THE ANATOMICAL RECORD 291:19–27 (2007) Versican Knock-Down Compromises Chondrogenesis in the Embryonic Chick Limb JOHN B. SHEPARD,1 DIANA A. GLIGA,1 AMANDA P. MORROW,1 STANLEY HOFFMAN,2 AND ANTHONY A. CAPEHART1* 1 Department of Biology, East Carolina University, Greenville North Carolina 2 Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, South Carolina ABSTRACT Mesenchymal cell aggregation is critical for cartilage formation in the vertebrate limb. The extracellular matrix (ECM) plays a critical role in governing cell behavior and cell phenotype in this tissue, and the hyalectin versican is highly expressed in the ECM of precartilage mesenchymal cells and developing synovial joints. Although several in vitro studies have been conducted in an attempt to address versican’s role during limb mesenchymal condensation, factors such as differences in cell density in culture, variations between chondrogenic cell lines, and the inability to prolong the viability of limb explants have led to conﬂicting data, mandating an in vivo analysis. By using a morpholino directed strategy in ovo, we performed knock-down of versican expression in the presumptive ulnar region of the developing chick wing at time points critical to skeletogenesis. These data indicate that in ovo misexpression of versican compromised mesenchymal condensation with resulting ulnar cartilages reduced in length distally by an average of 53% relative to contralateral control limbs. In select versican morphants the olecranon process was also reduced in size proximally and failed to cup the humerus, likely impairing joint morphogenesis. This study represents the ﬁrst report assessing the role of versican in the developing chick limb in ovo, further demonstrating the importance of versican proteoglycan expression during chondrogenesis and extending previous ﬁndings to suggest a role for versican during synovial joint development. Anat Rec, 291:19–27, 2007. Ó 2007 Wiley-Liss, Inc. Key words: versican; chick embryo; chondrogenesis; extracellular matrix; limb development; morpholino Mesenchymal cell condensation is a prerequisite for chondrogenesis in the developing vertebrate limb (Hall and Miyake, 1992, 2000), involving organized aggregation of mesenchymal cells in a proximal to distal direction. Strong versican expression has previously been localized in limb precartilage mesenchymal condensations (Kimata et al., 1986; Shinomura et al., 1990), suggesting an important role for versican in this process. Williams et al. (2005) demonstrated in vitro that chondrogenesis failed to occur in versican-deﬁcient matrix from hdf (heart defect) limb mesenchyme, and Kamiya et al. (2006) showed that versican knock-down also inhibited chondrogenesis in cartilage precursor cells in Ó 2007 WILEY-LISS, INC. vitro. Interestingly, Zhang et al. (1998a) demonstrated (at a cell density several-fold lower than Williams et al., 2005) that antisense oligonucleotide inhibition of versi- Grant sponsor: NIH; Grant number: HD040846-02A1. *Correspondence to: Anthony A. Capehart, Department of Biology, East Carolina University, Greenville, NC 27858. Fax: 252-328-4178. E-mail: email@example.com Received 13 August 2007; Accepted 3 October 2007 DOI 10.1002/ar.20627 Published online in Wiley InterScience (www.interscience.wiley. com). 20 SHEPARD ET AL. Fig. 1. Immunohistochemical staining in parafﬁn sections through Hamburger and Hamilton (HH) stage 25 chick wings 36 hr after morpholino transfection. A,B: Immunostaining demonstrates versican proteoglycan expression is knocked down in targeted distal mesenchyme transfected with 1.0 mM versican morpholino (A, asterisk) as compared with the contralateral control wing (B). C–E: Versican expression is normal in the chondrogenic core transfected with 1.0 mM control morpholino (D) as compared with the contralateral control wing (E). D: Arrow indicates ﬂuorescein isothiocyanate (FITC) -tagged control morpholino (CMO) in the versican-positive limb core in C. Scale bars 5 250 mm in A,B, 250 mm in C–E. can in HH stage 24 (Hamburger and Hamilton, 1951) chick limb mesenchyme in vitro had no effect on mesenchymal aggregation and enhanced chondrogenesis in committed chondrocytes. These discrepancies in vitro underscore the need to evaluate versican function during chondrogenesis using an in vivo model. Strong versican expression has also been detected at the epiphyseal ends of long bones (Yamamura et al., 21 VERSICAN KNOCK-DOWN IMPAIRS CHONDROGENESIS TABLE 1. Viability of morpholino transfected embryos and resulting phenotypes Transfected morpholino Versican morpholino Control morpholino a Embryos electroporated (Hamburger and Hamilton stage 22–25) Embryos surviving (Hamburger and Hamilton stage 33–39) Embryos with impaired skeletogenesis (%)a 36 18 22 13 11 (50%) 0 (0%) Reduction in length of one or more skeletal elements in the targeted area. 1997; Shibata et al., 2003) and in the presumptive joint of mice (Snow et al., 2005), and although ectopic versican expression has been demonstrated to modulate chondrocyte morphology in vitro by means of changes in cytoskeletal structure (Zhang et al., 2001), little else is known about versican function in this regard and versican’s potential role during synovial joint morphogenesis is not yet known. In the present study, we used a morpholino-directed strategy to ascertain the importance of matrix versican during avian limb development in ovo thereby building on prior work with the versican null hdf mouse in vitro (Williams et al., 2005). Results suggest that speciﬁc morpholino-mediated misexpression of versican inhibited chondrogenesis in limb mesenchyme in ovo. Skeletal elements in the targeted area were shortened distally with retardation of ossiﬁcation while cartilages outside the target area developed normally. Furthermore, if the ulnar condensation was transfected proximally, the olecranon process was inadequately developed, likely affecting elbow joint morphogenesis. MATERIALS AND METHODS Embryos Normal, viable chick embryos were harvested between HH stage 22 and 39 of development following East Carolina University IACUC approved guidelines, placed in icecold phosphate buffered saline (PBS), and preserved in Dent’s ﬁxative (4:1 methanol:dimethylsulfoxide; Dent and Klymkowsky, 1987) overnight at 48C or 4% paraformaldehyde for 1 hr at room temperature. Immunohistochemical staining for versican was similar with both ﬁxatives. Two different ﬁxatives were used because, although Dent’s ﬁxed tissues displayed superior tissue morphology as compared to paraformaldehyde ﬁxation, Dent’s ﬁxative did not preserve morpholino ﬂuorescence. Fixed embryos were dehydrated through a graded series of ethanols, followed by xylene, and then embedded in parafﬁn. Transfection of Chick Limbs In Ovo Eggs were incubated in a humidiﬁed atmosphere at 37.58C until the appropriate stage. To assess the effect of versican knock-down on precartilage mesenchymal condensation, the precartilage limb core was targeted at HH st22. To examine versican’s role during later stages of limb skeletal template formation and joint morphogenesis, the presumptive ulna or distal limb mesenchyme was targeted at HH stage 24 and 25, respectively. In ovo transfection experiments were performed using ﬂuorescein-conjugated morpholino oligonucleotides (GeneTools, LLC). Morpholino solutions (1 mM in distilled deionized water [ddH2O]) were kept at 2208C for long- term storage, heated at 658C for 5 min, and then allowed to cool to room temperature before use. Morpholino was loaded into glass micropipettes by drawing up the solution from the tip of the needle while mounted in the microinjection apparatus. Eggs were windowed, and vitelline membranes were removed using tungsten needles. Gold-plated 3-mm electrodes (Model 514, Harvard Apparatus Inc.) of a square wave generator (BTX830, Harvard Apparatus Inc.) were placed 1–2 mm on either side of the limb bud ﬂanking the dorsoventral axis before microinjection. Appropriately staged embryos were injected with 0.15–0.25 ml of versican morpholino (VsMO; 50 -CAACATCTTGATCTTAAAAGGTAGC-0 3) or ‘‘standard’’ control morpholino (CMO; 50 -CCTCTTACCT CAGTTACAATTTATA-0 3) at 1.0 mM in ddH2O as regulated by pneumatic pump (Model 820, WPI). Microinjection was immediately followed by electroporation. Following extensive trials, in our experience, six 100-msec square wave pulses of between 12 and 15 V were found to be optimal electroporation parameters for embryonic survival and consistent results. Injections were targeted to the precartilage limb core or distal limb mesenchyme. The transfected area had an approximate diameter of 500 mm as ascertained by morpholino ﬂuorescence in parafﬁn embedded sections. To ensure consistency, VsMO and CMO transfection experiments were run in parallel. Electrodes were cleaned between injections, and the egg was sealed with a small piece of tape, placed back into the incubator, and permitted to develop until the desired stage before ﬁxation and staining. We used epiﬂuorescence optics to verify consistent morpholino uptake and knock-down of versican protein expression in parafﬁn-embedded sections of selected embryos 24–48 hr after electroporation. Immunohistochemistry Primary antibodies used included polyclonal rabbit anti-chick versican and aggrecan (Zanin et al., 1999) and monoclonal mouse anti-chick type II collagen antibody (II-II6B3; Developmental Studies Hybridoma Bank, Iowa City, IA). Rhodamine-conjugated peanut agglutinin (PNA; Vector labs) at 20 mg/ml was used to detect precartilage condensations (Zimmermann and Thies, 1984). Double labeling was routinely performed with antiversican antibody in combination with type II collagen antibody or PNA. Before processing for immunohistochemistry, deparafﬁnized sections were incubated with 0.1% testicular hyaluronidase (Sigma) for 30 min at 378C to remove potentially masking hyaluronan or chondroitin sulfate glycosaminoglycan residues. Subsequent immunohistochemical staining procedures were a modiﬁcation of Capehart et al. (1999). Brieﬂy, sections were blocked with PBS 22 SHEPARD ET AL. containing 3% bovine serum albumin and 1% goat serum for 1 hr and incubated with primary immunoreagents overnight at 48C. Sections were washed with PBS and incubated with ﬂuorescein- or rhodamine-conjugated anti-mouse or -rabbit IgG secondary antibodies (ICNCappel), diluted 1:200 in blocking buffer, for 2 hr at room temperature. Primary antibodies were omitted from control specimens. Sections were washed with PBS, post-ﬁxed in 80% and 50% ethanols, re-equilibrated in PBS, and mounted in 10% PBS-90% glycerol containing 100 mg/ml 1,4-diazabicyclo(2,2,2)octane (Sigma). Slides were viewed with an Olympus BX-40 equipped with epiﬂuorescence optics and images were captured using a SPOT-RT camera and software (Diagnostic Instruments). In Situ Detection of Apoptosis Terminal transferase dUTP nick end labeling (TUNEL) assay for detection of apoptotic cells on parafﬁn embedded sections was performed using the ApopTag Peroxidase In Situ Kit (Chemicon) following manufacturer’s instructions. Brieﬂy, samples were post-ﬁxed and endogenous peroxidase quenched with 0.1% hydrogen peroxide. Sections were equilibrated in terminal transferase buffer before addition of reaction buffer containing digoxigenin-11-dUTP followed by detection using diaminobenzidine substrate. Whole-Mount Alcian Blue Histochemistry Embryos were ﬁxed in 95% EtOH overnight at 48C and stained for 24 hr at room temperature in 0.02% Alcian blue in acidiﬁed ethanol (pH 1.0). Embryos were then rinsed twice in 95% EtOH for 1 hr and macerated in 2% potassium hydroxide (KOH) for 3–6 hr. Samples were stained with 0.01% Alizarin red in 1% KOH for 1 hr and cleared 1 hr each in sequential KOH:glycerol solutions of 80:20, 60:40, 40:60, 20:80, and stored in 100% glycerol (Kuczuk and Scott, 1984). Embryos were viewed with an Olympus SZ60 microscope and images captured using a SPOT-RT camera and software. Developing limb skeletal structures were identiﬁed according to Bellairs and Osmond (1998). RESULTS To examine the role of versican during chick limb skeletogenesis, we used an in ovo morpholino transfection strategy. Morpholinos targeted to the translational start site function by binding mRNA in the cytosol and blocking ribosomal access (Heasman, 2002). Fluoresceinconjugated VsMO complementary to bases 219 to 13 surrounding the starting methionine codon (positions 126–150, GenBank accession no. NM_204787) or ‘‘stand- Fig. 2. Parafﬁn sections through Hamburger and Hamilton (HH) stage 24 chick wings 24 hr after 1.0 mM versican morpholino (VsMO) transfection. A–C: Staining with rhodamine-PNA (A, used to detect precartilage mesenchymal condensation) indicates VsMO transfection (B) inhibited mesenchymal aggregation (asterisk) compared with contralateral control (C). B: Arrow indicates ﬂuorescein isothiocyanate (FITC) -tagged VsMO in the same section as A. Scale bar 5 250 mm for all panels. Fig. 3. Whole-mount Alcian blue and Alizarin red staining demonstrating effects of targeted versican knock-down on limb skeletal development in ovo. ‘‘VsMO’’ and ‘‘CMO’’ indicate limbs transfected with 1.0 mM versican and control morpholinos, respectively; ‘‘CLC’’ is contralateral control limb for all panels. A,B: Targeted versican knockdown at Hamburger and Hamilton (HH) stage 24 as indicated (F) resulted in shortened skeletal elements (arrows; A,B) and reduction in size of the radial carpal (arrowhead; B) as compared to respective contralateral control wings. C,D: Targeted versican knock-down in the distal limb mesenchyme at HH stage 25 as indicated (G) resulted in shortened skeletal elements (arrows; C,D), reduction in size of the radial carpal (arrowhead; D), and digit 2 (asterisk; C,D), and the second phalangeal element of digit 3 (asterisk, C) failed to form as compared to their contralateral control wings. E: Targeted transfection of distal limb mesenchyme with CMO at HH stage 25 as indicated (G) had no effect on limb skeletal development. F,G: Schematic representation showing orientation of the microinjection apparatus (blue arrow) and electrode placement (,) for limbs A,B and C,D respectively. Scale bar 5 1 mm for all ﬁgures. 24 SHEPARD ET AL. TABLE 2. Ulnar length in millimeters of embryos transfected in the presumptive ulna Embryo age (n) Versican Morpholino HHc stage 33 (3) HH stage 35 (1) HH stage 37 (2) HH stage 38 (1) Control Morpholino HH stage 33 (1) HH stage 35 (1) HH stage 37 (2) HH stage 38 (2) HH stage 39 (1) Number of embryos exhibiting impaired joint morphogenesisb Contralateral control wing Morpholino transfected wing % Reduction 3 4 5.4 6 0.1 6 1.75 6 0.25 1.5 2.25 1.85 42 62 58 69 1 0 2 1 3 4 5.5 6 7 0 0 0 0 0 0 0 0 0 0 3 4 5.5 6 7 a a % Reduction represents the relative difference in length of the transfected ulna compared to its corresponding contralateral control. b Reduction in the size of the olecranon process. c HH, Hamburger and Hamilton. ard’’ CMO targeted to a human beta globin mutation were microinjected and electroporated into HH stage 22–25 chick wing mesenchyme in ovo. Thirty-ﬁve viable embryos from 12 microinjection experiments were evaluated from experimental and control groups to determine the most consistent results. Following transfection, VsMO, but not CMO, selectively knocked down versican expression in targeted distal regions of chick limb mesenchyme (Fig. 1). Overall viability of transfected embryos was approximately 60– 70% (Table 1), with VsMO and CMO groups sharing similar survival rates. At HH stage 24 speciﬁc reduction of rhodamine-conjugated PNA labeling in targeted limb core regions indicated that precartilage mesenchymal condensation was noticeably impaired by transfection with VsMO at HH stage 22 in ovo as compared to contralateral control limbs (Fig. 2). To examine effects of versican knock-down on later stages of limb skeletogenesis, transfection experiments were conducted using HH stage 24–25 wing mesenchyme in ovo. Alcian blue and Alizarin red histochemistry indicated that cartilage elements transfected with VsMO were abnormally short with retardation of Alizarin red positive ossiﬁcation (Fig. 3A,B). By HH stage 33 to 37, ulnar cartilages transfected with VsMO were reduced in length by an average of 53% compared with contralateral control wings (Table 2), whereas skeletal elements transfected with CMO were indistinguishable from contralateral controls (Figs. 3E, 4D). In limbs transfected with VsMO, ulnar phenotypes ranged from moderate reduction in length (less than 50%) to more severe reduction in length (60% or greater) as compared to contralateral control limbs. This is likely a result of slight variations in microinjection location and differential morpholino uptake between embryos handled in a similar manner. Targeting the distal limb mesenchyme also resulted in shortened, occasionally broader skeletal elements accompanied by the loss of distal phalangeal structure (Fig. 3C,D). Targeting the ulnar condensation commonly resulted in a ‘‘wrist drop’’ phenotype characterized by the length of the radius exceeding that of the ulna, causing the distal end of the radius to curve toward the ulna near the carpometacarpus (Figs. 3A,B, 4A–C). As shown in Table 2, 57% of VsMO trans- fected embryos exhibited reduced ulnar length and also a deﬁciency in formation of the olecranon process (Fig. 4A– C). In these specimens, the olecranon process appeared ﬂattened and failed to cup the humerus, likely impairing joint morphogenesis. TUNEL assay of parafﬁn-embedded limb sections was performed to examine whether electroporation, morpholino uptake, or versican knock-down increased mesenchymal apoptosis. At HH stage 25, TUNEL analysis showed only low numbers of apoptotic cells 36 hr after transfection that were similar between wings transfected with VsMO and respective contralateral controls (not shown). DISCUSSION Aggregation of mesenchymal cells is critical to chondrogenesis in the developing limb (Hall and Miyake, 1992, 2000) and versican proteoglycan is expressed strongly by condensing mesenchyme. However, in vitro experimentation has produced conﬂicting data regarding versican’s role in this process (Zhang et al., 1998a; Williams et al., 2005; Kamiya et al., 2006). In the present study, we examined the role of versican during chick limb skeletogenesis by using a morpholino directed knock-down strategy in ovo. Versican has been demonstrated to be highly expressed by limb mesenchymal cells undergoing condensation (Kimata et al., 1986; Shinomura et al., 1990) and strong versican expression is maintained in those cells that subsequently participate in synovial joint morphogenesis (Snow et al., 2005), suggesting potential roles for versican in these processes. Our results show that misexpression of versican proteoglycan inhibits mesenchymal condensation in ovo and also suggest that versican proteoglycan may contribute to formation of cartilage structures integral to synovial joint morphogenesis. Morpholinos have been used widely for targeted lossof-function experiments (Muramatsu et al., 1997; Ogino and Yasuda, 1998; Sakamoto et al., 2000; Yasuda et al., 2000; Yasugi and Nakamura, 2000; Tucker, 2001, 2002, 2004; Kos et al., 2003; Luo and Redies, 2005) and permitted us to circumvent the early embryonic lethality of the versican null hdf mouse, which occurs before skele- VERSICAN KNOCK-DOWN IMPAIRS CHONDROGENESIS Fig. 4. Whole-mount Alcian blue and Alizarin red staining demonstrating the effects of targeted versican knock-down on synovial joint morphogenesis in ovo. ‘‘VsMO’’ and ‘‘CMO’’ indicate limbs transfected with 1.0 mM versican and control morpholinos, respectively; ‘‘CLC’’ is contralateral control limb for all panels. A–C: Targeted in ovo versican knock-down at Hamburger and Hamilton (HH) stage 24 as indicated (E) resulted in shortened skeletal elements (arrows; A–C) and reduction in size of the olecranon process (arrowhead; A–C) as compared to respective contralateral control wings. Higher magniﬁcation inset of 25 elbow joint illustrating alteration of the olecranon process (arrowhead) in a VsMO-treated sample is shown in B. In severe cases, orientation of the limb was altered by the extent to which the radial cartilage curved toward the ulnar cartilage at the carpometacarpus (1; A). D: Targeted in ovo transfection of limb mesenchyme with CMO at HH stage 24 as indicated (E) had no effect on limb skeletal development. E: Schematic representation showing orientation of the microinjection apparatus (blue arrow) and electrode placement (, ) for all limbs. Scale bars 5 1 mm in A,B,D, 1 mm in C. 26 SHEPARD ET AL. togenesis (Williams et al., 2005). While morpholinos are not readily taken up through the plasma membrane, when electrophoresed, they will migrate a short distance toward the positive pole (Kos et al., 2003) as a result of the negatively charged ﬂuorescein tag. This slight charge has been shown sufﬁcient to drive morpholino uptake into cells into postgastrula stage embryos (Tucker, 2001, 2002, 2004; Kos et al., 2003; Luo and Redies, 2005). Electroporation parameters also affect morpholino transfection efﬁciency, thus we systematically varied electroporation conditions to achieve optimal parameters for embryonic survival and consistent results (six 100msec square wave pulses of between 12–15 V). Furthermore, other critical factors such as morpholino concentration, rapid dilution due to diffusion, and slight changes in positioning of electrodes may result in uneven morpholino uptake. Thus, as reported previously (Kos et al., 2003), we analyzed a large number of embryos and routinely veriﬁed morpholino uptake by ﬂuorescence optics. While concentrations of 500 mM have been reported to work well for microinjection and electroporation in embryonic areas with a lumen, such as neural tube (Kos et al., 2003; Tucker, 2002, 2004), we found this to be too low for efﬁcient transfection of limb mesenchyme due to rapid diffusion from the injection site. We used several morpholino concentrations and found that 1.0 mM was the lowest at which we were able to achieve consistent results. To rule out the possibility that this relatively high concentration was resulting in nonspeciﬁc defects, we tested the ‘‘standard’’ control morpholino (CMO) at the same concentration and found no observable difference between the injected limb and noninjected contralateral limb. This ﬁnding is similar to reports from Tucker (2002) and Kos et al. (2003) in which concentrations of CMO (0.1–1.0 mM) had no observable effect on neural tube development. The mechanism whereby versican may enhance mesenchymal cell condensation is poorly understood. Versican’s C-terminus bears a striking similarity to adhesive proteins and this combined with the hyaluronan (HA) binding element at its N-terminus suggest that versican could provide a mechanism to stabilize HA in close association with the cell membrane (Zimmermann and Ruoslahti, 1989) forming a HA/versican pericellular matrix around the cell that may affect adhesive and migratory capabilities. Previous in vitro studies (Williams et al., 2005; Kamiya et al., 2006) have reported that versican may function to create an extracellular environment conducive to mesenchymal condensation. Our results are consistent with this hypothesis, and in some embryos, reduction in ulnar length in VsMO-targeted limb regions is likely a result of impaired mesenchymal condensation alone. This ﬁnding is evident in limbs in which the proximal aspects of the ulna developed normally, yet the ulna appeared to terminate in a point distally (Fig. 3), suggesting transfection of a slightly more distal population of cells. Inhibition of mesenchymal condensation may also have resulted in reduction in size of the radial carpal and loss of distal phalangeal elements. Such may not be the case, however, for the observed elbow joint defects as mesenchymal condensation had already been initiated in the more proximal areas of the ulna at the time of transfection. It is possible that an ECM rich in versican proteoglycan may provide condens- ing mesenchymal cells and immature chondrocytes with a supporting environment for continued proliferation, thus facilitating the expansion of the chondrogenic rod into structures such as the olecranon process. Versican is highly expressed in the early cartilage comprising the olecranon process (Shepard et al., 2007) and our results are consistent with the hypothesis that inadequate development of the olecranon process as a result of versican misexpression may perhaps be attributable to reduced proliferation by cells committed to the chondrocyte lineage at the time of transfection. Versican expression is often observed in proliferative cells and strong versican expression has been detected in the presumptive joint (Snow et al., 2005; Shepard et al., 2007) and at the epiphyseal ends of long bones (Yamamura et al., 1997; Shibata et al., 2003). The G3 domain at the versican C-terminus consists of a C-type lectin adjacent to two EGF domains and a complement regulatory region (Shinomura et al., 1993), and versican participation in growth regulation has been shown by enhanced cell proliferation in ﬁbroblasts expressing an exogenous G3 domain (Zhang et al., 1998b) and versican G3 activation of EGF receptors (Xiang et al., 2006). The current study is in agreement with the prior in vitro work of Williams et al. (2005) and Kamiya et al. (2006) and provides further support for versican in facilitation of early chondrogenesis and, to our knowledge, shows for the ﬁrst time that versican knock-down inhibited mesenchymal condensation resulting in a limb phenotype in ovo. Furthermore, these data also extend previous ﬁndings to suggest a role for versican during synovial joint morphogenesis in the developing chick limb. LITERATURE CITED Bellairs R, Osmond M. 1998. The atlas of chick development. San Diego: Academic Press. 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