BMP receptor signaling is required for postnatal maintenance of articular cartilage.
ABSTRACT Articular cartilage plays an essential role in health and mobility, but is frequently damaged or lost in millions of people that develop arthritis. The molecular mechanisms that create and maintain this thin layer of cartilage that covers the surface of bones in joint regions are poorly understood, in part because tools to manipulate gene expression specifically in this tissue have not been available. Here we use regulatory information from the mouse Gdf5 gene (a bone morphogenetic protein [BMP] family member) to develop new mouse lines that can be used to either activate or inactivate genes specifically in developing joints. Expression of Cre recombinase from Gdf5 bacterial artificial chromosome clones leads to specific activation or inactivation of floxed target genes in developing joints, including early joint interzones, adult articular cartilage, and the joint capsule. We have used this system to test the role of BMP receptor signaling in joint development. Mice with null mutations in Bmpr1a are known to die early in embryogenesis with multiple defects. However, combining a floxed Bmpr1a allele with the Gdf5-Cre driver bypasses this embryonic lethality, and leads to birth and postnatal development of mice missing the Bmpr1a gene in articular regions. Most joints in the body form normally in the absence of Bmpr1a receptor function. However, articular cartilage within the joints gradually wears away in receptor-deficient mice after birth in a process resembling human osteoarthritis. Gdf5-Cre mice provide a general system that can be used to test the role of genes in articular regions. BMP receptor signaling is required not only for early development and creation of multiple tissues, but also for ongoing maintenance of articular cartilage after birth. Genetic variation in the strength of BMP receptor signaling may be an important risk factor in human osteoarthritis, and treatments that mimic or augment BMP receptor signaling should be investigated as a possible therapeutic strategy for maintaining the health of joint linings.
- SourceAvailable from: Esmeralda Blaney Davidson[Show abstract] [Hide abstract]
ABSTRACT: Transgenic mice are used to study the roles of specific proteins in an intact living system. Use of transgenic mice to study processes in cartilage, however, poses some challenges. First of all, many factors involved in cartilage homeostasis and disease are also crucial factors in embryogenesis. Therefore, meddling with these factors often leads to death before birth, and mice who do survive cannot be considered normal. The build-up of cartilage in these mice is altered, making it nearly impossible to truly interpret the role of a protein in adult cartilage function. An elegant way to overcome these limitations is to make transgenic mice time- and tissue-specific, thereby omitting side-effects in tissues other than cartilage and during embryology. This review discusses the potential building blocks for making an inducible cartilage-specific transgenic mouse. We review which promoters can be used to gain chondrocyte-specificity - all chondrocytes or a specific subset thereof - as well as different systems that can be used to enable inducibility of a transgene.Arthritis Research & Therapy 06/2014; 16(3):210. · 4.12 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Articular cartilage has poor capacity of self-renewal and repair. Insufficient number and activity of resident mesenchymal (connective tissue) progenitors is likely one of the underlying reasons. Chondroprogenitors reside not only in the superficial zone of articular cartilage but also in other zones of articular cartilage and in the neighboring tissues, including perichondrium (groove of Ranvier), synovium and fat pad. These cells may respond to injury and contribute to articular cartilage healing. In addition, marrow stromal cells can migrate through subchondral bone when articular cartilage is damaged. We should develop drugs and methods that correctly stimulate resident progenitors for improvement of repair and inhibition of degenerative changes in articular cartilage.Matrix Biology 10/2014; · 3.65 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The central importance of BMP signaling in the development and homeostasis of synovial joint of appendicular skeleton has been well documented, but its role in the development of temporomandibular joint (TMJ), also classified as a synovial joint, remains completely unknown. In this study, we investigated the function of BMPRIA mediated signaling in TMJ development in mice by transgenic loss-of- and gain-of-function approaches. We found that BMPRIA is expressed in the cranial neural crest (CNC)-derived developing condyle and glenoid fossa, major components of TMJ, as well as the interzone mesenchymal cells. Wnt1-Cre mediated tissue specific inactivation of BmprIa in CNC lineage led to defective TMJ development, including failure of articular disc separation from a hypoplastic condyle, persistence of interzone cells, and failed formation of a functional fibrocartilage layer on the articular surface of the glenoid fossa and condyle, which could be at least partially attributed to the down-regulation of Ihh in the developing condyle and inhibition of apoptosis in the interzone. On the other hand, augmented BMPRIA signaling by Wnt1-Cre driven expression of a constitutively active form of BmprIa (caBmprIa) inhibited osteogenesis of the glenoid fossa and converted the condylar primordium from secondary cartilage to primary cartilage associated with ectopic activation of Smad-dependent pathway but inhibition of JNK pathway, leading to TMJ agenesis. Our results present unambiguous evidence for an essential role of finely tuned BMPRIA mediated signaling in TMJ development.PLoS ONE 08/2014; 9(8):e101000. · 3.53 Impact Factor
BMP Receptor Signaling Is Required
for Postnatal Maintenance
of Articular Cartilage
Ryan B. Rountree1, Michael Schoor1¤, Hao Chen1, Melissa E. Marks1, Vincent Harley2, Yuji Mishina3, David M. Kingsley1*
1 Department of Developmental Biology and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, United States of America,
2 Prince Henry’s Institute of Medical Research, Monash Medical Centre, Clayton, Victoria, Australia, 3 National Institute of Environmental Health Sciences, National Institutes
of Health, Research Triangle Park, North Carolina, United States of America
Articular cartilage plays an essential role in health and mobility, but is frequently damaged or lost in millions of people
that develop arthritis. The molecular mechanisms that create and maintain this thin layer of cartilage that covers the
surface of bones in joint regions are poorly understood, in part because tools to manipulate gene expression
specifically in this tissue have not been available. Here we use regulatory information from the mouse Gdf5 gene (a
bone morphogenetic protein [BMP] family member) to develop new mouse lines that can be used to either activate or
inactivate genes specifically in developing joints. Expression of Cre recombinase from Gdf5 bacterial artificial
chromosome clones leads to specific activation or inactivation of floxed target genes in developing joints, including
early joint interzones, adult articular cartilage, and the joint capsule. We have used this system to test the role of BMP
receptor signaling in joint development. Mice with null mutations in Bmpr1a are known to die early in embryogenesis
with multiple defects. However, combining a floxed Bmpr1a allele with the Gdf5-Cre driver bypasses this embryonic
lethality, and leads to birth and postnatal development of mice missing the Bmpr1a gene in articular regions. Most
joints in the body form normally in the absence of Bmpr1a receptor function. However, articular cartilage within the
joints gradually wears away in receptor-deficient mice after birth in a process resembling human osteoarthritis. Gdf5-
Cre mice provide a general system that can be used to test the role of genes in articular regions. BMP receptor
signaling is required not only for early development and creation of multiple tissues, but also for ongoing maintenance
of articular cartilage after birth. Genetic variation in the strength of BMP receptor signaling may be an important risk
factor in human osteoarthritis, and treatments that mimic or augment BMP receptor signaling should be investigated
as a possible therapeutic strategy for maintaining the health of joint linings.
Thin layers of articular cartilage line the bones of synovial
joints and provide a smooth, wear-resistant structure that
reduces friction and absorbs impact forces (Brandt et al.
1998). Loss or damage to articular cartilage is a hallmark of
arthritic diseases and is one of the most common reasons that
both young and old adults seek medical care. Millions of
people are afflicted with arthritis, and it ultimately affects
more than half of people over the age of 65 (Badley 1995;
Yelin and Callahan 1995). A better understanding of the
molecular mechanisms that create and maintain articular
cartilage is crucial for discovering the causes of joint
disorders and providing useful medical treatments.
Joint formation begins during embryogenesis, when stripes
of high cell density called interzones form across developing
skeletal precursors (Haines 1947). Programmed cell death
occurs within the interzone, and a three-layered interzone
forms that has two layers of higher cell density flanking a
region of lower cell density. Non-joint precursors of the
skeleton typically develop into cartilage, which hypertrophies
and is replaced by bone. However, cells within the high-
density layers of the interzone are excluded from this process
and develop into the permanent layers of articular cartilage
found in the mature joint (Mitrovic 1978).
Studies over the last 10 y have begun to elucidate some of
the signaling pathways that contribute to the early stages of
joint formation. Wnt14 is expressed in stripes at the sites
where joints will form, and it is capable of inducing
expression of other joint markers when misexpressed at
new locations in the limb (Hartmann and Tabin 2001).
Several members of the bone morphogenetic protein (BMP)
family of secreted signaling molecules are also expressed in
stripes at sites where joints will form, including those
encoded by the genes Gdf5, Gdf6, Gdf7, Bmp2, and Bmp4
(Storm and Kingsley 1996; Wolfman et al. 1997; Francis-West
Received May 2, 2004; Accepted August 19, 2004; Published October 19, 2004
Copyright: ? 2004 Rountree et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Abbreviations: BAC, bacterial artificial chromosome; Bmpr1a, bone morphogenetic
protein receptor 1a; E[number], embyonic day [number]; ECM, extracellular matrix;
GAC, transgenic line carrying Gdf5-alkaline phosphatase-Cre construct; Gdf5,
growth differentiation factor 5; hPLAP, human placental alkaline phosphatase; IRES,
internal ribosome entry site; PFA, paraformaldehyde; R26R, lacZ ROSA26 Cre
reporter strain; TGF-b, transforming growth factor b; TUNEL, terminal deoxynu-
cleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling
Academic Editor: Lee Niswander, University of Colorado Health Sciences Center
*To whom correspondence should be addressed. E-mail: kingsley@cmgm.
¤ Current address: ARTEMIS Pharmaceuticals, an Exelixis Company, Ko ¨ln, Germany
PLoS Biology | www.plosbiology.orgNovember 2004 | Volume 2 | Issue 11 | e3551815
Open access, freely available online P PL Lo oS S BIOLOGY
et al. 1999; Settle et al. 2003). Of these, Gdf5 expression is most
strikingly limited to regions where joints will develop and is
one of the earliest known markers of joint formation.
Mutations in either Gdf5 or the closely related Gdf6 gene also
block formation of joints at specific locations, providing
strong evidence that these molecules are essential for the
joint formation process (Storm et al. 1994; Settle et al. 2003).
However, mutations in Bmp2 or Bmp4 cause early embryonic
lethality, making it difficult to test their role in joint
formation (Winnier et al. 1995; Zhang and Bradley 1996).
Much less is known about how signaling pathways function
during the subsequent maturation and maintenance of adult
joint structures. Importantly, BMP signaling components are
present in adult articular cartilage, suggesting that they may
function during the late development or maintenance of this
critical structure (Erlacher et al. 1998; Chubinskaya et al.
2000; Muehleman et al. 2002; Bau et al. 2002; Bobacz et al.
BMPs bind tetrameric complexes of two type I and two
type II transmembrane serine-threonine kinase receptors.
Upon BMP binding, these complexes transduce a signal by
phosphorylating members of the Smad family of tran-
scription factors (Massague 1996). Recent experiments have
implicated two different BMP type I receptors in skeletal
patterning, BMPR1A and BMPR1B. Both receptors can bind
BMP2, BMP4, and GDF5, although GDF5 shows higher
affinity for BMPR1B (Koenig et al. 1994; ten Dijke et al.
1994; Yamaji et al. 1994; Nishitoh et al. 1996; Chalaux et al.
1998). Both receptors are also expressed in dynamic patterns
during normal development. In limbs, Bmpr1a expression
becomes restricted to joint interzones, perichondrium,
periarticular cartilage, hypertrophic chondrocytes, and
interdigital limb mesenchyme. In comparison, Bmpr1b
expression is seen primarily in condensing precartilaginous
mesenchymal cells, regions flanking joint interzones, peri-
chondrium, and periarticular cartilage (Dewulf et al. 1995;
Mishina et al. 1995; Zou et al. 1997; Baur et al. 2000). Null
mutations in the Bmpr1b gene produce viable mice with
defects in bone and joint formation that closely resemble
those seen in mice missing Gdf5 (Storm and Kingsley 1996;
Baur et al. 2000; Yi et al. 2000). Null mutations in Bmpr1a
cause early embryonic lethality, with defects in gastrulation
similar to those seen in mice with mutations in Bmp4
(Mishina et al. 1995; Winnier et al. 1995). Recent studies with
floxed alleles suggest that Bmpr1a is also required for many
later developmental events, but its roles in bone and joint
formation have not yet been tested (Mishina 2003).
A genetic system for activating or inactivating genes
specifically in joint tissues would be particularly useful for
further studies of joint formation and maintenance. Here we
take advantage of the tissue-specific expression pattern of
the Gdf5 gene to engineer a Cre/loxP system (Nagy 2000),
Gdf5-Cre, that can be used to remove or ectopically express
genes in joints. Tests with reporter mice show that this
system is capable of modifying genes in all of the structures
of the mature synovial joint, including the ligaments of the
joint capsule, the synovial membrane, and the articular
cartilage. Gdf5-Cre recombination bypasses the early embry-
onic lethality of null mutations in Bmpr1a, and shows that
this receptor is required for early joint formation at some
locations and for initiation of programmed cell death in
webbing between digits. Interestingly, Bmpr1a is also re-
quired for postnatal maintenance of articular cartilage
throughout most of the skeleton. In Gdf5-Cre/Bmpr1afloxP
mice, articular cartilage initially forms normally, but
subsequently loses expression of several key cartilage
markers after birth. It ultimately fibrillates and degenerates,
resulting in severe osteoarthritis and loss of mobility. These
experiments suggest that BMP signaling is required for
normal maintenance of postnatal articular cartilage, and
that modulation of the BMP signaling pathway may play an
important role in joint disease.
Figure 1. A Genetic System to Drive Gene Recombination in Developing
(A) A 140-kb BAC from the Gdf5 locus was modified by inserting Cre-
IRES-hPLAP into the translation start site of Gdf5 and used to make
transgenic mice. Not to scale. See Materials and Methods for details.
(B–E) Visualization of Gdf5-Cre driven recombination patterns based
on activation of lacZ expression from the R26R Cre reporter allele. (B)
LACZ activity is visible as blue staining in the ear (ea) and the joints of
the shoulder (s), elbow (eb), wrist (w), knee (k), ankle (a), vertebra (vj),
andphalanges(blackarrowheads) ofanE14.5 mouseembryo.(C)E14.5
hindlimb double-stained to show both HPLAP expression from the
transgene (grey/purple staining) and LACZ expression from the
rearranged R26R allele (blue staining). Note that both markers are
visible in theoldest, proximal interphalangeal joint (black arrowhead),
only HPLAP activity is visible in the more recently formed medial
interphalangeal joint (black arrow), and neither HPLAP nor LACZ
arrowhead). (D) Newborn (P0) forelimb with skin partially removed
showing LACZ activity expressed in all phalangeal joints (red Salmon
gal staining, black arrowheads) and regions of some tendons (asterisk).
(E) Section through the most distal phalangeal joint of a P0 hindlimb
stained with Alcian blue to mark cartilage showing LACZ expression
(stained red) in all tissues of developing joints: articular cartilage
(black arrowhead), precursors of ligaments and synovial membranes
(black arrow), and cells where cavitation is occurring (asterisk).
PLoS Biology | www.plosbiology.orgNovember 2004 | Volume 2 | Issue 11 | e3551816
Joint-Specific Knockout of Bmpr1a
Genetic System for Testing the Function of Genes in Joint
To generate a general system capable of specifically
testing genes for functions in skeletal joint development, we
engineered transgenic mice to express Cre recombinase in
developing joints (Figure 1). Gdf5 is a gene strongly
expressed in stripes across developing skeletal elements
during embryonic joint formation. A bacterial artificial
chromosome (BAC) containing the Gdf5 locus was modified
by homologous recombination in bacteria to insert a
cassette encoding Cre-internal ribosome entry site (IRES)-
human placental alkaline phosphatase (hPLAP) into the
translation start site of Gdf5 (Figure 1A). This modified BAC
was then used to make lines of transgenic mice. The
resulting Gdf5-Cre transgenic mice were tested for transgene
expression and Cre recombinase activity by crossing them
to R26R reporter mice that activate the expression of lacZ
after Cre-mediated removal of transcriptional stop sequen-
ces (Soriano 1999). The resulting progeny were analyzed
both for expression of the transgene by assaying HPLAP
activity and for recombination of DNA by assaying LACZ
activity. The progeny from all three lines showed strong
LACZ expression primarily in joints, and in two of three
lines HPLAP expression could also be seen in joint regions.
Interestingly, HPLAP expression in the Gdf5-Cre transgenic
GAC(A) line used for all subsequent breeding experiments
was seen to precede LACZ expression during successive
development of joints in the digits (Figure 1C) (unpublished
data). These experiments clearly demonstrate that the Gdf5-
Cre transgene expresses Cre recombinase and causes DNA
recombination in developing joint regions.
GAC(A) mice were crossed with lacZ ROSA26 Cre reporter
strain (R26R) mice to analyze the pattern of Cre-mediated lacZ
recombination throughout development. Joints in developing
limbs begin forming in a proximal-distal pattern such that the
shoulder joint forms prior to the elbow joint. In addition,
three major stages of early joint development have been
defined by histology as (1) interzone formation, (2) three-layer
interzone formation, and (3) cavitation (Mitrovic 1978).
Consistent with the proximal-distal pattern of joint develop-
ment in the limbs, LACZ activity is seen at embryonic day 12.5
(E12.5) in the more proximal joints, including the shoulder
and knee (unpublished data). By E14.5, LACZ expression is
typically seen in all but the most distal joints of the limbs
(Figure 1B and 1C), but with some variability in both strength
and extent of expression from embryo to embryo. The
strongest-staining embryos often have additional staining in
fingertips (not seen in the E14.5 embryo in Figure 1C, but
clearly detectable in the E13.5 embryo shown in Figure 2).
Sections through developing joints show that LACZ is present
in many cells at the interzone stage (unpublished data).
However, expression of LACZ in nearly 100% of joint cells is
not achieved until the three-layer interzone stage (for
example, in the knee joint at E14.5 or in any of the phalangeal
joints at E16.5 (unpublished data). Within the developing
skeleton, Cre-mediated expression of LACZ remains strikingly
specific to joints throughout development. Furthermore, it is
seen in all the structures of postnatal synovial joints including
the articular cartilage, joint capsule, and synovial membrane
Figure 2. Bmpr1a Is Required for Webbing
Regression and Apoptosis in Specific Re-
gions of the Limb
(A and B) Control E14.5 forelimb (A)
compared to a, E14.5 mutant forelimb
(B) showing webbing between digits 1
and 2 (arrowheads) and extra tissue at
the posterior of digit 5 (arrows).
(C) Gdf5-Cre induced lacZ expression
from R26R in an E13.5 forelimb showing
LACZ staining (blue) in metacarpal-
phalangeal joints, between digits 1 and
2 (arrowhead), and in a region posterior
to digit 5 (arrow).
(D and E) Sections of E14.5 hindlimbs
showing apoptosis visualized by TUNEL
staining (green) and proliferation visual-
ized by staining for histone H3 phos-
phorylation (red). Controls show strong,
uniform TUNEL staining between digits
1 and 2 (D, arrowhead) while mutants
show patchy TUNEL staining inter-
spersed with mitotic cells in similar
regions (E). Scale bar = 200 lm.
(F) Quantitation of TUNEL staining and
mitotic cells in the posterior region of
the fifth digit shows apoptosis is reduced
30% while proliferation is increased
20% (asterisks indicate statistically sig-
(G and H) By E15.5, interdigital tissue
has regressed in controls (G, arrowhead).
In contrast, tissue remains in mutants at
this location, primarily derived from
cells that have undergone Gdf5-Cre-mediated recombination that inactivates Bmpr1a function and activates expression of LACZ (H). Scale bar
= 75 lm.
PLoS Biology | www.plosbiology.org November 2004 | Volume 2 | Issue 11 | e3551817
Joint-Specific Knockout of Bmpr1a
(Figure 1D and 1E) (unpublished data). These patterns are
consistent with the well-established expression of Gdf5 in
interzone regions during embryonic development (Storm and
Kingsley 1996). Adult expression patterns of the Gdf5 gene are
not as well characterized, but Gdf5 expression has previously
been detected in adult articular cartilage using both RT-PCR
and immunocytochemistry (Chang et al. 1994; Erlacher et al.
1998; Bobacz et al. 2002).
Other sites besides limb joints also have Cre-mediated lacZ
expression. Starting at E13.5, LACZ activity is detected in an
anterior and posterior domain of the limb bud (Figure 2C). At
E14.5, LACZ activity is detectable in the developing ear
pinnae, ribs, sternum, tissues in the face, and some regions of
the brain and spinal cord (Figure 1B) (unpublished data). At
birth, LACZ is also expressed in tendons running along the
vertebral column, regions of tendons in the wrist and ankle,
and some tendon insertions (Figure 1D) (unpublished data).
By 5 wk of age, LACZ is also expressed in the hair follicles, ear
cartilage, some cells in the growth plate of the long bones, and
portions of the brain and spinal cord (unpublished data).
Surprisingly, 23 of 63, or 37% of transgenic mice analyzed also
show some degree of wider ‘‘ectopic’’ LACZ expression, which
can extend throughout many different tissues in the animal.
However, sustained expression of the transgene itself, as
assayed by HPLAP activity, is still restricted primarily to joints
in animals that show evidence of more generalized recombi-
nation based on LACZ expression (unpublished data). This
suggests that in a fraction of animals, sporadic expression of
Cre at some time early in development is sufficient to lead to
both ectopic recombination and LACZ expression. While the
fraction of animals with broader recombination patterns
must be tracked and accounted for during experiments, these
animals offer the potential benefit of revealing additional new
functions of target genes that could be subsequently studied
with additional site-specific Cre drivers.
Gdf5-Cre/Bmpr1afloxPAnimals Survive to Adulthood with
Ear, Webbing, and Joint Defects
We next used the Gdf5-Cre system to test the role of BMP
signaling during normal joint development. Gdf5-Cre trans-
genic mice were bred to animals carrying a conditional floxed
allele of the Bmpr1a locus (Mishina et al. 2002), usually in the
presence of the R26R reporter allele to facilitate simulta-
neous visualization of Cre-mediated recombination patterns
(see typical cross in Figure 3). PCR amplification confirmed
that a key exon of the Bmpr1a gene was deleted in mice that
also carried the Gdf5-Cre transgene (unpublished data).
Previous studies have shown that the recombined Bmpr1afloxP
allele mimics a null allele of the Bmpr1a locus when
transmitted through the germline (Mishina et al. 2002). The
Gdf5-Cre/Bmpr1afloxPconditional knockout mice were viable
and survived to adulthood, showing that the Gdf5-Cre driver
can bypass the early embryonic lethality previously reported
in animals with a null mutation in the Bmpr1a locus (Mishina
et al. 1995).
The viable Gdf5-Cre/Bmpr1afloxPmice showed several pheno-
types. First, the conditional knockout mice had shorter ears
that often lay flatter against their heads than controls
(controls 13.1 6 0.1 mm long, n = 38; mutants 11.8 6 0.2
mm, n = 11; p , 0.0001). BMP signaling is known to be
required for growth of the external ear of mice (Kingsley et
al. 1992), and this phenotype likely reflects loss of Bmpr1a
Figure 3. Gdf5-Cre-Mediated Deletion of Bmpr1a
(A) Breeding strategy simultaneously deletes Bmpr1afloxPand allows
visualization of Gdf5-Cre-mediated recombination by lacZ expression
(B–E) 5-week-old mutant and control mice stained with Alcian blue to
mark cartilage and alizarin red to mark bone. (B) Ankle of control
with strong blue staining lining each joint (arrowheads). (C) Ankle of
mutant showing an absence of blue staining in most regions
(arrowheads) and a joint fusion between the central (c) and second
(2) tarsals (arrow). (D) Control and (E) mutant metatarsal/phalangeal
joint which lacks blue staining in articular regions (arrowheads) but
retains staining in the growth plate (asterisks).
(F) Control forelimb.
(G) Mutant forelimb with webbing between the first and second digit
PLoS Biology | www.plosbiology.orgNovember 2004 | Volume 2 | Issue 11 | e3551818
Joint-Specific Knockout of Bmpr1a
function in the fraction of ear cells that express the Gdf5-Cre
transgene. Most mutant mice also showed soft tissue
syndactyly or retention of webbing between the first and
second digits of their feet, a phenotype that was more
frequent and more severe in the forelimbs (201 of 220, or
91%, of forefeet and 109 of 220, or 50%, of hindfeet). Finally,
mutant animals showed obvious skeletal changes in whole-
mount skeletal preparations. At some sites in the ankles,
joints seemed to be missing entirely, with fusion of bones that
would normally be separate. For example, the second distal
tarsal was fused to the central tarsal bone in every conditional
knockout animal examined (18 of 18), a phenotype not
observed in controls (zero of 18) (Figure 3B and 3C). At other
locations, joints had clearly formed but showed dramatic loss
of staining with the cartilage matrix marker Alcian blue
(Figure 3B–3E) (unpublished data). Normal Alcian blue
staining was seen in non-articular regions, such as the
cartilaginous growth plate (Figure 3D and 3E, asterisk). These
data suggest that Bmpr1a function is required for the
formation of specific joints in the ankle region and for either
generation or maintenance of articular cartilage in most
other joints of the limb.
Developmental Origin of Webbing Phenotype
Interdigital mesenchyme is normally eliminated by apop-
tosis during embryonic development, a process that can be
stimulated by BMP beads, inhibited by Noggin, or blocked by
overexpression of dominant-negative BMP receptors (Gar-
cia-Martinez et al. 1993; Yokouchi et al. 1996; Zou and
Niswander 1996; Guha et al. 2002). Limbs of Gdf5-Cre/
Bmpr1afloxPmutant embryos showed obvious retention of
interdigital webbing between the first and second, but not
other, digits of E14.5 forelimbs (Figure 2A and 2B), a pattern
that corresponds to the presence or absence of webbing seen
in the adult limb. They also showed excess tissue on the
posterior margin of the fifth digit (Figure 2B, arrow).
Analysis of LACZ expression in Gdf5-Cre/R26R reporter
embryos showed that Cre-mediated recombination has
occurred by E13.5 in the metacarpal-phalangeal joints, and
in the interdigital region between the first and second, but
not other, digits. In addition, a domain of recombination
and expression of LACZ is also reproducibly seen in the
posterior half of the fifth digit (Figure 2C). Terminal
deoxynucleotidyl transferase–mediated deoxyuridine tri-
phosphate nick end labeling (TUNEL) staining of interdigital
mesenchyme between the first and second digits (Figure 2D
and 2E) and the fifth digit flanking mesenchyme showed a
decreased number of dying cells in the regions where excess
tissue is retained in the mutant limbs. Numbers of
phosphorylated histone H3-labeled proliferating cells were
also elevated in these regions (Figure 2F). Most cells found in
the webbed region between the first and second digits at
E15.5 strongly expressed LACZ in Gdf5-Cre/Bmpr1afloxPmu-
tant embryos (Figure 2H). These data suggest that regional
loss of BMPR1A receptor signaling blocks programmed cell
death in interdigital mesenchyme, and that the recombined
cells survive and proliferate in the absence of BMPR1A
Failure of Early Joint Formation in Ankle Regions
The Bmpr1a gene is expressed in the interzone region of
developing joints at E13.5 (Baur et al. 2000). In situ
Figure 4. Bmpr1a Is Expressed in Joints
and Is Required for Continued Joint For-
mation in the Ankle Region
(A) Diagram of ankle bones from a wild-
type mouse; bones fusing in mutant are
colored red. Roman numerals II–IV,
metatarsals; 2, 3, and 4/5, distal row of
tarsal bones; c, central tarsal bone; ta,
talus; ca, calcaneus.
(B and C) In situ hybridization at E15.5
showing that Bmpr1a is expressed in
ankle joint interzones (B, arrowheads)
and in the forming articular regions of
the phalangeal joints (C, arrowheads).
(D) Near adjacent section to (C) showing
Gdf5-Cre induced LACZ expression from
R26R in the forming joints of the digits
(E–J) Marker gene expression and R26R
LACZ staining patterns on near adjacent
sections of control and mutant embryos.
In control mice at E15.5 ankle joints are
clearly delineated as regions that have
down-regulated Col2 (E), express Gdf5
throughout (F), and express LACZ in
most cells (G; white arrowheads and
black arrows). In mutant embryos at the
same stage, joint formation is incom-
plete. Faint Col2 expression can be seen
connecting a medial region of tarsal 2
with metatarsal II (H, white arrowhead),
and Gdf5 expression does not extend all
the way across the joint at this location
(I, white arrowhead). Between tarsals c
and 2, mutants express Col2 across the normal joint-forming region (H, black arrow) and lack expression of Gdf5 at sites where skeletal fusions
are observed (I, black arrow and bracket). (J) Scale bar = 100 lm.
PLoS Biology | www.plosbiology.orgNovember 2004 | Volume 2 | Issue 11 | e3551819
Joint-Specific Knockout of Bmpr1a
hybridization showed that the gene is also expressed in the
interzones of ankle joints and prospective articular cartilage
regions of digit joints at E15.5 (Figure 4). LACZ staining
indicated that Cre-mediated recombination begins to occur
in ankle joints around E14.5, and is extensive by E15.5
(Figure 4G and 4J) (unpublished data). In the ankle joint
regions that were obviously fused in postnatal mutant
animals, alterations in early joint marker expression could
also be seen by E15.5. At this stage, the Gdf5 gene is normally
expressed in stripes that mark the sites of joint formation
(Figure 4F), and the gene for the major collagen protein of
cartilage matrix (Col2a1) is down-regulated in the interzone
region (Figure 4E). In contrast, Col2a1 staining extended
completely through the joint region between the second and
central tarsal of Gdf5-Cre/Bmpr1afloxPmutants (Figure 4H,
black arrow), and Gdf5 expression was seen only as a small
notch extending into where the joint should be forming
(Figure 4I, bracket). These data suggest that the fusions seen
between ankle bones in postnatal mutant skeletons are the
result of incomplete segmentation of skeletal precursors
during embryonic development, a defect confined to some
locations in the ankle.
Failure to Maintain Articular Cartilage in Other Joints
In most joints of Bmpr1a conditional knockout mice,
embryonic segmentation of skeletal precursors occurred
normally. Although Gdf5-Cre-mediated recombination was
seen as early as E13.5 in digit interzone regions (see Figure
2C), no changes in cell death or cell proliferation could be
seen in the metacarpal-phalangeal or metatarsal-phalangeal
joints at E13.5 or E14.5 (unpublished data). Similarly,
although clear LACZ expression was seen by E15.5 in
interphalangeal joints and periarticular regions (Figure 4D),
no difference in morphology or expression of Col2a1, Gdf5, or
Bmpr1b was seen in the articular regions of the phalanges at
these stages (unpublished data).
At birth, digit joints were generally indistinguishable from
those in control animals; chondrocytes were abundant in
articular regions and were surrounded by typical cartilage
matrix with normal staining by Safranin O, a histological
stain for proteoglycans (Figure 5). At this stage, both wild-
type and mutant cells in articular regions also expressed high
levels of Col2a1 and Aggrecan (Agg), the genes encoding the
major structural proteins of cartilage matrix (Figure 5B and
5G) (unpublished data). No alterations in cellular apoptosis or
proliferation were observed (unpublished data).
To determine whether articular cells were properly
specified in mutants, we also analyzed expression of Ma-
trilin-4 (Mat4), a gene expressed specifically in the periartic-
ular and perichondral regions of developing joints (Klatt et
al. 2001). In both control and mutant animals, transcription
of Mat4 was clearly detectable in the articular cartilage layers
of newborn joints (Figure 5D and 5I). In all experiments,
expression of LACZ throughout articular regions indicated
Figure 5. Bmpr1a Is Required to Maintain
Expression of ECM Components in Articu-
In situ hybridization or LACZ staining
on near adjacent sections of metacarpal-
phalangeal joints (A–C and F–H) and the
tarsal 2-metatarsal II joint (D–E and I–J)
of P0 mice. At birth, articular cartilage of
controls (A–E) and mutants (F–J) appears
similar by Safranin O staining (A and F),
and Col2 expression (B, G). Mat4 expres-
sion confirms that articular cartilage is
initially specified in mutants (D andI,
brackets). LACZ expression confirms
Cre-mediated recombination has oc-
curred in articular cartilage (C, H, E,
and J). (K–T) Near adjacent sections of
the metacarpal-phalangeal joints of P14
mice. Two weeks after birth, articular
cartilage of controls stains with pericel-
lular Safranin O (orange staining, K), and
expresses Col2 (L), Agg (M), and SOX9
(N). In contrast, mutant articular cells
are smaller and more densely packed,
lack pericellular Safranin O staining (P),
have reduced expression of Col2 (Q) and
Agg (R), but retain normal levels of SOX9
protein (S, brackets; dashed line marks
faint edges of articular surfaces). LACZ
expression confirms Cre-mediated re-
combination has occurred in articular
cells (O ansd T, brackets). (A and K) Scale
bar = 75 lm.
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Joint-Specific Knockout of Bmpr1a
that Cre-mediated recombination had occurred throughout
the articular regions (Figure 5C, 5H, 5E, and 5J). The normal
histological appearance, staining properties, and marker gene
expression patterns suggest that Bmpr1a is not required for
the initial formation or specification of articular cartilage.
By 1 wk after birth, obvious differences began to be
detected in the articular regions of mutant animals. The
expression of Col2a1 was reduced throughout the articular
surfaces of the carpals, metacarpals, and phalanges of the
forefeet (unpublished data). Less severe reductions were also
seen in articular cells of tarsals and metatarsals in the
hindfeet (unpublished data). By 2 wk of age, Col2a1 expression
was reduced in most cells of the articular region (Figure 5L
and 5Q), accompanied by markedly reduced Safranin O
staining (Figure 5K and 5P), and decreased expression of Agg
and two genes normally expressed in more mature articular
cartilage cells, Collagen 3 (Col3a1) and Collagen 10 (Col10a1)
(Figure 5M and 5R) (unpublished data) (Eyre 2002). Inhibition
of BMP signaling in cultured chondrocytes has previously
been reported to induce Collagen 1 (Col1a1) expression,
increase proliferation, and result in cells with flattened,
fibroblast-like morphology (Enomoto-Iwamoto et al. 1998).
However, we saw no increase in the expression of Col1a1 in
mutant articular cartilage, and no proliferation was detected
in articular cells of either mutant or control animals
(unpublished data). While recombined LACZ marker expres-
sion was detected in most articular cartilage cells, it was also
observed in scattered subarticular chondrocytes, growth plate
chondrocytes, and osteoblasts (Figure 5O and 5T) (unpub-
lished data). Although this implies that BMP signaling was
defective in multiple cell types, the observed defects were
confined to the articular cartilage. For example, Osteocalcin
and Col1a1 expression appeared normal in osteoblasts
(unpublished data). Together, these data suggest that
BMPR1A activity is required in postnatal joint articular
cartilage to maintain expression of many genes encoding
structural components of cartilage matrix.
Previousstudieshaveshown thatSox9isrequired fornormal
cartilage differentiation, for expression of cartilage extrac-
ellular matrix (ECM) genes including Agg, and is a direct
transcriptional regulator of the key cartilage matrix gene
et al. 2000). Notably, despite reduced expression of many
cartilage matrix marker genes in Bmpr1a mutant mice, the
SOX9 protein was present at normal levels in articular regions
at all stages examined, including newborn, 2-wk-old, 7-wk-old,
and 9-mo-old mice (Figure 5N and 5S) (unpublished data).
Synovial Hypertrophy, Cartilage Erosion, and Accelerated
synovial membrane in the joint capsule of some joints,
particularly in the ankle region. In the most severely affected
joints, the expanded synovial membrane grew into the joint
space and was associated with obvious loss or erosion of the
articular cartilage (Figure 6A and 6B, asterisks, arrows).
Accelerated cartilage maturation and increased expression
of Col10a1 was frequently seen in the chondrocytes underlying
the articular erosions (Figure 6C and 6D, brackets) (unpub-
lished data). Interestingly, the regions of increased Col10a1
expression did not correspond to the regions that had
undergone Cre-mediated recombination. Instead, increased
expression of Col10a1 was seen in a zone of largely LACZ-
negative cells stretching from the cartilage adjacent to the
ossification front (where Col10a1 is normally expressed in
maturing cartilage cells), toward the regions where surface
articular cartilage was severely eroded or missing (Figure 6A
and6B,arrowheads). Previous studies suggestthat parathyroid
hormone-related protein, a diffusible signal made in the
articular surface, may normally inhibit maturation of under-
lying cartilage (Vortkamp et al. 1996; Weir et al. 1996). Local
loss of the articular surface could remove this inhibition and
lead to a cell-nonautonomous acceleration of maturation in
chondrocytes underlying points of articular erosion.
This synovial hypertrophy is associated with increased
numbers of mononuclear cells resembling synoviocytes or
macrophages, cell types that are difficult to distinguish even
with surface markers at early postnatal stages. However, no
Figure 6. Synovial Membrane Expansion, Articular Surface Erosion, and
Accelerated Maturation of Underlying Cartilage in Ankles of Bmpr1a
Near adjacent sections from the tarsal 2-metatarsal II joint of 7-d-old
mice. (A and B) LACZ staining (blue) shows Cre-mediated recombi-
nation is largely restricted to articular (arrowheads) and synovial cells
(asterisks) in both controls and mutants. (C and D) In situ hybrid-
ization shows Col10 expression expands in mutants toward regions of
synovial membrane expansion and articular surface erosion (brackets
and arrows). This may be a cell nonautonomous effect of joint
damage, since the LACZ expressing cells at the articular surface do
not show upregulation of Col10 (arrowheads) and the region of
expanded Col10 expression is largely made up of cells that have not
undergone Cre-mediated recombination. Note the formation of a
cartilaginous bridge along the joint capsule of the mutant where joint
formation is disrupted at earlier stages (B, white arrowhead, and
Figure 3, white arrowheads). (A) Scale bar = 75 lm.
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Joint-Specific Knockout of Bmpr1a
neutrophils were observed, suggesting that there is little
inflammation. At later stages synovial hypertrophy is re-
duced. Further work will be needed to determine whether
synovial development is regulated by BMP signaling, or
whether the synovium becomes enlarged as a response to
nearby skeletal malformations (such as fusion of the second
and central tarsals or defects in the articular cartilage).
Noninflammatory Degeneration of Articular Cartilage in
Digit and Knee Joints
Outside of the ankle region, little or no evidence was seen
for expansion of the synovial membrane. Instead, mutant
mice showed histological signs of osteoarthritis, such as
fibrillation of the articular surface (Figure 7). As previously
seen in 1- and 2-wk-old animals, Safranin O staining and Agg
and Col10 expression were all reduced in mutant articular
regions of the forefeet and hindfeet by 7 wk of age, and the
beginning signs of cartilage loss were observed (unpublished
data). By 9 mo of age, many regions of articular cartilage were
completely missing or extremely fibrillated, leaving regions of
exposed bone on the surface (Figure 7A–7D). No alterations
were seen in the expression of Osteocalcin, Col1a1, or matrix
metalloprotease-13 at either 7 wk or 9 mo.
The major weight-bearing joint of the hindlimb, the knee,
showed changes that closely paralleled that seen in the foot
joints. All markers of cartilage matrix looked similar to
controls at E16.5, suggesting that early stages of joint
formation were not disrupted (unpublished data). By post-
natal day 7, Safranin O staining and Col2a1 and Agg
expression were clearly reduced in the mutant, despite
continued expression of Sox9 (unpublished data). The overall
shape of mutant knee skeletal elements appeared similar to
controls, although the fibrocartilaginous meniscus that re-
sides between the femur and tibia appeared much less dense
in mutants at E16.5. Some cartilage formed in the meniscus
region, but the size of these elements was greatly reduced and
contained abundant cells with fibrous, noncartilaginous
appearance (unpublished data). This reduction of the
meniscus can also be seen in sections from 7-wk- and 9-mo-
old animals (Figure 7E, 7H, 7K, and 7N, arrows).
At 7 wk of age the normally domed tibial epiphysis was
flattened and depressed in the knees of mutant animals,
markedly reducing the distance between the growth plate and
articular surface (Figure 7E and 7H, vertical bar). Articular
cartilage was also thinner than in control animals, showed
nearly complete absence of Safranin O staining, and was
either acellular or beginning to fibrillate in many regions
(Figure 7F and 7I). The few large Safranin O-stained cells still
apparent in mutant articular regions appeared to correspond
in position to rare LACZ-negative cells in adjacent sections,
suggesting that Bmpr1a is required cell-autonomously in
articular cartilage (Figure 7I and 7J, white arrowheads). By 9
mo, large areas of mutant knees were devoid of articular cells,
and the bones of the femur and tibia appeared to rub directly
Figure 7. Loss of Bmpr1a Signaling Leads
to Articular Cartilage Fibrillation and De-
generation in Digits and Knees of Aging
(A–D) Near adjacent sections of meta-
tarsal-phalangeal joints from 9 month
old mice. Articular cartilage of controls
is complete and stains strongly with
Safranin O (A, orange stain). In contrast,
articular cells of mutants are severely
fibrillated or absent with much reduced
staining of Safranin O (C, arrowheads).
LACZ expression confirms Cre-mediated
recombination has occurred in articular
cells (B and D).
(E–P) Sagittal sections through knee
joints of 7-wk- (E–J) or 9-mo-old animals
(K–P); fe, femur; ti, tibia; gp, growth
plate. Seven weeks after birth, the height
of the tibial epiphysis is reduced in
mutants (E and H, bars), and their
articular layer stains poorly with Safra-
nin O, is fibrillated, and is strikingly
thinner (F and I, black arrowhead, and
brackets). Near adjacent sections with
LACZ staining confirm Cre-mediated
recombination has occurred in articular
cells (G and J). Note that in mutants,
LACZ is absent in cells adjacent to those
that do stain with Safranin O, suggesting
Bmpr1a may act cell autonomously (I and
J, white arrowheads). At 9 mo old, the
mutant tibial epiphysis is extremely thin
(K and N, bars), and the articular layer is
completely absent, leaving bone to rub
directly on bone (L and O, bracket).
LACZ staining shows Cre-mediated re-
combination occurred in articular cells
of controls (M) and in some remaining skeletal tissue of mutants (P). Also note aberrantly formed meniscal cartilage in mutants (E, H, K, and N,
arrows), and increased sclerosis in mutant epiphyses (E, H, K, and N, asterisks).
(A and K) Scale bar = 50 lm; (I) scale bar = 300 lm.
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Joint-Specific Knockout of Bmpr1a
against each other. Furthermore, the epiphysis of the tibia
was extremely depressed, to the point that growth plate
cartilage was almost exposed through the surface of the bone
(Figure 7K, 7L, 7N, and 7O). In addition, mutants at 7 wk and
9 mo showed subchondral sclerosis, especially in the epiphysis
of the femur (Figure 7E, 7H, 7K, and 7N, asterisks). While
subchondral sclerosis is commonly seen in cases of osteo-
arthritis, it is unclear in this case whether the sclerosis is
mainly a response of bone formation to compensate for
decreased articular cartilage, or whether it is the effect of loss
of Bmpr1a signaling in some LACZ-positive cells that are also
observed in these regions (unpublished data).
functional impairments in both grasping ability and range of
motion in mutant animals. Gdf5-Cre/Bmpr1afloxPmutant ani-
mals showed a highly significantly reduced ability to grasp and
remain suspended on a slender rod (mean suspension time:
controls 38 6 6 s, n = 39; mutants 6 6 3 s, n = 11; p , 0.0001).
Mutant mice also showed a clear decrease in the maximum
range of mobility of two different joints in the digits, as
assayed by passive manipulation (MT/P1 joint: controls 100 6
08, n = 26; mutants 82 6 38, n = 8; p , 0.0003; P1/P2 joint:
controls 152 6 18, n = 23; mutants 140 6 58, n = 6; p , 0.05).
The structural, histological, marker gene expression, and
functional changes in mutant mice demonstrate that BMPR1A
is required for normal postnatal maintenance of articular
Previous studies suggest that BMP signaling is involved in a
large number of developmental events. Many of these events
occur early in embryogenesis, and complete inactivation of
BMP receptors causes death by E9.5 (Mishina et al. 1995). The
Gdf5-Cre recombination system bypasses the early embryonic
lethality of Bmpr1a mutations, and provides important new
information about the role of this receptor in limb and
The three major limb phenotypes revealed by eliminating
Bmpr1a with Gdf5-driven Cre include webbing between digits,
lack of joint formation at specific locations in the ankle, and
failure to maintain articular cartilage after birth, resulting in
severe arthritis. Previous studies have shown that manipu-
lation of BMP signaling alters interdigital apoptosis during
development of the limb, but no experiment has identified a
specific member of the BMP signaling pathway that is
required for this process (Yokouchi et al. 1996; Zou and
Niswander 1996; Zou et al. 1997; Guha et al. 2002). Our new
loss-of-function data confirm that BMP signaling is required
for interdigital apoptosis and suggests that Bmpr1a is a critical
component for mediating this signal.
At some sites, loss of Bmpr1a function leads to a defect in
the early stages of joint formation, resulting in a complete
failure to form a joint and fusion of bones in the ankle.
Mutations in two different ligands in the BMP family, Gdf5
and Gdf6, the Bmpr1b receptor, and in the human Noggin locus
(Storm and Kingsley 1996; Gong et al. 1999; Baur et al. 2000;
Yi et al. 2000; Settle et al. 2003) also produce defects in joint
formation at specific locations in the limbs. The joint defects
associated with multiple components of the BMP pathway
provide strong evidence that BMP signaling is required for
early stages of joint formation at some anatomical locations.
Most joints still form normally when Bmpr1a is knocked out
in Gdf5 expression domains. The lack of joint fusions outside
the ankle region could be due to differences in requirement
for BMP signaling in different joints, to compensating
expression of other BMP receptors outside the ankles, or to
differences in the detailed timing of Gdf5-Cre stimulated gene
inactivation in ankles and other joint regions. Comparison of
the expression of the HPLAP marker (driven directly by Gdf5
control elements) and the R26R LACZ marker (expressed
following Gdf5-Cre recombination) suggests that recombina-
tion-stimulated changes in gene expression may be delayed
for a 0.5–1 d in the digit region (see Figure 1C). In addition,
levels of Bmpr1a mRNA and protein may persist for some
time following Gdf5-Cre stimulated recombination, making it
possible to bypass an early requirement for Bmpr1a in joint
formation at some locations.
Following the decay of Bmpr1a mRNA and protein, the
Gdf5-Cre strategy should result in permanent inactivation of
Bmpr1a function in recombined cells. This system thus
provides one of the first strong genetic tests of Bmpr1a
function at later stages of joint development. Despite the
normal appearance of articular regions and gene expression
immediately after birth, Bmpr1a-deficient animals are unable
to maintain the normal differentiated state of articular
cartilage as they continue to develop and age. These results
suggest that BMP receptor signaling is essential for continued
health and integrity of articular cartilage in the postnatal
Articular cartilage is a key component of synovial joints
and is one of the few regions in the skeleton where cartilage is
maintained into adulthood. Despite the importance of
articular cartilage in joint health and mobility, little is known
about the factors that create and maintain it in thin layers at
the ends of long bones. In our experiments, articular cartilage
lacking Bmpr1a retains some normal characteristics, in that it
maintains a very low proliferation rate, does not express
Col1a1, and continues to express SOX9, a major transcription
factor regulating expression of structural components of
cartilage matrix. However, several of the most prominent
structural components of cartilage matrix fail to be main-
tained in mutant animals, resulting in decreased synthesis of
Col2a1, Agg, and proteoglycans. Therefore, BMPR1A appears
to maintain articular cartilage primarily through inducing
expression of key ECM components.
It is interesting that the SOX9 transcription factor
continues to be expressed in mutant cartilage despite loss
of Col2a1, a direct target of this transcription factor (Bell et al.
1997; Lefebvre et al. 1997). Previous studies suggest that
SOX9 activity can be modified by protein kinase A (PKA)-
dependent protein phosphorylation, or by coexpression of
two related proteins, L-SOX5 and SOX6 (Lefebvre et al. 1998;
Huang et al. 2000). In addition, close examination of the
order of genes induced during chicken digit formation
reveals that Sox9 turns on first, followed by Bmpr1b with L-
Sox5, and then Sox6 and the cartilage matrix structural
components Col2a1 and Agg (Chimal-Monroy et al. 2003).
These results, together with the altered pattern of gene
expression seen in our Bmpr1a-deficient mice, suggest that
BMPR1A signaling may normally act to stimulate SOX9 by
post-translational protein modification, or to induce L-Sox5
or Sox6 in cartilage to maintain expression of ECM
components. These models are consistent with the ability of
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Joint-Specific Knockout of Bmpr1a
BMP2 to both increase PKA activity and induce expression of
Sox6 in tissue culture cells (Lee and Chuong 1997; Fernandez-
Lloris et al. 2003). Although we have tried to monitor the
expression of L-Sox5 or Sox6 in postnatal articular cartilage,
and test the phosphorylation state of SOX9 using previously
described reagents (Lefebvre et al. 1998; Huang et al. 2000),
we have been unable to obtain specific signal at the late
postnatal stages required (unpublished data). Furthermore,
null mutations in L-Sox5 or Sox-6 cause lethality at or soon
after birth, and no effect on cartilage maintenance has been
reported (Smits et al. 2001). However, it seems likely that
these or other processes regulated by BMP signaling
cooperate with SOX9 to induce target genes in articular
Mutation of Smad3 or expression of dominant negative
transforming growth factor b (TGF-b) type II receptor also
disrupts normal articular cartilage maintenance (Serra et al.
1997; Yang et al. 2001). Both manipulations should disrupt
TGFb rather than BMP signaling, and both manipulations
cause articular cartilage to hypertrophy and be replaced by
bone. In contrast, our analysis of Bmpr1a mutant articular
cartilage showed a loss of ECM components, but no signs of
hypertrophy or bone replacement. Therefore, TGFb and BMP
signaling are playing distinct but necessary roles to maintain
Although BMPs were originally isolated on the basis of
their ability to induce ectopic bone formation, their presence
in articular cartilage and strong effect on cartilage formation
has stimulated interest in using them to repair or regenerate
cartilage defects in adult animals (Chang et al. 1994; Erlacher
et al. 1998; Edwards and Francis-West 2001; Chubinskaya and
Kuettner 2003). The failure to maintain articular cartilage in
the absence of normal BMPR1A function suggests that ligands
or small molecule agonists that interact specifically with this
receptor subtype may be particularly good candidates for
designing new approaches to maintain or heal articular
cartilage at postnatal stages.
Lack of Bmpr1a function in articular cartilage results in
severe fibrillation of the articular surface and loss of joint
mobility. The development of severe arthritis symptoms in
Bmpr1a-deficient mice raises the possibility that defects in
BMP signaling also contribute to human joint disease.
Osteoarthritis is known to have a significant genetic
component, but it likely involves multiple genetic factors
that have been difficult to identify (Spector et al. 1996; Felson
et al. 1998; Hirsch et al. 1998). Humans that are heterozygous
for loss-of-function mutations in BMPR1A are known to be at
risk for juvenile polyposis (Howe et al. 2001; Zhou et al. 2001),
but the risk of osteoarthritis for these people has not been
reported. However, the control mice used in this study were
heterozygous for a null allele of Bmpr1a, and they showed
little sign of osteoarthritis even late in life. Several chromo-
some regions have been previously linked to arthritis
phenotypes in humans using either association studies in
populations or linkage studies in families. It is interesting to
note that several of these chromosome regions contain genes
encoding different members of the BMP signaling pathway,
including the BMP5 gene on human chromosome 6p12
(Loughlin et al. 2002), the MADH1 gene on human chromo-
some 4q26–4q31 (Leppavuori et al. 1999; Kent et al. 2002),
and the BMPR2 receptor on human chromosome 2q33
(Wright et al. 1996). The complex nature of human osteo-
arthritis suggests that interactions between multiple genes
may be involved in modifying susceptibility to the disease.
The inclusion of genetic markers near BMP signaling
components may help identify additional osteoarthritis
susceptibility loci and facilitate the search for causative
Development and disease processes in synovial joints have
been difficult to study genetically, because synovial joints are
generated and function at relatively late stages of vertebrate
development. The Gdf5-Cre system provides a new method for
restricting gene expression or inactivation primarily to
articular regions, thus avoiding the pleiotropic functions of
many genes in other tissues. Depending on the configuration
of the floxed target gene, this system can be used to either
activate the expression of a gene primarily in developing
joints (ssee Figure 1B–1D), or to inactivate gene function in
articular regions (see Figure 3). Additional studies with this
system should greatly enhance our knowledge of the develop-
ment, function, and disease mechanisms of joints, and may
bring us closer to better prevention and treatment of joint
Materials and Methods
Generation of Gdf5-Cre transgenic mice. A mouse 129x1/SvJ BAC
library (Invitrogen) was screened to identify a 140-kb BAC from the
Gdf5 locus. This BAC wasmodified using a homologous recombination
system in E. coli (Yang et al. 1997) to place nuclear-localized Cre
hPLAP (from plasmid 1726, gift of Oliver Bogler) directly behind the
removedand no functional GDF5proteinis predicted to be produced.
The 59 homology arm was subcloned from a PCR product tailed with
XhoI and Bsp120I restriction sites that contains 781 bp of 59 genomic
59-CTGTCTCGAGATGAGGTGGAGGTGAAGACCCC-39; reverse 59-
GTTTGGGCCCATCCTCTGGCCAGCCGCTG-39). Cre was subcloned
from a 1.1-kb Bsp120I/EcoRI fragment of pML78. IRES hPLAP was
subcloned from a 2.1-kb PCR product tailed with EcoRI and SpeI sites
that contains the hPLAP translation stop site (forward primer 59-
TAGTGG-39). The 39 homology arm was subcloned from a 0.8-kb
PCR product amplified from a 0.9-kb XhoI Gdf5 genomic subclone
containing part of the first exon and downstream intron. The forward
the reverse primer is from the T7 promoter of the vector containing
the 0.9-kb subclone and flanks the intronic XhoI site (forward primer
59-CTAAACTAGTCACCAGCTTTATTGACAAAGG-39; reverse 59-
GATTTCTAGAGTAATACGACTCACTATAGGGC-39). The targeting
construct was built and verified in pBSSK (Stratagene, La Jolla,
California, United States), then digested with XhoI and subcloned into
pSV1, the vector used for homologous recombination (Yang et al.
1997). Southern blotting, PCR, and DNA sequence analysis confirmed
Before the modified BAC was injected to produce transgenic
animals, a loxP site present in the BAC vector, pBeloBAC11, was
removed to prevent the addition of undesired Cre target sites into the
genome. To do this, BAC DNA was prepared by CsCl separation,
digested with NotI to free the insert from the vector, and size-
fractionated over a sucrose gradient.Aliquotsof fractions wererun on
a pulse-field gel and Southern blotted using vector-specific DNA as a
vector DNA were dialyzed in microinjection buffer (10 mM Tris [pH
7.4] with 0.15 mM EDTA [pH 8.0]) using Centriprep-30 concentrators
(Millipore, Billerica,Massachusetts, UnitedStates).Thispurifiedinsert
DNA was adjusted to 1 ng/ll and injected into the pronucleus of
fertilized eggs from FVB/N mice by the Stanford Transgenic Facility.
Transgenic founder mice were identified by PCR using Cre-specific
primers 59-GCCTGCATTACCGGTCGATGCAACGA-39 and 59-
GTGGCAGATGGCGCGGCAACACCATT-39, which amplify a 725-bp
product, and were assessed for absence of BAC vector using vector-
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Joint-Specific Knockout of Bmpr1a
specific primers 59-CGGAGTCTGATGCGGTTGCGATG-39 and 59-
AGTGCTGTTCCCTGGTGCTTCCTC-39, which amplify a 465-bp
product.Three lines ofGdf5-Cre micewereestablishedandmaintained
on the FVB background. Matings with R26R Cre-inducible LACZ
reporter mice (Soriano 1999) were used to test for Cre activity.
Staining for LACZ and HPLAP on whole embryos or sections of
embryos was accomplished following established protocols (Lobe et
al. 1999). The red LACZ substrate (see Figure 1E) is 6-chloro-3-
indoxyl-beta-D-galactopyranoside (Biosynth International, Naper-
ville, Illinois, United States).
General characterization of Bmpr1a mutant mice. Bmpr1a null and
floxed alleles (Ahn et al. 2001; Mishina et al. 2002) were obtained on a
mixed 129 and C57BL/6 background and maintained by random
breeding. Mice carrying the null and floxed alleles were typically
mated to Gdf5-Cre mice as shown in Figure 3. The resulting mice are
on a mixed 129; C57Bl/6; FVB/N background, with both controls and
mutant animals generated as littermates from the same matings.
Whole-mount skeletal preparations were made from 34- to 36-d-old
mice (Lufkin et al. 1992). Pairs of ears from euthanized 6-mo-old
animals were removed, pinned, photographed, projected, and
measured from the base of the curve formed between the tragus
and antitragus to the farthest point at the edge of the pinnae.
Grasping ability in 6-mo-old mice was measured by placing animals
on a slender rod and timing how long they could remain suspended
on the rod, to a maximum time allowed of 2 min. Data from five
consecutive trials for each mouse were averaged. Range of motion
assays were conducted on the MT/P1 and P1/P2 joints of the second
hindlimb digit from euthanized 18-wk-old animals. Forceps were used
to bend the joint to its natural stopping position, and the resulting
angle was measured to the nearest 108 under 12.53 magnification
using a 3608 reticule. Analysis described in this section occurred on
animals lacking R26R. Control mice included all nonmutant
genotypes generated by Parent 1 being heterozygous for Gdf5-Cre
and Bmpr1anulland Parent 2 being heterozygous for Bmpr1afloxP(see
Figure 3). All statistical analysis used the Student’s t-test or Welch’s t-
test, and values listed are mean 6 standard error of the mean.
Cell death and proliferation assays. Limbs from mutant and
control animals at E13.5 and E14.5 were dissected and frozen in OCT
(Sakura Finetek,Torrence, CA, United States). Cryosections of tissue
were assayed by TUNEL using the In Situ Cell Death Detection Kit,
Fluorescein (Roche, Basel, Switzerland). Following TUNEL, slides
were washed in PBS, blocked with PBSþ0.05% Tween-20þ5% goat
serum, washed again, and incubated with a 1:200 dilution of a rabbit
anti-phospho-histone-H3 antibody called Mitosis Marker (Upstate
Biotechnology, Lake Placid, New York, United States) to identify cells
in mitosis. Cy3-labeled anti-rabbit secondary antibody was used to
detect the antibody. Cell nuclei were labeled with DAPI, and slides
were mounted in Vectamount (Vector Laboratories, Burlingame,
California, United States) and visualized at 1003 magnification. The
area of selected anatomical sites were measured, and the number of
TUNEL-labeled nuclear fragments and the number of Cy3-labeled
nuclei were counted from three 10-lm sections spanning 50 lm, from
three control and three mutant animals. The number of labeled cells
in the metacarpal-phalangeal and metatarsal-phalangeal joints was
counted in a 290 lm3365 lm rectangle placed around the center of
the joint. The posterior region of the fifth digit was defined by
drawing a line from the tip of the digit down 2.15 mm and across to
the lateral edge of the tissue. For this analysis, the R26R Cre reporter
was not present.
Histology and histochemistry. Tissue from animals ranging from
stages E14.5 to P14 was prepared for analysis by fixing in 4%
paraformaldehyde (PFA) in PBS for 45 min to 4 h depending on the
stage; washing three times in PBS, once in PBSþ15% sucrose for 1 h,
and once in PBSþ30% sucrose for 2 h to overnight depending on the
stage; and then freezing in OCT. Tissue from animals aged 7 wk to 9
mo was processed similarly to earlier stages except that it was
decalcified in 0.5 M EDTA (pH 7.4) for 4 d prior to incubating in
sucrose. All solutions were prechilled and used at 4 8C with agitation,
and skin from tissues of P0 or older mice was lacerated or removed
prior to processing.
Tissue was then cryosectioned at 12 lm and processed. Staining of
sections with Safranin O, Fast Green, and Harris’ hematoxylin was
carried out using standard histological procedures. Detection of
LACZ activity with X-Gal was performed as described (Lobe et al.
1999) and was followed by refixing in 4% PFA, rinsing with deionized
water, counterstaining with Nuclear Fast Red (Vector Labs), rinsing
with water again, and then mounting in Aquamount (Lerner Labs,
Pittsburgh, Pennsylvania, United States).
RNA in situ hybridization was performed as described (Storm and
Kingsley 1996), with the following modifications: (1) Prior to the
acetylation step, sections were incubated with 10–20 lg/ml proteinase
K for 30 s to 7 min at room temperature (depending on the
developmental stage), followed by refixing in 4% PFA and washing
three times in PBS; (2) prehybridization step was skipped, and (3)
embryonic tissue sections used a different color development mix
(Thut et al. 2001). Probes for the following genes have been published
previously: Bmpr1a (Mishina et al. 1995), Col2a1 (Metsaranta et al.
1991), Col10a1 (Apte et al. 1992), Gdf5 (Storm and Kingsley 1996),
Osteocalcin (Celeste et al. 1986), and Sox5 and Sox6 (Lefebvre et al.
1998). The following probe templates were gifts: Agg, Dr. Vicki Rosen,
Genetics Institute; Bmp2 and Bmp4, Arend Sidow, Stanford University;
Col1a1, Bjorn Olsen, Harvard Medical School; Bmpr1b, Col3a1, and
Mat4 probes were made from ESTs with IMAGE clone numbers
5056341, 478480, and 406027, respectively (Invitrogen, Carlsbad,
California, United States).
Sections for immunohistochemistry were fixed in 4% PFA, then
digested with 942–2,000 U/ml type IV-S bovine hyaluronindase
(Sigma, St. Louis, Missouri, United States) in PBS (pH 5) at 37 8C
for 30 min to 2 h depending on the stage. Slides were then washed in
PBS, treated with 0.3% hydrogen peroxide in 100% methanol for 30
min, washed, blocked with PBS þ 0.05% Tween20 þ 5% goat or fetal
bovine serum, washed again, and incubated with primary antibodies
in PBSþ0.05% Tween 20þ1% goat or fetal bovine serum overnight
at 4 8C. Biotin-labeled secondary antibodies (Vector Labs) were
tagged with HRP using the Vectastain Elite ABC kit (Vector Labs)
followed by detection with DAB (Vector Labs). Primary antibodies
and dilutions used were: goat anti-mouse MMP13, 1:100 (Chemicon
International, Temecula, California, United States); rabbit anti-
human SOX9, 1:500 (Morais da Silva et al. 1996); rabbit anti-
phosphorylated-SOX9 (SOX9.P), 1:10–1:250 (Huang et al. 2000).
GenBank (http://www.ncbi.nih.gov/Genbank/) accession numbers for
the genes discussed in this paper are Gdf5 (AC084323) and Bmpr1a
We thank Gail Martin for the Cre construct (plasmid pML78) and
Oliver Bogler for the IRES-hPLAP construct (plasmid 1726); Bjorn
Olsen and Benoit de Crombrugghe for antibodies; the following
individuals for in situ probe templates: Sophie Candille, Arend Sidow
(Bmp2 and Bmp4), Vicki Rosen (Agg), and Bjorn Olsen (Col1a1);
Ve ´ronique Lefebvre for Sox5 and Sox6 probe templates and useful
discussions; Michelle Johnson for help with phenotypic assays on
mice; Dr. Corrine Davis for help in evaluating synovial sections;
Rebecca Rountree for Adobe Photoshop and Illustrator tips; and
members of the Kingsley lab for helpful comments on the manu-
script. This work was supported by an NIH predoctoral training grant
(RR), a postdoctoral fellowship from the Arthritis Foundation (MS),
and grants from the National Institutes of Health (DK). Dr. Kingsley is
an associate investigator of the Howard Hughes Medical Institute.
Conflicts of interest. The authors have declared that no conflicts of
Author contributions. RBR, MS, and DMK conceived and designed
the experiments. RBR and MEM performed the experiments. RBR,
HC, and DMK analyzed the data. MS, HC, VH, and YM contributed
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