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Address correspondence to Véronique Lefebvre, Dept. of Biomedical Engineering, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., ND-20, Cleveland, OH 44195. Tel.: (216) 445-0762. Fax: (216) 444-9198. email: lefebvrv{at}bme.ri.ccf.org
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Abstract |
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Key Words: chondrogenesis; development; differentiation; mouse; transcription factor
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Introduction |
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Cartilage primordia arise upon condensation of prechondrogenic mesenchymal cells. These cells differentiate sequentially into prechondrocytes and chondroblasts. Prechondrocytes activate Col2a1 (collagen type 2), Agc1 (aggrecan), and other cartilage-specific extracellular matrix genes. Chondroblasts up-regulate these genes and activate others like Mat1 (matrilin-1). They produce large amounts of extracellular matrix and actively proliferate. Several types of signaling molecules and transcription factors control skeleton patterning and cell differentiation at these early stages (DeLise et al., 2000; Hall and Miyake, 2000; de Crombrugghe et al., 2001; Karsenty and Wagner, 2002). The latter include three Sry-related HMG box-containing proteins, L-Sox5, Sox6, and Sox9. L-Sox5 (a long product of Sox5) and Sox6 are highly identical to each other but related to Sox9 only in the HMG box DNA-binding domain (Lefebvre, 2002). Sox5, Sox6, and Sox9 are coexpressed in all cartilage primordia of the mouse embryo and may cooperate to directly activate Col2a1 (Ng et al., 1997; Zhao et al., 1997; Lefebvre et al., 1998). Sox9 is required for prechondrogenic cell condensation, prechondrocyte and chondroblast differentiation, and activation of Sox5, Sox6, and cartilage matrix genes (Bi et al., 1999; Akiyama et al., 2002). Sox5 and Sox6 have essential, redundant roles in early chondroblasts (Smits et al., 2001). Although Sox5-/- and Sox6-/- mice are born with minor cartilage defects, Sox5-/-/Sox6-/- embryos develop a severe, generalized chondrodysplasia due to considerable delay and impairment of chondroblast proliferation and expression of cartilage matrix genes. They die around embryonic day 16.5 (E16.5) with rudimentary and matrix-deficient cartilage primordia.
The fate of each chondroblast is determined by its location in cartilage primordia (Karsenty and Wagner, 2002; Kronenberg, 2003). Although epiphyseal chondroblasts are maintained at an early differentiation stage throughout gestation, diaphyseal and metaphyseal chondroblasts mature to form growth plates and induce the formation of primary ossification centers. Upon undergoing prehypertrophy, they stop proliferating and sequentially activate Ppr (receptor for parathyroid hormone and parathyroid hormonerelated peptide), Ihh (Indian hedgehog), and Col10a1 (collagen type 10). They also induce the flanking perichondrium to form a bone collar. Next, they undergo hypertrophy, stop expressing Col2a1, Agc1, and other early markers, progressively down-regulate Ppr and Ihh, up-regulate expression of Col10a1, and induce cartilage matrix mineralization. As they terminally differentiate, they stop expressing Col10a1, activate Opn (osteopontin) and Mmp13 (matrix metalloproteinase-13), and undergo apoptosis. Bone collar chondro/osteoclasts, osteoblasts, and blood vessels then invade and replace the cartilage by bone. Various signaling molecules control these processes. They include Ihh, PTHrP (parathyroid hormonerelated peptide), and PPR (Karaplis et al., 1994; Lanske et al., 1996; Vortkamp et al., 1996; St-Jacques et al., 1999), FGFR3 (fibroblast growth factor receptor-3) and its ligand FGF18 (Colvin et al., 1996; Deng et al., 1996; Liu et al., 2002; Ohbayashi et al., 2002), bone morphogenetic proteins (BMPs; Zou et al., 1997), and Wnt proteins (Hartmann and Tabin, 2000; Yang et al., 2003). Few transcription factors have yet been demonstrated to control these processes. Cbfa1/Runx2 is expressed in late chondroblasts and in prehypertrophic and hypertrophic chondrocytes (Inada et al., 1999; Kim et al., 1999). It induces prehypertrophy as shown by a block of chondrocyte differentiation before prehypertrophy in several skeletal elements of Runx2-/- mouse embryos and by maturation of transgenic chondroblasts forced to express Runx2 (Takeda et al., 2001). However, because Runx2-/- chondrocytes properly mature in some skeletal elements, additional factors must contribute to induce chondrocyte maturation. Sox5, Sox6, and Sox9 are turned off upon chondrocyte prehypertrophy (Lefebvre et al., 1998). Sox9+/- embryos precocious mineralize cartilage primordia and develop enlarged hypertrophic zones (Bi et al., 2001). Thus, Sox9 might delay chondrocyte hypertrophy. When Sox5-/-/Sox6-/- fetuses die, their cartilage primordia contain few prehypertrophic chondrocytes but no hypertrophic cells. They are being invaded by thick bone collars but feature no cartilage growth plates and no ossification centers (Smits et al., 2001). Therefore, these fetuses do not allow for a full assessment of the roles of Sox5 and Sox6 in the growth plate.
To determine such roles, we mostly analyzed here embryos with three null alleles (3NA) of Sox5 and Sox6 (Sox5+/-/Sox6-/- and Sox5-/-/Sox6+/-). These embryos live until birth and show severe growth plate chondrocyte defects. We show that Sox5 and Sox6 are needed to form and maintain a pool of highly proliferating chondroblasts between epiphyses and metaphyses, to form columnar chondroblasts, delay chondrocyte prehypertrophy but promote hypertrophy, and to delay terminal differentiation of chondrocytes on contact with ossification fronts. Sox5 and Sox6 act in part by controlling the domain of action and expression of major regulatory factors.
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Results |
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Reduced proliferation and precocious prehypertrophy of 3NA growth plate chondroblasts
The size of the columnar region is determined by the rate of cell proliferation within this zone and by the rate at which chondroblasts exit this zone to become hypertrophic. It may also depend on the rate at which epiphyseal chondroblasts enter this zone (Kobayashi et al., 2002). To measure the rate of chondroblast proliferation, we labeled proliferating cells in vivo with BrdU and counted the percentages of positive cells in small segments juxtaposed from the presumptive joint to the center of cartilage primordia or to the hypertrophic zone of growth plates (Fig. 5). We found that control chondroblasts proliferated at highly variable rates in different subregions of epiphyses and growth plates. They proliferated at the highest levels at the top of metaphyses both in E13.5 (Fig. 5 A) and in E16.5 humeri (Fig. 5 B). Interestingly, 3NA chondroblasts were consistently found to proliferate slower than control cells in this region. At E13.5, the rate of cell proliferation decreased from this peak toward the presumptive joint, where prechondrocytes were still differentiating, and toward the center of the cartilage primordia, where chondroblasts were undergoing prehypertrophy. 3NA cells were proliferating at a lower rate than controls in epiphyses, likely reflecting their chondroblastic delay. At E16.5, chondroblasts were proliferating more slowly in the middle than in the periphery of epiphyses, but no major difference was seen between controls and mutants. The columnar zone was characterized by a progressive decline in cell proliferation, and this decline occurred twice as fast in 3NA humeri as in controls. Thus, 3NA chondroblasts formed shorter columnar zones because they failed to proliferate fast enough at the top of this zone and because they growth arrested faster than normal. The unaffected proliferation rate of 3NA epiphyseal chondroblasts was consistent with the normal growth rate of epiphyses, indicating that these cells unlikely contributed to the shortness of the columnar zone.
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Discussion |
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Sox5/Sox6 3NA mice and Sox9 mutant mice
This work would not have been possible using only 4NA mice because these mice die at E16.5 with no true growth plates (Smits et al., 2001). 3NA embryos constituted a good complementary model because their generalized chondrodysplasia is mainly due to growth plate defects. Their chondrocytes are defective at many differentiation steps but do not arrest at any specific step and do complete their differentiation program before mouse death. Therefore, they have allowed us to study the roles of Sox5 and Sox6 at each of these steps. The two types of 3NA mice, Sox5-/-/Sox6+/- and Sox5+/-/Sox6-/- mice, exhibit virtually identical growth plate phenotypes, indicating that Sox5 and Sox6 are redundant in all steps of chondrocyte maturation. They are more affected than 2NA mice but less affected than 4NA mice, demonstrating that the expression dosage of Sox5 and Sox6 is critical for proper chondrogenesis. Sox5/Sox6 3NA mice resemble Sox9+/- mice (Bi et al., 2001) by the developmental delay and matrix deficiency of cartilage primordia as well as by ectopic mineralization and bending of endochondral elements. However, major differences can be noticed. The onset of cartilage mineralization is precocious in Sox9+/- fetuses but delayed in Sox5/Sox6 3NA fetuses; endochondral elements are short but not thin in Sox5/Sox6 3NA newborns and thin but not always short in Sox9+/- newborns; columnar zones are short in Sox5/Sox6 3NA fetuses but normal in Sox9+/- fetuses; and hypertrophic zones are short in Sox5/Sox6 3NA fetuses but long in Sox9+/- fetuses. These differences are unlikely due to the degree of severity of the phenotypes because Sox5/Sox6 2NA, 3NA, and 4NA mice have qualitatively identical defects. Thus, Sox5, Sox6, and Sox9 must have distinct functions during chondrocyte maturation.
Source chondroblasts
The presence of a major chondroblast proliferation peak between epiphyses and metaphyses in fetal long bones leads us to revisit the main cellular source of the growth plate. Most studies with fetal growth plates have assumed that epiphyseal chondroblasts (often referred to as reserve, resting, or periarticular chondrocytes) give rise to columnar chondroblasts. However, no definitive experimental evidence has validated this model. In contrast, our data strongly suggest that epiphyses elongate through cell proliferation mainly in the periarticular region and epiphyseal side of the major proliferation peak, and that columnar zones are generated and renewed mainly by cells originating in the metaphyseal side of this peak. Two pieces of data support our model. First, sternum growth plates develop and are maintained in the absence of epiphyses. They present in pairs oriented back-to-back and sharing a pool of intersternebral chondroblasts. This pool is the likely equivalent of the major proliferation peak in long bones. Second, long bone growth plates become postnatally restricted to the region of the major proliferation peak and metaphysis. They are physically separated from articular cartilage by the secondary ossification centers that develop in epiphyses. Therefore, their cellular source cannot be in articular cartilage. Consistent with our model, a study in rabbits has suggested that postnatal growth plates find their cell source just above the columnar zone, in a zone (called resting zone) that likely corresponds to the fetal chondroblast proliferation peak (Abad et al., 2002).
Epiphyseal chondroblasts
When 4NA fetuses die, their epiphyses are still largely precartilaginous. In contrast, epiphyseal chondroblasts differentiate with only a slight delay in 3NA embryos, and 3NA epiphyses are essentially normal at birth. These data indicate that epiphyseal chondroblasts require Sox5 and Sox6 for development, but only at a low dosage. PTHrP/PPR signaling prevents ectopic maturation of epiphyseal chondroblasts (Lanske et al., 1996; Chung et al., 1998; Kobayashi et al., 2002). Ectopic maturation of epiphyseal chondroblasts does not occur in 3NA cartilages, which is consistent with the normal expression of Ppr and Pthrp detected in these cartilages. Therefore, PTHrP/PPR signaling does not require high levels of Sox5 and Sox6.
Columnar chondroblasts
3NA chondroblasts fail to proliferate at maximum levels at the top of the columnar zone and decrease their proliferation rate much faster than control cells. Thus, columnar chondroblasts require a high dosage of Sox5 and Sox6 to function optimally, in contrast to epiphyseal chondroblasts. Nevertheless, Sox5 and Sox6 appear expressed at similar levels from the joint area to the prehypertrophic zone (Lefebvre et al., 1998; unpublished data), which means that their expression levels do not directly dictate the cell proliferation changes. Ihh signaling stimulates chondroblast proliferation (St-Jacques et al., 1999). However, because Ihh levels are likely inversely proportional to the gradient of cell proliferation in the columnar zone, Ihh signaling probably attenuates rather than generates this gradient. Interestingly, Ihh expression is not substantially altered in 3NA cartilages, and Ihh signaling actually functions better in these cartilages than in control cartilages. This result was revealed by up-regulation and expansion of the expression domain of Ptc1. It has been shown that proteoglycans or glycosaminoglycans facilitate the movement of Ihh through tissues (Gritli-Linde et al., 2001). Therefore, it is possible that the 3NA cartilage matrix deficiency allows for a better diffusion of Ihh, and hence, for increased signaling. The other possibility, that 3NA chondroblasts are more responsive to Ihh signaling, is not supported by the fact that Ptc1 is also up-regulated in 3NA perichondrium and bone cells, which do not express Sox5 and Sox6.
Minina et al. (2001) have shown that BMP signaling is required in addition to Ihh signaling to maintain chondroblast proliferation in limb explants, but a definitive role for BMP signaling in columnar chondroblasts has not been demonstrated in vivo. Nevertheless, inhibition of early chondroblast proliferation in Bmpr1b-/- mice (Baur et al., 2000; Yi et al., 2000) and continued expression of Bmpr1b and several BMP genes in the growth plate and perichondrium strongly suggest that BMP signaling must contribute to columnar chondroblast proliferation. Expression of BMP and BMP receptor genes is not altered in 3NA cartilages, except for Bmp6, which is expressed in prehypertrophic chondrocytes. Although the exact role of Bmp6 in growth plates has not been fully determined (Solloway et al., 1998), Sox5 and Sox6 appear to act upstream of BMP6 signaling, possibly in controlling chondroblast proliferation.
Fgfr3 and Fgf18 negatively regulate chondroblast proliferation, acting at least in part by down-regulating Ihh expression and signaling (Colvin et al., 1996; Deng et al., 1996; Naski et al., 1998; Liu et al., 2002; Minina et al., 2002; Ohbayashi et al., 2002). Fgf18 is expressed in the perichondrium, and its expression is unaffected in 3NA elements. Fgfr3 is expressed in the columnar zone, and we found that its expression follows a positive gradient from the top to the bottom of this zone. This finding suggests that FGFR3 signaling generates the negative gradient of columnar chondroblast proliferation. Interestingly, 3NA chondroblasts up-regulate Fgfr3 earlier than control cells in the columnar zone, strongly suggesting that Sox5 and Sox6 control columnar chondroblast proliferation at least in part by delaying Fgfr3 up-regulation.
Chondrocyte prehypertrophy
In the cartilage primordia of future long bones, 3NA and 4NA chondroblasts activate the prehypertrophic marker Ppr later than control cells when considering the gestation time. However, when considering the time at which the cells become chondroblastic, 3NA chondroblasts activate Ppr faster than control cells. In the sternum primordium, activation of Ppr occurs so early and is so widespread that it is truly ectopic. Thus, Sox5 and Sox6 are needed to delay chondrocyte prehypertrophy. In seeking for the underlying mechanism, we found that Sox5 and Sox6 are needed to delay and down-regulate expression of the prehypertrophy inducer Runx2. However, their mode of action is likely indirect because Runx2 is also up-regulated in the perichondrium where Sox5 and Sox6 are not expressed. Significant changes in expression of Pthrp and Ppr, which delay prehypertrophy, are not detected in 3NA growth plates, indicating that Sox5 and Sox6 unlikely act upstream of PPR signaling. FGF signaling accelerates the onset and the pace of prehypertrophic chondrocyte differentiation in limb explants in vitro and BMP signaling antagonizes the effect of FGF signaling (Minina et al., 2002). Because Sox5 and Sox6 might control chondroblast proliferation at least in part by delaying up-regulation of Fgfr3 in columnar chondroblasts, they may similarly delay prehypertrophy and may decrease the effect of FGF signaling by up-regulating Bmp6.
Chondrocyte hypertrophy and terminal differentiation
Chondrocytes become hypertrophic with a significant delay in 3NA cartilage primordia, and then form short hypertrophic zones because they terminally differentiate prematurely. In 4NA embryos, chondrocytes skip hypertrophy as seen namely by failure to express Col10a1, the most specific marker of hypertrophic chondrocytes, and directly differentiate from early prehypertrophy to the terminal stage. This critical role of Sox5 and Sox6 in allowing chondrocytes to undergo hypertrophy and in delaying terminal differentiation appears opposed to that of Sox9, which may prevent chondrocyte hypertrophy and accelerate terminal differentiation (Bi et al., 2001). Sox9 is expressed at a normal level in 3NA and 4NA chondroblasts and is correctly turned off in 3NA hypertrophic cells (Smits et al., 2001; unpublished data). Therefore, it is not involved in the phenotype of Sox5/Sox6 mutants. Because Sox5, Sox6 and Sox9 are no longer expressed in hypertrophic chondrocytes, their actions on these cells must be indirect. Runx2 is needed for chondrocyte prehypertrophy and may still be needed for hypertrophy because it was proposed to directly activate Col10a1 (Zheng et al., 2003). Therefore, its up-regulation in 3NA cells is at odds with the impairment of hypertrophy, unless Runx2 promotes prehypertrophy but delays hypertrophy, or cooperates with another yet unknown factor to specify chondrocyte hypertrophy. The signaling pathways that promote chondrocyte hypertrophy are still unknown, but the possibility that chondroblasts depend on Sox5 and Sox6 to express signaling molecules mediating hypertrophy of neighboring chondrocytes is not supported by observations made in Sox9/Col2-Cre mutants (Akiyama et al., 2002). In these mutants, cells in the core of cartilage primordia appear to undergo normal differentiation up to and including hypertrophy, whereas all other cartilage cells remain prechondrocytic and fail to activate Sox5 and Sox6. This suggests that chondrocyte hypertrophy is controlled cell autonomously. A mechanism whereby Sox5 and Sox6 might be required cell autonomously but indirectly for chondrocyte hypertrophy might be that chondroblasts need to surround themselves with a proper cartilage matrix, a function promoted by Sox5 and Sox6, to be able to undergo hypertrophy.
Despite hypertrophy impairment, 3NA and 4NA chondrocytes undergo terminal differentiation, a step that is not yet fully understood at the regulatory level. Several works have shown a block of chondrocyte differentiation at the mineralizing stage, for instance upon inactivation of Mmp9 (Vu et al., 1998), Vegf (Gerber et al., 1999), Runx2 (Komori et al., 1997; Otto et al., 1997), or Osx (Nakashima et al., 2002), but these works have not demonstrated whether chondrocytes are blocked at the hypertrophic or terminal stage. We have shown that 3NA and 4NA chondrocytes terminally differentiate only on contact with ossification fronts, strongly suggesting that chondrocyte terminal differentiation is induced at least in part by ossification fronts.
3NA cells undergo terminal differentiation even if they do not reach full hypertrophy, and 4NA cells do so immediately after reaching prehypertrophy, indicating that hypertrophy is a dispensable step in the chondrocyte pathway. Thus, Sox5 and Sox6 allow chondrocytes to add hypertrophy in their pathway, a step determinant for the proper elongation of endochondral elements. Here, they may also function by allowing cells to accumulate extracellular matrix and thereby protect themselves against bone invasion. In agreement with this model, 3NA sternum chondrocytes may be able to become hypertrophic because they undergo prehypertrophy so massively that ossification fronts cannot invade them all at once.
Conclusions
This work has demonstrated essential, redundant roles for Sox5 and Sox6 in chondrocyte differentiation and proliferation (Fig. 9) beyond their critical role in promoting differentiation of prechondrocytes into early chondroblasts (Smits et al., 2001). Sox5 and Sox6 ensure the development of early chondroblasts into epiphyseal, source, and columnar chondroblasts and delay chondrocyte prehypertrophy. They stimulate the proliferation of source chondroblasts, keep columnar chondroblasts proliferating, and prevent precocious prehypertrophy at least in part by delaying up-regulation of Fgfr3 and by down-regulating Runx2. They are also needed to allow development of hypertrophic chondrocytes and to prevent precocious terminal differentiation of prehypertrophic and hypertrophic chondrocytes. Because they are no longer expressed in chondrocytes beyond prehypertrophy, their actions in hypertrophic and terminal chondrocytes must be indirect, possibly extracellular matrix mediated. Thus, Sox5 and Sox6 are required for the establishment of cartilage growth plates and thereby for the proper and timely development of endochondral bones.
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Materials and methods |
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Skeleton staining, histology analysis, and RNA in situ hybridization
Whole-mount staining of embryos, skeletal preparations, and histology were performed as described previously (Smits et al., 2001). Samples from control and mutant littermates were embedded in the same paraffin blocks to minimize experimental variation. Sections stained with Alcian blue or with the von Kossa reagent were counterstained with nuclear fast red. The Bmp6 probe was an 893-bp EcoRISacI fragment of the mouse cDNA (a gift from Y. Furuta, University of Texas, Houston, TX), the Mmp13 probe was the first 699 nucleotides of the mouse cDNA, the Opn probe was a 1.3-kb EcoRI fragment of the rat cDNA (a gift from K. Nakashima and B. de Crombrugghe, University of Texas, Houston, TX), and the Ptc1 probe was the full-length mouse cDNA (a gift from R.L. Johnson, University of Texas, Houston, TX). The Agc1 and Col10a1 probes (gifts from E. Vuorio, University of Turku, Turku, Finland), the Fgfr3 probe (a gift from C.X. Deng, National Institutes of Health, Bethesda, MD), the Ihh probe (a gift from A.P. McMahon, Harvard University, Cambridge, MA), the Mat1 probe (a gift from A. Aszódi, Max-Planck Institute, Martinsried, Germany), the Mmp9 probe (a gift from Z. Werb, University of California, San Francisco, San Francisco, CA), the Ppr probe (a gift from H.M. Kronenberg, Massachusetts General Hospital, Boston, MA), and the Runx2 probe (a gift from G. Karsenty, Baylor College of Medicine, Houston, TX) were as described previously (Smits et al., 2001).
Cell proliferation assay
Pregnant mice were injected with BrdU reagent (Zymed Laboratories; 10 µl/g of mouse) and killed 2 h later. BrdU-labeled nuclei were detected by immunostaining (Zymed Laboratories), and sections were counterstained with hematoxylin, as described previously (Smits and Lefebvre, 2003). Cell proliferation rates were determined by counting the percentages of BrdU-positive cells in consecutive segments distributed from the periarticular to the hypertrophic zone of growth plates. Reproducible data were obtained with three sets of control and mutant littermates.
Image acquisition and manipulation
Samples were visualized on a microscope (model BX50; Olympus) equipped with Uplanapo 10x/0.40 and Uplanapo 20x/0.70; /0.17 lenses (Olympus). Images were captured with a digital camera (model DMC2; Polaroid) using accompanying software. In situ hybridization images were taken under dark field using a red filter for RNA signals and under blue fluorescence for nuclei stained with Hoechst 33258 dye. Figures were composed using Adobe Photoshop 7.0.
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Acknowledgments |
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This work was supported by National Institutes of Health grant AR46249 to V. Lefebvre, by the Lerner Research Institute of The Cleveland Clinic Foundation, and by a postdoctoral fellowship from the Arthritis Foundation to P. Smits.
Submitted: 5 December 2003
Accepted: 16 January 2004
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