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Correspondence to T. Michael Underhill: tunderhi{at}interchange.ubc.ca
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Abbreviations used in this paper: atRA, all-trans RA; BMP, bone morphogenetic protein; BMPR, BMP receptor; DEAB, diethyl aminobenzaldehyde; E, embryonic age; GDF, growth and differentiation factor; IDR, interdigital region; RA, retinoic acid; RAR, RA receptor; RARE, RA response element; Tp, distal tip; WL, whole limb.
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Introduction |
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Other signaling molecules that influence the chondrogenic program include vitamin A and its metabolites, the retinoids (Weston et al., 2003a,b). The retinoids act through the modulation of the transcriptional activity of the nuclear receptors for retinoic acid (RA), the RA receptors (RARs), and the retinoid X receptors. The transcriptional activity of these receptors is governed by RA availability, which is regulated largely by the combined actions of the enzymes involved in its synthesis and degradation, the ALDH1As and CYP26s, respectively. Indeed, the Rars, and other genes encoding enzymes involved in RA metabolism, are dynamically expressed during chondrogenesis. Furthermore, compound null mutants of the Rars and null mutants of Aldh2a1, Cyp26a1, or b1 present with a spectrum of skeletal abnormalities (Lohnes et al., 1994; Abu-Abed et al., 2001; Niederreither et al., 2002b; Weston et al., 2003b; Yashiro et al., 2004). The formation of precartilaginous condensations and the subsequent appearance of chondroblasts require the activity of Sox9, which is a transcription factor belonging to the Sry-related HMG box gene family (Bi et al., 1999; Akiyama et al., 2002). In accordance with a proposed role for RA signaling in chondrogenesis, RA influences the expression and/or activity of Sox9 (Weston et al., 2000, 2002).
In earlier studies, we demonstrated that mesenchymal cells isolated from a transgenic animal overexpressing a weak, constitutively active Rara transgene exhibit skeletal defects, and this, in part, results from delayed or inhibited chondroblast differentiation (Cash et al., 1997). Subsequent studies have indicated that the chondrogenic defect within the transgene mesenchyme is not rescued by the addition of BMP2 or -4, leading to the postulation that retinoid signaling may operate downstream of the BMP signaling pathway within this program (Cash et al., 1997; Weston et al., 2000). Further series of experiments demonstrated that skeletal progenitor differentiation requires RAR-mediated repression, and that antagonism of RAR signaling in primary cultures of limb mesenchyme is accompanied by increased Sox9 expression and activity (Weston et al., 2002). In this study, we demonstrate that BMPs decrease RA availability, and that this is required for their prochondrogenic function. Furthermore, during autopod development, Aldh1a2 expression is dynamically expressed and influenced by BMP signaling. During this process, Aldh1a2 expression becomes progressively restricted to nonchondrogenic regions, where it likely serves to inhibit or suppress expression of a chondroblast phenotype.
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12-fold reduction); further analysis revealed that Aldh1a2 expression is substantially reduced as early as 6 h (an approximately sixfold reduction), and at 12 h there is an 38-fold differential between treated and untreated cultures (Fig. 3 A).
As discussed earlier, BMP has the potential to recruit cells with low chondrogenic potential to the chondrocytic lineage. Based on this information, we sought to determine the distribution of Aldh1a2-expressing cells within limb mesenchymal cultures. Whole-mount in situ hybridization revealed that in WL cultures, Aldh1a2 is expressed in discrete foci in a pattern reminiscent of Col2a1 expression (Fig. 3 B). Furthermore, addition of BMP4 abolishes Aldh1a2 expression, whereas, consistent with previous observations, Col2a1 expression is intensified and its domain of expression expanded (Fig. 3 B). Thus, it appears that BMP4 expands cartilage formation through down-regulation of Aldh1a2.
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18 h), and Aldh1a2 transcript abundance was quantified thereafter at 4 h intervals for 12 h. To determine if BMP4 decreased Aldh1a2 mRNA stability, BMP4-treated and untreated cultures were incubated with 5 µg/ml of the transcription inhibitor actinomycin D. In the presence of actinomycin D, Aldh1a2 mRNA levels decline in control cultures, and the presence of BMP4 does not accelerate this decline (Fig. 3 C). In contrast, treatment of cultures with the protein synthesis inhibitor cycloheximide abolished down-regulation of Aldh1a2 by BMP4 (Fig. 3 C). There is an initial slight downward trend in the BMP4 and cycloheximide-treated cultures at the 4-h time point, but by 8 and 12 h there is no significant difference between the cycloheximide-treated control and BMP4 cultures. Thus, down-regulation of Aldh1a2 by BMP4 requires protein synthesis, indicating that BMP4 probably acts indirectly through the regulation of factors to down-regulate Aldh1a2.
Endogenous RA signaling regulates expression of the chondroblast phenotype
To further demonstrate the potential importance of ALDH1a2 in BMP4 action, we first examined the role of endogenous RA synthesis and degradation in chondrogenesis. Catabolic inactivation of RA is mediated by the CYP26 subfamily of cytochrome P450 enzymes (Niederreither et al., 2002a). Inhibition of RA degradation with an inhibitor of cytochrome P450 enzymes, ketoconazole (Osanai and Petkovich, 2005), leads to increased endogenous RA signaling, as evaluated with an RA-responsive reporter gene. Under these conditions, even small changes in endogenous RA availability (approximately fourfold) lead to a >10-fold reduction in SOX9 activity and a decrease in the formation of cartilage nodules (Fig. 4 A, insets).
Importantly, the effects of ketoconazole on the retinoid and SOX9-responsive reporter genes can be completely reversed through addition of a pan-RAR antagonist, demonstrating that the observed effects of ketoconazole in this system reside in its ability to inhibit CYP26 enzyme activity (Fig. 4 B). Conversely, RA degradation increases through heterologous expression of Cyp26a1, and this is accompanied by a decrease in RA response element (RARE) reporter gene activity and an approximately fourfold increase in SOX9 activity (Fig. 4 C). Consistent with these observations, inhibition of ALDH1a2 activity with the ALDH1 inhibitor DEAB (Perz-Edwards et al., 2001) results in an approximately sevenfold reduction in "RA availability" and an approximately fivefold increase in SOX9 activity; this is associated with increased cartilage nodule formation (Fig. 4 D, insets). To follow Col2a1 expression in live cultures, WL cultures were established from transgenic embryos that contain a 6.3-kb portion of the Col2a1 promoter, driving expression of EGFP (Grant et al., 2000). In control cultures, weak transgene expression is apparent within 24 h, and by 96 h an abundance of EGFP-positive nodules can be observed (Fig. 4 E). Modulation of endogenous retinoid signaling via treatment with ketoconazole significantly impacts transgene expression, with fewer EGFP-positive cells being evident in these cultures. In contrast, attenuation of RA synthesis with DEAB leads to precocious and widespread transgene expression in comparison to control cultures (Fig. 4 E). Together, these results demonstrate that small changes in endogenous retinoid status profoundly influence expression of the chondroblast phenotype.
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. Indeed, heterologous expression of Ncor2 or Ncor2 and Rara together increases SOX9 activity by 2- and 10-fold, respectively. Treatment with BMP4 alone leads to a 3.6-fold increase in reporter gene activity, whereas treatment with BMP4 combined with expression of Ncor2 or Ncor2 and Rara increases reporter activity by 10- and 30-fold, respectively (Fig. 5 B). Importantly, SOX9 activity correlates inversely with the extent of the decrease in RARE reporter activity (Fig. 5 B). Consistent with our observations, overexpression of either of the BMP receptor (Bmpr) type I receptors, a or b, stimulates SOX9 activity, and this is associated with a reduction in RARE-luc activity (Fig. 5 C). These effects are potentiated by BMP4 addition. Together, these observations suggest that down-regulation of retinoid signaling in primary limb mesenchymal cells favors expression of a chondrogenic cell fate. Furthermore, addition of 100 nM atRA completely abrogates the prochondrogenic effects of BMP4 on the aforementioned reporter genes and on cartilage nodule formation (Fig. 5, C and D). Interestingly, treatment with ketoconazole on either day 0 or 1 of culture also inhibits the prochondrogenic action of BMP4 (Fig. 5 F), again indicating that even small changes in endogenous retinoid signaling affect expression of the chondrogenic phenotype.
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RA availability is regulated by BMP signaling in vitro and in vivo
In the forelimb, the autopod cartilages, as determined by Col2a1 expression, first become evident around early E11, with the hindlimb following 0.5 d later (Fig. 7 A).
At the beginning of autopod development, Col2a1 expression in precartilaginous digital rays is quite weak, but quickly intensifies, such that by late E12 all the digits exhibit strong Col2a1 expression. In contrast, Aldh1a2 exhibits a dynamic pattern of expression that quickly changes from a band of expression through the early autopod to a fragmented appearance a few hours later (Fig. 7 A). Bifurcation of Aldh1a2 expression in the autopod occurs over a relatively short period of time (9 h) and is closely associated with digit formation. As previously reported, Aldh1a2 expression appears between the developing zeugopodal elements, within the regions flanking the digit cartilages, and within the interdigital region (IDR; Niederreither et al., 1997).
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During early appendicular skeletal development, the BMPs, GDFs, and their receptors exhibit a complex pattern of expression, appearing at various times and locations within the cartilaginous anlagens. In this regard, activated BMP signaling has been observed in the developing cartilages, and an absence of the Bmpr1a and -1b severely compromises precartilaginous condensation formation and chondroblast differentiation (Yoon et al., 2005). Activation of either of these two receptors leads to a stage-dependent increase in cartilage formation (Zou et al., 1997). In contrast, activated retinoid signaling inhibits chondrogenesis, whereas decreased retinoid signaling, either through antagonism of the receptors or in compound Rar knockouts, promotes chondrogenesis in vitro and in vivo (Zou et al., 1997; Weston et al., 2003a,b). Together, these observations would suggest that Aldh1a2 and liganded Rars should be excluded from developing cartilages. To examine the relationship of Aldh1a2-expressing cells and developing cartilages, double in situ hybridization was used in vitro and in vivo in WL cultures and E11.25–12.5 embryos, respectively. Aldh1a2 and Col2a1 are expressed in spatially distinct regions (Fig. 8, A and B). More specifically, Aldh1a2-expressing cells are excluded from Col2a1-expressing regions and from the apical ectodermal ridge and subjacent mesenchyme. During autopod development, small rays of Col2a1-expressing cells can be observed to bisect the band of Aldh1a2 expression (Fig. 8 A). Consistent with these observations, Aldh1a2-expressing cells are excluded from Col2a1-expressing regions in WL cultures. Furthermore, the distribution of Aldh1a2-positive cells would be expected to cause localized activation of the retinoid signaling pathway. This is, indeed, the case because establishment of WL cultures from a transgenic mouse harboring an RA-responsive reporter gene (RARE-hsp68-lacZ; Rossant et al., 1991) demonstrates foci of transgene-expressing cells, which are distinct from the developing cartilages (Fig. 8 C). Transgene expression is markedly increased and decreased in response to 50 or 100 nM of exogenous atRA or the addition of 100 nM of 310, respectively (unpublished data). In both culture systems, BMP4 leads to expansion of Col2a1-expressing cells and increased cartilage formation, as well as substantial reduction in Aldh1a2 expression and RARE activity (Fig. 8 B, C). In aggregate, these findings suggest that during autopod development BMP signaling regulates expression of the chondrogenic phenotype by controlling RA availability.
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Function of BMP signaling in chondrogenesis: involvement of the retinoid signaling pathway
BMP2, -4 and -7, along with GDF5, bind and activate the BMPR type I a and b receptors. Several hallmarks of activated BMP/GDF signaling are observed in the developing skeleton, including the phosphorylation of Smads (1, 5, or 8; Brunet et al., 1998; Yoon et al., 2005). Noggin is also often up-regulated in response to the activation of BMPRIs (Canalis et al., 2003); this, too, was observed in this study (Fig. 2). An essential requirement for BMPRI-mediated signaling in endochondral ossification has been elegantly demonstrated by Yoon et al. (2005) through generation of compound null mutants of Bmpr1a and -1b. In this study, mutant animals were found to exhibit severe deficiencies in cartilage formation. More specifically, these animals present with poorly defined precartilaginous condensations that weakly express Sox9. Similarly, excess Noggin in vitro or in vivo leads to reduced cartilage formation, and decreased Sox9 expression and activity (Pizette and Niswander, 2000; Weston et al., 2000; unpublished data). Reduced activity of BMPs or GDFs through targeted or spontaneous mutations also negatively impacts the formation of precartilaginous condensations and subsequent cartilage formation. In contrast, expression of wild-type (Fig. 5 C) or constitutively active versions of the BMPR type I receptors promotes chondrogenesis (Zou et al., 1997; unpublished data). Furthermore, ectopic BMP signaling in vivo and in vitro expands cartilage formation, likely through recruitment of cells not destined to become chondrocytes (Duprez et al., 1996a,b). Thus, BMP signaling appears to act at multiple stages within the chondrogenic program, including the establishment, maintenance, and differentiation of chondroprogenitors. The ability of BMPs to stimulate chondroblast differentiation is influenced by retinoid signaling (Figs. 4, 5, and 6), suggesting that at least a subset of these activities involves BMP-mediated attenuation of the retinoid signaling pathway.
Cells throughout the early autopod exhibit high chondrogenic potential, and these cells can be directed under a variety of conditions to form cartilage in vitro and in vivo (Lee et al., 1994; Ganan et al., 1996; Rodriguez-Leon et al., 1999). As autopod development ensues, the chondrogenic potential of many of these cells is lost, especially those within the IDRs, i.e., prechondrogenic mesenchyme loses its ability to become cartilage or is inhibited from doing so. In the presence of BMP signals, however, these cells express a chondrogenic fate (Fig. 7 A); this is especially apparent in the Noggin-null mice, which present with very broad digits and greatly limited IDR (Brunet et al., 1998). Importantly, during the establishment of these distinct regions, Aldh1a2 is dynamically expressed within these domains. Before overt chondrogenesis and robust expression of Col2a1, Aldh1a2 appears as a broad band throughout the autopod. As digit formation proceeds, Aldh1a2 expression is quickly extinguished in cartilage-forming regions, becoming localized to the nonchondrogenic flanking regions. At this embryonic stage, the developing limb is exquisitely sensitive to changes in retinoid status (Weston et al., 2003b), and the presence of Aldh1a2 is congruent with a role for retinoid signaling in inhibiting expression of the chondrogenic phenotype in cells flanking the developing digits. Moreover, activation of a RA-responsive reporter in transgenic mice can be observed within the early IDR (Rossant et al., 1991), and RARE-expressing cells are excluded from developing cartilages (Fig. 8 C). In contrast, numerous studies have demonstrated that diminished retinoid signaling promotes chondrogenesis in vitro and in vivo (Weston et al., 2003a,b). In this study, we show that manipulation of endogenous RA production and signaling significantly impacts chondrogenesis (Figs. 4 and 5). In aggregate, these results suggest dual roles for the RARs in regulating chondrogenesis. Unliganded receptors promote expression of the chondroblastic phenotype, whereas liganded receptors inhibit differentiation of prechondrogenic cells. In this regard, local activation of the retinoid signaling pathway in the developing autopod would specify nonchondrogenic regions. Consequently, under conditions where retinoid signaling is diminished in the developing autopod, widened phalanges and/or cartilage formation in the IDR would be expected. Interestingly, Rara/g double-null mice present with both of these phenotypes. More specifically, a bulbous phalange abnormality is consistently observed, along with a less frequent appearance of ectopic cartilages in the IDRs (Lohnes et al., 1994). These mice also exhibit ectopic cartilage at several extraskeletal sites (Lohnes et al., 1994; Mendelsohn et al., 1994).
Parallels in axial and appendicular skeletogenesis: overlapping players involving different mechanisms
In the axial skeleton, BMPs play a role in the establishment and maintenance of an autoregulatory loop between the transcriptional repressor NKX3.2 and SOX9, thereby promoting axial chondrogenesis (Zeng et al., 2002). Interestingly, even though BMPs are sufficient and necessary for axial and appendicular chondrogenesis, their regulation of Sox9 appears to operate through an NKX3.2-independent manner in appendicular skeletogenesis (Zeng et al., 2002). In axial chondrogenesis, expression of a chondrogenic fate in somitic mesoderm appears to involve derepression of Sox9 by NKX3.2-mediated down-regulation of gene X, which is a transcriptional repressor of Sox9. Likewise, the retinoid pathway appears to be operating in a similar manner in the appendicular skeleton to regulate Sox9 and expression of a chondrogenic fate. In this scenario, activation of the retinoid signaling pathway up-regulates the expression of a negative regulator of Sox9 expression, gene Y, leading to decreased Sox9 expression and reduced cartilage formation. Conversely, antagonism of RAR-mediated signaling down-regulates the expression of Y, leading to derepression of Sox9 and increased cartilage formation, and, thus, supporting a chondrogenic-permissive condition. Thus, at least in the limb, BMPs function by attenuating the expression of Aldh1a2, thereby stimulating Sox9 expression and activity, and the elaboration of a chondroblastic phenotype. Both programs appear to converge on negative regulators of Sox9, genes X and Y. Further complicating an understanding of BMP action in the limb is that BMPs do not appear to act directly to regulate Aldh1a2 expression, as protein synthesis is required for this activity. Thus, BMPs possibly act to increase the expression of a negative regulator of Aldh1a2 expression, or rather that BMPs may act directly through a relatively labile protein. Evidence for this latter scenario comes from the observation that in the absence of protein synthesis (Fig. 3 C), BMP4 addition leads to an initial transient decrease in Aldh1a2 expression. Collectively, these findings demonstrate that BMP4-mediated establishment and maintenance of Sox9 expression in limb chondrogenesis involves a cascade of factors with the retinoid signaling pathway figuring prominently in this program. Further elucidation of the factors operating upstream and downstream of the RA signaling pathway will provide additional important insights into the molecular mechanisms underlying the chondrogenic program.
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Materials and methods |
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Plasmid constructs
Reporter plasmids containing SOX9 binding sites (pGL3[4X48]) or trimerized RARE-luc were previously described (Weston et al., 2002). pG5, which is a reporter that contains five GAL4 binding domains, was obtained from Promega. To generate GAL4–RAR(DEF) fusions, the C-terminal region of Rara encompassing the ligand-binding domain was subcloned in-frame into pBIND (Promega). For expression in primary cells, full-length versions of murine Cyp26a1 (provided by M. Snead, University of California, Los Angeles, Los Angeles, CA) and Rara1 were subcloned into a modified pSG5 (Weston et al., 2002), and the construct containing an N-terminal EGFP fusion to full-length Ncor2 (provided by M. Privalsky, University of California, Davis, Davis, CA) was as previously described (Goodson et al., 2005). Mammalian expression plasmids containing BMPR1a and Bmpr1b were provided by J. Wrana (University of Toronto, Toronto, Canada).
Establishment and transfection of primary mesenchymal cultures
Primary mesenchymal cultures from CD-1 E11.5 mouse limb buds were established as previously described (Weston et al., 2000) with the following modifications. For microarray analyses and other specified experiments, distal limb bud tips (subridge region) were dissected from E11.5 mouse embryos (the morning of the plug was considered E0.5) as described by Gay and Kosher (1984), with the excised region extending 0.3–0.4 mm from the distal apex of the limb to the proximal cut edge. After proteolytic digestion, cells were filtered through a cell strainer (40 µM; BD Biosciences) to obtain a single cell suspension. Cells were pelleted and resuspended at a density of 2 x 107/ml and 12–15 10-µl aliquots of this suspension were plated into a 35-mm tissue culture dish (Nunc) and allowed to adhere for 1 h. After this period, 2 ml of culture medium consisting of 60% F12/40% DME and supplemented with 10% FBS (Qualified; Invitrogen) was added to each well with or without 20 ng/ml BMP4 (R&D Systems); this time was considered T = 0. Cultures were maintained for a period of up to 3 d; to minimize handling, culture media was replaced on alternate days. Cultures from either RARE-hsp68-lacZ (CD-1 background; provided by J. Greer, University of Alberta, Edmonton, CA) or Col2-EGFP mice (CD-1 background; derived from breeding of heterozygous transgenic males with CD-1 females) were established in a similar manner. Alcian blue staining, ß-galactosidase staining, and the establishment and transient transfection of cultures derived from WL buds was performed as described in Weston et al. (2000, 2002). In brief, cultures were transfected at day 0 and extracts for luciferase analysis were collected at day 2 unless indicated otherwise. For experiments involving primary limb mesenchymal cells alone or cells transfected with reporter genes only, factors or compounds were added at the time of media addition (T = 0). For experiments involving cotransfection with expression plasmids, factors and compounds were added 24 h after transfection.
Limb bud organ culture and bead implantation
WL buds were collected in PSA from E11.5 Col2-EGFP embryos. Affi-Gel blue beads (Bio-Rad Laboratories) soaked in either vehicle or BMP4 (20 ng/µl) for 2 h were transferred into the IDR of the limb buds. Limb buds were cultured on Nucleopore Track-Etch membranes (Whatman) at the air–liquid interface on top of stainless steel mesh in 12-well tissue culture plates. PSA was aspirated from each well and replaced with BGJb medium (Invitrogen) containing 10% FBS and antibiotics. The level of culture medium should not exceed the height of the membranes. Limbs were incubated under standard tissue culture conditions. After a 24-h incubation, EGFP expression was visualized using a dissection microscope (model MZ12; Leica) with epifluorescence. Limbs were subsequently fixed in 4% PFA before processing for whole-mount in situ hybridization.
Transcriptional profiling with microarrays: experimental design and analysis
RNA was harvested from primary cultures using RNAeasy (QIAGEN) according to the manufacturer's instructions. For the zero time point, cells were allowed to attach for 1 h and were subsequently processed for RNA isolation. For other cultures, the media was gently aspirated, and any remaining media was blotted from the well before the addition of the lysis reagent. After isolation, the RNA was precipitated, and resuspended at 2 µg/ml; RNA quality was examined on a Bioanalyzer 2100 (Agilent), and the expression of Sox9 and Col2a1 were measured using real-time PCR.
For each time point, a minimum of two biological replicates were analyzed on U74V2 chips A and B. 10 µg of RNA was labeled and hybridized to the chips using the manufacturer's recommended protocol. Gene expression profiles were subsequently analyzed using MAS 5.0 (Affymetrix) and GeneTraffic UNO bioinformatics programs (Stratagene). All datasets were initially filtered to remove genes called absent by MAS 5.0, and were further filtered as indicated in the text using GeneTraffic. Hierarchical clustering was performed in GeneTraffic using gene clustering and a Pearson correlation distance metric.
Real-time PCR and whole-mount in situ hybridizations
To follow the expression of transcripts for Aldh1a2, Cyp26a1, Col2a1, and Sox9, quantitative real-time PCR was performed using the 7900HT Sequence Detection System (Applied Biosystems). Primers and MGB probes (TaqMan) were designed using PrimerDesigner 2.0 (Applied Biosystems). The primer/probe sets used for detection of Sox9 and Col2a1 were as described in Weston et al. (2002) and the following primer/probe sets were used for quantifying Cyp26a1 transcript abundance: forward primer, 5'-CTCCAACCTGCACGATTCCT-3', reverse primer, 5'-CGGCTGAAGGCCTGCAT-3', probe 6FAM-5'-CAGCGAAAGAAGGTG-3'-MGBNFQ. Aldh1a2 transcripts were detected using the forward primer, 5'-GGTATCCTCCGCAATGCAA-3', reverse primer, 5'-GCGCATTTAAGGCATTGTAACA-3', and probe, 6FAM-5'-CTGGGACAGTTTGGATC-3'-MGBNFQ. Primer and probe concentrations were optimized according to the manufacturer's instructions. Total RNA was isolated from primary cultures as described above. Quantification was performed using 25 ng of total RNA and the expression of all genes relative to endogenous rRNA was determined using TaqMan Ribosomal Control Reagents (Applied Biosystems) and the comparative CT method as described in User Bulletin #2 (Applied Biosystems).
Whole-mount in situ hybridization was performed on primary mesenchymal cultures as previously described (Weston et al., 2000) with the following modifications. After permeabilization using 10 µg/ml proteinase K in PBS supplemented with 0.05% Triton X-100, cells were postfixed in 4% PFA and 2% glutaraldehyde in PBS. Blocking and antibody incubations were performed in 1% blocking reagent (Roche) in 1x maleic acid buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5). Riboprobes were synthesized in the presence of UTP-digoxigenin with the appropriate RNA polymerase and linearized template DNA, according to manufacturer's instructions (Roche). Riboprobe complementary to Col2a1 was generated from BamHI-linearized pBluescript containing 1.1 kb of the C-propeptide–encoding region of Col2, and transcribed in vitro with T7 RNA polymerase. Aldh1a2 riboprobe was transcribed from EcoRI-linearized modified pBluescript containing a 400-bp fragment of the Aldh1a2 coding sequence using T3 RNA polymerase. Double in situ hybridizations were performed as above, but with the following modifications. Hybridization was performed using both DIG- and fluorescein-labeled RNA probes. Cultures were subsequently incubated overnight in fluorescein-labeled antibody (for detection of Aldh1a2). After incubation in NBT/BCIP/10% PVA–staining buffer, cultures were rinsed twice with PBS, and then fixed for 10 h in a 4% PFA/PBS solution. Cultures were again briefly rinsed twice in PBS, and the blocking antibody and staining steps were repeated, using an anti–DIG-AP antibody and INT/BCIP for detection of Col2a1.
Single and double whole-mount in situ hybridization on limb buds and cultured limbs was performed as described by Hargrave and Koopman (2000). Double in situ hybridizations were performed as described in the previous paragraph, except that BCIP alone was used for detection of Col2a1. To enhance visualization of the light blue (BCIP) and purple staining (NBT/BCIP), all images were adjusted to maximum hue in Photoshop (Adobe).
Microscopy and image acquisition
Images of fixed cultures (in 70% ethanol or Tris-buffered salt solution with 0.1% Triton X-100 [TBTX]) were collected at room temperature using either a dissection microscope (Stemi SV11 Apo, S1.6x objective; Carl Zeiss MicroImaging, Inc.) or an inverted microscope (Axiovert S100; Carl Zeiss MicroImaging, Inc.) fitted with a CP-Achromat 5x, 0.12 NA, CP-Achromat 10x, 0.25 NA, LD Achrostigmat, 0.55 NA, 10x Fluar, 0.5 NA, and an LD Achroplan 40x, 0.6 NA. Monochromatic and color images were acquired from a QImaging Retiga Exi (12-bit) and a QImaging Retiga 1300i (12-bit) camera, respectively, using Openlab 4 software (Improvision). Live cell and organ culture images were collected at room temperature in culture media with either an Axiovert S100 microscope or a dissection microscope (MZ FLIII; Plan 1.0x objective; Leica). Photoshop was used to adjust some image levels.
Statistical analysis
All luciferase assays were performed in quadruplicate and repeated using three distinct preparations of primary cells. Real-time PCR analysis was performed in duplicate and repeated a minimum of two times with independent RNA samples. Real-time PCR and luciferase reporter data were analyzed by one-way analysis of variance, followed by a Bonferroni posttest for multiple comparisons using GraphPad Prism, Version 4.0 (GraphPad Software, Inc.). Significance is represented as follows: *, P < 0.05; **, P < 0.01; #, P < 0.001. One representative experiment is shown for all luciferase and real-time PCR results.
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Acknowledgments |
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L.M. Hoffman and K. Karamboulas were supported by a postdoctoral and doctoral fellowship, respectively, from the Canadian Arthritis Network. K. Garcha was supported by a doctoral fellowship from the Stem Cell Network. L.M. Hoffman was supported by a postdoctoral fellowship from the Canadian Arthritis Network, and this research was funded by grants to T.M. Underhill from the Canadian Institutes of Health Research (CIHR) and the Canadian Arthritis Network. T.M Underhill holds a New Investigator award from the CIHR and The Arthritis Society.
Submitted: 25 April 2006
Accepted: 30 May 2006
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