Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.     

The Wnt antagonist Frzb-1 regulates chondrocyte maturation and long bone development during limb skeletogenesis

The Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, we determined Frzb-1 gene expression patterns during chick long bone development and carried out gain- and loss-of-function studies by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression was quite strong in mesenchymal prechondrogenic condensations and then characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression caused shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintained the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promoted chondrocyte maturation, hypertrophy, and calcification. Immunostaining revealed that the distribution of endogenous Wnt mediator beta-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevented beta-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action.     

Differentiation and mineralization in chick chondrocytes maintained in a high cell density culture: A model for endochondral ossification

Summary Chondrocytes isolated from the proliferative and differentiating zones of 3-wk-old chick growth plates were cultured in the presence of 10% fetal bovine serum (FBS) and ascorbic acid for up to 21 d in a high cell density culture within Eppendorf tubes. The proliferative, differentiating, and calcification properties of the chondrocytes were examined by immunolocalization and by enzyme histochemical and biochemical methods. The cells maintained a chondrocyte phenotype throughout culture: they were round in shape and synthesized both collagen type II and proteoglycans. The expression of a hypertrophic phenotype was evident by Day 3 of culture and from this time onwards characteristics of terminal differentiation were observed. The cells were positive for both alkaline phosphatase (ALP) activity and c-myc protein and the surrounding matrix stained strongly for collagen type X. Small foci of mineralization associated with individual chondrocytes were first evident by Day 6 and more widespread areas of mineralization occupying large areas of matrix were present by Day 15. Mineralization occurred without the addition of exogenous phosphate to the medium. This culture system displays characteristics that are similar in both morphological and developmental terms to that of chick chondrocyte differentiation and calcification in vivo and therefore offers an excellent in vitro model for endochondral ossification.     

Growth and shaping of metacarpal and carpal cartilage anlagen: application of morphometry to the development of short and long bone. A study of human hand anlagen in the fetal period

A histological and morphometric analysis of human metacarpal and carpal anlagen between the 16th and 22nd embryonic weeks was carried out with the aim of studying the establishment of the respective anlage architecture. No differences in the pattern of growth were documented between the peripheral and central zones of the metacarpal epiphyses and those of the carpals. The regulation of longitudinal growth in long bone anlagen occurred in the transition zone between the epiphysis and the diaphysis (homologous to the metaphyseal growth plate cartilage in more advanced developmental stage of the bone). Comparative zonal analysis was conducted to assess the chondrocyte density, the mean chondrocyte lacunar area, the paired chondrocyte polarity in the orthogonal longitudinal and transverse planes, and the lacunar shape transformation in the metacarpal. In transition from epiphysis to diaphysis chondrocyte density decreased and mean lacunar area increased. No significant differences in the chondrocyte maturation cycle were observed between proximal/distal metacarpal epiphyses and the carpal anlagen. The number of paired chondrocyte oriented along the growth vector was significantly higher in both proximal/distal transition zones between epiphysis and diaphysis. Human metacarpals shared with experimental models (like mice and nonmammal tetrapods) an early common chondrocyte maturation cycle but with a different timing due to the slower embryonic and fetal developmental rate of human anlagen.     

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation.

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT-PCR indicates that FGF-2 and FGF-4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF-8 and FGF-9 are not. Thus, FGF-2 and FGF-4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF-2 and FGF-4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth.     

High density micromass cultures of embryonic limb bud mesenchymal cells: An in vitro model of endochondral skeletal development

Summary To study the mechanisms regulating endochondral skeletal development, we examined the characteristics of long-term, high density micromass cultures of embryonic chicken limb bud mesenchymal cells. By culture Day 3, these cells underwent distinct chondrogenesis, evidenced by cellular condensation to form large nodules exhibiting cartilage-like morphology and extracellular matrix. By Day 14, extensive cellular hypertrophy was seen in the core of the nodules, accompanied by increased alkaline phosphatase activity, and the limitation of cellular proliferation to the periphery of the nodules and to internodular areas. By Day 14, matrix calcification was detected by alizarin red staining, and calcium incorporation increased as a function of culture time up to 2 to 3 wk and then decreased. X-ray probe elemental analysis detected the presence of hydroxyapatite. Analogous to growth cartilage developing in vivo, these cultures also exhibited time-dependent apoptosis, on the basis of DNA fragmentation detected in situ by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), ultrastructural nuclear morphology, and the appearance of internucleosomal DNA degradation. These findings showed that cellular differentiation, maturation, hypertrophy, calcification, and apoptosis occurred sequentially in the embryonic limb mesenchyme micromass cultures and indicate their utility as a convenient in vitro model to investigate the regulatory mechanisms of endochondral ossification.     

Role of Runx genes in chondrocyte differentiation

Runx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, we cloned chicken Runx2 and overexpressed it in chick embryos using a retroviral system. Infected chick wings showed multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation was not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, we have generated transgenic mice that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, we performed in situ hybridization with Runx2- and Runx3-specific probes. Both genes were coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs.     

Circular RNA circANAPC2 mediates the impairment of endochondral ossification by miR-874-3p/SMAD3 signalling pathway in idiopathic short stature

Idiopathic short stature (ISS) is a main reason for low height among children. Its exact aetiology remains unclear. Recent findings have suggested that the aberrant expression of circRNAs in peripheral blood samples is associated with many diseases. However, to date, the role of aberrant circRNA expression in mediating ISS pathogenesis remains largely unknown. The up-regulated circANAPC2 was identified by circRNA microarray analysis and RT-qPCR. Overexpression of circANAPC2 inhibited the proliferation of human chondrocytes, and cell cycle was arrested in G1 phase. The expressions of collagen type X, RUNX2, OCN and OPN were significantly down-regulated following circANAPC2 overexpression. Moreover, Von Kossa staining intensity and alkaline phosphatase activity were also decreased. Luciferase reporter assay results showed that circANAPC2 could be targeted by miR-874-3p. CircANAPC2 overexpression in human chondrocytes inhibits the expression of miR-874-3p. The co-localization of circANAPC2 and miR-874-3p was confirmed in both human chondrocytes and murine femoral growth plates via in situ hybridization. The rescue experiment demonstrated that the high expression of miR-874-3p overexpression antagonized the suppression of endochondral ossification, hypertrophy and chondrocyte growth caused by circANAPC2 overexpression. A high-throughput screening of mRNA expression and RT-qPCR verified SMAD3 demonstrated the highest different expressions following overcircANAPC2. Luciferase reporter assay results indicated that miR-874-3p could be targeted by Smad3, thus down-regulating the expression of Smad3. Subsequent rescue experiments of SMAD3 further confirmed that circANAPC2 suppresses endochondral ossification, hypertrophy and chondrocyte growth through miR-874-3p/Smad3 axis. The present study provides evidence that circANAPC2 can serve as a promising target for ISS treatment.     

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.     

Relationship between formation of the femoral bicondylar angle and trochlear shape: independence of diaphyseal and epiphyseal growth

During hominin evolution, an increase in the femoral bicondylar angle was the initial change that led to selection for protuberance of the lateral trochlear lip and the elliptical profile of the lateral condyle. No correlation is found during ontogeny between the degree of femoral obliquity and of the prominence of the lateral trochlear lip. Might there be a relationship with the elliptical profile of the lateral condyle? On intact femoral diaphyses of juvenile humans and great apes, we compared the anteroposterior length of the lateral and medial sides of the distal metaphysis. The two diaphyseal pillars remain equal during postnatal growth in great apes, while the growth of the lateral pillar far exceeds that of the medial pillar in humans. Increase in bicondylar angle is correlated with disproportionate anteroposterior lengthening of the lateral pillar. The increased anteroposterior length of the lateral side of the metaphysis would contribute to increasing the radius of the curvature of the lateral condyle, but not to the projection of the lateral trochlear lip. The similar neonatal and adult femoro‐patellar joint shape in humans prompted an assessment of the similarity during growth of the entire neonatal and adult epiphyses. We showed that the entire epiphysis undergoes drastic changes in proportions during postnatal growth. Finally, we emphasize the need to distinguish the cartilaginous phenotype and the ossified phenotype of the distal femoral epiphysis (and of any epiphysis) during postnatal growth. This crucial distinction applies to most postcranial bones, for they almost all develop following the process of endochondral ossification. Am J Phys Anthropol, 2006. © 2006 Wiley‐Liss, Inc.     

Cross-talk between FGF and other cytokine signalling pathways during endochondral bone development

FGF (fibroblast growth factor)/FGFR (FGF receptor) signalling plays an essential role in both endochondral and intramembranous bone development. FGF signalling pathways are important for the earliest stages of limb development and throughout skeletal development. The activity and the outcome of this signalling pathway during bone development are also influenced by many other intracellular and extracellular signals. In this review, we focus on the interplay between FGF signalling and other pathways, which is tightly regulated both spatially and temporally during endochondral skeletal development.     

Brief communication: infracranial maturation in the skeletal collection from Coimbra, Portugal: new aging standards for epiphyseal union

Age at death of a single skeletal individual or a group is essential information in archaeological, paleoanthropological, and forensic contexts. Dental remains are the most commonly used age indicators, but when the dentition is not available, or too few teeth are present for an accurate age assessment, other age indicators such as skeletal maturation must be used. Of particular utility in this regard is the fusion of the epiphyses of the infracranial skeleton. Here we present new aging standards based on the infracranial maturation of individuals from the known age and sex collection from Coimbra, Portugal. We scored infracranial epiphyseal fusion and spheno-occipital synchondrosis closure (64 loci of ossification in total) on 137 skeletons from individuals between 7 and 29 years old. We further discuss developmental differences between the sexes and similarities and differences between the Coimbra documented collection and other published aging standards.     

Chronology of fusion of the primary and secondary ossification centers in the human sacrum and age estimation in child and adolescent skeletons

Little is known about fusion times of the primary and secondary centers of ossification in the sacrum, particularly from dry bone observations. In this study, the timing of union of these centers was studied in a sample of modern Portuguese skeletons (90 females and 101 males) between the ages of 0 and 30 years, taken from the Lisbon documented skeletal collection. A three‐stage scheme was used to assess fusion status between ossification centers as unfused, partially fused and completely fused. Posterior probability tables of age, given a certain stage of fusion, were calculated for most anatomical locations studied using both reference and uniform priors. Partial union of primary centers of ossification was observed from 1 to 8 years of age and partial union of secondary centers of ossification was observed from 15 to 21 years of age. The first primary centers of ossification to complete fusion are the neural arch with the centrum of the fifth sacral vertebrae and the last are the costal element with the centrum of the first sacral vertebra. The annular and sacroiliac epiphyses are the first, among the secondary centers of ossification observed, to complete fusion, after which the lateral margin fuses. This study offers information on timing of fusion of diverse locations in the developing sacrum useful for age estimation of complete or fragmented immature human skeletal remains and fills an important gap in the literature, by adding to previously published times of fusion of primary and secondary ossification centers in this sample. Am J Phys Anthropol 153:214–225, 2014. © 2013 Wiley Periodicals, Inc.     

Study on oocyte maturation and activation of the common prawn palaemon serratus (Pennant): Relationship between oocyte maturation and the molt cycle cytological aspects

A detailed chronology of the cytological events related to maturation that take place within the reproduction molt cycle has been established. It has been shown that oocytes, initially arrested at prophase I, resume meiosis when approaching stage D₁? of the molt cycle, ie, 4–5 days before molting. The following steps characterize this premolt period of oocyte maturation: nuclear envelope folding, nucleolar dissociation, condensation of the chromosomes, and beginning of the breakdown of the nuclear envelope (GVBD). At the ultrastructural level, it has been confirmed that GVBD actually takes place at the D₁??D₂ stage transition, when the germinal vesicle still occupies a central position in the oocyte. The migration of the chromosome takes only a few hours and begins approximately 4 hr before molting. It is only 1–2 hr before molting that the divalent chromosomes that are not yet organized in a metaphase plate become visible at the surface of the oocyte. They lay in a nucleoplasmic area no longer limited by the nuclear envelope. Metaphase I is reached a few minutes after molting. A second meiotic block appears at this stage, which persists until spawning, ie, for about 24 hr. Fertilization occurs at the moment of spawning. In vitro fertilization experiments demonstrated that fertilization normally triggers the release of the second meiotic block. Extrusion of the two polar bodies can be easily observed using a method for clearing and staining the oocytes in toto.     

Indian hedgehog: its roles and regulation in endochondral bone development

Normal endochondral bone development requires the coordination of chondrocyte proliferation and differentiation. Indian hedgehog (Ihh) is a morphogen produced by chondrocytes in the early stage of terminal differentiation and plays several key roles in this process. Ihh regulates growth of adjacent proliferative chondrocytes and can also regulate the rate of differentiation of chondrocytes indirectly through its stimulation of parathyroid hormone-related protein (PTHrP). In this review, we focus on recent studies that have identified new functions of Ihh and how Ihh itself is being regulated.