RESEARCH ARTICLE
307
Development 134, 307-316 (2007) doi:10.1242/dev.02732
Intraflagellar transport is essential for endochondral bone
formation
Courtney J. Haycraft1,*, Qihong Zhang1,*,†, Buer Song2, Walker S. Jackson3,‡, Peter J. Detloff3, Rosa Serra1 and
Bradley K. Yoder1,§
While cilia are present on most cells in the mammalian body, their functional importance has only recently been discovered. Cilia
formation requires intraflagellar transport (IFT), and mutations disrupting the IFT process result in loss of cilia and mid-gestation
lethality with developmental defects that include polydactyly and abnormal neural tube patterning. The early lethality in IFT
mutants has hindered research efforts to study the role of this organelle at later developmental stages. Thus, to investigate the role
of cilia during limb development, we generated a conditional allele of the IFT protein Ift88 (polaris). Using the Cre-lox system, we
disrupted cilia on different cell populations within the developing limb. While deleting cilia in regions of the limb ectoderm had no
overt effect on patterning, disruption in the mesenchyme resulted in extensive polydactyly with loss of anteroposterior digit
patterning and shortening of the proximodistal axis. The digit patterning abnormalities were associated with aberrant Shh
pathway activity, whereas defects in limb outgrowth were due in part to disruption of Ihh signaling during endochondral bone
formation. In addition, the limbs of mesenchymal cilia mutants have ectopic domains of cells that resemble chondrocytes derived
from the perichondrium, which is not typical of Indian hedgehog mutants. Overall these data provide evidence that IFT is essential
for normal formation of the appendicular skeleton through disruption of multiple signaling pathways.
INTRODUCTION
Cilia are microtubule-based organelles that are expressed on the
surface of most cells in the mammalian body. Intraflagellar transport
(IFT), the process by which cilia are formed and maintained, was
first described in Chlamydomonas, and proteins required for IFT
concentrate at the base of cilia, where they assemble into large
protein complexes called IFT particles (Kozminski et al., 1995;
Piperno and Mead, 1997). The IFT particles are trafficked along the
axoneme by a heterotrimeric kinesin-II and a cytoplasmic dynein in
the anterograde and retrograde directions, respectively. Cilia and
flagella have diverse functions ranging from fluid and cell
movement to mechanosensation and sensory perception (Davenport
and Yoder, 2005; Scholey, 2003).
In mammals, Kif3a is a component of the kinesin-II motor protein
complex required for cilia assembly while Ift88 (also known as
Tg737 or polaris) is a core component of the IFT particle (Cole et
al., 1998; Pazour et al., 2000; Taulman et al., 2001). Mice
homozygous for mutations in Kif3a or any of the IFT proteins
identified to date, including Ift88, die during mid-gestation and have
randomization of the left-right body axis, neural tube closure and
patterning defects, as well as polydactyly (Marszalek et al., 1999;
Murcia et al., 2000; Nonaka et al., 1998; Takeda et al., 1999). The
severe phenotype of homozygous mutants and the expression of cilia
on most cells throughout the body have complicated research
1
Department of Cell Biology, 2Department of Pathology and 3Department of
Biochemistry and Molecular Genetics, University of Alabama at Birmingham,
Birmingham, AL 35294-0005, USA.
*These authors contributed equally to this work
Present address: Department of Pediatrics, University of Iowa, Iowa City, IA, USA
‡
Present address: Whitehead Institute for Biomedical Research, Cambridge, MA,
USA
§
Author for correspondence (e-mail: [email protected])
†
Accepted 7 November 2006
directed at understanding the function of cilia on specific cell types
or during distinct stages of development, as well as their role in
normal tissue function in postnatal life.
Recent work from several groups has shown that disruption of IFT
results in abnormal patterning of the developing murine limb and
neural tube and that this is due to impaired sonic hedgehog (Shh)
signal transduction (Haycraft et al., 2005; Huangfu and Anderson,
2005; Huangfu et al., 2003; Liu et al., 2005). Hedgehog signal
transduction is both positively and negatively regulated, and
disruption of this pathway leads to severe developmental defects
(Huangfu and Anderson, 2006; Ingham and McMahon, 2001). In the
absence of ligand, the pathway is repressed through the inhibition of
the signal transducer smoothened (Smo) by the hedgehog receptor
patched (Ptch1). The Glioma family of transcription factors, Gli1,
Gli2 and Gli3, are the main transducers of signaling. In the absence
of ligand, Gli3 is proteolytically processed to generate a potent
transcriptional repressor (Gli3R) of the pathway (Ding et al., 1999;
Dunaeva et al., 2003; Stone et al., 1999; Wang et al., 2000). Whereas
the major role of Gli3 appears to be repression of target gene
transcription in the absence of ligand, Gli2 is predicted to act as the
main transcriptional activator upon pathway induction (Bai and
Joyner, 2001; Litingtung and Chiang, 2000; Persson et al., 2002;
Ruiz i Altaba, 1999). Unlike Gli2 and Gli3, which are regulated
post-translationally, Gli1 is predicted to act only as a transcriptional
activator after pathway activation (Park et al., 2000).
Normal IFT function is required in the Shh signaling pathway, as
Smo, the Gli transcription factors and Sufu have all been localized
to the cilium axoneme (Corbit et al., 2005; Haycraft et al., 2005;
May et al., 2005). While Sufu and the Gli proteins are found at the
distal tip of cilia, Smo translocation to the cilium axoneme is
induced in response to pathway activation (Corbit et al., 2005;
Haycraft et al., 2005). In mice with congenital loss of Kif3a or Ift
proteins required for anterograde trafficking, such as Ift88, the Shh
signaling pathway remains inactive, despite the fact that the
processing of Gli3 to the repressor form is severely impaired
DEVELOPMENT
KEY WORDS: Cilia, Limb patterning, Hedgehog, Bone development, IFT, Mouse
RESEARCH ARTICLE
(Haycraft et al., 2005; Huangfu and Anderson, 2005; Liu et al.,
2005). In addition, exogenously expressed Gli2 is unable to activate
signaling in cells lacking IFT (Haycraft et al., 2005). By contrast,
Gli1 function is independent of IFT (Haycraft et al., 2005); however,
its expression is dependent on hedgehog pathway activation and thus
is also lost in IFT mutants.
During development and patterning of the mammalian limb,
signaling molecules are secreted from three major signaling centers
(Tickle, 2003). Cells on the apical ectodermal ridge (AER) secrete
fibroblast growth factors (Fgfs) to promote proper proximodistal
outgrowth of the limb, whereas the dorsal and ventral ectoderm
secrete molecules including Wnts and Bmps essential for
dorsoventral patterning. The formation of five digits is regulated by
secretion of signaling molecules from mesenchymal cells in the zone
of polarizing activity (ZPA). The main ligand secreted by cells in the
ZPA is Shh, which acts to promote development of five patterned
digits along the anteroposterior limb bud axis through inhibition of
Gli3 proteolytic processing to generate Gli3R. This leads to the derepression of genes such as the Bmp antagonist gremlin in the
anterior mesenchyme of the limb bud (Litingtung et al., 2002; te
Welscher et al., 2002).
In addition to patterning of the digits in the mammalian limb, the
hedgehog signaling pathway is also required for proper formation of
other tissues, including the long bones of the appendicular skeleton
(Razzaque et al., 2005; St-Jacques et al., 1999; Vortkamp et al.,
1996). The long bones are formed by the aggregation and
differentiation of cells from the lateral plate mesoderm to generate
a cartilage template of the future bone (Erlebacher et al., 1995; Olsen
et al., 2000). Following condensation of cells to generate the
template of the future bone, chondrocytes proliferate to contribute
to the longitudinal growth of the bone. Prehypertrophic
chondrocytes adjacent to the proliferating chondrocytes secrete Ihh,
which is necessary for proper proliferation of the chondrocytes. The
prehypertrophic chondrocytes undergo hypertrophic maturation and
secrete extracellular matrix proteins, which are mineralized to form
the trabecular bone. In Ihh null mice, the chondrocytes show reduced
proliferation as well as premature hypertrophy, leading to severe
shortening of the skeletal elements (Karp et al., 2000; St-Jacques et
al., 1999). While Ihh signaling appears to directly regulate
chondrocyte proliferation, its effects on hypertrophic differentiation
are mediated through induction of PTHrP (PthIh – Mouse Genome
Informatics) expression in the presumptive articular cartilage and
perichondrium (Alvarez et al., 2002; Karp et al., 2000; Vortkamp et
al., 1996).
Ihh secreted from prehypertrophic chondrocytes is also essential
for formation of the bone collar from the perichondrium (Long et al.,
2004; Razzaque et al., 2005; St-Jacques et al., 1999; Vortkamp et al.,
1996). Mesenchymal cells forming the perichondrium surround the
cartilage template and differentiate to generate osteoblasts, which
form the bone collar and contribute to increased diametrical growth
of the bone throughout postnatal life. Ihh signaling in the
perichondrium leads to differentiation of osteoblasts through
induction of canonical Wnt signaling (Hilton et al., 2005; Hu et al.,
2005; Long et al., 2004).
To examine the role of cilia and IFT in the developing limb, we
generated a new conditional mutant allele (Ift88fl) of the IFT protein
Ift88. We flanked essential exons with loxP recombination sites to
disrupt IFT in specific cell types in the mouse using transgenic
strains expressing Cre recombinase. Following Cre expression, the
level of Ift88 protein was severely reduced and cilia were no longer
detected on cells. Surprisingly, in light of the reciprocal signaling
events that occur between Shh-expressing cells of the ZPA, the AER
Development 134 (2)
and ectoderm, the loss of cilia and/or Ift88 on cells of the AER and
ventral ectoderm did not result in major defects in limb outgrowth
or in dorsoventral patterning. By contrast, mice lacking cilia and/or
IFT in the mesenchyme due to expression of Cre recombinase under
control of the Prx1 (Prrx1 – Mouse Genome Informatics) limb
enhancer developed multiple ectopic digits and showed a
progressive loss of Shh signal transduction, preceded by expansion
of gremlin expression in the anterior limb bud mesenchyme.
Additionally, at later stages of limb development, endochondral
bone formation in prx1cre;Ift88fl/n conditional mutants was severely
affected, and Ihh signaling was disrupted. The long bones displayed
defects in chondrocyte differentiation and loss of the bone collar
adjacent to the metaphysis. Surprisingly, ectopic chondrocyte-like
cells were observed between the perichondrium and diaphysis.
Overall, these results suggest that cilia and/or IFT are required in the
mesenchyme at early stages of limb morphogenesis for Shh
signaling to determine anteroposterior patterning of the digits and at
later stages for proper endochondral bone formation.
MATERIALS AND METHODS
Mouse strains
All mice were maintained on a mixed genetic background. Transgenic
prx1cre and msx2cre mice have been previously described, and both
transgenic strains were genotyped by PCR using primers for the Cre coding
region (Logan et al., 2002; Sun et al., 2000). The Kif3a conditional allele has
been previously described (Lin et al., 2003). The standard Ift88⌬2-3␤gal null
allele has been previously described (Murcia et al., 2000).
Embryonic stem (ES) cells for targeting were derived from HPRTdeficient 129/Ola mice. Chimeric mice were generated from targeted ES
cells by the UAB transgenic animal core facility. Germline transmission of
the targeted allele was determined by coat color and resulting offspring were
genotyped for the presence of the loxP-containing Ift88 allele (Ift88fl) using
PCR. The null allele of Ift88 was generated using the prx1cre transgenic
strain, which expresses Cre in the female germline. Ift88 conditional mice
were genotyped by PCR using primers designed to amplify a region of
genomic DNA flanking one of the loxP sites (wild-type and fl alleles) or
spanning the region deleted upon Cre-mediated recombination (null allele;
Ift88n). Primer sequences are available on request. All mice were maintained
in AALAC certified mouse facilities at UAB with protocols approved by the
IACUC. For staged embryos, noon of the day of the vaginal plug was
designated as embryonic day (E) 0.5.
Immunofluorescence
Immunofluorescence on semi-thin frozen sections of limbs was performed
on 15 ␮m-thick frozen sections as previously described (Haycraft et al.,
2005; Taulman et al., 2001) using monoclonal antibodies to acetylated ␣tubulin (Sigma Aldrich, St Louis, MO) or polyclonal antiserum to detect
Ift88 (Haycraft et al., 2005). For aggrecan immunolocalization, E18.5 limbs
were fixed and frozen and sectioned as indicated for histology. Sections were
incubated with 1 mg/ml hyaluronidase (Sigma Aldrich, St Louis, MO) at
37°C for 45 minutes before incubation with anti-aggrecan antibodies
(Millipore, Billerica, MA).
Histology and skeletal staining
Limbs were dissected from E18.5 embryos and fixed in 4% PFA in
phosphate buffered saline (PBS) overnight at 4°C. Fixed tissues were
washed with several changes of PBS and infiltrated with 30% sucrose in PBS
overnight at 4°C. Sucrose-equilibrated samples were frozen in OCT and 15
␮m sections were cut on a Leica CM1900 cryostat (Leica Microsystems
GmbH, Wetzlar, Germany). Sections were fixed in 4% PFA in PBS and
stained with Hematoxylin and Eosin Yellow (H&E) or Safranin O and Fast
Green using standard protocols. For alkaline phosphatase staining, limbs
from E18.5 wild-type and conditional mutant embryos were isolated, snap
frozen in OCT, sectioned and fixed as described for immunofluorescence.
Fixed sections were washed twice with Tris buffered saline (TBS), twice
with NTMT (0.1 mol/l Tris pH 9.4, 0.1 mol/l NaCl, 0.05 mol/l MgCl2, 0.1%
Tween-20) and incubated with BM Purple precipitating alkaline phosphatase
DEVELOPMENT
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substrate (Roche Diagnostics Corp., Indianapolis, IN) in the dark at room
temperature for 10 minutes. After staining, sections were washed twice with
TBS and nuclei were stained with Nuclear Fast Red (Sigma Aldrich, St
Louis, MO). H&E- and alkaline phosphatase-stained sections were
visualized on a Nikon TE2000 inverted microscope or a Nikon SMZ-800
dissecting scope and images were captured with a MicroPublisher 3.3 color
digital camera (Q Imaging, Burnaby, BC).
For skeletal analysis, staged embryos were stained with Alizarin Red and
Alcian Blue to identify mineralized bone and cartilage, respectively, as
previously described (McLeod, 1980). Postnatal mice were euthanized, and
skeletal elements were stained with Alizarin Red as previously described
(Selby, 1987).
In situ hybridization
For both whole-mount and radioactive in situ analyses, staged embryos were
dissected and fixed in 4% PFA in PBS overnight at 4°C followed by washing
with PBS. Whole-mount in situ hybridization was performed with
digoxygenin (DIG)-labeled antisense probes using standard protocols
(Wilkinson, 1992). In situ hybridization was performed on 10 ␮m frozen
sections as previously described (Pelton et al., 1990). Ihh, Ptch1, Gli1,
PTHrP and gremlin probes have been previously described (Alvarez et al.,
2001; Bitgood and McMahon, 1995; Goodrich et al., 1996; Hui et al., 1994;
Lu et al., 2001).
Western blotting
Whole protein lysates from E11.5 limbs were generated by placing limb
buds in RIPA buffer (150 mmol/l NaCl, 1% NP-40, 0.5% Sodium
deoxycholate, 1% SDS, 50 mmol/l Tris pH 8.0) followed by brief sonication.
Equal volumes of wild-type and mutant lysate were separated by SDSPAGE, transferred to nitrocellulose membranes and blotted with anti-Ift88
antiserum and ␤-tubulin antibodies (H-235) (Santa Cruz Biotechnologies,
Santa Cruz, CA).
RESULTS
Generation of a conditional allele of Ift88
As Ift88 is expressed in most cell types throughout the mammalian
body, and congenital loss of IFT proteins results in severe
developmental defects and mid-gestation lethality, the analysis of
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309
cilia and/or IFT function in specific tissues and cell types has not
been tenable (Murcia et al., 2000; Zhang et al., 2003). To circumvent
these problems, we generated an allele of Ift88 containing loxP sites
flanking exons 4-6 to allow disruption of Ift88 by Cre-mediated
recombination (Fig. 1A). Removal of these three exons is predicted
to result in a translational frame shift and loss of all Ift88 function.
Mice were genotyped using DNA from tail biopsies and PCR
primers designed to amplify a region of DNA containing one of the
loxP sites or spanning the region deleted upon Cre-mediated
recombination (Fig. 1B). To verify that the recombined allele
results in loss of Ift88 function, we generated embryos carrying the
deleted allele (Ift88n, germline mutation derived from the floxed
allele) and the previously described targeted congenital null
mutation (Ift88⌬2-3␤gal). At E11.5, embryos carrying both
alleles (Ift88n/⌬2-3␤gal) showed phenotypes characteristic of
Ift88⌬2-3␤gal/⌬2-3␤gal embryos, including misalignment of the neural
tube, failure of neural tube closure and cardiac sac ballooning (Fig.
1C and data not shown). The identical phenotype in the Ift88⌬23␤gal/⌬2-3␤gal
embryos confirmed that Cre-mediated deletion of the
floxed allele results in a null mutation.
As prx1cre has been shown to be expressed specifically in the
mesenchyme, but not the overlying ectoderm, of the developing
limbs beginning at E9.5 (Logan et al., 2002), we analyzed the
mesenchyme and ectoderm of wild-type and conditional mutants at
E11.5 for the presence of cilia by immunofluorescence (Fig. 2A-C).
In wild-type samples, cilia visualized by acetylated ␣-tubulin and
Ift88 immunostaining, are found on cells throughout the
mesenchyme of the developing limb (Fig. 2A). By contrast, cilia
were present on very few cells in prx1cre;Ift88fl/n conditional mutant
limb mesenchyme (Fig. 2B), while cilia on the cells of the overlying
ectoderm in the same section were normal (Fig. 2C).
To verify that the loss of cilia and Ift88 immunostaining correlates
with the loss of Ift88 protein, we isolated whole limb buds from
E11.5 prx1cre;Ift88fl/n conditional mutants and compared the
amount of Ift88 protein to that found in limb buds from a control
Fig. 1. Generation of a conditional allele of Ift88.
(A) Schematic representation of the targeting construct
and the Ift88 genomic region, showing the location of
the exons and the loxP sites. The deletion allele (Ift88n)
was generated by leaky germline Cre activity in prx1cre
females. The diagram is not drawn to scale. (B) Wildtype, floxed and null alleles (generated from the floxed
allele) of Ift88 were distinguished by PCR using
genomic DNA isolated from tail biopsies of mice. The
approximate locations of the primers used for
genotyping are indicated in A. (C) At E11.5, mice
heterozygous for the (Ift88n) deleted allele and the
previously described targeted Ift88⌬2-3␤-gal null allele of
Ift88 (right) exhibited phenotypes characteristic of the
congenic Ift88⌬2-3␤-gal null mice, including defects in
the neural tube when compared with a wild-type
littermate (left).
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Development 134 (2)
Fig. 2. Cre-mediated recombination of the conditional allele
results in loss of cilia and Ift88 protein. (A-C) Cilia were identified in
frozen sections of E11.5 wild-type (A) and prx1cre;Ift88fl/n conditional
mutant (B,C) limbs by immunofluorescence staining with antibodies to
detect acetylated ␣-tubulin (green) and Ift88 (red). Nuclei are shown in
blue. In wild-type samples (A), cilia were visible on most cells in the
mesenchyme. By contrast, cilia were rarely observed on cells in the
mesenchyme of conditional mutants (B). The presence of cilia on the
ectoderm of prx1cre;Ift88fl/n mutant limbs (C) was not affected.
(D) Protein lysate from conditional mutant limbs at E11.5 (prx1cre)
showed a dramatic reduction in the amount of Ift88 expressed when
compared with lysate from a control littermate (WT). Samples were
simultaneously blotted for ␤-tubulin as a loading control. Approximate
molecular weights are shown to the left of the blot. (E,F) At E11.5, cilia
are apparent in the dorsal ectoderm of the hindlimb in msx2cre;Ift88fl/n
mice (E) by immunofluorescence staining for acetylated ␣-tubulin
(green) and Ift88 (red), but are absent from the ventral ectoderm of the
same limb (F).
littermate by western blot analysis. In agreement with the
immunofluorescence data, the level of Ift88 protein was dramatically
reduced in conditional mutants (Fig. 2D). The low level of Ift88
remaining in prx1cre;Ift88fl/n conditional mutants is probably due to
Ift88 expression in the limb bud ectoderm, where the Cre transgene
is not expressed, and a small number of limb bud mesenchymal
cells, where recombination has not occurred. The blot was
simultaneously probed for ␤-tubulin to ensure similar loading of
control and mutant samples.
prx1cre;Ift88fl/n mutants exhibit severe
polydactyly
By contrast to the normal limb patterning observed in
msx2cre;Ift88fl/n mutants, prx1cre;Ift88fl/n mutant mice developed
eight non-patterned digits on each forelimb and a single extra
preaxial digit on each hindlimb (Fig. 3E-H). The minimal effect
observed in the hindlimbs is probably due to the previously reported
weak expression of prx1cre in the hindlimb at early stages of limb
development (Logan et al., 2002). In addition to the patterning
defects in the autopod, all four limbs of prx1cre;Ift88fl/n mutants
were severely shortened along the proximodistal axis (Fig. 3I).
Although the conditional mutants were smaller than wild-type
littermates postnatally, the decreased length in the limbs was evident
as early as E13.5, whereas the total size of the embryos was
comparable to wild-type littermates (data not shown). An identical
phenotype was seen using a conditional allele of the IFT kinesin-II
subunit Kif3a (data not shown). Analysis of Alizarin Red-stained
skeletons at postnatal day 11 indicate that all skeletal elements of the
limb, including the humerus, radius and ulna, were formed properly
in the conditional mutants (Fig. 3I), indicating that proximodistal
patterning of the limb is not dependent on Ift88 function in the
mesenchyme.
As congenital loss of Ift88 results in defects in Shh signaling
during limb bud formation (Haycraft et al., 2005; Liu et al., 2005),
we examined the level of Gli1 expression in the developing
forelimbs of conditional prx1cre;Ift88fl/n mutants by whole-mount
in situ hybridization. Although prx1cre expression was detectable
by E9.5, the expression of Gli1 appeared normal in the forelimbs at
E10.5 (Fig. 4A,B); however, at E11.5, expression of Gli1 was nearly
abolished in the conditional mutants (Fig. 4C,D). Small patches of
cells were occasionally observed that retained Gli1 expression
(arrowhead in Fig. 4D) and probably represent cells in which Ift88
expression was maintained due to inefficient Cre-mediated
recombination.
DEVELOPMENT
Loss of cilia and/or IFT in the AER and ventral
ectoderm cells has no gross effect on limb
patterning
As cilia are expressed on cells of the AER and ectoderm of the
developing limb, we generated conditional mutants as described
above using transgenic mice expressing Cre recombinase under
the control of the Msx2 promoter (msx2cre). During early limb
development, expression of Cre in this transgenic line is
restricted to the AER and the ventral ectoderm (Sun et al., 2000).
To verify that the msx2cre transgene is sufficient to disrupt cilia
formation on the ventral ectoderm and AER of the limb bud, we
performed immunofluorescence on frozen sections of E11.5
hindlimb buds. Cells in the dorsal ectoderm of msx2cre;Ift88fl/n
conditional mutants showed cilia when stained with antibodies
for acetylated ␣-tubulin and Ift88 (Fig. 2E), whereas the ventral
ectoderm of the same limb bud was nearly devoid of cilia (Fig.
2F). Despite the disruption of cilia on the ventral ectoderm, limbs
of msx2cre;Ift88fl/n mice showed no overt defects in outgrowth or
patterning and were indistinguishable from their wild-type
littermates (Fig. 3A-D). Identical results were seen with a
conditional allele of the IFT kinesin subunit Kif3a, which
functions along with Ift88 in anterograde IFT (data not shown).
Although these data suggest that Ift88/IFT on the ectoderm has
no function in limb outgrowth or patterning, we cannot exclude
a role for IFT at stages before Cre expression in the AER or in
the dorsal ectoderm, because the transgene is not expressed in
these regions.
Fig. 3. msx2cre;Ift88fl/n mice show no phenotype but
prx1cre;Ift88fl/n mice develop polydactyly. (A-D) Images of the
dorsal (A,B) and ventral (C,D) surfaces of the hindlimbs of wild-type
(A,C) and msx2cre;Ift88fl/n conditional mutant (B,D) adult mice. No
defects in limb patterning were visible in the conditional mutants.
(E-H) Alizarin Red and Alcian Blue staining of forelimbs (E,F) and
hindlimbs (G,H) from wild-type (E,G) and prx1cre;Ift887fl/n conditional
mutant (F,H) mice at E18.5. Forelimbs of prx1cre;Ift88fl/n (F) develop
eight or more nonpatterned digits. While the forelimb exhibited
extensive polydactyly, the hindlimb (H) typically showed a duplication of
digit I (asterisk). (I) Alizarin Red-stained forelimb of wild-type (bottom)
and prx1cre;Ift88fl/n (top) conditional mutant at postnatal day 11. The
proximodistal length of the limbs is shortened in conditional mutants,
but the patterning of the long bones is largely unaffected. Anterior is
up in all panels.
By contrast to the normal expression of Gli1 at E10.5, expression
of the Bmp antagonist gremlin was expanded into the anterior half
of the developing limb bud of prx1cre;Ift88fl/n conditional mutants
(Fig. 4E,F). At E11.5, gremlin expression was nearly absent in wildtype forelimbs, whereas conditional mutant forelimbs expressed
significant levels of gremlin on the anterior side of the limb bud (Fig.
4G,H). These results suggest that while Shh signaling is retained for
some time after loss of Ift88, processing of Gli3 to generate Gli3R
is probably impaired before E10.5, resulting in de-repression of
gremlin transcription in the anterior limb bud. In addition, as the Cre
recombinase is expressed only in the developing mesenchyme, these
results, along with the lack of a phenotype in msx2cre;Ift88fl/n
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Fig. 4. Expression of Gli1 and gremlin is altered in the forelimbs
of prx1cre;Ift88fl/n conditional mutants. Whole-mount in situ
hybridization analysis of Gli1 (A-D) and gremlin (E-H) expression in
wild-type (A,C,E,G) and prx1cre;Ift88fl/n (B,D,F,H) forelimbs at E10.5
(A,B,E,F) and E11.5 (C,D,G,H). At E10.5, Gli1 expression is similar in
conditional mutants (B) and wild-type controls (A). At E11.5, the
expression of Gli1 is retained in wild-type controls (C) but is almost
completely absent in conditional mutants (D), except small patches of
cells expressing Gli1 can occasionally be seen in prx1cre;Ift88fl/n
mutants (arrowhead). Gremlin expression is restricted to the posterior
mesenchyme at E10.5 in wild-type forelimbs (E) but is expanded into
the anterior mesenchyme in prx1cre;Ift88fl/n conditional mutants
(arrowhead in F). Expression of gremlin is minimal in wild-type forelimbs
at E11.5 (G). By contrast, in prx1cre conditional mutant forelimbs (H),
ectopic anterior expression of gremlin is maintained (arrowhead in H).
Anterior is up in all panels.
conditional mutants, suggest that Ift88 function is required in the
limb bud mesenchyme only to direct proper anteroposterior
patterning.
Conditional mutants exhibit defects in
endochondral bone formation
As chondrocyte precursors of the long bones are present in the limb
mesenchyme when Cre expression is active in prx1cre mice, and as
the appendicular skeletal elements in the prx1cre;Ift88fl/n mutants
show reduced length, we hypothesized that this phenotype was due
to a defect in endochondral bone formation. To evaluate this
possibility, we isolated the limbs from conditional mutants and
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Development 134 (2)
control littermates at E18.5 and stained frozen sections with
Hematoxylin and Eosin (H&E) to analyze the histology of the long
bones. Sections from the tibia are shown, but similar results were
seen for all long bones in the appendicular skeleton. While
conditional mutants exhibited some variability in the severity of
endochondral bone defects between litters, all prx1cre;Ift88fl/n and
prx1cre;Kif3afl/n mutants showed a dramatic reduction in the overall
length of the skeletal elements relative to controls (Fig. 5A,C,E),
although the overall size of the embryos was unchanged. The growth
region of the tibia in conditional mutants contained only a small
disorganized area of round and flat proliferating chondrocytes,
suggesting accelerated hypertrophic differentiation. Although
vascularization did occur, it was delayed in conditional mutant
embryos and hypertrophic cells persisted (Fig. 5D,F).
To confirm that cilia are disrupted on the chondrocytes, we
performed immunofluorescence on frozen sections of wild-type and
conditional mutant tibia at E18.5. Cilia were apparent on the
chondrocytes in the growth region of the tibia in wild-type embryos
Fig. 6. Ihh signaling is disrupted in the long bones of
prx1cre;Ift88fl/n conditional mutants. (A-D) Radioactive in situ
hybridization analysis of Ihh expression in the fibula of wild-type (C) and
conditional mutant (D) mice at E18.5. Strong expression of Ihh is seen
in the prehypertrophic chondrocytes in the wild-type sample (C). Ihh
expression is also evident in the prehypertrophic chondrocytes of
conditional mutants (D) although at reduced levels. (E-L) Analysis of
Ptch1 (E-H) and Gli1 (I-L) expression in wild-type (G,K) and conditional
mutant (H,L) tibiae. In wild-type samples, Ptch1 and Gli1 expression is
increased in the proliferating and prehypertrophic regions (asterisks) in
addition to the perichondrium (arrowhead). By contrast, no significant
expression of Ptch1 or Gli1 is seen in conditional mutant samples.
(M-P) Expression of PTHrP in wild-type (M,O) and conditional mutant
(N,P) radii at E14.5. PTHrP is expressed in both wild-type (O) and
conditional mutant (P) radii at E14.5. Bright field images of each section
(A,B,E,F,I,J,M,N) show histology.
DEVELOPMENT
Fig. 5. Endochondral bones in prx1cre;Iftfl/n conditional mutant
limbs are shorter than in wild-type littermates. Sections of
hindlimbs from E18.5 wild-type (A,B), prx1cre;Ift88fl/n (C,D), and
prx1cre;Kif3afl/n (E,F) conditional mutant embryos were stained with
H&E to analyze bone formation. Frozen sections through the tibia are
shown. The overall size of the growth region in conditional mutants is
reduced (double-headed arrows in B,D,F). Vascularization is evident in
conditional mutant tibiae (asterisks in D,F), although the marrow cavity
is poorly formed. (G,H) Cilia, as visualized by localization of acetylated
␣-tubulin (green) and Ift88 (red), are apparent on the chondrocytes in
control animals (G). By contrast, few chondrocytes in the developing
tibia of prx1cre;Ift88fl/n embryos (H) expressed a cilium.
(Fig. 5G). By contrast, only small cilia were occasionally observed
on the chondrocytes of the developing tibia in prx1cre;Ift88fl/n
conditional mutants (Fig. 5H).
Ihh signaling is disrupted in the bones of
prx1cre;Ift88fl/n mutants
The loss of Shh signaling during early limb patterning in
prx1cre;Ift88fl/n conditional mutants prompted us to examine Ihh
signal transduction in the long bones of conditional mutants. We
analyzed the expression of Ihh and two downstream targets of the
hedgehog pathway, Ptch1 and Gli1, in E18.5 conditional mutants by
radioactive in situ hybridization. As seen for Shh in the autopod of
Ift88 null embryos, Ihh expression was maintained in the
prehypertrophic chondrocytes in the fibula of prx1cre;Ift88fl/n
conditional mutants although at reduced levels (Fig. 6C,D). A
similar decrease in Ihh expression is seen in mice lacking Gli2 and
may be due to the positive feedback loop required to maintain high
levels of Ihh expression (Miao et al., 2004). Alternatively, the
decrease in Ihh expression may reflect fewer prehypertrophic
chondrocytes in conditional mutants. Despite expression of Ihh, no
significant expression of Ptch1 or Gli1 was detected in the
perichondrium flanking the prehypertrophic chondrocytes or the
region of proliferating chondrocytes in the tibia of conditional
mutants (Fig. 6H,L). These findings were evident in all skeletal
elements in the developing limbs, suggesting that Ift88 is required
for Ihh signaling during embryonic endochondral bone formation.
By contrast to the loss of Ptch1 and Gli1 expression, PTHrP was
expressed in the conditional mutants at E14.5 (Fig. 6O,P).
RESEARCH ARTICLE
313
Development of the bone collar is altered in
conditional mutants
The perichondrium consists of multipotent mesenchymal cells lining
the outer edge of the bone anlagen. During development, Ihh
secreted from prehypertrophic chondrocytes results in the activation
of canonical Wnt signaling and subsequent differentiation of cells in
the inner layer of the perichondrium to osteoblasts, thus forming the
bone collar (Hu et al., 2005). By contrast to the uniform spindleshaped appearance of perichondrial cells along the tibia in wild-type
samples, the perichondrium in prx1cre;Ift88fl/n conditional mutants
was disorganized and cells did not adopt the characteristic flattened
morphology (Fig. 7A-D). In addition to the loss of normal cell
architecture, the perichondrium exhibited uneven thickness along
the length of the bone (Fig. 7B). The most severe defects in
perichondrial organization were observed flanking the diaphysis,
where cells adjacent to the perichondrium resembled chondrocytes
rather than osteoblasts (arrowheads in Fig. 7B). While it is unclear
if these cells originated from the perichondrium or the growth plate
of the developing bone, they appeared to be continuous with the
perichondrium in some sections, suggesting that they may have
originated there (arrowheads in Fig. 7B). The bone collar develops
along the metaphysis of the bone anlagen and extends to the level of
the prehypertrophic and hypertrophic chondrocytes in wild-type
tibiae (Fig. 7C). In prx1cre;Ift88fl/n mutant embryos, no bone collar
was apparent adjacent to the corresponding region (Fig. 7D).
The loss of perichondrial architecture and bone collar
development suggested a potential defect in osteoblast
differentiation, prompting us to examine the presence of osteoblasts
Fig. 7. Perichondrial architecture and bone collar
development are affected in prx1cre;Ift88fl/n
conditional mutant mice. (A-D) H&E-stained sections
from E18.5 wild-type (A,C) and conditional mutant (B,D)
tibiae. The outer and inner edges of the perichondrium
are marked by green lines. The perichondrium in wildtype samples (A) is a compact layer flanking the bone
anlagen. By contrast, the cells adjacent to the diaphysis in
conditional mutant samples (B) are disorganized. The
perichondrium varies in thickness along the length of the
bone and is flanked by cells that more closely resemble
chondrocytes than osteoblasts (green arrowheads in B).
Metaphysis and perichondrium of tibia sections stained
with H&E from E18.5 wild-type (C) and prx1cre;Ift88fl/n
conditional mutant (D) tibiae, show loss of bone collar
formation in conditional mutants. (E,F) Sections of wildtype (E) and conditional mutant (F) tibiae at E18.5 stained
for alkaline phosphatase activity (blue) to identify the
bone collar (arrow). Sections were counterstained with
Nuclear Fast Red. Alkaline phosphatase expression is
visible in the cells of the bone collar flanking the
proliferating and prehypertrophic chondrocytes in wildtype samples (E), but is absent in the perichondrial region
of conditional mutants (F). Insets in E and F show higher
magnification views of the regions indicated by the
arrows. The distal end of the tibia is left in all panels. HC,
hypertrophic chondrocytes.
DEVELOPMENT
IFT and bone formation
RESEARCH ARTICLE
in prx1cre;Ift88fl/n conditional mutants. Osteoblasts that generate the
bone collar express specific markers, including alkaline phosphatase
(AP), which can be easily detected using biochemical analyses. To
determine if osteoblasts were present where the bone collar is
normally located, we stained sections of the radius of wild-type and
conditional mutant mice for AP activity using a colorimetric AP
substrate. By contrast to the normal expression of AP adjacent to the
proliferating chondrocytes in the radius of wild-type mice, no AP
expression was observed in the perichondrium in the conditional
mutant, whereas staining in the hypertrophic chondrocytes was
unaffected (Fig. 7E,F).
Ectopic chondrocytes develop in the
perichondrium of prx1cre;Ift88fl/n mice
To determine the position of bone and cartilage matrix in the
conditional mutants, we stained sections of the long bones with
Safranin O and Fast Green. In wild-type tibiae, Safranin O staining
was present along the growing epiphysis, whereas trabecular and
cortical bone stained with Fast Green (Fig. 8A). Intriguingly, Safranin
O staining was evident in regions of the diaphysis in conditional
mutants (Fig. 8B), suggesting that some of the cells in the
perichondrium of conditional mutants are differentiating along the
chondrocyte lineage rather than the appropriate osteoblastic lineage.
Despite the defects in perichondrial architecture and bone collar
formation, some regions of prx1cre;Ift88 conditional mutant tibiae did
contain mineralized bone (blue-green staining in Fig. 8B). To further
investigate the ectopic perichondrial cells in conditional mutants, we
performed immunofluorescence to determine the localization of the
proteoglycan aggrecan. In wild-type tibiae, aggrecan is localized to
the chondrocytes but is absent from the perichondrium and developing
bone collar. By contrast, prx1cre;Ift88fl/n mutant tibiae showed strong
localization of aggrecan in the perichondrium (Fig. 8C,D).
Overall, these results suggest that prx1cre;Ift88 conditional
mutant long bones share some features of Ihh;Gli3 mutants,
including loss of the bone collar and restoration of PTHrP
expression relative to Ihh mutant mice (Hilton et al., 2005; StJacques et al., 1999). However, the development of ectopic
chondrocytes along the diaphysis is not seen in Ihh;Gli3 mutants.
Rather, this is characteristic of defects in canonical Wnt signaling
(Hu et al., 2005).
DISCUSSION
Cilia are small microtubule-based organelles found on most cells in
the mammalian body. The embryonic lethality and severe
developmental defects associated with loss of cilia or IFT have
Development 134 (2)
hindered efforts to investigate the role of IFT in the development of
specific tissues and postnatal tissue homeostasis (Davenport and
Yoder, 2005; Scholey, 2003). We addressed this issue in the limb bud
using the Cre-lox system and a conditional allele of the IFT gene
Ift88. Whereas conditional loss of cilia on the ventral ectoderm and
AER had no overt effect on limb patterning, loss of Ift88 in the limb
mesenchyme resulted in extensive polydactyly on the forelimb and
shortening of the long bones in all four limbs. In addition to
abnormalities in chondrocyte differentiation in the prx1cre;Ift88fl/n
conditional mutants, the perichondrium was disorganized, ectopic
cells that resemble chondrocytes developed along the diaphysis and
the bone collar was not properly formed.
Although cilia are present on the ectodermal cells of the
developing limb (Haycraft et al., 2005), the conditional loss of cilia
on the ventral ectoderm and AER of the limb bud with msx2cre did
not significantly affect limb patterning. This indicates that ciliary
function is not required on these cell populations for normal limb
development, although a role for cilia and/or IFT before AER
formation or in the dorsal ectoderm cannot be excluded, as msx2cre
is expressed in the ventral ectoderm and AER.
Despite the fact that Gli1 expression appears normal at E10.5 in
the prx1cre conditional cilia mutants, gremlin, which is normally
restricted to the posterior region of the limb, is expanded anteriorly.
This expanded domain of gremlin is also observed in the Gli3 and
Shh;Gli3 double mutants (Litingtung et al., 2002; te Welscher et al.,
2002). These data suggest that by E10.5, loss of IFT or the cilia has
already impaired the formation of Gli3R in the anterior, leading to
de-repression of gremlin. This is in agreement with previous work
demonstrating that Ift88 is essential for efficient processing of Gli3
(Haycraft et al., 2005; Liu et al., 2005).
In addition to a role in Shh signaling, our data indicate that cilia
are required for normal Ihh signaling activity based on the loss of
Ptch1 and Gli1 expression in the long bones of prx1cre;Ift88fl/n
conditional cilia mutants. However, the phenotype of the conditional
mutants is distinct from that seen in mice with congenital loss of Ihh,
which exhibit premature hypertrophic differentiation due to loss of
PTHrP expression in the presumptive articular cartilage and
perichondrium, decreased chondrocyte proliferation and loss of
vascularization of the long bones (St-Jacques et al., 1999). Recent
work has shown that congenital loss of Gli3 in the developing bones
of Ihh null mice can restore the formation of proliferating
chondrocytes in Ihh null mutants, although at reduced levels, as well
as restore PTHrP expression in both the presumptive articular
cartilage and perichondrium and vascularization of the bones (Hilton
et al., 2005; Koziel et al., 2005). While prx1cre;Ift88fl/n conditional
Fig. 8. Chondrocytes develop in the perichondrium of
prx1cre;Ift88fl/n conditional mutant tibiae. (A,B) In sections of
wild-type E18.5 tibia (A), cartilage matrix, identified with Safranin
O staining (red), is only seen in the growth region of the long
bone, whereas bone, identified with Fast Green staining (bluegreen), is present along the diaphysis. In conditional mutants (B),
regions of cartilage matrix are evident along the diaphysis of the
tibia (arrowheads) and appear to be derived from the overlying
perichondrium. (C,D) In sections of the wild-type tibia (C) at
E18.5, aggrecan is localized to the hypertrophic region and
excluded from the perichondrium by immunofluorescence.
Aggrecan expression is found ectopically in the perichondrium of
the conditional mutant tibia (asterisk in D). Nuclei are stained
blue and the epiphysis is up in both panels. (E) An adjacent
section of the conditional mutant tibia shown in D was stained
with Safranin O and Fast Green to confirm the presence of
cartilage matrix in the perichondrium of the mutant sample.
DEVELOPMENT
314
mutants share characteristic phenotypes with Ihh null mutants,
including accelerated chondrocyte hypertrophy and loss of proper
bone collar development, PTHrP is expressed in conditional mutants
potentially through disruption of Gli3R formation as seen for
gremlin in the developing limb. Additionally, vascularization occurs
in the conditional mutants, although it is delayed.
Osteoblast differentiation and development of the bone collar also
requires canonical Wnt signaling downstream of Ihh. In agreement
with this, conditional loss of ␤-catenin in the developing
perichondrium results in defective differentiation of the bone collar
(Hilton et al., 2005; Hu et al., 2005). While undifferentiated cells
accumulated between the perichondrium and diaphysis of Ihh;Gli3
double mutants and in mice with conditional loss of ␤-catenin, the
ectopic cells observed in Ihh;Gli3 mutants expressed markers of preosteoblasts. By contrast, mice with conditional loss of ␤-catenin in
the developing skeleton exhibited ectopic chondrocyte
differentiation in the perichondrium. A similar accumulation of cells
between the perichondrium and diaphysis was seen in
prx1cre;Ift88fl/n conditional mutants. The ectopic cells in conditional
mutants expressed aggrecan and morphologically resembled
chondrocytes, suggesting that they are similar to the ectopic
chondrocytes observed due to loss of canonical Wnt signaling.
Taken together, the endochondral bone phenotype seen in
prx1cre;Ift88fl/n conditional mutants has similarities with several
mouse models, including Ihh, Ihh;Gli3 double mutants and mice
with conditional disruption of ␤-catenin in the skeleton, but the
phenotype is not identical to any one mouse model. This suggests
that the role of IFT and cilia during endochondral bone development
is not restricted to the known role for IFT in hedgehog signaling and
may include a role for cilia or IFT in additional signaling pathways
such as canonical Wnt signaling. Indeed, previous reports have
pointed to a role for cilia or centrosomes in canonical and
noncanonical Wnt signaling (Cano et al., 2004; Ross et al., 2005;
Simons et al., 2005).
Overall, the generation of a conditional allele of the IFT protein
Ift88 provides a valuable resource to allow the study of IFT function
in specific cell types during development, as well as normal tissue
homeostasis. Using the Cre-lox system we have uncovered a role for
Ift88 in Ihh signal transduction, similar to that previously reported in
Shh signaling during limb and neural tube patterning (Haycraft et al.,
2005; Huangfu and Anderson, 2005; Huangfu et al., 2003; Liu et al.,
2005) in addition to a possible role in multiple signaling pathways
required for endochondral bone formation. Together these data
demonstrate an essential requirement for cilia or IFT function in
normal patterning of the autopod during early stages of limb
development in addition to a requirement during endochondral bone
formation in the appendicular skeleton at later stages of limb
morphogenesis. Analysis of ciliary function during long bone
development was previously untenable due to the early lethality of
IFT null mutants. The use of these conditional cilia-IFT mutant
alleles in combination with additional strains expressing Cre
recombinase under the control of alternative and inducible promoters
will allow a more thorough analysis of the functional importance of
IFT and cilia throughout development and during postnatal life.
We would like to thank Dr G. Marqués and members of our laboratories for
critical reading of the manuscript and helpful discussions. We would like to
thank M. Irwin and the UAB Transgenic Animal Resource facility for generation
of chimeric mice. We would also like to thank V. Roper and M. Croyle for
providing excellent technical assistance. This work was supported by grant
DK65655 from NIH and March of Dimes research grant 1-FY04-95 to B.K.Y.,
and grants AR46982 and AR45605 from the NIH to R.S. Additional support
was provided to C.J.H. through an NIH T32 training grant (HL07553) to J.
Yother.
RESEARCH ARTICLE
315
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