated with gain of function in multiple signaling pathways in the epiphyseal chondrocytes, such as those derived by WNT, BMP, and PTHrP/IHH molecules, suggesting that the FAM20B-catalyzed proteoglycans are critical mediators for a signaling balance in the regulatory network controlling chondrocyte differentiation and proliferation. In particular, we demonstrated that the WNT inhibitor was able to rescue part of the bone defects in Osr2-Cre;Fam20Bfl/fl mice, indicating that FAM20B-catalyzed proteoglycans regulate postnatal endochondral ossification partially through the mediation of WNT signaling.

Phlpp1 facilitates post-traumatic osteoarthritis and is induced by inflammation and promoter demethylation in human osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability. OA is characterized by articular chondrocyte deterioration, subchondral bone changes and debilitating pain. One strategy to promote cartilage regeneration and repair is to accelerate proliferation and matrix production of articular chondrocytes. We previously reported that the protein phosphatase Phlpp1 controls chondrocyte differentiation by regulating the activities of anabolic kinases. Here we examined the role of Phlpp1 in OA progression in a murine model. We also assessed PHLPP1 expression and promoter methylation. Knee joints of WT and Phlpp1(-/-) mice were surgically destabilized by transection of the medial meniscal ligament (DMM). Mice were assessed for signs of OA progression via radiographic and histological analyses, and pain assessment for mechanical hypersensitivity using the von Frey assay. Methylation of the PHLPP1 promoter and PHLPP1 expression were evaluated in human articular cartilage and chondrocyte cell lines. Following DMM surgeries, Phlpp1 deficient mice showed fewer signs of OA and cartilage degeneration. Mechanical allodynia associated with DMM surgeries was also attenuated in Phlpp1(-/-) mice. PHLPP1 was highly expressed in human articular cartilage from OA patients, but was undetectable in cartilage specimens from femoral neck fractures (FNFxs). Higher PHLPP1 levels correlated with less PHLPP1 promoter CpG methylation in cartilage from OA patients. Blocking cytosine methylation or treatment with inflammatory mediators enhanced PHLPP1 expression in human chondrocyte cell lines. Phlpp1 deficiency protects against OA progression while CpG demethylation and inflammatory cytokines promote PHLPP1 expression.

Elevated expression of microRNA-30b in osteoarthritis and its role in ERG regulation of chondrocyte.

ERG (ETS-related gene) belongs to the ETS family of transcription factors, and has been recently reported to contribute to homeostatic balance in skeleton cell plasticity. MicroRNA-30 (miR-30) family is also demonstrated to play a role in controlling chondrocyte differentiation. The current study investigated the miR-30b and ERG expression in articular cartilage of osteoarthritis (OA) patients. A total of 20 subjects, with 10 OA patients and 10 healthy participants, were included in this study. Human chondrosarcoma cell line SW1353 was used to explore the relationship of miR-30b and ERG in vitro. In OA patients, a significant increase of miR-30b and a decrease of ERG were observed in articular cartilage compared with Normal ones. MiR-30b mimic down-regulated the ERG mRNA and protein expression levels, while miR-30b inhibitor up-regulated ERG expression. In addition, miR-30b mimic also decreased the mRNA expression of COL2a and aggrecan, while miR-30b inhibitor had the opposite effect. Luciferase reporter assay confirmed that miR-30b targeted ERG. In conclusion, miR-30b was involved in the process of OA, and it probably functioned through its target gene ERG.

MicroRNA-140 Provides Robustness to the Regulation of Hypertrophic Chondrocyte Differentiation by the PTHrP-HDAC4 Pathway.

Growth plate chondrocytes go through multiple differentiation steps and eventually become hypertrophic chondrocytes. The parathyroid hormone (PTH)-related peptide (PTHrP) signaling pathway plays a central role in regulation of hypertrophic differentiation, at least in part, through enhancing activity of histone deacetylase 4 (HDAC4), a negative regulator of MEF2 transcription factors that drive hypertrophy. We have previously shown that loss of the chondrocyte-specific microRNA (miRNA), miR-140, alters chondrocyte differentiation including mild acceleration of hypertrophic differentiation. Here, we provide evidence that miR-140 interacts with the PTHrP-HDAC4 pathway to control chondrocyte differentiation. Heterozygosity of PTHrP or HDAC4 substantially impaired animal growth in miR-140 deficiency, whereas these mutations had no effect in the presence of miR-140. miR-140-deficient chondrocytes showed increased MEF2C expression with normal levels of total and phosphorylated HDAC4, indicating that the miR-140 pathway merges with the PTHrP-HDAC4 pathway at the level of MEF2C. miR-140 negatively regulated p38 mitogen-activated protein kinase (MAPK) signaling, and inhibition of p38 MAPK signaling reduced MEF2C expression. These results demonstrate that miR-140 ensures the robustness of the PTHrP/HDAC4 regulatory system by suppressing MEF2C-inducing stimuli. © 2014 American Society for Bone and Mineral Research © 2015 American Society for Bone and Mineral Research.

Notch Suppresses Nuclear Factor of Activated T Cells (Nfat) Transactivation and Nfatc1 Expression in Chondrocytes

Notch1 to Notch4 transmembrane receptors determine cell fate, and release of the Notch intracellular domain (NICD) in the cytoplasm induces gene expression. Notch regulates endochondral ossification, but it is not clear whether Notch interacts with signals controlling chondrocyte differentiation. Nuclear factor of activated T cells (Nfatc) transcription factors regulate chondrogenesis, and we asked whether Notch modifies Nfat signaling in chondrocytes. Notch was induced in teratocarcinoma ATDC5 chondrogenic cells infected with a retroviral vector, where the cytomegalovirus (CMV) promoter directs NICD expression. NICD suppressed chondrocyte differentiation and inhibited Nfat transactivation and Nfatc1 expression. Notch was activated in chondrocytes from Rosa(Notch) mice, where the Rosa26 promoter is upstream of a loxP-flanked STOP cassette and NICD. To excise the STOP cassette and express NICD, Rosa(Notch) chondrocytes were infected with an adenoviral vector where the CMV promoter directs Cre expression (Ad-CMV-Cre). Notch1 and Notch2 mediate the effects of Notch in skeletal cells, and to inhibit Notch signaling, chondrocytes from mice homozygous for Notch1 and Notch2 alleles targeted with loxP sites were infected with Ad-CMV-Cre. NICD suppressed chondrogenic nodules formation and expression of selected chondrocyte gene markers, induced Col10a1 and Mmp13, and suppressed Nfat transactivation and Nfatc1 expression, whereas inactivation of Notch1 and Notch2 did not affect chondrocyte differentiation. To investigate Nfatc1 function in chondrocytes, Nfatc1 was induced in Rosa(Notch) chondrocytes overexpressing NICD or controls. Nfatc1 suppressed chondrocyte differentiation and opposed Col10a1 induction by Notch. In conclusion, Notch suppresses Nfat transactivation in chondrocytes and Notch and Nfatc1 regulate chondrocyte differentiation.

Hoxa2 controls chondrocyte differentiation through Sox9 inhibition

Hoxa2 controls chondrocyte differentiation through Sox9 inhibition

Lysyl Oxidase-like-2 (LOXL2) Is a Major Isoform in Chondrocytes and Is Critically Required for Differentiation

The lysyl oxidase family is made up of five members: lysyl oxidase (LOX) and lysyl oxidase-like 1-4 (LOXL1-LOXL4). All members share conserved C-terminal catalytic domains that provide for lysyl oxidase or lysyl oxidase-like enzyme activity; and more divergent propeptide regions. LOX family enzyme activities catalyze the final enzymatic conversion required for the formation of normal biosynthetic collagen and elastin cross-links. The importance of lysyl oxidase enzyme activity to normal bone development has long been appreciated, but regulation and roles for specific LOX isoforms in bone formation in vivo is largely unexplored. Fracture healing recapitulates aspects of endochondral bone development. The present study first investigated the expression of all LOX isoforms in fracture healing. A remarkable coincidence of LOXL2 expression with the chondrogenic phase of fracture healing was found, prompting more detailed analyses of LOXL2 expression in normal growth plates, and LOXL2 expression and function in developing ATDC5 chondrogenic cells. Data show that LOXL2 is expressed by pre-hypertrophic and hypertrophic chondrocytes in vivo, and that LOXL2 expression is regulated in vitro as a function of chondrocyte differentiation. Moreover, LOXL2 knockdown studies in vitro show that LOXL2 expression is required for ATDC5 chondrocyte cell line differentiation through regulation of SNAIL and SOX9, important transcription factors that control chondrocyte differentiation. Taken together, data provide evidence that LOXL2, like LOX, is a multifunctional protein. LOXL2 promotes chondrocyte differentiation by mechanisms that are likely to include roles as both a regulator and an effector of chondrocyte differentiation.

A feedback loop between xylt1 and sox9 controls chondrocyte differentiation

A feedback loop between xylt1 and sox9 controls chondrocyte differentiation

The effect of weight loading and subsequent release from loading on the postnatal skeleton

Introduction The relationship between load and the structure and mechanical properties of mature bones has been thoroughly described. In contrast, this relationship has been studied much less in immature bones, which consist of bony tissue and cartilaginous growth plate, during the postnatal period. This paper describes the effect of an externally applied load on the bones of young fast-growing chicks; in particular, we examine the effect on the growth plate, which regulates longitudinal bone growth, and the consequences in terms of bone structural and mechanical properties. Materials and methods The tibial growth plates from chicks subjected to external load and control chicks, immediately after loading and following 5 days of load release, were studied by histological staining and quantitative PCR. The contralateral tibiae were mechanically tested by three-point bending and their structural features determined by micro-CT. Results At the end of the external loading period, the tibias of the experimental group were shorter and their growth plate narrower than in controls. However, at this time point, effects were not yet apparent in the bones' structural or mechanical parameters. After a further 5 days of no external load, bones and growth plates of the experimental group demonstrated the phenomenon of ‘catch-up’: the thickness of the growth plate exceeded that of the control; however the relative expression of genes controlling chondrocyte differentiation (collagen II and X) did not change, while the expression of factors related to growth-plate ossification (osteopontin, alkaline phosphatase) and cartilage and bone calcification (matrix and bone Gla proteins) was upregulated as a result of the catch-up process. At this time, however, the tibiae of the experimental group showed inferior mechanical and structural properties relative to the control group. Conclusion External loading during bone elongation negatively affects the mechanical and structural properties of the skeleton. The effect is first noticeable in the growth plate, which regulates bone growth, and is exhibited in the bone phenotype after a lag period.

Regulator of G‐protein signaling (RGS) proteins differentially control chondrocyte differentiation

Control of chondrocyte differentiation is attained, in part, through G-protein signaling, but the functions of the RGS family of genes, well known to control G-protein signaling at the Galpha subunit, have not been studied extensively in chondrogenesis. Recently, we have identified the Rgs2 gene as a regulator of chondrocyte differentiation. Here we extend these studies to additional Rgs genes. We demonstrate that the Rgs4, Rgs5, Rgs7, and Rgs10 genes are differentially regulated during chondrogenic differentiation in vitro and in vivo. To investigate the roles of RGS proteins during cartilage development, we overexpressed RGS4, RGS5, RGS7, and RGS10 in the chondrogenic cell line ATDC5. We found unique and overlapping effects of individual Rgs genes on numerous parameters of chondrocyte differentiation. In particular, RGS5, RGS7, and RGS10 promote and RGS4 inhibits chondrogenic differentiation. The identification of Rgs genes as novel regulators of chondrogenesis will contribute to a better understanding of both normal cartilage development and the etiology of chondrodysplasias and osteoarthritis.

Interactions between Sox9 and beta-catenin control chondrocyte differentiation.

Chondrogenesis is a multistep process that is essential for endochondral bone formation. Previous results have indicated a role for beta-catenin and Wnt signaling in this pathway. Here we show the existence of physical and functional interactions between beta-catenin and Sox9, a transcription factor that is required in successive steps of chondrogenesis. In vivo, either overexpression of Sox9 or inactivation of beta-catenin in chondrocytes of mouse embryos produces a similar phenotype of dwarfism with decreased chondrocyte proliferation, delayed hypertrophic chondrocyte differentiation, and endochondral bone formation. Furthermore, either inactivation of Sox9 or stabilization of beta-catenin in chondrocytes also produces a similar phenotype of severe chondrodysplasia. Sox9 markedly inhibits activation of beta-catenin-dependent promoters and stimulates degradation of beta-catenin by the ubiquitination/proteasome pathway. Likewise, Sox9 inhibits beta-catenin-mediated secondary axis induction in Xenopus embryos. Beta-catenin physically interacts through its Armadillo repeats with the C-terminal transactivation domain of Sox9. We hypothesize that the inhibitory activity of Sox9 is caused by its ability to compete with Tcf/Lef for binding to beta-catenin, followed by degradation of beta-catenin. Our results strongly suggest that chondrogenesis is controlled by interactions between Sox9 and the Wnt/beta-catenin signaling pathway.

Establishing the protein MIA (melanoma inhibitory activity) as a marker for chondrocyte differentiation

Melanoma inhibitory activity (MIA), also referred to as cartilage derived retinoic acid-sensitive protein (CD-RAP), is detected physiologically in cartilage tissue and pathologically in malignant melanomas. To measure MIA/CD-RAP quantitatively we developed a sensitive ELISA system. Recently, we described diagnostic applications of the MIA-ELISA in patients with cartilage diseases. The study described herein was performed to determine whether there is any relation between MIA/CD-RAP levels and the degree of chondrocyte differentiation in tissue culture and to analyse whether MIA/CD-RAP may serve as a useful marker to control chondrocyte differentiation in in vitro tissue engineering. Our data provide evidence that measuring MIA in tissue culture supernatant by a quantitative ELISA can be used as a marker for differentiated chondrocytes.

Bone defects in latent TGF-beta binding protein (Ltbp)-3 null mice; a role for Ltbp in TGF-beta presentation.

The latent transforming growth factor (TGF)-beta binding proteins (LTBP)-1, -3 and -4 bind the latent form of the multipotent cytokine TGF-beta. To examine the function of the LTBPs, we made a null mutation of Ltbp-3 by gene targeting. The homozygous mutant animals developed cranio-facial malformations by 12 days. By three months, there was a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. The mutant animals developed osteosclerosis of the long bones and vertebrae as well as osteoarthritis between 6 and 9 months of age. These latter phenotypic changes were similar to those described for mice that have impaired TGF-beta signaling. Thus, we suggest that Ltbp-3 plays an important role in regulating TGF-beta bioavailability as the phenotype of the Ltbp-3 null mouse appears to result from decreased TGF-beta signaling. Histological examination of the skulls from null animals revealed no effects on calvarial suture closure. However, the synchondroses in the skull base were obliterated within 2 weeks of birth. This is in contrast to the wild-type synchondroses, which remain unossified throughout the life of the animal and enable growth of the skull base through endochondral ossification. Histological changes in mutant basooccipital-basosphenoid synchondrosis were observed 1.5 days after birth. Compared with wild-type or heterozygous littermates, the basooccipital-basosphenoid synchondrosis of Ltbp-3 null mice contained increased numbers of hypertrophic chondrocytes. The expression of bone sialoprotein-1 (a marker for osteoblasts) was observed in cells surrounding the synchondrosis at postnatal day 1.5 indicating ectopic ossification. The expression of Indian hedgehog (Ihh) (a marker for chondrocytes committed to hypertrophic differentiation) was found through the basooccipital-basosphenoid synchondrosis, whereas the expression of parathyroid hormone related protein (PTHrP), which inhibits chondrocyte differentiation, appeared to be diminished in Ltbp-3 null mice. This suggests that Ltbp-3 may control chondrocyte differentiation by regulating TGF-beta availability. TGF-beta may regulate PTHrP expression either downstream of Ihh or independently of Ihh signaling.

A novel cell culture model of chondrocyte differentiation during mammalian endochondral ossification.

Endochondral ossification (EO) occurs in the growth plate where chondrocytes pass through discrete stages of proliferation, maturation, hypertrophy, and calcification. We have developed and characterized a novel bovine cell culture model of EO that mirrors these events and will facilitate in vitro studies on factors controlling chondrocyte differentiation. Chondrocytes derived from the epiphyses of long bones of fetal calves were treated with 5-azacytidine (aza-C) for 48 h. Cultures were maintained subsequently without aza-C and harvested at selected time points for analyses of growth and differentiation status. A chondrocytic phenotype associated with an extensive extracellular matrix rich in proteoglycans and collagen types II and VI was observed in aza-C-treated and -untreated cultures. aza-C-treated cultures were characterized by studying the expression of several markers of chondrocyte differentiation. Parathyroid hormone-related protein (PTHrP) and its receptor, both markers of maturation, were expressed at days 5-9. Type X collagen, which is restricted to the stage of hypertrophy, was expressed from day 11 onward. Hypertrophy was confirmed by a 14-fold increase in cell size by day 15 and an increased synthesis of alkaline phosphatase during the hypertrophic period (days 14-28). The addition of PTHrP to aza-C-treated cultures at day 14 led to the down-regulation of type X collagen by 6-fold, showing type X collagen expression is under the control of PTHrP as in vivo. These findings show that aza-C can induce fetal bovine epiphyseal chondrocytes to differentiate in culture in a manner consistent with that which occurs during the EO process in vivo.

A molecular analysis of matrix remodeling and angiogenesis during long bone development.

The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a 'molecular map' of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.

The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation.

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (-/-) cells, and analyzed cell-cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.