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DEVELOPMENTAL DYNAMICS 218:394 – 400 (2000)
BRIEF COMMUNICATION
Col2-GFP Reporter Marks Chondrocyte Lineage and
Chondrogenesis During Mouse Skeletal Development
T. DAWN GRANT,1,2 JAY CHO,1,2 KILEY S. ARIAIL,1 NICOLE B. WEKSLER,1 RANDALL W. SMITH,3
1,2*
AND WILLIAM A. HORTON
1
Research Center, Shriners Hospital for Children, Portland, Oregon
2
Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon
3
Department of Pathology, Oregon Health Sciences University, Portland, Oregon
ABSTRACT
Mice were generated in which a
Col2-GFP transgene serves as a reporter for the
chondrocyte lineage and for chondrogenesis in
live embryos and newborn pups. Cells actively
engaged in chondrogenesis were identified by
confocal optical sectioning within their native
environments in embryos and in thick tissue
slices. Chondrocytes exhibiting GFP fluorescence were purified from rib cages by high-speed
cell sorting of crude cell suspensions. Intensity of
fluorescence correlated with biosynthesis of procollagen II in these cells. The use of these mice
and their cells provides a novel approach for
studying chondrocyte differentiation and chondrogenesis during skeletal development. Dev Dyn
2000;218:394 – 400. © 2000 Wiley-Liss, Inc.
Key words: transgenic mice; reporter mice; green
fluorescent protein; type II collagen;
chondrocytes; chondrogenesis; skeletal development; confocal microscopy; cell sorting
INTRODUCTION
Chondrocytes play a key role in formation and maintenance of the vertebrate skeleton (Tickle and Eichele,
1994; Erlebacher et al., 1995; Johnson and Tabin, 1997;
Beier et al., 1999; Neame et al., 1999; St-Jacques et al.,
1999; Stevens and Williams, 1999). They differentiate
in response to signals that determine where the future
skeleton will form. They proliferate, terminally differentiate and die in the growth plate in response to
signals that regulate linear bone growth. They synthesize and maintain cartilage matrices that serve as templates for endochondral ossification, account for the
functional properties of permanent cartilages and contribute to the repair of cartilage injuries. In short,
chondrocytes exhibit a number of adaptive behaviors in
response to changing environments in skeletal tissues.
Despite their importance to skeletal development
and homeostasis, chondrocyte behaviors are difficult to
study because the microenvironments responsible for
them are difficult to reproduce experimentally. Indeed,
© 2000 WILEY-LISS, INC.
loss of typical chondrocyte characteristics, i.e., dedifferentiation, has been an obstacle for study of chondrocytes, especially mammalian chondrocytes, in vitro
(Benya et al., 1978; Benya and Shaffer, 1982; Aulthouse et al., 1989; Ramdi et al., 1993). Even if chondrocyte phenotypes can be produced in vitro, it is difficult to be certain that in vitro behaviors accurately
reflect in vivo behaviors given the substantial differences in microenvironment. Consequently, investigators often turn to in situ approaches in which experiments are carried out in living animals or in organ
cultures that maintain native environments. These tissues are examined after fixation with probes that can
be detected microscopically. Such analyses provide
snapshots of relevant behaviors, but dynamic aspects
of chondrocyte behavior can not be studied directly
because preparation of tissues for conventional microscopy kills cells.
We have developed an approach to study chondrocyte
biology during skeletal development that allows analysis of live cells within tissues with the potential for
real-time studies of adaptive behaviors relevant to
skeletal biology. Transgenic mice were generated in
which expression of enhanced Green Fluorescent Protein (GFP) is controlled by the murine type II collagen
promoter/enhancer (Col2-GFP) to produce a fluorescent reporter for chondrocytes. Expression of type II
collagen is widely accepted as a chondrocyte marker
and its promoter/enhancer has been well characterized
(von der Mark, 1980; Horton et al., 1987; Mendler et
al., 1989; Vikkula et al., 1992; Ala-Kokko et al., 1995;
Metsaranta et al., 1995). GFP has been utilized as a
lineage marker in many cell types (Doevendans et al.,
1996; Zhuo et al., 1997; Chen et al., 1998; Fleischmann
et al., 1998; van den Pol and Ghosh, 1998; Minegishi et
Grant sponsor: Shriners Hospitals for Children; Grant number:
8650.
*Correspondence to: William A. Horton, M.D., Research Center,
Shriners Hospital for Children, 3101 S.W. Sam Jackson Park Road,
Portland, OR 97201. E-mail: wah@shcc.org
Received 21 December 1999; Accepted 17 February 2000
Col2-GFP REPORTER MICE
Fig. 1. Composite images of cartilaginous skeletons of live embryos
transgenic for Col2-GFP (A, E14.5; B, E17.5). Many structures that
exhibit fluorescence at E14.5 no longer fluoresce at E17.5, including
395
diaphyses of radius and ulna (arrow) and femur and fibula (double
arrows), posterior ribs and vertebral bodies (arrowhead) except for those
in the tail. Double arrowheads mark external ear in A. Scale bars ⫽ 1 mm.
Fig. 2. Distal femur from transgenic embryo (E 17.5) imaged by three different modes. A is a paraffin
section stained with alcian blue and H&E. B is a paraffin section immunostained with antibody to collagen II.
C is a thick section imaged by confocal microscopy. Note that fluorescence is restricted to matrix in B and to
cells in C. Scale bars ⫽ 200 ␮.
al., 1999; Ono et al., 1999), but not for chondrocytes to
our knowledge.
Previous studies have utilized ␤-galactosidase (␤gal)
as a reporter protein for the Col2 promoter. The
present study documents that Col2-GFP protein identifies chondrocytes throughout development in a pattern comparable to that of Col2-␤gal reporter protein
(Metsaranta et al., 1995). However, the Col2-GFP reporter system is more versatile since it can be visualized directly in living tissues by conventional fluorescence or by laser-scanning confocal microscopy. The
latter technique minimizes background fluorescence
and offers a potential means to investigate chondrocytic behaviors relevant to skeletal development in intact tissues and in real time. GFP fluorescence provides
an in situ assay of type II collagen synthesis during
processes, such as bone growth and cartilage repair.
Finally, the presence of the Col2-GFP protein also provides an endogenous label for high-speed cell sorting
that allows quick and efficient isolation of chondrocytes
for in vitro studies.
We report here the generation of the transgenic
mouse, document the expression of the reporter during
mouse development, correlate GFP fluorescence with
biosynthesis of type II collagen and describe a protocol
developed to isolate chondrocytes by cell sorting.
RESULTS AND DISCUSSION
Transgenic Mice
Eight founder mice were positive for the Col2-GFP
transgene by PCR and Southern blot analysis of tail
gDNA. Transgenic newborn progeny of three of these
founders exhibited fluorescence of skeletal elements of
the paw when examined using epifluorescence microscopy. No fluorescence was observed for wild-type offspring. These three strains were expanded based on
fluorescence phenotyping and PCR genotyping. All observations were made on embryos and newborn mice
heterozygous for the transgene.
No differences were noted in general development,
health, and fertility of transgenic versus wild-type
mice. Crown-rump lengths and weights from E12.5–
396
GRANT ET AL.
E19.5 were comparable, and no differences were specifically observed in skeletal development.
Imaging of Whole Embryos by Epifluorescence
Fluorescence of the cartilaginous skeleton was visualized in whole live embryos by epifluorescence microscopy. It was not diminished by paraformaldehyde fixation, which made subsequent dissection much easier
for embryos younger than E15.5. Wild-type embryos
exhibited no fluorescence. The expected progression of
skeletal development is shown at low magnification in
composites of E14.5 and E17.5 transgenic embryos in
Figure 1.
Confocal Imaging of Skeletal Elements
The distribution of GFP-positive cells matched
closely the distribution collagen II detected by immunostaining (Fig. 2). However, fluorescence in the
former case was restricted to cells, whereas it localized
to matrix in the latter case. The distribution closely
resembled the pattern observed in previous studies
using in situ hybridization and ␤gal reporter transgene
strategies to demonstrate expression of Col2a1 (Cheah
et al., 1991; Metsaranta et al., 1995). This was expected
since we used the same murine Col2a1 promoter/enhancer to drive GFP expression that had been used by
Metsaranta et al. to direct the ␤gal expression (Metsaranta et al., 1995).
GFP fluorescence was first detected at E10.5 (Fig.
3A,B). This is one day earlier than that observed in the
Col2-␤gal mice (Metsaranta et al., 1995). However, it
corresponds to the earliest detection of Col2a1 transcripts by in situ hybridization (Cheah et al., 1991) and
is consistent with other reports that GFP is a more
sensitive reporter than ␤gal in transgenic mice (Chiocchetti et al., 1997).
GFP fluorescence was localized in E10.5 embryos to
axial structures, including the otic capsule, chondrogenic regions of developing maxillary and mandibular
processes and sclerotome condensations of pre-vertebrae. Figure 3B shows high magnification of the sclerotomal cells in a pre-vertebra. By E14.5, most of the
endochondral skeleton shows GFP fluorescence (Fig
1A, 3C, 3D) as do nonskeletal cartilages, such as nasal
and external ear cartilages (Fig 1A).
Less GFP fluorescence was present by E17.5 as many
bones are mostly or partially ossified (Fig. 1B). This
was most notable for the spine (except for tail), posterior ribs and diaphyses of long bones of the limbs.
Figures 3E and 3F demonstrate this phenomenon in
the lower extremity at E16.5, where the tibia is partially ossified while the bones of the feet are primarily
cartilaginous. The proximal tibial growth plate is seen
at higher magnification in Figure 3F. There are fewer
fluorescing cells in the hypertrophic zone than in the
resting and proliferating zones of the growth plate.
GFP fluorescence tended to be more intense in the
nucleus than in the cytoplasm of cells.
The intensity of GFP fluorescence was not the same
in all chondrocytes within a given cartilaginous struc-
ture. This variability was consistently more evident in
cartilaginous structures from older embryos, especially
beyond E16.5. Image analysis of z-series of confocal
images showed that this was not an artifact of optical
sectioning.
Cell Sorting
Figure 4A shows a confocal image of a rib cage from
a transgenic newborn prior to collagenase digestion.
The results of typical high-speed sorting runs of cells
derived from wild-type (left) and transgenic (right) rib
cages are shown in Figure 4B. The threshold established from sorting the wild-type cells was used to
isolate GFP fluorescence-positive cells in this experiment. The characteristics of cells isolated by sorting
were compared to cells from a comparable cell suspension that had not been sorted after 48 hr of routine
culture on plastic (Fig. 5). Most of the sorted cells
displayed a rounded morphology and GFP fluorescence
typical of chondrocytes, whereas most of the non-sorted
cells exhibited a spread-out “fibroblastic” morphology
and were not fluorescent. Fluorescence was restricted
to rounded cells in both instances. Thus, sorting produced considerable enrichment for round, fluorescing
cells. To further demonstrate that GFP fluorescence
marks expression of the chondrocyte phenotype and
indicates expression of endogenous Col2a1, crude cell
suspensions from ribs of transgenic newborn mice were
sorted twice. The first sort separated non-fluorescing
from fluorescing cells; the second sort separated high
from low-moderate fluorescing cells. The three cell populations were compared by western blot analysis of
procollagen II. Figure 6 shows that intensity of fluorescence correlated well with the level of procollagen II
synthesis.
Thus, fluorescence derived from the Col2-GFP transgene provides an in situ cellular assay for type II collagen synthesis. One can assess the level of type II
collagen synthesis of one or a group of cells relative to
other cells within intact skeletal structures. This capability would be useful for studying biologic phenomena
in which synthesis of type II collagen is considered
important, such as bone development and growth and
repair of bone fractures and cartilage injuries. However, one must be aware that GFP is a stable protein
that may survive in cells for up to 24 hours making the
assay most useful for assessing collagen synthesis over
many hours to days rather than for shorter periods (Li
et al., 1998).
The mouse is becoming the experimental model of
choice to study mammalian skeletal development.
However, a significant drawback is the difficulty isolating chondrocytes for in vitro studies. The small size
of embryonic and newborn mouse bones makes it almost impossible when isolating chondrocytes to avoid
substantial contamination of nonchondrocytic cells,
which in turn, may compromise analyses being carried
out. High-speed flow cytometric cell sorting of GFPpositive cells circumvents this problem. Indeed, our
results demonstrate that an almost pure population of
Col2-GFP REPORTER MICE
chondrocytes can be obtained by sorting of crude collagenase cell suspensions of ribs in less than 24 hr. A
similar strategy has been used successfully to isolate
dermal papilla cells from the skin of newborn mice
transgenic for a versican-GFP transgene (Kishimoto et
al., 1999).
Confocal imaging of endogenous fluorescence in skeletal elements has many advantages over conventional
microscopy. Artifacts related to processing and staining are avoided because the tissues are sectioned optically rather than mechanically, and probes or treatments with other agents are not needed. Background
fluorescence of non-cartilaginous tissues, such as bone
and perchondrium, is minimal in optical sectioning of
even fixed tissues. This presumably reflects the much
thinner optical sections (0.5␮) imaged by confocal microscopy compared to thicker sections (8␮) imaged conventionally, the relatively high intensity of GFP fluorescence compared to the intensity of background
fluorescence and the avoidance of paraffin embedment.
Most important is the ability to image single cells in
intact, live tissues such as individual bones or limbs in
culture or in whole embryos. At low magnification,
images resemble X-rays, except that cartilage rather
than bone is identified.
A potential use of this strategy will be to study the
effects of various mutations on skeletal development in
mice and to isolate chondrocytes harboring these mutations. In this case the relevant mutants would be
genetically crossed with the Col2-GFP reporter mice to
produce mice that carry both the mutant gene of interest and the reporter transgene. Such mice could be
studied microscopically and cell sorting would yield
chondrocytes bearing the mutation for analyses. We
have begun to exploit this strategy to study the consequences of receptor mutations on chondrocyte signaling pathways.
The use of the Col2-GFP reporter mice as reported
here provides a new tool to study murine chondrocyte
biology. It offers a potential window through which to
observe the dynamic events in which chondrocytes participate and a quick access to murine chondrocytes.
EXPERIMENTAL PROCEDURES
Generation of Transgenic Mice
The pCol2a1-GFP vector was constructed using standard cloning procedures. The Col2a1 promoter was excised from the plasmid p3000i3020Col2a1 by Cla I-Not
I-Asp 718 digestion (Zhou et al., 1995). After filling in
the ends with Klenow, the 6.3 kb Col2a1 promoter was
blunt-end ligated into the filled-in BamHI site of
pEGFP-1 (Clontech, Palo Alto, CA). Orientation was
verified by Not I-Xba I digestion. The entire 7.35 kb
Col2a1-GFP transgene was excised by Asp718-Afl II
digestion and gel-purified. Microinjection and other
methods used to generate the transgenic mice have
been described previously (Garofalo et al., 1991; Metsaranta et al., 1995).
397
Detection of Transgenic Mice and Embryos
Pregnant mice were sacrificed following anesthesia
using CO2 inhalation. Embryos were removed by Csection at 9.5–18.5 days p.c. Transgenic embryos and
neonatates were identified by fluorescence microscopy
and confirmed by PCR and Southern blot analysis of
gDNA using probes and primers for GFP.
Tissue Preparation and Staining
The whole skeletons of some embryos were cleared
and stained for cartilage and bone using Alizarin red S
and alcian blue (McLeod, 1980). For alcian blue/hematoxylin and eosin (H&E) staining, tissues were fixed 24
hr in 4% paraformaldehyde, embedded in paraffin, cut
into 8 ␮m sections and stained using standard protocols.
Immunostaining for collagen II was done on 8-␮m
paraffin sections fixed according to the AMeX method
(Sato et al., 1986). Deparaffinized sections were treated
with chondroitinase ABC (0.25 units/ml, Sigma, St.
Louis, MO) for 30 min at RT, washed in PBS and
incubated in primary antibody (Collagen Type II Ab-2,
Clone 2B1.5, Neomarkers, Inc., Fremont, CA) diluted
1:20 in PBS overnight in a humidified chamber at 4°C.
Secondary antibody (Alexa 546, 1:50, Molecular
Probes, Inc., Eugene, OR) was applied at RT for 30 min.
Finally, sections were washed with PBS, mounted with
glycerol and imaged by epifluorescence.
For confocal microscopy, whole embryos and embryonic tissues were embedded in 7% agarose (Gibco-BRL,
Gaithersburg, MD) either unfixed or after overnight
fixation in 4% paraformaldehyde before embedment.
For newborn pups, bones were dissected free, cut in
half and in some cases fixed overnight in 4% paraformaldehyde. In both instances skeletal tissues were cut
into 200 –300 ␮m sections using a D.S.K. DTK-2000
Microslicer (Ted Pella, Inc., Irvine, CA),
Imaging
GFP fluorescence was visualized in whole animals
using a Nikon E800 microscope equipped with epifluorescence, a SenSys digital camera (Photometrics, Tucson, AZ), and Metamorph imaging system (Universal
Imaging, West Chester, PA). Confocal images were
captured using a Nikon Diaphot 300, attached to a
Biorad MRC 1024 confocal system. The digitized images were assembled in Photoshop 5.02 (Adobe Systems, Seattle, WA).
Isolation of Chondrocytes and Cell Sorting
One- to two-day-old mice were anesthetized by CO2
inhalation. Rib cages were removed and in a few cases
imaged confocally to demonstrate GFP fluorescence.
The rib cages were treated with pronase (Sigma, 2
mg/ml in PBS) at 37°C for 30 min washed in PBS
washes and transferred to a bacteriological dish. Collagenase B was added (Boehringer Mannheim; 2 mg/ml
in cell culture medium: DMEM without phenol red
supplemented with 10% fetal calf serum, 100 units/ml
398
GRANT ET AL.
Fig. 3. Confocal images of chondrogenic/cartilaginous structures of
transgenic embryos E10.5 (A, B), E14.5 (C, D), and E16.5 (E, F).
A shows otic vesicle (arrow), chondrogenic regions of developing maxillary and mandibular processes (double arrows) and sclerotome condensations of pre-vertebrae (arrowhead). B is a high magnification of the
sclerotomal cells in a pre-vertebra. C shows thoracic vertebral bodies.
D shows vertebral bodies in the lower lumbar area. E and F show the
middle segment of the lower limb at low and higher magnification. Arrow
points to fibula, double arrows to ossified portion of tibia and arrowhead
to the proximal tibial growth plate. Note the cellular distribution of fluorescence in B–F. Scale bars ⫽ 100 ␮ for A, 10 ␮ for B–D, 1 mm for E and
500 ␮ for F.
penicillin, 100 ug/ml streptomycin, and 2 mM sodium
pyruvate; Gibco-BRL) and incubation was carried out
overnight at 37°C with gentle shaking to dissociate the
ribs. The cell suspension formed by pipeting with PBS
was strained through a 70 ␮ filter. The dishes were
rinsed with an equal volume of PBS and the solution
also filtered. After the filtrate was centrifuged for 5 min
at 1,000 rpm, the supernatant was discarded and the
pellet was resuspended in cell culture medium. Viable
cells were counted using trypan blue solution, and the
cells were resuspended to 5 ⫻ 105 cells/ml in cell culture medium with 0.5% Collagenase-B to prevent
clumping. Cells were then sorted into fluorescing and
non-fluorescing populations. In some cases the fluorescing cell population was sorted again into low-tomoderate and high fluorescing cell subpopulations.
Col2-GFP REPORTER MICE
399
Fig. 4. Isolation of chondrocytes by high-speed flow cytometry. A shows ribs from transgenic newborn
prior to digestion. Cellular distribution of fluorescence is indicated in inset. B shows results of a sorting
experiment for cells from a wild-type mouse on left and from a transgenic mouse on the right.
Fig. 5. Cells cultured on plastic chamber slides 48 hr after isolation
from transgenic newborn mouse ribs. Cells in A and B were not sorted;
fluorescence-positive cells in C and D were selected by sorting. A and C
were imaged by DIC to show morphology; B and D by epifluorescence.
Sorted cells exhibit predominantly a rounded morphology and most cells
fluoresce. In contrast, many of the non-sorted cells have a spread-out
appearance and there are many fewer fluorescing cells. Fluorescence is
restricted to non-spread out, rounded cells in both instances.
Cell Culture
Some cells were cultured on Permanox plastic chamber
slides (NUNC, Naperville, IL) and photographed using
DIC optics to show morphology and fluorescence.
After sorting, each population of cells was plated in
cell culture medium at a density of 1 ⫻ 106 cells per 60
mm dish. After 24 hr, the cell layer was washed with
PBS and incubated for 24 hr in DMEM supplemented
with 0.05% fetal calf serum and 50 ␮g/ml ascorbic acid
phosphate (Wako Biochemicals, Richmond, VA). The
collected media were diluted in 5X SDS sample buffer
(325 mM Tris pH6.8, 10% SDS, 25% glycerol, and 5%
bromophenol blue) and used for immunoblot analysis.
Immunoblot Analysis
Equal volumes of media samples were electrophoresed on 7.5% SDS polyacrylamide gels and proteins
were transferred to PVDF membranes (Millipore, Bedford, MA). After 1 hr incubation at RT in blocking
buffer (1X Tris-buffered saline with 0.05% Tween-20
400
GRANT ET AL.
Fig. 6. GFP fluorescence intensity correlates with procollagen II synthesis. Cells were sorted into populations exhibiting no (lane 1), lowmoderate (lane 2) and high intensity (lane 3) fluorescence and cultured
as described in the Experimental Procedures. Equivalent aliquots of
media from these cells were immunoblotted for procollagen II. Pepsindigested collagen II served as a control (C). Increasing amounts of
procollagen II were detected in lanes 2 and 3. The slightly smaller band
of much lower intensity in lane 3 is probably partially processed pC
collagen II.
and 4% BSA), the membranes were incubated in primary antibody diluted in blocking buffer overnight at
4°C. They were washed 3 times in blocking buffer and
incubated in secondary antibody (anti-mouse IgG
linked to horseradish peroxidase; Amersham) diluted
in blocking buffer without BSA for 1 hr at RT. After 3
more washes, membranes were developed for chemiluminescence using SuperSignal (Pierce, Rockford, IL).
ACKNOWLEDGMENTS
The work was supported by a grant from the Shriners Hospitals Research Program (WAH). We wish to
thank Silvio Garofalo for plasmid p3000i3020Col2a1,
Megan Nguyen for technical assistance and Peter Hurlin and Gregory Lunstrum for helpful suggestions.
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