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Versican G1 Domain and V3 Isoform Overexpression Results in Increased Chondrogenesis in the Developing Chick Limb In Ovo.

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THE ANATOMICAL RECORD 293:1669–1678 (2010)
Versican G1 Domain and V3 Isoform
Overexpression Results in Increased
Chondrogenesis in the Developing Chick
Limb In Ovo
KARLA S. HUDSON,1 KRISTEN ANDREWS,1 JUNE EARLY,1 COREY H.
MJAATVEDT,2 AND ANTHONY A. CAPEHART1
1
Department of Biology, East Carolina University, Greenville, North Carolina
2
Department of Cell Biology and Anatomy, Medical University of South Carolina,
Charleston, South Carolina
ABSTRACT
Previous work has shown that versican proteoglycan is highly
expressed in the extracellular matrix of precartilage limb mesenchyme.
Although much of versican’s role in chondrogenesis has been attributed
to its glycosaminoglycan complement, N- and C-terminal G1 and G3
domains of versican have been shown to possess distinct functions when
expressed ectopically. This study was undertaken to test the hypothesis
that overexpression of the versican G1 domain and short V3 isoform, comprised of only G1 and G3, in the chick wing in ovo would result in
increased chondrogenesis, suggesting function for discrete versican
domains in limb skeletal development. Recombinant adenoviruses encoding G1 and V3 proteins were microinjected into proximal HH19–25 chick
wing buds which resulted in significant enlargement of humeral primordia at HH35. Enhanced cartilage deposition appeared due to increased
chondrogenic aggregation as a result of recombinant G1 or V3 overexpression, further implicating versican in early stages of limb development.
C 2010 Wiley-Liss, Inc.
Anat Rec, 293:1669–1678, 2010. V
Key words: versican; limb development; chondrogenesis;
extracellular matrix; chick embryo
INTRODUCTION
Limb chondrogenesis and patterning of the appendicular skeleton are multistep processes in which each phase
involves a complex array of cellular interactions. The
first stage of overt chondrogenesis involves formation of
precartilage mesenchymal condensations, a pivotal step
(Fell, 1925; Mackie et al., 1987; Daniels and Solursh,
1991; Hall and Miyake, 1995, 2000; Olsen et al., 2000).
Mesenchymal condensations are regulated and formed
by cell–cell and cell–extracellular matrix (ECM) interactions (Mackie et al., 1987; Knudson et al., 1996) coupled
with cell movement and mitotic activity (DeLise et al.,
2000; Hall and Miyake, 2000; Barna and Niswander,
2007). ECM molecules implicated in initiation of cellular
aggregation include collagen type I (Dessau et al., 1980),
hyaluronan (Knudson, 2003), fibronectin (Kulyk et al.,
1989; Downie and Newman, 1995; Chimal-Monroy and
Diaz, 1999), tenascin (Hall and Miyake, 2000), and synC 2010 WILEY-LISS, INC.
V
decan proteoglycan (Shimizu et al., 2007). Cooperative
action of ECM constituents in early stages of aggregation may lead to cross-bridging of cells via N-cadherin
(Shum et al., 2003; Shimizu et al., 2007) and N-CAM
(Chimal-Monroy and Diaz, 1999; Hall and Miyake, 2000;
Shimizu et al., 2007) which triggers chondrogenesis.
Versican is a predominant chondroitin sulfate proteoglycan in the prechondrogenic limb that has also been
shown important during early stages of chondrogenesis
Grant sponsor: NIH; Grant number: HD040846.
*Correspondence to: Anthony A. Capehart, Department of
Biology, East Carolina University, Greenville, NC 27858. Fax:
328-4178. E-mail: capehartt@ecu.edu
Received 22 March 2010; Accepted 2 July 2010
DOI 10.1002/ar.21235
Published online 20 August 2010 in Wiley Online Library
(wileyonlinelibrary.com).
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HUDSON ET AL.
(Zhang et al., 2001; Williams et al., 2005; Kamiya et al.,
2006; Shepard et al., 2008) and is later restricted to the
perichondrium and epiphysis of developing cartilages
(Shinomura et al., 1990; Yamamura et al., 1997; Shibata
et al., 2003; Snow et al., 2005; Shepard et al., 2007).
Chondrogenesis fails to occur in limb mesenchyme or in
cartilage precursor cells that lack versican in vitro (Williams et al., 2005; Kamiya et al., 2006). Furthermore,
versican knockdown resulted in targeted inhibition of
chondrogenesis of the embryonic chick limb in ovo (Shepard et al., 2008).
As with other members of the hyalectin family, the
core protein of versican is modular with amino- and carboxy-terminal G1 and G3 domains separated by chondroitin sulfate glycosaminoglycan attachment regions
(GAG-a and –b). Alternative splicing of the GAG-a and
-b domains yields four isoforms, termed V0-V3 that may
regulate cellular behavior (Shinomura et al., 1993). V0
possesses both GAG attachment regions, while V1 contains only GAG-b, V2 contains GAG-a, and V3 has neither GAG attachment sequence (Zimmermann and
Rouslahti, 1989; Zako et al., 1995). All four variants
express the G1 hyaluronan-binding domain and G3 carboxy terminal domain consisting of a C-type lectin-like
domain, two epidermal growth factor (EGF)-like repeats,
and complement regulatory domain (Shinomura et al.,
1993).
Versican has been implicated in developmental processes such as eliciting cell shape changes, regulation of
migration, and proliferation (Wight, 2002) and loss of
mature versican results in an embryonic lethal phenotype (hdf mutant) due to defects in cardiac morphogenesis (Mjaatvedt et al., 1998). In addition to the
hyaluronan-binding G1 domain, the G3 domain interacts
with fibronectin, fibrillin, fibulin, collagen type I, as well
as tenascin and may also interact directly with EGF
receptors (Yamagata et al., 1986; LeBaron et al., 1992;
Aspberg et al., 1995; Zhang et al., 1998; Isogai et al.,
2002). Versican’s chondroitin sulfate chains also bind
CD44 and L- and P-selectins (Kawashima et al., 2001;
Kamiya et al., 2006). V0 and V1 isoforms are expressed
in heart and blood vessels where cell shape is important
(Landolt et al., 1995) and also in vitro during early
stages of chondrogenesis when cell shape changes are
occurring (Kamiya et al., 2006). On the other hand, V2
is expressed in the brain (Schmalfeldt et al., 1998) and
at later stages of chondrogenesis where cell shape is
more stable (Kamiya et al., 2006). Furthermore, V2 promotes cell elongation and subsequent inhibition of chondrogenesis in vitro, whereas V1 promotes rounding
(Sheng et al., 2006). Ectopic expression of the V3 isoform
in arterial smooth muscle increases cell adhesion resulting in decreased growth and migration (Lemire et al.,
2002). V3 overexpression in the embryonic cardiac outflow tract also caused thickening of the myocardium as a
result of increased adhesion among cardiomyocytes
(Kern et al., 2007).
Interestingly, proteolytic cleavage of versican liberates
domains that may regulate its function in some tissues
(Sandy et al., 2001; Russell et al., 2003; Kern et al.,
2006, 2007; Capehart, 2010). Indeed, increasing evidence
has shown that individual domains of versican may
function independently of the intact proteoglycan when
expressed ectopically (Zhang et al., 1998, 2001; Ang
et al., 1999; Yang et al., 1999; Kern et al., 2007). This is
especially important when taken with the fact that individual versican domains have been reported to possess
differing activities. Ectopic expression of the G3 domain
in vitro promotes elongation of chondrocytes, inhibiting
chondrogenesis through its EGF domain (Zhang et al.,
2001). On the other hand, expression of the G1 domain
facilitates cell rounding by reducing cell–substrate interactions by binding to hyaluronan (Daniels and Solursh,
1991; Kohda et al., 1996; Toole, 2001). Versican/hyaluronan complexes create a loose highly hydrated environment with a destabilizing effect on adhesion that may
influence cell migration (Yamagata et al., 1986; Landolt
et al., 1995; Lee et al., 1999; Yang et al., 1999). In other
cases, however, the activity of different versican domains
may be complementary. Ectopic G1 expression in vitro
enhances cellular proliferation (Zhang et al., 1998, 1999)
through reduction in cell adhesion via hyaluronan binding (Ang et al., 1999). The G3 domain also stimulates
cellular proliferation via its EGF domains (Zhang et al.,
1998) and has been shown to protect against apoptosis
(Wu et al., 2002). Thus, in vitro data suggest that versican domains have diverse functions leaving open the
question regarding effects that individual domains of
versican may have during limb chondrogenesis in vivo.
In the present study, this led us to ask if individual versican domains are able to function independently of the
mature proteoglycan during chick limb morphogenesis
in ovo. As G1 and G3 domains are found in all versican
isoforms we reasoned that their use would provide valuable insight into versican function. This is also particularly interesting because little is known regarding the
V3 isoform with respect to its expression or potential
role in limb skeletogenesis. Because full length versican
has previously been implicated in precartilage mesenchymal condensation in vitro (Williams et al., 2005;
Kamiya et al., 2006) and deficits (Shepard et al., 2008)
or delay (Choocheep et al., in press) in formation in limb
cartilages have been reported through loss of versican in
vivo, we hypothesized that expression of individual G1
domains and V3 isoform, containing only G1 and G3,
would facilitate limb chondrogenesis in vivo. Recombinant adenoviruses encoding G1 and V3 versican were
utilized in these studies as viral-mediated constitutive
expression has provided a reliable and powerful tool to
investigate protein function in several model organisms,
including the developing chick (Yajima et al., 2002; Kern
et al., 2007; Li et al., 2007). Our results demonstrate
that expression of recombinant versican G1 domain and
V3 variant in the chick wing in ovo led to additional
chondrogenic aggregation resulting in enlargement of
cartilage at sites along the humeral primordium.
MATERIALS AND METHODS
Recombinant Adenoviruses
Recombinant adenoviruses expressing a C-terminal
hemaglutinin (HA)-tagged G1 domain and V3 isoform of
murine versican were prepared using the Adeno-X vector system (Clontech) as described previously by Kern
et al. (2007). Control viral constructs expressing b-galactosidase contained the same cytomegalovirus promoter
and viral backbone. Recombinant adenoviruses were
amplified in low passage HEK 293 cells with virions
purified and titered using Adeno-X virus Purification
and Rapid Titer kits (Clontech) according to
RECOMBINANT VERSICAN G1 AND V3 EXPRESSION
manufacturer’s instructions. Immunohistochemistry and
Western analysis of lysates prepared from transfected
HEK 293 cells using an anti-hemaglutinin tag antibody
(HA-7; Sigma) were used to confirm expression of proteins with the predicted molecular weights of 40 and
70 kDa, respectively, for recombinant G1 and V3 versican proteins (Kern et al., 2007).
Microinjection of Recombinant Adenoviruses
Fertilized, viral-free White Leghorn chick eggs (SPF;
Charles River) were incubated at 37.5 C in a humidified
egg incubator until Hamburger and Hamilton (HH)
stages 19–25 (Hamburger and Hamilton, 1951). Eggs
were ‘‘windowed,’’ overlying membranes removed, and
right wing buds of appropriately staged, viable embryos
microinjected with recombinant G1, V3, LacZ adenoviruses, or with adenoviral vehicle alone. Viral microinjections targeting the proximal limb core were performed
utilizing a pneumatic pump (Model 820, WPI). G1 and
V3 adenoviruses were injected at a 1:1 ratio with LacZencoding virus to track and localize the area of viral
infection (Kern et al., 2007). Injections were performed
by inserting the microinjection capillary needle at a 45to 50-degree angle along the proximodistal axis just
proximal to the center of the wing bud at HH19-23 and
midway between the trunk and presumptive elbow joint
at HH24-25. Each microinjection delivered 1.5 106
ifu of G1 or V3 adenoviruses (G1/V3 titers, 2 109 ifu/
mL) in a total volume of 1.5 lL (0.75 lL G1 or V3:0.75
lL LacZ). LacZ control viral injections (6 109 ifu/mL)
were performed using the same total volume. Following
microinjection, eggs were sealed with tape, returned to
the incubator and allowed to develop up until HH35.
Embryos assessed in this study received a nonlethal
injection of viruses as defined by survival beyond 48 hr
postinfection (Kern et al., 2007). Experimental and control embryos were subject to identical handling and
treatment. Findings represented here are based on analysis of a minimum of 30 experimental embryos for each
of the recombinant G1, V3, and LacZ adenoviral injection series.
Whole-mount Histochemical Staining
Alcian blue and alizarin red histochemistry followed a
modification of Kuczuk and Scott (1984). Embryos were
fixed overnight in 95% ethanol, followed by staining
overnight in 0.02% alcian blue in acidified ethanol (pH
1.0) at room temperature. After washing in 95% ethanol
embryos were macerated in 2% potassium hydroxide
(KOH) until tissue was translucent and skeletal elements were clearly visible (3 hr). Overnight staining in
0.01% alizarin red in 1% KOH was followed by a series
of 1 hr KOH:glycerol exchanges (80:20, 60:40, 40:60,
20:80%). Embryos were stored in 70% glycerol:30% phosphate-buffered saline (PBS).
b-galactosidase staining protocol was adapted from
Kern et al. (2007). Briefly, embryos were fixed on ice in
4% paraformaldehyde for 30 min, rinsed with PBS, and
washed in 0.02% sodium deoxycolate and 0.01% Tergitoltype NP-40 in PBS. Specimens were stained in 0.1% Xgal in 0.02% magnesium chloride, 0.1% potassium-ferrocyanide, 0.1% potassium-ferricyanide at 37 C, washed in
1671
PBS, post fixed in 4% paraformaldehyde, and stored at
4 C.
Statistical Analysis
Measurements of three humeral sites were taken (Fig.
2F): across the length of the ventral tuberculum (proximal), at the center of the humeral primordium (mid),
and from the dorsal to ventral condyle (distal). Anatomical structures were identified according to Bellairs and
Osmond (1998). Examination of data included calculation of means, standard deviations and P-values for
proximal, mid and distal regions of injected limbs and
uninjected contralateral controls using mean matchedpairs analysis and t-test (Triola, 2005). Mean value of
differences between injected and uninjected contralateral
control limbs of individual embryos at specific humeral
sites was used to test the following null hypothesis: no
differences between humeral elements of the injected
and uninjected contralateral control. Statistical significance was set at P < 0.05 with analyses performed using
STATDISK software (Triola, 2005).
Immuno- and Histochemical Staining of Tissue
Sections
Mouse anti-hemaglutinin (HA-7, Sigma) was used at
3.3lg/mL to detect recombinant G1 and V3 proteins and
rabbit antiphosphohistone H3 (Ser10) (Cell Signaling
Technology) at 1:100 as recommended by the manufacturer to identify mitotic cells Rhodamine-conjugated peanut agglutinin (PNA; Vector Labs) was used at 30 lg/mL
to detect precartilage mesenchymal aggregates (Zimmerman and Thies, 1984) and biotinlylated hyaluronic acid
binding protein (HABP; Associates of Cape Cod) at 2.5
lg/mL to localize endogenous hyaluronan. Double labeling was routinely performed with the hemaglutinin tag
antibody and other reagents when possible.
Paraformaldehyde-fixed specimens were paraffin embedded, sectioned at 7 lm, dewaxed, and subjected to
antigen unmasking based on a high-temperature citric
acid formula (Vector Labs) for 20 min (Snow et al.,
2005). To remove potentially masking hyaluronan or
chondroitin sulfate glycosaminoglycan residues sections
were incubated with 0.1% testicular hyaluronidase
(Sigma) in 30 mM sodium acetate, pH 5.2, 125 mM sodium chloride for 30 min at room temperature (Capehart
et al., 1999). Sections receiving HABP were incubated in
0.25 U/mL chondroitinase ABC (Sigma) in 50 mM Tris,
pH8.0, 60 mM sodium acetate with 0.1% bovine serum
albumen (BSA; Sigma). Sections were blocked with 3%
BSA, 1% goat serum in PBS 1 hour and incubated with
primary immunoreagents overnight at 4 C. Sections
were washed with PBS and incubated with FITC (2 lg/
mL)- or rhodamine (6 lg/mL)-conjugated goat antimouse and -rabbit IgG secondary antibodies (Cappel) for
1–2 hr at room temperature. FITC-strepavidin (Vector
Labs) was used at 5 lg/mL. Primary antibodies were
omitted from control specimens. Sections were washed
with PBS, postfixed in 80% and 50% ethanol, re-equilibrated in PBS, and mounted in DABCO anti-fading
agent [10% PBS-90% glycerol containing 100 mg/mL 1,4diazabicycle(2,2,2)octane; Sigma].
1672
HUDSON ET AL.
from HH25 chick heart cDNA for ligation into pGEMTEasy (Promega). Digoxygenin-labeled RNA anti-sense
and sense probes generated with the DIG RNA Labeling
kit (Roche) according to manufacturer’s instructions.
RESULTS
Fig. 1. Expression of endogenous versican V3 isoform transcripts
in the chick limb. A: RT-PCR at Hamburger and Hamilton (HH) stages
25, 28, 34 shows the predicted 313 bp amplicon in the developing
limb and heart. Heart cDNA was used to generate riboprobes for in
situ hybridization. Position of the 298 base pair standard is marked by
arrowhead and actin loading control for each lane at the bottom. B: In
situ hybridization with antisense probe shows low level expression of
V3 mRNA in the wing core at HH25 (asterisk). C: Sense control probe
in an adjacent section shows lack of signal in the limb core. Note
staining in the dorsal mesenchyme underlying the ectoderm represents nonspecific signal in panels (B) and (C).
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 postfixed 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
antidigoxigenin and diaminobenzidine substrate.
Detection of Endogenous Versican V3 Variant
mRNA in Developing Limbs
Reverse transcriptase-polymerase chain reaction (RTPCR) was performed to detect endogenous V3 transcripts
between HH25–34. RNA was isolated using RNaqueous
4-PCR (Applied Biosystems, Foster City, CA) and cDNA
prepared with Superscript III (Invitrogen, Carlsbad, CA)
kits. Primers used to amplify chick V3 cDNA were:
and
1447F-50 -TACCTCAGACTGTCACAGATGG-30
10067R-50 -ATACATGTGGCTCCATTGCGGCA-30 . Primer
sequences were derived from the 30 end of chick versican
‘‘Plus’’ and 50 end of G3 domains (Zako et al., 1995) to
yield a predicted amplicon of 313 base pairs.
In situ hybridization was performed to localize endogenous V3 transcripts following Cronin and Capehart
(2007). The V3 primer set was used to prepare inserts
Although much is known regarding versican expression, particularly of V0/V1 isoforms, during chick limb
skeletogenesis (Kimata et al., 1986; Shinomura et al.,
1990; Landolt et al., 1995; Shepard et al., 2007), little information is available on V3 (comprised of only G1 and
G3 domains) expression during developmental stages
relevant to the present study. As such, RT-PCR and in
situ hybridization were utilized to detect endogenous V3
transcripts in the developing chick limb. RT-PCR showed
expression of the predicted 313bp V3 amplicon in the
wing between HH25-34 (Fig. 1A) and in situ hybridization localized low levels of V3 transcripts in chondrogenic areas of the limb core (Fig. 1B) that coincided with
expression of endogenous full-length versican protein as
described previously (Shinomura et al., 1990; Landolt
et al., 1995; Shepard et al., 2007).
Expression of recombinant versican G1 and V3 variant
were selected for use in this study because all four versican isoforms contain G1 and G3 and differ in inclusion
or deletion of the GAG-a and GAG-b chondroitin sulfate
attachment regions. We did not attempt to express
adenoviral constructs containing all or part of GAG-a or
GAG-b domains as there was no certainty that chondroitin sulfate glycosylation would occur appropriately,
potentially
making
experimental
interpretation
extremely difficult.
To investigate whether the versican G1 domain or
short V3 isoform could function independently during
limb morphogenesis and/or if one molecule was particularly critical for versican function, recombinant adenoviral constructs encoding these polypeptides were
microinjected into the developing chick limb bud at
HH19-25. Adenoviral titers of 2 103 ifu were reported
to work well for functional studies of G1 and V3 effects
on cardiac outflow tract development in which recombinant adenoviruses were microinjected into the anterior
heart field (Kern et al., 2007), but in the present study
we found this too low for efficient infection of limb mesenchyme due to rapid diffusion from the injection site.
We systematically varied viral injection volume in order
to determine the lowest titer needed to maintain embryonic viability and still obtain a consistent phenotype and
thus used G1 and V3 adenoviruses at 1.5 106 ifu in
combination with control LacZ encoding viruses in a
total volume of 1.5 lL. In control specimens, we routinely injected the LacZ encoding adenovirus of higher
titer and at the same volume without effect on the embryonic limb to rule out possible non-specific viral
effects.
A minimum of 30 embryos from at least six separate
experiments for each experimental and control group
were evaluated for each treatment to validate the resulting phenotype (Table 1). Adenoviral-mediated G1 and V3
overexpression resulted in statistically significant peripheral expansion of humeral primordia at HH35 (Table
2) due to injections into the proximal limb bud at prechondrogenic and early chondrogenic stages. In some
specimens in which the microinjection needle was
1673
RECOMBINANT VERSICAN G1 AND V3 EXPRESSION
withdrawn and reinserted into the limb during the same
experiment, slight movement of injection site resulted in
cartilage enlargement at different sites along the same
humeral element. Two different phases of limb development were used for injection of recombinant adenoviruses, precartilage mesenchyme (HH19-21) and
committed chondrogenic tissue (HH22-25). Injections
during both intervals resulted in similar increases in
skeletal anlagen thickness suggesting that overexpression of versican domains can have an effect on skeletal
development even after mesenchymal cells have committed to chondrogenic fate.
Alcian blue and alizarin red whole mount histochemistry
were used to evaluate gross morphological changes in the
skeletal template. As shown in Fig. 2, at HH35 there was
significant enlargement in the proximal humeral primordia
of wings overexpressing the G1 domain or along its length,
particularly toward the distal end (Fig. 2A and B; Table 2).
Proximal and mid regions of limbs overexpressing the V3
isoform also exhibited areas of enlarged humeral growth
(Fig. 2C and D; Table 2) with evidence of localized enlargement of cartilage first observed as early as HH31. Limbs
TABLE 1. Viability and resulting phenotypes of
embryos (HH35) injected with experimental G1, V3,
and control LacZ adenoviruses
Embryos with
Percentage of
increased
Adenovirus
Number
thickness of embryos resulting
in phenotype
and injection of embryos
skeletal
at HH35 (%)b
Stage
injected (n) anlagen (n)a
G1
HH19-21
HH22-25
V3
HH19-21
HH22-25
LacZ Control
HH19-21
HH22-25
12
22
4
6
33
27
15
15
4
3
26
20
18
14
0
0
0
0
a
Embryos included only if contributed to a statistically significant phenotype.
b
Phenotype defined as increase in thickness of skeletal anlagen at HH35 of proximal, mid or distal humerus.
injected with the b-galacotsidase-encoding control adenovirus were indistinguishable from respective uninjected contralateral controls (Fig. 2E). On average, V3-injected
embryos had slightly larger increases in thickness of the
skeletal anlagen but when taken in consideration relative
to the contralateral control measurements this may be
attributed to the variation of overall thickness of the skeletal template among individual embryos. Proximal humeral
phenotypes ranged from moderate (G1-26%, V3-22%) to
more dramatic expansion (G1-130%, V3-88%) as compared
with contralateral control limbs and to LacZ-adenovirus
injected limbs which showed an insignificant enlargement
of 2%–8%. A similar pattern in extent of humeral expansion
was consistent among sites. This variation in humeral
enlargement is likely a result of slight variations in adenoviral uptake by limb mesenchyme in individual embryos
which were handled in a similar manner.
Ectopic expression of recombinant G1 and V3 proteins
in areas where increases of thickness were observed was
validated by immunolocalizing an antibody directed
against the C-terminal hemaglutinin tag of both G1 and
V3 constructs in HH35 limb sections (Fig. 3). Whole
mount b-galactosidase staining was performed in order to
facilitate localization of limb areas with viral infection
prior to embedding/sectioning and subsequent antibody
labeling (Fig. 3A and C). b-galactosidase staining has previously been demonstrated to provide reliable indication
of adenoviral infection when co-injected with experimental constructs (Kern et al., 2007). In the present study,
location of b-galactosidase reactivity and hemaglutinin
tag-positive adenoviral expression consistently overlapped and LacZ staining also found to be a reliable
marker of infection sites (Fig. 3). Expression of recombinant G1 and V3 proteins correlated with areas of
increased cartilage deposition in mid-(G1, Fig. 3B) and
proximal-(V3, Fig. 3D) humeral anlagen observed in
whole mount alcian blue stained embryos.
To suggest a possible mechanism for observed increases
in chondrogenesis in areas expressing recombinant G1
and V3 proteins several parameters were evaluated,
including increased mitosis, decreased apoptosis, as well
as changes in hyaluronan acculmulation and/or cellular
aggregation. Phosphohistone H3 antibody-labeling was
used to visualize mitotic cells in sections from G1 or V3/
LacZ adenoviral-infected limbs at HH21 to determine
TABLE 2. Humeral measurements of limbs injected with G1, V3, or LacZ adenoviruses at HH19-25 and
corresponding contralateral control (CLC) limbs (mean 6 SD in mm)
Adenovirus
Humeral area (n)
Injected
CLC
P-value
Proximal (6)
Mid (5)
Distal (4)
0.66 0.14
0.42 0.10
0.93 0.33
0.45 0.14
0.35 0.06
0.83 0.34
0.008
0.024
0.0302
Proximal (6)
Mid (4)
Distal (3)
0.86 0.22
0.49 0.07
0.86 0.22
0.61 0.19
0.45 0.07
0.57 0.19
0.002
0.018
NS
Proximal (16)
Mid (16)
Distal (16)
0.61 0.17
0.48 0.04
0.62 0.10
0.62 0.16
0.47 0.04
0.62 0.10
NS
NS
NS
G1
V3
LacZ
Data represent mean of individual wings measured at each humeral area (n) exhibiting cartilage enlargement from 6 different experiments. Position of humeral measurements is shown in Figure 2F. Embryos (Table 1) in some instances exhibited humeral enlargement at 2 sites. In LacZ control experiments wings were measured at each of the humeral sites.
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HUDSON ET AL.
Fig. 2. Whole-mount alcian blue and alizarin red staining of the
HH35 wing demonstrating effects of recombinant versican G1 domain
and V3 isoform expression in wings injected with recombinant adenoviruses at HH20 (C), HH21 (A, B, E) and HH22 (D). Targeted adenoviral injection resulted in enlargement of skeletal primordia in the
proximal (A, C), distal (B) or mid humerus (D). There was little or no
enlargement of the skeletal anlagen observed with the LacZ adenovirus control (E). Humeral sites from which measurements in Table 2
were taken are shown in panel (F). CLC indicates uninjected contralateral control limbs. Scale bar ¼ 0.5 mm.
whether increased cell proliferation contributed to the
observed thickening of skeletal primordia. In a series of
11 immunohistochemical experiments on 23 wing samples
from 9 embryos as represented in Fig. 4, no correlation
with changes in proliferation at sites of recombinant G1
or V3 adenoviral infection were observed at HH23-31 or
later at HH35. Similar results were noted at HH35 for G1
and V3 adenovirus infected limbs at HH22-25 (not
shown). Reduction in apoptosis during the postinfection
incubation period as a result of recombinant G1 or V3
expression that could account for possible increases in
chondrocyte number were also examined by TUNEL
assay. As only very low levels of apoptosis have been
observed previously in the wild-type chick wing at HH25
(Shepard et al., 2008), TUNEL was performed at HH30/31
when limb chondrogenesis had further progressed. Low
numbers of scattered apoptotic cells were noted, but no
correlation with changes in apoptotic cell number at sites
of adenoviral infection were detected (not shown).
Because of its ability to bind hyaluronan, the effect of
recombinant G1 expression on hyaluronan distribution
in the developing chick limb was also assessed. Although
less obvious at earlier stages, by HH35 definitive pericellular hyaluronan signal was noted surrounding individ-
ual recombinant G1-positive chondrocytes (Fig. 5A and
B). G1-positive cells could also be observed in association
with noninfected cells in areas of hyaluronan accumulation. In limbs coinjected with V3/LacZ adenoviruses, we
were unable to ascertain localized changes in hyaluronan associated specifically with V3-positive chondrocytes at the stages examined.
Rhodamine PNA labeling of the forming humerus at
HH25 in wings injected at HH19-21 showed staining of
cell aggregates within early chondrogenic primordia as
expected, but in addition, PNA labeling displayed an overlapping distribution with recombinant G1 (Fig. 6A–C) and
V3 (Fig. 6D–G) expression in several areas. Interestingly,
multiple small PNA-positive cell clusters not observed in
the contralateral control (Fig. 6H and I) were also G1- and
V3-hemaglutinin tag-positive in addition to PNA/hemaglutinin tag staining in the limb core, suggesting that
additional mesenchymal aggregates were undergoing
incorporation into the cartilage template.
DISCUSSION
It is well-documented that versican is expressed during early stages of limb development (Shinomura et al.,
RECOMBINANT VERSICAN G1 AND V3 EXPRESSION
Fig. 3. Whole mount b-galactosidase staining and hemaglutinin tag
immunostaining shows localization of recombinant versican G1 domain and V3 isoform expression in enlarged humeral cartilages at
HH35 in wings injected with recombinant adenoviruses at HH23 (C, D)
and HH24 (A, B). Whole mount b-galactosidase reactivity localized
sites of adenoviral infection (arrow, A; arrowhead, C). Immunostaining
of hemaglutinin tagged G1 (B) and V3 (D) constructs correlated well
with b-galactosidase reactivity in enlarged areas of the mid (mh; A,)
and proximal (ph; C) humeral template. Inset in panel (A) shows higher
magnification image of b-galactosidase positive cells indicated by the
arrow. Scale bars ¼ 250lm (A, B and C, D); 125lm, inset in A.
Fig. 4. Recombinant versican V3 isoform expression does not correlate with increased mitosis in humeral cross section of the HH31
wing following coinjection with recombinant V3/LacZ adenovirus at
HH23. Change in overall number of antiphosphohistone H3 (ser10)
positive cells (B) did not show correlation with V3/LacZ infected areas
as indicated by b-galactosidase staining (boxed area in A) in either
pre- and early chondrogenic stages HH23-31 or in later stage cartilaginous tissue (HH35). Scale bar ¼ 100lm.
1990; Shibata et al., 2003; Snow et al., 2005). Endogenous versican is expressed in the ECM during formation
of prechondrogenic condensations (Kimata et al., 1986)
with expression reduced and replaced by aggrecan as
mesenchymal cells differentiate into chondrocytes. As
overt limb chondrogenesis continues, versican expression
persists in perichondrial and joint interzone tissues (Shinomura et al., 1990; Yamamura et al., 1997; Shibata
et al., 2003; Snow et al., 2005; Shepard et al., 2007).
Although several studies have reported the importance
of versican to early stages of limb skeletogenesis, the
mechanism by which versican is involved is yet unclear.
1675
Fig. 5. Cross section through the proximal humeral element of the
chick wing coinjected with recombinant versican G1/LacZ at HH23
displays pericellular hyaluronan localization in peripheral limb cartilages at HH35. A: G1-hemaglutinin tag localization (arrows) associated
with individual hyaluronan-positive cells or uninfected hyaluronan-positive chondrocytes (B). C: Area of infection shown by b-galactosidase
reactivity. Asterisk, perichondrium. Scale bar ¼ 25lm.
We undertook the present study to determine whether
N- and C-terminal versican domains could function independently of the mature proteoglycan when overexpressed during chick limb skeletogenesis and
hypothesized that expression of individual G1 domains
and V3 isoform would facilitate limb chondrogenesis in
vivo. Our results showed that overexpression of G1 and
V3 versican had growth promoting effects on humeral
skeletal anlagen suggesting that individual versican
domains may function during establishment of cartilages
in early stages of wild type limb skeletogenesis. Replication-incompetent recombinant adenoviruses were used to
express the versican G1 domain and V3 variant, comprised of only G1 and G3 domains, in the proximal limb
at HH19-21 (mesenchymal cell stages) and HH22-25
(precartilage condensation and early chondrogenic
stages). At both intervals adenoviral-mediated expression of either the G1 domain or V3 variant of versican
resulted in enlargement of humeral cartilages and skeletal primordia due to localized increases in cartilage formation. Versican domains have been shown previously
to function independently when expressed ectopically
(Zhang et al., 1998, 2001; Ang et al., 1999; Yang et al.,
1999; Kern et al., 2007) and increasing evidence suggests that versican may be proteolytically processed in
different tissues which may enable individual versican
domains to function apart from the intact proteoglycan
(Sandy et al., 2001; Kern et al., 2006, 2007, Capehart,
2010). Indeed, versican proteolysis by ADAMTS family
members liberates a fragment containing the G1 domain
(Sandy et al., 2001).
In the present study, several potential mechanisms that
could lead to increased cartilage size due to recombinant
versican G1 and V3 overexpression were investigated. G1
and G3 domains have both been shown to enhance proliferation (Zhang et al., 1998, 1999; Ang et al., 1999; Wu
et al., 2002) and localized increases in mitosis of precartilage mesenchyme and/or chondrocytes could possibly
account for increased humeral size. Proliferative cells
were found scattered throughout the proximal limb at several developmental stages but distribution of mitotic cells
1676
HUDSON ET AL.
Fig. 6. Recombinant hemaglutinin-tagged versican G1 and V3 proteins codistribute with PNA-binding materials in sagittal sections
through humeral cartilage condensations of the HH25 wing following
injection with recombinant adenoviruses at HH19 (A–I). Double labeling with antihemaglutinin shows G1 (A) and V3 (D) overlapping with
PNA in chondrogenic areas (B, E; overlay in C, F). Small clusters of
cells colabeled with both antihemaglutinin tag and PNA are noted at
the edges of chondrogenic regions in both G1 and V3 infected limbs
(arrowheads). Enlarged view of area indicated by arrowheads in (F) is
shown in (G). No hemaglutinin-tag immunoreactivity (H) is noted in the
PNA-positive chondrogenic core (I) of noninjected contralateral control
limbs (CLC). Scale bars ¼ 100lm in C, 25lm in G.
did not show significant correlation with areas of recombinant G1 and V3 expression as was also reported in the
embryonic outflow tract (Kern et al., 2007). Reduction in
number of apoptotic cells was also examined and though
small numbers of scattered apoptotic cells were detected
in the forming humerus, no obvious changes were noted
in G1-, V3-, or LacZ- infected areas. This does not rule out
possible reduction in apoptosis at other stages, but apoptotic level in relevant areas of the proximal limb appear low
overall during the period spanned by the present study
and thus is unlikely. In addition, by subjective assessment
of chondrocyte-chondrocyte proximity in 10 fields from
three separate experimental treatments only slight differences in accumulation of interstitial cartilage matrix
between individual chondrocytes in areas of recombinant
G1 and V3 expression at HH35 were apparent (see Fig.
3), suggesting that increased overall matrix deposition by
individual chondrocytes alone was not responsible for
increased local cartilage growth. The absence of data to
support increased cell proliferation or decreased apoptosis
in these studies points to a third mechanism in which versican G1 domain and V3 isoform facilitate aggregation of
cartilage progenitors within the humeral primordium. As
there is growing appreciation of the role of extracellular
matrix in regulating gene expression and pattern formation, it is possible that expression of these recombinant
matrix proteins by adenovirally infected cells in developing skeletal primordia could impact cellular behavior in
the surrounding cell population.
Versican has been shown important for chondrogenesis
in vitro, particularly with regard to formation of precartilage mesenchymal aggregates (Williams et al., 2005;
Kamiya et al., 2006). In contrast, inhibition of versican
in chick limb mesenchyme in vitro had no effect on mesenchymal aggregation (Zhang et al., 1998). Several factors can affect direct comparison of these studies, for
example, different cell lines, plating densities, and use of
protein knockdown techniques. The discrepancies and
difficulty in interpreting the in vivo relevance of work in
vitro emphasized the importance of attempting use of an
in vivo model in which to evaluate versican function in
the developing limb. One such study showed that targeted morpholino-mediated knockdown of versican in
ovo inhibited precartilage mesenchymal condensation in
the embryonic chick limb (Shepard et al., 2008).
PNA has been widely used as a marker for identifying
aggregating precartilage limb mesenchyme and early
chondrogenic foci (Zimmermann and Theis, 1984; Aulthouse and Solursh, 1987; Capehart et al., 1997). PNA
labeling of the developing humerus at HH25 showed
overlapping localization with recombinant G1- and V3
expression in the proximal limb core and also in ‘‘extra’’
small clusters of cells around the edges of the forming
humerus. Colocalization of PNA-binding with G1 and
V3-positive cells suggests that expression of recombinant
versican domains led to localized increases in PNA-positive aggregates, resulting in additional cartilage formation that could account for chondrogenic expansion at
specific humeral sites. Our observations are in agreement with the results of other studies in which virally
mediated ectopic V3 isoform expression was reported to
increase cell adhesion in vitro (Lemire et al., 2002; Kern
et al., 2007) and thickness of the outflow tract myocardium in vivo (Kern et al., 2007). On the other hand, ectopic V3 expression reduced cartilage formation in a
chondrogenic cell line in vitro, perhaps by competing
with endogenous versican (Kamiya et al., 2006). In the
present study, however, recombinant V3 overexpression
continued well beyond the period when endogenous V0/
V1 versican is found in proximal limb cartilage matrix
in vivo, and so perhaps facilitated continued aggregation
of cartilage-forming progenitors into the peripheral humeral primordium.
In the present study, obvious differences in hyaluronan were not readily observed in areas of recombinant
versican domain expression until later chondrogenic
stages where increased pericellular hyaluronan was
noted about well-rounded G1-positive chondrocytes; thus
it is uncertain whether recombinant versican G1 domain
expression impacted significant hyaluronan accumulation at earlier stages. On the other hand, if hyaluronan
was incrementally stabilized through increased binding
of virally-expressed G1 domain, it is possible that additional mesenchyme were gradually incorporated into
RECOMBINANT VERSICAN G1 AND V3 EXPRESSION
cellular aggregates that contributed to an overall
increase in chondrocyte number in specific humeral locations. Indeed, versican/hyaluronan complexes in vitro
have been shown to enhance recruitment of stromal cells
during mammary tumor neovascularization (Koyama
et al., 2007).
To our knowledge, this study is the first attempt to
examine versican G1 domain and V3 isoform effects on
limb skeletogenesis in vivo. It is interesting to note that
although versican G1 and G3 domains have been
reported to have differing activities in vitro, our data
show that G1 and V3 both have gain-of-function effects
on chondrogenesis in vivo and suggests that the mechanism by which endogenous versican is involved in limb
development occurs by way of effecting chondrogenic
condensations as shown in previous studies in vitro (Williams et al., 2005; Kamiya et al., 2006) and in vivo (Shepard et al., 2008). Alternatively, expression of
recombinant versican domains could perhaps bind to
other endogenous molecules within the limb resulting in
their loss-of-function with similar overall effect. The
observed enlargement of skeletal primordia resulting
from overexpression of both recombinant G1 and V3 also
suggests that versican domains are capable of acting independently of intact V0 and V1 isoforms during embryonic limb development.
ACKNOWLEDGMENTS
The authors thank Dr. Said Said for assistance with
statistical analysis.
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