According to current dogma, chondrocytes and osteoblasts are considered independent lineages derived from a common osteochondroprogenitor. In endochondral bone formation, chondrocytes undergo a series of differentiation steps to form the growth plate, and it generally is accepted that death is the ultimate fate of terminally differentiated hypertrophic chondrocytes (HCs). Osteoblasts, accompanying vascular invasion, lay down endochondral bone to replace cartilage. However, whether an HC can become an osteoblast and contribute to the full osteogenic lineage has been the subject of a century-long debate. Here we use a cell-specific tamoxifen-inducible genetic recombination approach to track the fate of murine HCs and show that they can survive the cartilageto-bone transition and become osteogenic cells in fetal and postnatal endochondral bones and persist into adulthood. This discovery of a chondrocyte-to-osteoblast lineage continuum revises concepts of the ontogeny of osteoblasts, with implications for the control of bone homeostasis and the interpretation of the underlying pathological bases of bone disorders.osteoblast ontogeny | chondrocyte lineage | bone repair I n vertebrates, the endochondral bones of the axial and appendicular skeleton (1) develop from mesenchymal progenitors that form condensations in the approximate shape of the future skeletal elements. These progenitors differentiate into chondrocytes, which proliferate, mature, and undergo hypertrophy, forming an avascular cartilaginous template surrounded by a perichondrium. The first osteoblasts differentiate from mesenchymal precursors in the perichondrium and produce a bone collar, which will become the future cortical bone (1). Blood vessels then invade through the bone collar into the hypertrophic cartilage, bringing in osteoblast progenitors from the perichondrium (2), which lay down bone matrix to form the primary ossification center (POC); the cartilage matrix is degraded; and the proximal and distal growth plates, comprising layers of differentiating chondrocytes and spongy/trabecular bone (the primary spongiosa), form (2). Thereafter, linear bone growth continues by endochondral ossification mediated by the growth plate, whereas osteoblasts in the perichondrium form cortical bone on the outer circumference.Chondrocytes and osteoblasts are regarded as separate lineages in development, being derived from common mesenchymal progenitors that express the transcription factors sex determining region Y (SRY)-box 9 (Sox9) and runt related transcription factor 2 (Runx2) (1). Lineage determination toward the chondrocyte or osteoblast fate is controlled by the relative expression of Sox9 and Runx2 (3-5) (Fig. 1A). Sox9 controls chondrocyte proliferation and their progression into hypertrophy (6). Collagen X is the most specific marker of hypertrophic chondrocytes (HCs), the Col10a1 gene being expressed only in prehypertrophic and hypertrophic chondrocytes in the growth plate (7). By contrast, Runx2 is essential for specifying the osteoblast lineage an...

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded α1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

Dielectric relaxation and dynamic light scattering of small molecule glass-forming liquids invariably show that the fractional exponent beta(alpha) of the Kohlrausch-Williams-Watts correlation function, exp[-(t/tau(alpha))(beta(alpha))], used to fit the alpha-relaxation data is temperature dependent, increasing towards the value of unity as temperature is raised and the relaxation time tau decreased. Comparing different glass formers, another property is the existence of a correlation between the value of (1-beta(alpha)) at the glass temperature, T(g), and the T(g)-scaled temperature dependence of tau(alpha). We analyze a system of interacting arrays of globally coupled nonlinear oscillators. Each array has its oscillators coupled among themselves with a coupling strength K. The coupling between arrays is characterized by the interarray coupling strength K'. The decay of the phase coherence r for each array is slowed down by the interarray coupling and its time dependence is well approximated at sufficiently long times by exp[-(t/tau)(beta)]. For a fixed K', on increasing K the results exhibit a decrease of tau and a concomitant increase of beta, similar to the properties of dielectric relaxation and dynamic light scattering of glass-forming liquids on increasing temperature. For each K' we define K(g) to be the value of K at which tau is equal to an arbitrarily chosen long time. We find that beta(K(g)) is correlated with the K(g)-scaled K dependence of tau. The results obtained in this manner at various fixed values of K' reproduce the relaxation properties and temperature dependencies of strong, intermediate, and fragile glass-forming liquids.

We analyze a system of interacting arrays of globally coupled nonlinear oscillators. The relaxation in the interacting arrays with different interaction strengths is compared to that in an array not subject to interaction with others. The relaxation of the latter is found to be an exponential function of time. On the other hand the relaxation of the interacting arrays is slowed down and departs from an exponential of time. There exists a crossover time, t c , before which relaxation of the interacting arrays is still an exponential function. However, beyond t c relaxation is no longer exponential but well approximated by a stretched exponential exp͓Ϫ(t/) ␤ ]. The fractional exponent ␤ decreases further from unity with increasing interaction strength. The result bears strong similarity to the basic features suggested by the coupling model and seen experimentally by neutron scattering for relaxation in densely packed interacting molecules in glass-forming liquids.

Cells in multicellular organisms are surrounded by a complex three-dimensional macromolecular extracellular matrix (ECM). This matrix, traditionally thought to serve a structural function providing support and strength to cells within tissues, is increasingly being recognized as having pleiotropic effects in development and growth. Elucidation of the role that the ECM plays in developmental processes has been significantly advanced by studying the phenotypic and developmental consequences of specific genetic alterations of ECM components in the mouse. These studies have revealed the enormous contribution of the ECM to the regulation of key processes in morphogenesis and organogenesis, such as cell adhesion, proliferation, specification, migration, survival, and differentiation. The ECM interacts with signaling molecules and morphogens thereby modulating their activities. This review considers these advances in our understanding of the function of ECM proteins during development, extending beyond their structural capacity, to embrace their new roles in intercellular signaling.

Missense, nonsense and frame-shift mutations in the collagen X gene (COL10A1) result in metaphyseal chondrodysplasia type Schmid (MCDS). Complete degradation of mutant COL10A1 mRNA by nonsense-mediated decay in human MCDS cartilage implicates haploinsufficiency in the pathogenesis for nonsense mutations in vivo. However, the mechanism is unclear in situations where the mutant mRNA persist. We show that nonsense/frame-shift mutations can elicit a gain-of-function effect, affecting chondrocyte differentiation in the growth plate. In an MCDS proband, heterozygous for a p.Y663X nonsense mutation, the growth plate cartilage contained 64% wild-type and 36% mutant mRNA and the hypertrophic zone was disorganized and expanded. The in vitro translated mutant collagen X chains, which are truncated, were misfolded, unable to assemble into trimers and interfered with the assembly of normal alpha1(X) chains into trimers. Unlike Col10a1 null mutants, transgenic mice (FCdel) bearing the mouse equivalent of a human MCDS p.P620fsX621 mutation, displayed typical characteristics of MCDS with disproportionate shortening of limbs and early onset coxa vara. In FCdel mice, the degree of expansion of the hypertrophic zones was transgene-dosage dependent, being most severe in mice homozygous for the transgene. Chondrocytes in the lower region of the expanded hypertrophic zone expressed markers uncharacteristic of hypertrophic chondrocytes, indicating that differentiation was disrupted. Misfolded FCdel alpha1(X) chains were retained within the endoplasmic reticulum of hypertrophic chondrocytes, activating the unfolded protein response. Our findings provide strong in vivo evidence for a gain-of-function effect that is linked to the activation of endoplasmic reticulum-stress response and altered chondrocyte differentiation, as a possible molecular pathogenesis for MCDS.

SummaryDisturbances to the balance of protein synthesis, folding and secretion in the endoplasmic reticulum (ER) induce stress and thereby the ER stress signaling (ERSS) response, which alleviates this stress. In this Commentary, we review the emerging idea that ER stress caused by abnormal physiological conditions and/or mutations in genes that encode client proteins of the ER is a key factor underlying different developmental processes and the pathology of diverse diseases, including diabetes, neurodegeneration and skeletal dysplasias. Recent studies in mouse models indicate that the effect of ERSS in vivo and the nature of the cellular strategies induced to ameliorate pathological ER stress are crucial factors in determining cell fate and clinical disease features. Importantly, ERSS can affect cellular proliferation and the differentiation program; cells that survive the stress can become 'reprogrammed' or dysfunctional. These cellautonomous adaptation strategies can generate a spectrum of context-dependent cellular consequences, ranging from recovery to death. Secondary effects can include altered cell-extracellular-matrix interactions and non-cell-autonomous alteration of paracrine signaling, which contribute to the final phenotypic outcome. Recent reports showing that ER stress can be alleviated by chemical compounds suggest the potential for novel therapeutic approaches. (1) Physiological ER stress during development is mild and controllable. The cell can recover from and/or adapt to the protein-folding stress; it can also differentiate into a specialized cell type in a manner that depends on components of ERSS and appropriate developmental signals. (2) In severe pathological ER stress, ERSS is initially an adaptive response, but, if stress remains unresolved, perhaps because of continuous and/or cyclical expression of mutant protein, it can lead to interference with developmental signals and disruption of cellular gene expression patterns. The cell can then display altered proliferation and differentiation status, and become dysfunctional. (3) Under conditions of extreme stress, an apoptotic signal is triggered and becomes dominant, leading to cell death. This can occur through excessive production of reactive oxygen species (ROS) caused by enhanced protein-folding activity, which relates to levels of CHOP, GADD34 and ERO1 expression. Journal of Cell Science Attenuation of translationOne of the early responses to ER stress is the modulation of translation, a highly conserved adaptation mechanism (Yamasaki and Anderson, 2008). Phosphorylation of eukaryotic initiation factor 2 (eIF2) by PERK reduces protein translation and shifts the translation machinery to favor alternative modes of initiation, including selective translation of activating transcription factor 4 (ATF4) . Phosphorylated eIF2 also activates the conversion of microtubule-associated protein 1 light chain 3 (LC3-I), an essential protein for autophagy, to LC3-II and hence promotes autophagosome formation Kouroku et al., 2007), as well as t...

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.