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Site-1 protease is essential for endochondral bone formation in mice
Correspondence to D. Patra: patrad{at}wudosis.wustl.edu; or L.J. Sandell: sandelll{at}wudosis.wustl.edu
Site-1 protease (S1P) has an essential function in the conversion of latent, membrane-bound transcription factors to their free, active form. In mammals, abundant expression of S1P in chondrocytes suggests an involvement in chondrocyte function. To determine the requirement of S1P in cartilage and bone development, we have created cartilage-specific S1P knockout mice (S1Pcko). S1Pcko mice exhibit chondrodysplasia and a complete lack of endochondral ossification even though Runx2 expression, Indian hedgehog signaling, and osteoblastogenesis is intact. However, there is a substantial increase in chondrocyte apoptosis in the cartilage of S1Pcko mice. Extraction of type II collagen is substantially lower from S1Pcko cartilage. In S1Pcko mice, the collagen network is disorganized and collagen becomes entrapped in chondrocytes. Ultrastructural analysis reveals that the endoplasmic reticulum (ER) in S1Pcko chondrocytes is engorged and fragmented in a manner characteristic of severe ER stress. These data suggest that S1P activity is necessary for a specialized ER stress response required by chondrocytes for the genesis of normal cartilage and thus endochondral ossification.
E.B. Hunziker's current address is Department of Cranio-Maxillofacial Surgery and Department of Clinical Research, University of Bern, Bern 3010, Switzerland.
Abbreviations used in this paper: Agc1, aggrecan; ATF6, activating transcription factor 6; BiP, immunoglobulin heavy chain binding protein; BSP, bone sialoprotein; Col I, type I collagen; CREBH, cAMP-responsive element binding protein H; E, embryonic day; ERSS, ER stress signaling; IHC, immunohistochemistry; Ihh, Indian hedgehog; MMP, matrix metalloproteinase; OASIS, old astrocyte specifically induced substance; PECAM-1, platelet/endothelial cell adhesion molecule 1; Ptc-1, Patched-1; PTHrP, parathyroid hormone-related peptide; S1P, site-1 protease; SCAP, SREBP cleavage–activating protein; SREBP, sterol regulatory element binding protein; WT, wild type.
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In this paper, we have identified site-1 protease (S1P) as a new player involved in regulating endochondral ossification. S1P (also known as membrane-bound transcription factor protease, site 1) is a proprotein convertase and a key member of the regulated intramembrane proteolysis pathway involved in the unfolded protein response and cholesterol homeostasis (Brown et al., 2000). A role for S1P in cartilage development was shown through the study of the zebrafish gonzo mutant (Schlombs et al., 2003), which has both lipid and skeletal abnormalities. S1P plays a critical role in the processing of the sterol regulatory element binding proteins (SREBP-1a, -1c, and -2; Eberle et al., 2004). SREBPs are membrane-bound transcription factors in the ER and regulate cholesterol and fatty acid biosynthesis and uptake. When cholesterol levels are high, SREBP is retained in the ER membrane as a complex with the sterol-sensing protein SREBP cleavage–activating protein (SCAP) and the retention protein INSIG (insulin induced gene). When cholesterol levels drop, the SREBP–SCAP complex dissociates from INSIG and translocates to the Golgi bodies, where SREBP is sequentially cleaved by S1P and S2P to release the amino-terminal domain of SREBP containing the basic helix-loop-helix leucine zipper region. The basic helix-loop-helix leucine zipper region translocates to the nucleus to bind to cis-acting sterol responsive elements. In a similar fashion, S1P is also involved in the activation of other ER membrane-bound proteins such as activating transcription factor 6 (ATF6; Haze et al., 1999; Ye et al., 2000), old astrocyte specifically induced substance (OASIS; Murakami et al., 2006), and cAMP-responsive element binding protein H (CREBH; Zhang et al., 2006), which are transcription factors for the unfolded protein response target genes.
To elucidate the role of S1P in all aspects of skeletal development, we created cartilage-specific S1P knockout mice (S1Pcko) using a Col2a1 promoter. S1Pcko mice die shortly after birth and exhibit severe chondrodysplasia. The cartilage matrix is abnormal in S1Pcko mice with defects in Col II protein secretion and assimilation into the matrix, and endochondral bone formation is completely absent. This is the first example of a defect in a regulated intramembrane proteolysis enzyme that affects cartilage development and endochondral ossification in mice. Deletions of various matrix metalloproteinases (MMPs), such as MMP13 or MMP9 (Inada et al., 2004; Stickens et al., 2004), thought to be important in bone morphogenesis did not abolish endochondral ossification. Thus, S1P is a unique enzyme that plays an integral role in skeletal development.
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Indian hedgehog (Ihh) signaling is intact in S1Pcko mice
Because of a lack of endochondral ossification in S1Pcko mice, we examined Ihh signaling, which is known to be an essential regulator of chondrocyte proliferation and hypertrophic differentiation. Ihh's function is thought to be dependent on lipophilic modifications critical for the spatially restricted localization of Ihh signaling (Gritli-Linde et al., 2001). In the growth plate, Ihh is distributed in a gradient that is necessary for coordinated chondrocyte proliferation and differentiation (Koziel et al., 2004). As cholesterol and fatty acid synthesis is likely to be affected in chondrocytes because of the absence of S1P, it could lead to defects in Ihh signaling. A defect in Ihh signaling is associated with a decrease in chondrocyte proliferation (St-Jacques et al., 1999). Thus, to analyze the nature of Ihh signaling in our model, we labeled proliferating chondrocytes with BrdU at E14.5 (Fig. 4, A and B).
Notably, at E14.5, analysis of the proliferating BrdU-labeled cells revealed that both WT and S1Pcko mice exhibit nearly identical rates of proliferation (Fig. 4 C). Moreover, WT and S1Pcko proliferating chondrocytes displayed the ability to exit the cell cycle, as seen by a lack of BrdU incorporation as they progressed toward hypertrophic differentiation (Fig. 4, A and B).
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Hypertrophic chondrocyte differentiation is incomplete in S1Pcko mice
Hypertrophic differentiation begins with the proliferating chondrocytes exiting the cell cycle and beginning to express Ihh and Runx2 to form the prehypertrophic cells, which exhibit a decreased expression of Col2. The fully differentiated hypertrophic chondrocytes are large, no longer express Col2, and are characterized by the expression of Col10 and VEGF. In S1Pcko mice, hypertrophic differentiation is initiated as chondrocytes exit the cell cycle and no longer incorporate BrdU (Fig. 4 B). However, under higher magnification, this region in E14.5 S1Pcko mice does not show the same morphology as in the WT (Fig. 5).
In the WT humerus (Fig. 5 A), there is a clear definition of the hypertrophic zone. Above the hypertrophic zone, proliferating chondrocytes are seen organized in groups of columnar chondrocytes. Between these groups, there is an abundant matrix that stains a deep red. In S1Pcko mice (Fig. 5 B), however, columnar organization of the cells is absent. Chondrocytes are present in a random configuration and are more densely packed. Although a few enlarged cells are present, a distinct hypertrophic zone is absent.
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These results indicate that the loss of S1P does not prevent the molecular program necessary for hypertrophic differentiation. Rather, the loss of S1P appears to affect cell morphology and may affect matrix production. Therefore, we analyzed the expression of the matrix genes Col2 and aggrecan (Agc1) and MMP13 and Col10 in the E14.5 humerus (Fig. 6). At E14.5, chondrocyte hypertrophy is clearly established in the WT, characterized by the expression of Col10 (Fig. 6 A) and the lack of Col2 (Fig. 6 C) and Agc1 (Fig. 6 E) expression in hypertrophic cells. In S1Pcko mice, however, expression of Col2 (Fig. 6 D) and Agc1 (Fig. 6 F) is maintained into the zone of Col10-expressing cells (Fig. 6 B). Thus, in S1Pcko mice, expression of hypertrophic markers is seen concomitantly with expression of markers for proliferating cells. However, the high levels of Col2 and Agc1 expression seen in the columnar cells in WT mice (Fig. 6, C and E, brackets) are absent in S1Pcko mice. Exit from the cell cycle and the expression of Runx2 and Col10 suggest hypertrophic differentiation. But the lack of characteristic hypertrophic morphology and the persistence of Ihh, PTHrP-R, Col2, and Agc1 into the Col10-expressing zone suggest that organized transitions between various cell types are missing; the cells do not respond cohesively to differentiation signals and are therefore unable to organize themselves into structurally discrete zones. Finally, we checked for expression of MMP13 at E14.5 in S1Pcko cells. MMP13 is normally expressed by the terminal hypertrophic cells in the growth plate (Colnot et al., 2004). WT mice showed considerable expression of MMP13 (Fig. 6 G) in the terminal hypertrophic chondrocytes. However, S1Pcko mice exhibit a dramatic decrease in MMP13 expression in the humerus at E14.5 (Fig. 6 H). This decrease is not a reflection of delay in differentiation but is maintained at E15.5 in S1Pcko mice (Fig. 6 J), which suggests a defect in terminal hypertrophic differentiation. However, MMP13 expression was observed in the perichondrium of S1Pcko mice at E15.5 presumably in the osteoblasts in the exuberant bone collar. These data suggest that S1P is required for the completion of hypertrophic differentiation and organization of the hypertrophic zone.
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Abnormal Col II properties in S1Pcko mice
Several lines of evidence suggest that matrix abnormalities are primarily responsible for the S1Pcko phenotype. First, chondrocytes are more densely packed in S1Pcko skeletal elements. Second, Ihh signaling is intact, as indicated by Ptc-1 and PTHrP expression, although Ptc-1 expression appears to be slightly disorganized. Third, it is unlikely that the reduced MMP13 expression seen in S1Pcko mice is responsible for the lack of endochondral bone, as the targeted inactivation of MMP13 in mice does not prevent endochondral bone formation (Inada et al., 2004; Stickens et al., 2004). Finally, the increase in chondrocyte apoptosis in S1Pcko mice that is suggestive of an abnormal matrix is also seen in transgenic mice lacking Col II (Yang et al., 1997). S1P may thus play a role in the deposition of a normal cartilage. To assess abnormalities in the matrix, we studied the properties of the Col II protein, which is a major cartilage component. First, we attempted to extract the Col II protein from E17.5 WT and S1Pcko cartilage by pepsin digestion after extraction of the proteoglycans by 4 M Gu-HCl. After pepsin digestion, the extract was concentrated and equal concentrations of protein from each sample were subjected to SDS-PAGE and analyzed by Western blotting for Col II (using the IIF antibody that detects the triple helical domain of Col II) and Col X protein (Fig. 8 A).
We were able to extract the full-length Col II from the WT head (Fig. 8 A, lane 3) and the rest of its skeleton (Fig. 8 A, lane 1). Notably, we were not able to extract the full-length Col II from any of the S1Pcko skeletal elements (Fig. 8 A, lanes 2 and 4). In some preparations, some lower molecular weight proteins with immunoreactivity to the IIF antibody are seen (unpublished data). But we were unable to extract comparable amounts of the full-length Col II protein in any of our attempts. By Western blotting analysis of twofold dilutions of the pepsin-digested material, we determined that we were extracting 
64-fold less of the full-length Col II protein from the S1Pcko mice as compared with WT (unpublished data). In contrast, we were able to extract comparable amounts of the Col X protein from both WT and S1Pcko mice.
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To pursue this observation further, we performed double-labeled immunofluorescence analysis with the IIA and IIF antibodies (Zhu et al., 1999) to study the organization and localization of the Col II protein (Fig. 9). In the WT, the secretion and incorporation of Col IIA into the matrix is seen as a well-formed and organized green lattice network. Localization of the collagen triple helical domains is seen as red immunofluorescence, which in the WT is seen as a distinct red lattice network with hardly any yellow colocalization signals from the two antibodies, suggesting that the red lattice is almost entirely made of Col IIB protein. As expected in the WT, the presence of Col IIA is stronger in the matrix surrounding the early immature chondrocytes (Fig. 9 B) and the presence of Col IIB is stronger around the more mature columnar chondrocytes (Fig. 9 C). In S1Pcko mice (Fig. 9, F and G), the organization of the Col IIA lattice network appears normal, though it is also prominent in the matrix surrounding mature chondrocytes (Fig. 9 G). However, in the matrix surrounding early chondrocytes (Fig. 9 F), well-formed organization of Col IIB fibrils (red) is considerably reduced and the majority of the signal appears to be caused by colocalization (yellow), suggesting that both antibodies are detecting primarily Col IIA and very little Col IIB. In the matrix surrounding more mature chondrocytes (Fig. 9 G), the lattice network is primarily green with very little incorporation of Col IIB (red signaling) into the lattice network of the ECM. Most of the Col IIB protein (Fig. 9 G, arrows) is seen trapped inside the cell. The lack of matrix accumulation can be seen more clearly in cross sections of the ribs (at E18.5) in S1Pcko mice (Fig. 9 H). Again, primarily colocalization signals (yellow) and the substantial absence of matrix when compared with the WT (Fig. 9 D) is observed, which showed abundant matrix (red) with yellow colocalization signals restricted to the rim of the rib.
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The lack of endochondral bone seen in S1Pcko mice is also seen in Ihh (St-Jacques et al., 1999) and Runx2 null mice (Komori et al., 1997; Otto et al., 1997). In both these mutants, endochondral bone development is hampered because of a defect in osteoblastogenesis. Both Ihh and Runx2 null mice also suffer from varying degrees of an abnormal chondrocyte differentiation program (Inada et al., 1999; Kim et al., 1999). In S1Pcko mice, we initially expected that the phenotype could have arisen because of a defect in the Ihh signaling pathway. However, our data suggest that S1Pcko mice have an intact Ihh signaling pathway, as indicated by the expression of Ptc-1 and PTHrP in S1Pcko skeletal elements. However, the mislocalization of Ptc-1–expressing cells leaves open the possibility that S1P has an effect on the activity level and/or diffusion of Ihh. The morphological phenotype that sets S1Pcko mice apart from Ihh and Runx2 null mice is that S1Pcko mice exhibit an exuberant bone collar showing normal osteoblastogenesis. Elimination of S1P results in an uncoupling of normal endochondral bone formation from cortical bone formation. Thus, like Ihh and Runx2, S1P is a major positive regulator of endochondral ossification. Expression of S1P is observed in the perichondrium in the WT. However, in S1Pcko mice, S1P activity would be expected to be absent in the perichondrium because of active Col2-Cre activity in these cells. These data suggest that S1P is not necessary for cortical bone formation. Mice with double knockouts of the transcription factors L-Sox5 and Sox6 also exhibit a lack of endochondral ossification with development of a thick cortical bone (Smits et al., 2001). However, it is unlikely that this pathway is affected in S1Pcko mice, as they exhibit abundant expression of L-Sox5 and Sox6 (unpublished data).
The lack of endochondral bone in S1Pcko mice appears to be caused by an inability of the blood vessels to invade the abnormal, mineralized cartilage in spite of apparently adequate VEGF expression. According to the tenets of organogenesis, where development is guided by tissue interactions, the developmental history of a tissue plays an important role in its ability to respond to instructive cues (De Robertis, 2006). Thus, it would be expected that an abnormal cartilage would constrain its ability to respond to instructions from the perichondrium and/or vascular tissue. The genesis of a normal matrix is necessary for proper hypertrophic differentiation and endochondral ossification. Our data suggest that the defects in hypertrophic differentiation and endochondral ossification can be traced to an abnormal matrix in S1Pcko mutants. In S1Pcko mice, the chondrocytes are densely packed and randomly oriented with very little matrix between them. The increased chondrocyte apoptosis seen in S1Pcko mice is also highly indicative of an abnormal cartilage that is unable to support chondrocyte survival. This is similar to the increased apoptosis seen in transgenic mice lacking Col II (Yang et al., 1997). The absence of a proper cartilage could hinder not only the supply of nutrients to the avascular ECM (Hunziker and Herrmann, 1990) but also have profound effects on integrin-mediated pathways and distribution of growth factors (Yoon and Fisher, 2006). The inability to extract normal amounts of full-length Col II from cartilage, the decreased birefringence of Col II network, the lack of the Col IIB lattice network, and the considerable reduction in collagen fibrillar density as seen by electron microscopy all attest to the fact that the cartilage is abnormal in S1Pcko mice. S1Pcko mice also exhibit abnormal spine development in that the intervertebral discs are not well formed and lack the gelatinous nucleus pulposus in the center. This is also seen in transgenic mice with a null mutation in the Col2a1 gene (Aszodi et al., 1998).
An abnormal cartilage ECM could mask angiogenic signaling by VEGF in spite of the adequate VEGF expression that is seen in S1Pcko mice. Given the abnormal characteristics of the cartilage matrix, even if VEGF is not masked, the MMPs may not be able to process and break down the alien matrix in S1Pcko mice. An inability to degrade the matrix is suggested by the persistence of Col IIA in the S1Pcko cartilage, as Col IIA is seen throughout the length of the humerus. These observations are highly suggestive of a lack of structurally intact cartilage matrix in S1Pcko mice that is normally a forerunner for endochondral bone development. Some of the phenotypes seen in S1Pcko mice also bear resemblance to the phenotypes seen in mutants for other matrix proteins. For example, a lack of columnar hypertrophic chondrocytes, disorganized growth plates, chondrodysplasia, and incomplete nucleus pulposus are seen in mice lacking perlecan (Arikawa-Hirasawa et al., 1999; Gustafsson et al., 2003), Agc1 (Watanabe et al., 1994; Wai et al., 1998), and/or link protein (Watanabe and Yamada, 1999). These observations clearly attest to the importance of a properly organized matrix for chondrocyte differentiation. However, these mutations exhibit only reduced or delayed endochondral ossification. Thus, it is noteworthy that only the disruption of the Col2a1 gene (Li et al., 1995) results in a complete lack of endochondral ossification as seen in S1Pcko mice. These data suggest a convergence of S1P activity with a property of Col II in relation to the cartilage matrix.
Thus, our paper shows that S1P is essential to endochondral ossification. The much accepted role of S1P is that of a proprotein convertase required to activate the transcription factors SREBPs, ATF6, OASIS, and CREBH, the latter three playing vital roles during ER stress response. Given that the knockout of the SREBP molecules has not exhibited any known defect in bone morphogenesis in mice that escape embryonic lethality (Shimano et al., 1997; Liang et al., 2002), it seems unlikely that the phenotype in S1Pcko mice is manifested through SREBPs. Furthermore, in the zebrafish gonzo phenotype, knockdown of SCAP (the lipid sensor) results only in strong lipid phenotypes with normal cartilage (Schlombs et al., 2003). These data suggest that the abnormal cartilage phenotype in S1Pcko mice is mediated by lipid-independent pathways. Given the engorgement and fragmentation of the ER in S1Pcko mice, it seems likely that S1P is required to activate ER stress signaling (ERSS) in response to the hyperproduction of complex cartilage matrix proteins seen in chondrocytes. Unlike other studies (Tsang et al., 2007), it is noteworthy that the ER stress observed in S1Pcko mice is not caused by the introduction of any mutant matrix protein but by the inability of the chondrocyte to respond to ER stress because of a lack of S1P activity. Based on these data we propose that the ER stress response is part of the vital protein processing machinery in normal chondrocytes and is essential for normal matrix production (Fig. 10). In a normal chondrocyte differentiation program, chondroprogenitor cells do not require ER stress response because of the relatively low level of protein synthesis and secrete Col IIA into the matrix. However, on differentiation into chondrocytes, to alleviate the stress caused by the high demand for complex matrix protein synthesis, the chondrocytes initiate ERSS, a mechanism that requires S1P to activate ATF6, OASIS, or CREBH (currently the three known ER stress signal transducers that require activation by S1P). This allows for the deposition of normal cartilage followed by endochondral ossification. In S1Pcko mice, chondroprogenitor cells do not show any defect in the secretion of Col IIA either because of the level of Col IIA synthesis or the different dynamics of Col IIA secretion. However, upon differentiation into chondrocytes and a switch to the alternatively spliced form Col IIB and the concomitant increase in protein synthesis and secretion, S1Pcko mice are unable to initiate the ER stress response because of an inability to activate the appropriate transcription factors in the absence of S1P. This results in the engorgement and fragmentation of the ER followed by poor Col IIB secretion into the matrix, which results in the matrix acquiring the properties typically seen on disruption of the Col2a1 gene. Other cell types, such as the periosteal cells, which lack S1P but do not require the same ER stress response, are able to synthesize bone, as seen in S1Pcko mice. Thus, chondrocytes in this respect are like the differentiating plasma cells, which turn on the unfolded protein response to alleviate ER stress and optimize antibody secretion (Gunn et al., 2004), or exocrine glands, where the ER stress response is necessary for the full development of the secretory machinery (Lee et al., 2005).
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Isolation of collagens from WT and S1Pcko mice
The isolation of collagens was done from E17.5 WT and S1Pcko mice using a modification of previously published protocols (Mayne et al., 1995). In brief, E17.5 embryos were skinned and eviscerated, and the head was processed separately from the rest of the body. The spine/limbs and head were frozen and crushed, and the proteoglycans were extracted with 4 M Gu-HCl in 0.05 M Tris-HCl, pH 7.5, containing 0.01 M EDTA and a 1x complete protease inhibitor cocktail (Roche), for 36 h at 4°C. The cartilage residues left after proteoglycan extraction were washed with cold water and digested with 1 mg/ml pepsin in 0.5 M acetic acid for 24 h at 4°C. The digest was then concentrated with centricon30 (Millipore) to 250 µl, dialyzed against 0.05 M Tris-HCl, pH 7.5, buffer containing 0.4 M NaCl and 5 mM EDTA, and stored at –20°C. Equal concentrations of protein preparations (determined by Bio-Rad Laboratories protein assay) from WT and S1Pcko mice were separated by SDS-PAGE followed by Western blotting for Col II (using the previously described rat antibody IIF against bovine Col II triple helical domain; Zhu et al., 1999) and Col X proteins (EMD). The molecular weight marker used in SDS-PAGE was BenchMark prestained protein ladder (Invitrogen).
Primary chondrocyte cultures from WT and S1Pcko mice
To study the biosynthesis and secretion of the Col II protein in vitro, primary cultures from WT and S1Pcko mice were established. Chondrocytes were isolated from the sternum and ribs of WT and S1Pcko mice as described previously (Lefebvre et al., 1994). In brief, rib cages were taken from WT or S1Pcko mice and treated with 2 mg/ml pronase (Roche) at 37°C for 30 min followed by 3 mg/ml collagenase (Roche) treatment in the presence of 8% CO2 for 1.5 h at 37°C or until all soft tissues are detached from the cartilage. The cartilage was then separated from the soft tissues and treated again with 3 mg/ml collagenase for 4 h at 37°C in the presence of 5% CO2. The digest was filtered through a cell strainer to remove undigested bony parts and the cells were then pelleted by centrifugation, washed with 1x PBS, and plated at a density of 2.5 x 105 per cm2 in DME with 10% heat-inactivated serum, 2% penicillin/streptomycin (Sigma-Aldrich), and 0.25 mM sodium ascorbate. 50 µl of media from the WT and S1Pcko chondrocyte cultures was harvested at regular intervals and separated by 7.5% SDS-PAGE (Bio-Rad Laboratories), and the type II procollagen was detected by Western blotting using the IIF antibody.
Morphological and histological analysis
Whole-mount skeletal staining of embryos by alcian blue and alizarin red was performed as described previously (McLeod, 1980). For histological and in situ hybridization analyses on sections, embryonic tissues were collected at various embryonic time points and processed and sectioned as described previously (Hilton et al., 2005). Histological analyses were done primarily by Safranin O, Fast green, and hematoxylin staining. Detection of mineralization was performed by staining with 1% silver nitrate as per the von Kossa method, followed by counterstaining with Methyl green. In situ hybridization analyses were performed using 35S-labeled riboprobes as described previously (Long et al., 2001). All in situ probes with the exception of Agc1 were provided by F. Long (Washington University School of Medicine) and have been described by Hilton et al. (2005) and references therein. The Agc1 in situ probe (a gift of E. Vuorio, University of Turku, Turku, Finland) covering the IGD sequence of the mouse Agc1 has been described previously (Glumoff et al., 1994). The full-length S1P and the S1P exon 2 in situ probes were derived from a full-length cDNA clone (American Type Culture Collection). For BrdU analyses, pregnant females were injected with BrdU as described previously (Hilton et al., 2005), and proliferation of chondrocytes was analyzed using a kit (Invitrogen). For PECAM-1 IHC, embryos were fixed using 4% formaldehyde followed by 30% sucrose infiltration. 10-µm cryostat sections were derived from tissues embedded in OCT (Tissue-Tek; Thermo Fisher Scientific) and IHC was performed using a monoclonal rat anti–mouse PECAM-1 antibody (BD Biosciences). Detection of PECAM-1 was done using HRP-conjugated anti–rat IgG and DAB. Methyl green was used for counterstaining. Detection of apoptosis was performed by a TUNEL assay using the in situ cell death detection kit (Roche) according to the manufacturer's instructions. Nuclei were counterstained with DAPI and sections were examined by fluorescence microscopy. Tartrate-resistant acid phosphatase–positive cells were stained using standard procedures with methyl green as counterstain. Study of the birefringence of collagen fibers by polarized light microscopy was done on 5-µm-thick paraffin-embedded sections as described previously (Li et al., 1995). Detection of total Col II protein by IHC was done on 5-µm-thick paraffin-embedded sections at E15.5 and 18.5 stages using the IIF antibody and procedure described previously (Zhu et al., 1999), with the exception that detection was done using an HRP-conjugated goat anti–rat secondary antibody and a DAB substrate (Invitrogen). Detection of Col IIA protein was done similarly with the previously described rabbit antisera against recombinant human type IIA-GST (IIA; Oganesian et al., 1997; Zhu et al., 1999) and an HRP-conjugated goat anti–rabbit secondary antibody. Double-labeled immunofluorescence with IIF and IIA antibodies was performed as described previously (Zhu et al., 1999) on paraffin embedded sections, with the exception that the IIA and IIF antibodies (and their corresponding secondary antibodies) were used sequentially with the IIA antibody and its secondary antibody applied first. The secondary antibodies used were goat anti–rabbit Alexa Flour 488 (Invitrogen) and goat anti–rat Alexa Fluor 546 (Invitrogen). Images for double immunofluorescence were collected with a 60x, 1.4 NA oil immersion objective using either a scanning laser confocal microscope (for E18.5 ribs cross sections; MRC600; Bio-Rad Laboratories) mounted on a microscope (Eclipse E800; Nikon) and LaserSharp 2000 software (Bio-Rad Laboratories) or a camera (2000R Fast1394; Retiga)and Q Capture Pro (for limbs; QImaging). All other sections (in situ/histology) were viewed with a microscope (BX51; Olympus) using a 10x objective (all in situ analysis) or 20 and 40x objectives (for stained sections or IHC), and images were captured with the digital camera (DP70; Olympus) using DP controller software (Olympus). For in situ analysis, pictures of hybridization signals were hued red and superimposed on toluidine blue–counterstained images using Photoshop (Adobe).
Online supplemental material
Fig. S1 shows the lack of endochondral ossification in the femur, ribs, sternum, and spine of S1Pcko mice. Fig. S2 shows the abnormal hypertrophic differentiation in S1Pcko mice. Fig. S3 shows normal VEGF and MMP9 expression and normal osteoblastogenesis in S1Pcko mice. Fig. S4 shows normal Col2 mRNA splicing in S1Pcko mice. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200708092/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health grants RO1 AR50847 and R01 AR36994 to L.J. Sandell and Swiss National Science Foundation grant 320000-118205 to E.B. Hunziker.
Submitted: 13 August 2007
Accepted: 15 October 2007
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