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Versican Knock-Down Compromises Chondrogenesis in the Embryonic Chick Limb.

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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: capehartt@ecu.edu
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 ([2],[1]) 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 ([2], [1]) 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.
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