Recently identified hereditary skeletal disorders associated with extracellular matrix gene mutations in humans and animal models are providing insights into the roles that the affected molecules play in skeletal development and growth (Schwartz, 2004
). However, most of these studies have analyzed the fully developed mature GP of matrix-deficient models (Maddox et al., 1997
; Savontaus et al., 1996
; Wai et al., 1998
; Watanabe and Yamada, 1999
); therefore, little insight has been provided on the function of the matrix during GP formation or its influence on GP cell signaling. One recent study of a gene trap mutation in the chondroitin-4-sulfotransferase 1 (C4st1
) gene which causes down-regulation of 4-O- sulfated chondroitin synthesis identified a severe chondrodysplasia that is characterized by strong up-regulation of TGFß signaling and down-regulation of BMP signaling (Kluppel et al., 2005
). Furthermore, in the brachymorphic (bm) mouse, where levels of CS sulfation are reduced due a natural inactivating mutation of one of the isoforms of PAPS synthetase, PAPSS2 (Kurima et al., 1998
), alterations in IHH signaling have been observed (Cortes M. et al, 2009
), demonstrating that modification of the chondroitin sulfate component of aggrecan can adversely affect distinct signaling pathways during GP morphogenesis.
We have now obtained intriguing data from the avian aggrecan-deficient nm model, which revealed severe alterations in the genetic and developmental programs occurring in the nm GP concomitant with appearance of atypical morphology (-). Strikingly, absence of aggrecan, the major proteoglycan component of cartilage matrix, is accompanied by dysregulation of several genes previously shown to be critical to the maturation process of hypertrophic chondrocytes and osteogenesis. First, although there appears to be both initiation and progression through the normal states of differentiation by nm chondrocytes (i.e., to expression of COL10A1), the hypertrophic zone is small and disorganized by the time a mature GP (E12) is formed. Secondly, based on the expression of a number of GP markers, the zones of pre-hypertrophic and hypertrophic cells that characterize a normal GP appear to overlap in the nm mutant (-); a phenomenon which starts at the earliest stages of GP formation (E7) concomitant with the lack of aggrecan accumulation (-). Thirdly, both in vivo and in vitro, lack of aggrecan in the ECM is associated with significant differences in numbers of cells in mutant versus wt GP caused by fluctuations in the proliferative and apoptotic pathways ( and ), a phenomenon that also changes as GP maturation proceeds.
A role for IHH dysregulation underlying these changes in the nm GP was verified by analysis of PTCH expression in the mature growth plate which showed a narrow epiphyseal gradient in the nm mutant, suggesting that the lack of matrix could impair the normal IHH gradient (). The gene expression patterns of modulators critical to GP development in the E12 nm chick, in particular the overlapping expression of COL10A1, BMP6 and IHH mRNAs which obscures the normal distinctions between the pre-hypertrophic and hypertrophic regions, suggest acceleration of hypertrophy (-). Also, strong expression of OPN mRNA, a marker of late hypertrophic chondrocytes and osteoblasts, observed in a pattern that completely overlaps that of COL10A1 mRNA, suggests that acceleration of hypertrophy induces precocious bone formation in the nm mutant. Interestingly, advanced hypertrophy was already observed in the mutant when the hypertrophic chondrocytes are first detected in wt GP (E7-8) (). Most importantly, IHH-expressing cells rapidly proceed to hypertrophy (expression of COL10A1) without down-regulating expression of IHH (), indicating loss of control of this critical switch. Consequently, i) the nm GP does not exhibit the characteristic pre-hypertrophic and hypertrophic zone demarcation; ii) separation and appositional growth of the twin GPs of each element is delayed; iii) vascular invasion becomes accelerated (up-regulation of OPN) and osteogenesis occurs prematurely; and iv) longitudinal as well as lateral growth is retarded.
Since the IHH-expressing domain is reduced from E7 onward, we tested whether this deficiency was driven by an even earlier (E6) abnormal setting of the IHH expression domain. Although, IHH appears to be turned on normally in the condensations (), arguing against precocious maturation of chondroblasts to pre-hypertrophic chondrocytes, the domain of expression of the hedgehog effector PTCH is broader in the perichondrium of the E6 nm elements, indicating a disturbance in the range of IHH signaling () beyond the area where aggrecan is normally expressed, thus potentially modifying a key source of GP signaling, the developing periosteum. Several other important changes were observed in the aggrecan-deficient mutant at this critical period of early GP development. In contrast to the increased cell division observed during GP maturation in the nm mutant (E12), early stage (E6) nm epiphyseal cartilage exhibits a slightly lower rate of cell division. This cell division arrest observed in the area where IHH is expressed may be only slightly accelerated in the nm GP even though the levels of FGFR3, an important regulator of cell division and hypertrophic differentiation (Ornitz and Marie, 2002
), are already strongly down-regulated in the forming pre-hypertrophic zone of the nm mutant ().
The changes observed in FGFR3 transcription levels and localization are difficult to explain since little is known about FGFR3 mRNA regulation in the developing GP. It is possible that the altered IHH signaling could elicit changes in FGF signaling, since our organ culture data () indicate changes in patterning and levels of FGFR3 expression when IHH signaling was inhibited by cyclopamine. Alternatively, is possible that early down-regulation of FGFR3 in the nm maturation zone might be an indicator of FGF signaling mis-regulation, as it has been reported that FGF-2 could down-regulate FGFR3 expression in culture (Ling et al., 2006
). Both possibilities are supported by our finding that levels of phosphorylated ERK are up-regulated in E6 nm cartilage, as a result of either saturation of FGFR3 activation due to its down-regulation or alteration of FGF2 diffusion due to the lack of matrix. Thus, increased FGF signaling could explain the early down-regulation of cell division in the nm GP and the early depletion of FGFR3 in the nm pre-hypertrophic zone. This depletion of FGF signaling then could push IHH-expressing precursor cells to prematurely differentiate to hypertrophic chondrocytes, quickly depleting the pool of IHH-expressing cells and further driving cells into hypertrophy. Although only moderate reduction in BMP6 expression in the forming GP (E6) was observed, later (E9) increases in the level of BMP signaling in the hypertrophic zone was seen as determined by increased SMAD 1-5-8 phosphorylation (); perhaps this occurs in response to the accelerated hypertrophy, since BMPs have been described as negative regulators of chondrocyte maturation (Minina et al., 2002
; Minina et al., 2001
). Increased BMP signaling could be a reaction directed toward moderating the acceleration, or alternatively, simply a reflection of the accelerated terminal differentiation observed in the nm GP. Thereafter, the combined effects on the developing periosteum and continued down-regulation of FGFR3 ultimately could reduce FGF signaling, thereby accelerating cell division. It has been suggested that FGF and BMP signaling act in antagonistic and independent fashions upstream of IHH/parathyroid hormone-like protein signaling to define the zone where pre-hypertrophy will be initiated and also to regulate the rate of progression to hypertrophy. While FGF signaling is a negative regulator of cell division and suppresses hypertrophy, BMP signaling acts in parallel to IHH to induce proliferation and hinder hypertrophic differentiation (Minina et al., 2002
). This last point has been contested in the literature since knock-outs of Bmpr1a in mouse suggest that BMP signaling is required for completion of terminal hypertrophic differentiation (Yoon et al., 2006
). In the nm mutant, these three integrated signaling pathways appear to lose their balance-controlling influence after the initial IHH domain of expression is formed, possibly due to absence of a matrix in which to establish the respective gradient fields.
The lack of aggrecan in the nm matrix also may affect access by growth factors necessary for signaling between the perichondrium and the developing chondrocytes. Since part of the altered phenotype was retained in dispersed cell cultures, i.e. increased cell division and cell death, altered diffusion, or availability of signaling molecules in the nm mutant could be a factor. However, accelerated differentiation was not observed when the cytoarchitecture was destroyed by plating single cells from nm cartilage into culture, indicating that positional information and zonal signaling paradigms, as well as the integrity of the matrix and an intact perichondrium, are likely critically important to establishment of the hypertrophic phenotype (, ). As well, we have not ruled out the contribution of changes caused by mechanical stresses due to muscle contraction in the developing limb; however, since clear modifications of signaling-molecule expression patterns are observed as early as E6, when limb size in the wt and nm are comparable, it is expected that these influences are secondary to the defects in GP formation.
Lastly, aggrecan-induced matrix deficiency may lead to premature and/or increased invasion of the nm GP hypertrophic region by blood vessels and osteoblasts; indeed, an anti-angiogenic function for the aggrecan matrix has been proposed (Fenwick et al., 1997
). GP chondrocytes produce angiogenic factors (e.g., bFGF and VEGF) that recruit endothelial cells to migrate towards the hypertrophic cells (Carlevaro et al., 2000
; Gerber et al., 1999
), but this process may be altered in the absence of aggrecan, a known diffusion barrier to many factors in cartilage (Grodzinsky, 1983
). The developmental expression pattern of aggrecan mRNA throughout the GP of normal limbs indicates that different states of chondrocyte differentiation require different levels of aggrecan in the extracellular matrix. The fact that pre-hypertrophic chondrocytes express the highest levels of aggrecan message in the wt GP is congruent with our finding in the nm mutant that the lack of aggrecan throughout the GP affects predominantly the transition of chondrocytes from the pre-hypertrophic to the hypertrophic phenotype. Also, the altered PTCH gradient in the nm limb suggests that aggrecan is essential for establishing a proper IHH gradient; these data are supported by the observations that IHH binds chondroitin sulfate chains in vitro
and that in the undersulfated CSPG matrix of the bm mouse GP, a reduction in the extent of the IHH gradient has also been observed (Cortes M. et al. 2009
). If IHH long range diffusion is impaired in matrix depleted of the major CSPG, aggrecan, such as in the nm limb, it may explain an increased PTCH signal towards the developing periostium at E6 and a reduced PTCH gradient in the fully formed GP (E12). Early changes in the IHH gradients could trigger changes in the other signaling pathways, or alternatively other ligand gradients could also be influenced by the altered matrix. In sum, our results indicate that an aggrecan-rich matrix is necessary for appropriate morphogen gradient formation; thereby indirectly modulating the coordination of several distinct signaling pathways during GP morphogenesis. These findings take us a step closer to understanding the pathogenesis of chondrodysplasias, especially human disorders associated with mutations in the aggrecan gene (Gleghorn et al., 2005
; Tompson et al., 2009