Disproportional skeletal growth and markedly decreased bone mineral content in growth hormone receptor -/- mice

Growth hormone (GH) is important for skeletal growth as well as for a normal bone metabolism in adults. The skeletal growth and adult bone metabolism was studied in mice with an inactivated growth hormone receptor (GHR) gene. The lengths of femur, tibia, and crown-rump were, as expected, decreased in GHR-/- mice. Unexpectedly, GHR-/- mice displayed disproportional skeletal growth reflected by decreased femur/crown-rump and femur/tibia ratios. GHR-/- mice demonstrated decreased width of the growth plates in the long bones and disturbed ossification of the proximal tibial epiphysis. Furthermore, the area bone mineral density (BMD) as well as the bone mineral content (BMC)/body weight were markedly decreased in GHR-/- mice. The decrease in BMC in GHR-/- mice was not due to decreased trabecular volumetric BMD but to a decreased cross-sectional cortical bone area In conclusion, GHR-/- mice demonstrate disproportional skeletal growth and markedly decreased bone mineral content.     

Skeletal Development of Mice Lacking Bone Sialoprotein (BSP) - Impairment of Long Bone Growth and Progressive Establishment of High Trabecular Bone Mass

Adult Ibsp-knockout mice (BSP−/−) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP−/− mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP−/− newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP−/− mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP−/− than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP−/− mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP−/− mice, while impairing primary mineralization.     

Bone morphogenetic protein signaling through ACVR1 and BMPR1A negatively regulates bone mass along with alterations in bone composition

Bone quantity and bone quality are important factors in determining the properties and the mechanical functions of bone. This study examined the effects of disrupting bone morphogenetic protein (BMP) signaling through BMP receptors on bone quantity and bone quality. More specifically, we disrupted two BMP receptors, Acvr1 and Bmpr1a, respectively, in Osterix-expressing osteogenic progenitor cells in mice. We examined the structural changes to the femora from 3-month old male and female conditional knockout (cKO) mice using micro-computed tomography (micro-CT) and histology, as well as compositional changes to both cortical and trabecular compartments of bone using Raman spectroscopy. We found that the deletion of Acvr1 and Bmpr1a, respectively, in an osteoblast-specific manner resulted in higher bone mass in the trabecular compartment. Disruption of Bmpr1a resulted in a more significantly increased bone mass in the trabecular compartment. We also found that these cKO mice showed lower mineral-to-matrix ratio, while tissue mineral density was lower in the cortical compartment. Collagen crosslink ratio was higher in both cortical and trabecular compartments of male cKO mice. Our study suggested that BMP signaling in osteoblast mediated by BMP receptors, namely ACVR1 and BMPR1A, is critical in regulating bone quantity and bone quality.     

Knockout of Nuclear High Molecular Weight FGF2 Isoforms in Mice Modulates Bone and Phosphate Homeostasis

We previously reported that targeted overexpression of the fibroblast growth factor 2 (FGF2) high molecular weight (HMW) isoforms in osteoblastic lineage cells in mice resulted in phenotypic changes, including dwarfism, rickets, osteomalacia, hypophosphatemia, increased serum parathyroid hormone, and increased levels of the phosphatonin FGF23 in serum and bone. This study examined the effects of genetically knocking out the FGF2HMW isoforms (HMWKO) on bone and phosphate homeostasis. HMWKO mice were not dwarfed and had significantly increased bone mineral density and bone mineral content in femurs and lumbar vertebrae when compared with the wild-type (WT) littermates. Micro-computed tomography analysis of femurs revealed increased trabecular bone volume, thickness, number, and connective tissue density with decreased trabecular spacing compared with WT. In addition, there was significantly decreased cortical porosity and increased cortical thickness and sub-periosteal area in femurs of HMWKO. Histomorphometric analysis demonstrated increased osteoblast activity and diminished osteoclast activity in the HMWKO. In vitro bone marrow stromal cell cultures showed there was a significant increase in alkaline phosphatase-positive colony number at 1 week in HMWKO. At 3 weeks of culture, the mineralized area was also significantly increased. There was increased expression of osteoblast differentiation marker genes and reduced expression of genes associated with impaired mineralization, including a significant reduction in Fgf23 and Sost mRNA. Normal serum phosphate and parathyroid hormone were observed in HMWKO mice. This study demonstrates a significant negative impact of HMWFGF2 on biological functions in bone and phosphate homeostasis in mice.     

Effects of spirulina, a blue-green alga, on bone metabolism in ovariectomized rats and hindlimb-unloaded mice

The safety and effectiveness were examined of the spirulina alga on bone metabolism in ovariectomized estrogen-deficient rats and hindlimb-unloaded mice. The dosage range was from an amount equal to that recommended in so-called health foods for humans (0.08 g/kg BW/day) to a 100-fold higher dose. The bone mineral density (BMD) of the whole femur and tibia of ovariectomized rats in the any spirulina-treated groups was not significantly different from that of the ovariectomized group, although BMD of the distal femur and proximal tibia was significantly lower in the spirulina-treated groups than in the ovariectomized group after a 6 week-experimental period. BMD of the femur and tibia was not affected by treatment with any dose of spirulina in hindlimb-unloaded mice. These results suggest that the intake of spirulina decreased BMD in the trabecular bone of rodents under estrogen-deficient conditions.     

A potential role for the myeloid lineage in leptin-regulated bone metabolism

Leptin influences bone formation centrally through the hypothalamus and peripherally by acting on osteoblasts or their precursors. However, neither mechanism explains the divergent, gender-specific correlation between leptin and bone mineral density in humans. Although leptin is a potent regulator of pro-inflammatory immune responses, a potential role for leptin as an osteoimmunologic intermediate in bone metabolism has not been tested. Mice with myeloid-specific ablation of the long-form leptin receptor (ObRb) were generated using mice expressing cre-recombinase from the lysoszyme M promoter. At 12 weeks of age, the conditional knockout mice did not display any appreciable phenotype. However, at 52 weeks 2 changes were noted. First, there was a mild increase in liver inflammation. Second, a gender-specific, divergent bone phenotype was observed. Female mice displayed a consistent trend toward decreased trabecular bone parameters including reductions in bone volume fraction, trabecular number, and bone mineral content as well as a significant increase in marrow adipogenesis. Conversely, male mice lacked trabecular changes, but had statistically significant increases in cortical bone volume, thickness, and bone mineral density with equivalent total cortical volume. Since the year 2000, over 25 studies on more than 10,000 patients have sought to determine the correlation between leptin and bone mineral density. The results revealed a gender-specific correlation similar to that observed in our LysM transgenic animals. We hypothesize and show new evidence that regulation of myeloid lineage cells by leptin may facilitate their actions as an osteoimmunologic intermediate and contribute to leptin-regulated bone formation and metabolism in a gender-specific manner.     

动态调节Rad GTPase可以控制骨形成的平衡

小GTP结合蛋白Rad (Ras-related associated with diabetes)是小GTPases的RGK亚家族成员,其在心脏之外的细胞和生理功能仍有待阐明,本研究旨在探讨Rad对小鼠骨密度、破骨细胞分化和骨量的调节作用。本研究以Rad基因敲除小鼠为动物模型,野生(WT)小鼠为对照,通过微计算机断层摄影术(microscopic computed tomography,μCT)分析雄性和雌性小鼠的股骨小梁骨体积分数和骨小梁数量,以抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase, TRAP)染色和抗酒石酸酸性磷酸酶(TRAP)+多核细胞(multinucleated cell, MNC)计数检测破骨细胞的分化和表面积,使用组织形态计量学来考察骨形成速率。结果显示,与WT野生型小鼠相比,雌性Rad基因敲除小鼠的股骨表现出显著较低的小梁骨体积分数(BV/TV)。Rad缺失使小鼠股骨的皮质骨面积明显低于WT小鼠。抗酒石酸酸性磷酸酶(TRAP)染色和TRAP+MNCs计数表明Rad的缺失显著增强了体外破骨细胞的分化。与正常野生小鼠相比,Rad缺失使小鼠的破骨细胞表面积减少。在Rad基因敲除小鼠中矿物沉积率(MAR)显著降低,矿化表面百分比(MS/BS)升高,骨形成速率/骨表面(BFR/BS)下降。本研究初步结论表明,Rad GTPase在骨代谢的调节中起着重要的作用,在小鼠中敲除Rad可导致骨密度降低,对Rad作用和调节机制的研究可能会找到骨质疏松症治疗的潜在靶点。     

Accelerated Growth Plate Mineralization and Foreshortened Proximal Limb Bones in Fetuin-A Knockout Mice

The plasma protein fetuin-A/alpha2-HS-glycoprotein (genetic symbol Ahsg) is a systemic inhibitor of extraskeletal mineralization, which is best underscored by the excessive mineral deposition found in various tissues of fetuin-A deficient mice on the calcification-prone genetic background DBA/2. Fetuin-A is known to accumulate in the bone matrix thus an effect of fetuin-A on skeletal mineralization is expected. We examined the bones of fetuin-A deficient mice maintained on a C57BL/6 genetic background to avoid bone disease secondary to renal calcification. Here, we show that fetuin-A deficient mice display normal trabecular bone mass in the spine, but increased cortical thickness in the femur. Bone material properties, as well as mineral and collagen characteristics of cortical bone were unaffected by the absence of fetuin-A. In contrast, the long bones especially proximal limb bones were severely stunted in fetuin-A deficient mice compared to wildtype littermates, resulting in increased biomechanical stability of fetuin-A deficient femora in three-point-bending tests. Elevated backscattered electron signal intensities reflected an increased mineral content in the growth plates of fetuin-A deficient long bones, corroborating its physiological role as an inhibitor of excessive mineralization in the growth plate cartilage matrix - a site of vigorous physiological mineralization. We show that in the case of fetuin-A deficiency, active mineralization inhibition is a necessity for proper long bone growth.     

CARM1 deficiency inhibits osteoblastic differentiation of bone marrow mesenchymal stem cells and delays osteogenesis in mice

Bone repair remains a clinical challenge due to low osteogenic capacity. Coactivator associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that mediates arginine methylation and endochondral ossification. However, the roles of CARM1 in osteoblastic differentiation and bone remodeling have not been explored. In our study, heterozygous CARM1-knockout (KO) mice were generated using the CRISPR-Cas9 system and a model of femoral defect was created. At day 7 postsurgery, CARM1-KO mice exhibited obvious bone loss compared with wild type (WT) mice, as evidenced by reduced bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Deletion of CARM1 in mice lowered synthesis and accumulation of collagen at the injury sites. The alkaline phosphatase (ALP) activity and osteogenic-related gene expression were declined in CARM1-KO mice. To further understand the role of CARM1 in osteoblastic differentiation, bone marrow mesenchymal stem cells (BMSCs) were isolated from the tibia and femur of WT or CARM1-KO mice. CARM1 deletion decreased histone arginine methylation and inhibited osteoblastic differentiation and mineralization. The mRNA sequencing of CARM1-KO BMSCs revealed the possible regulatory molecules by CARM1, which could deepen our understanding of CARM1 regulatory mechanisms. These data could be of interest to basic researchers and provide the direction for future research into bone-related disorders.     

Infrared spectroscopic assessment of the inflammation-mediated osteoporosis (IMO) model applied to rabbit bone

A model of osteoporosis based on induced inflammation (IMO) was applied on rabbit bones. The structural heterogeneity and molecular complexity of bone significantly affect bone mechanical properties. A tool like Fourier transform infrared spectroscopy, able to analyze both the inorganic and organic phase simultaneously, could provide compositional information regarding cortical and trabecular sections under normal and osteoporotic conditions. In this study, we assessed the mineral/matrix ratio, carbonate and phosphate content and labile (i.e., non-apatitic) species contribution to bone mineral and collagen cross-linking patterns. Clear differences were observed between cortical and trabecular bone regarding mineral and carbonate content. Induced inflammation lowers the mineral/matrix ratio and increases the overall carbonate accumulation. Elevated concentrations of labile species were detected in osteoporotic samples, especially in the trabecular sections. Collagen cross-linking patterns were indirectly observed through the 1660/1690 cm^− 1 ratio in the amide I band and a positive correlation was found with the mineralization index. Principal component analysis (PCA) applied to female samples successfully clustered trabecular and osteoporotic cases. The important role played by the phosphate ions was confirmed by corresponding loadings plots. The results suggest that the application of the IMO model to rabbit bones effectively alters bone remodeling and forms an osteoporotic bone matrix with a dissimilar composition compared to the normal one.     

Spag17 Deficiency Results in Skeletal Malformations and Bone Abnormalities

Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.     

Adenosine A2B receptors play an important role in bone homeostasis

Bone homeostasis is a finely regulated mechanism involving different molecular pathways including adenosine signaling. The aim of this study is to determine the bone phenotype of adenosine A2B receptor knockout (A2BRKO) mice and to measure their ability to form new bone. Moreover, we analyzed the functionality of osteoclasts and osteoblasts from A2BRKO mice. Microcomputed tomography (μCT) analysis revealed a decrease of bone substance, bone mineral density, and trabecular number in A2BRKO mice compared to the WT mice at the same age. We measured the new bone formation by injecting fluorescent markers: it was reduced in femur and tibia of A2BRKO mice compare to the WT. A2BRKO young mice have fewer osteoblasts and an increase of osteoclasts was measured in the hind limbs of young and adult mice. A2BRKO osteoclasts are also more active in vitro, showing an increase of pit formation in dentin discs. Surprisingly in mature osteoblasts from A2BRKO mice, we measured an increase of calcified matrix production, collagen deposition, and alkaline phosphatase activity. These results demonstrate that A2BR on osteoblasts and osteoclasts regulate bone homeostasis.     

Influence of early qualitative feed restriction and environmental temperature on long bone development of broiler chickens

This investigation was carried out to study the influence of early qualitative feed restriction and environmental rearing temperature on long bone development in broiler. Energy and protein restriction reduced femur width and humerus weight, but did not affect tibia parameters. Broilers kept at cold environmental temperature showed reduced femur, tibia and humerus length and tibia weight, but the calculated density was not affected by rearing temperature. These findings suggest that qualitative feed restriction and environmental temperature influenced the normal long bone growth; however, bone weight/bone length index (calculated density) was not affected by rearing temperature.     

The effects of luteolin on osteoclast differentiation, function in vitro and ovariectomy-induced bone loss

Flavonoids, a group of polyphenolic compounds abundant in plants, are known to prevent bone loss in ovariectomized (OVX) animal models. Inhibition of osteoclast differentiation and bone resorption is considered as an effective therapeutic approach in the treatment of postmenopausal bone loss. Luteolin, a plant flavonoid, has potent anti-inflammatory properties both in vivo and vitro. In this study, we found that luteolin markedly decreased the differentiation of both bone marrow mononuclear cells and Raw264.7 cells into osteoclasts. Luteolin also inhibited the bone resorptive activity of differentiated osteoclasts. We further investigated the effects of luteolin on ovariectomy-induced bone loss using micro-computed tomography, biomechanical tests and serum markers assay for bone remodeling. Oral administration of luteolin (5 and 20 mg/kg per day) to OVX mice caused significant increase in bone mineral density and bone mineral content of trabecular and cortical bones in the femur as compared to those of OVX controls, and prevented decreases of bone strength indexes induced by OVX surgery. Serum biochemical markers assays revealed that luteolin prevents OVX-induced increases in bone turnover. These data strongly suggest that luteolin has the potential for prevention of bone loss in postmenopausal osteoporosis by reducing both osteoclast differentiation and function.     

A comparison of the fatigue behavior of human trabecular and cortical bone tissue

The fatigue properties of trabecular bone tissue (single trabeculae) and similarly sized cortical bone specimens from human tibia were experimentally determined on a microstructural level using four-point bending cyclic tests, and they were compared based on modulus, mineral density, and microstructural characteristics. The results showed that trabecular specimens had significantly lower moduli and lower fatigue strength than cortical specimens, despite their higher mineral density values. Fracture surface and microdamage analyses illustrated different fracture and damage patterns between trabecular and cortical bone tissue, depending upon their microstructural characteristics. Based on the results from mechanical tests and qualitative observations, a possible mechanical role of the cement lines in trabecular tissue microfracture was suggested.     

Effects of whole-body vibration on bone properties in aged rats

Objective:This study aimed to explore optimal conditions of whole-body vibration (WBV) for improving bone properties in aged rats.Methods:Eighty-week-old rats were divided into baseline control (BC), age-matched control (CON) and experimental groups, which underwent WBV (0.5 g) at various frequencies (15, 30, 45, 60 or 90 Hz) or WBV (45 Hz) with various magnitudes (0.3, 0.5, 0.7 or 1.0 g) for 7 weeks. After interventions, femur bone size, bone mechanical strength and circulating bone formation/resorption markers were measured, and trabecular bone microstructure (TBMS) and cortical bone geometry (CBG) of femurs were analyzed by micro-CT.Results:Several TBMS parameters and trabecular bone mineral content were significantly lower in the 15 Hz WBV (0.5 g) group than in the CON group, suggesting damage to trabecular bone. On the other hand, although frequency/magnitude of WBV did not influence any CBG parameters, the 0.7 g and 1.0 g WBV (45 Hz) group showed an increase in tissue mineral density of cortical bone compared with the BC and CON groups, suggesting the possibility of improving cortical bone properties.Conclusion:Based on these findings, it should be noted that WBV conditions are carefully considered when applied to elderly people.     

Prolonged increase in dietary phosphate intake alters bone mineralization in adult male rats

Excessive intake of dietary phosphate without the company of calcium causes serum parathyroid hormone (s-PTH) concentration to rise. We investigated the effect of a modest but prolonged increase in dietary intake of inorganic phosphate on the bone quantitative factors of mature male rats. Twenty Wistar rats were divided into two groups and fed a high-phosphate diet (1.2% phosphate) or a control diet (0.6% phosphate) for 8 weeks. In the beginning and at the end of the study period, femur and lumbar bone mineral density (BMD), bone mineral content and area were measured using DXA, s-PTH was analyzed from the blood sample, and after sacrifice, right femur was cut loose and processed into paraffin cuts. Bone diameter, inner diameter and cortical width was measured from the hematoxylin- and eosin-dyed femur cuts. Tibias were degraded and calcium and phosphate content was analyzed by inductively coupled plasma-mass spectrometer. Femoral BMD increased significantly more in the control group than in the phosphate group (P=.005). Lumbar BMD values decreased in both groups, and the fall was greater in the control group (P=.007). The phosphate group had significantly higher s-PTH values (P=.0135). Femoral histomorphometric values or tibial mineral contents did not differ between groups. In conclusion, increase in dietary phosphate intake caused s-PTH to rise and hindered mineral deposition into cortical bone, leading to lower BMD. The effect on trabecular bone was opposing as mineral loss was less in the lumbar spine of phosphate group animals. These results are in concurrence with the data stating that skeletal response to PTH is complex and site dependent.     

The role of estrogen receptor-beta, in the early age-related bone gain and later age-related bone loss in female mice

The molecular and cellular mechanism of estrogen action in skeletal tissue remains unclear. The purpose of this study was to understand the role of estrogen receptor-beta, (ERbeta) on cortical and cancellous bone during growth and aging by comparing the bone phenotype of 6- and 13-month-old female mice with or without ERbeta. Groups of 11-14 wild-type (WT) controls and ERbeta knockout (BERKO) female mice were necropsied at 6 and 13 months of age. At both ages, BERKO mice did not differ significantly from WT controls in uterine weight and uterine epithelial thickness, indicating that ERbeta does not regulate the growth of uterine tissue. Femoral length increased significantly by 5.5% at 6 months of age in BERKO mice compared with WT controls. At 6 months of age, peripheral quantitative computerized tomography (pQCT) analysis of the distal femoral metaphysis (DFM) and femoral shafts showed that BERKO mice had significantly higher cortical bone content and periosteal circumference as compared with WT controls at both sites. In contrast to the findings in cortical bone, at 6 months of age, there was no difference between BERKO and WT mice in trabecular density, trabecular bone volume (TBV), or formation and resorption indices at the DFM. In 13-month-old WT mice, TBV (-41%), trabecular density (-27%) and cortical thickness decreased significantly. while marrow cavity and endocortical circumference increased significantly compared with 6-month-old WT mice. These age-related decreases in cancellous and endocortical bone did not occur in BERKO mice. At 13 months of age, BERKO mice had significantly higher total, trabecular and cortical bone, while having significantly lower bone resorption, bone formation and bone turnover in DFM compared with WT mice. These results indicate that deleting ERbeta protected against age-related bone loss in both the cancellous and endocortical compartments by decreasing bone resorption and bone turnover in aged female mice. These data demonstrate that in female mice, ERbeta plays a role in inhibiting periosteal bone formation, longitudinal and radial bone growth during the growth period, while it plays a role in stimulating bone resorption, bone turnover and bone loss on cancellous and endocortical bone surfaces during the aging process.     

Impaired endochondral bone development and osteopenia in Gli2-deficient mice

Mice homozygous for targeted disruption of the zinc finger domain of Gli2 (Gli2(zfd/zfd)) die at birth with developmental defects in several organ systems including the skeleton. The current studies were undertaken to define the role of Gli2 in endochondral bone development by characterizing the molecular defects in the limbs and vertebrae of Gli2(zfd/zfd) mice. The bones of mutant mice removed by cesarian section at E16.5 and E18.5 demonstrated delayed endochondral ossification. This was accompanied by an increase in the length of cartilaginous growth plates, reduced bone tissue in the femur and tibia and by failure to develop the primary ossification centre in vertebral bodies. The growth plates of tibiae and vertebrae exhibited increased numbers of proliferating and hypertrophic chondrocytes with no apparent alteration in matrix mineralisation. The changes in growth plate morphology were accompanied by an increase in expression of FGF2 in proliferating chondrocytes and decreased expression of Indian hedgehog (Ihh), patched (Ptc) and parathyroid-hormone-related protein (PTHrP) in prehypertrophic cells. Furthermore, there was a reduction in expression of angiogenic molecules in hypertrophic chondrocytes, which was accompanied by a decrease in chondroclasts at the cartilage bone interface, fewer osteoblasts lining trabecular surfaces and a reduced volume of metaphyseal bone. These results indicate that functional Gli2 is necessary for normal endochondral bone development and that its absence results in increased proliferation of immature chondrocytes and decreased resorption of mineralised cartilage and bone formation.