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First published online 13 December 2006
doi: 10.1242/dev.02732
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1 Department of Cell Biology, University of Alabama at Birmingham, Birmingham,
AL 35294-0005, USA.
2 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL
35294-0005, USA.
3 Department of Biochemistry and Molecular Genetics, University of Alabama at
Birmingham, Birmingham, AL 35294-0005, USA.
Author for correspondence (e-mail:
Byoder{at}uab.edu)
Accepted 7 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cilia, Limb patterning, Hedgehog, Bone development, IFT, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 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 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 (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 de-repression 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 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.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sun et al., 2000). The
Kif3a conditional allele has been previously described
(Lin et al., 2003). The
standard Ift88
2-3ßgal 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 µm-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 µm 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 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 µm 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 SDS-PAGE, transferred to
nitrocellulose membranes and blotted with anti-Ift88 antiserum and
ß-tubulin antibodies (H-235) (Santa Cruz Biotechnologies, Santa Cruz,
CA).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
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
(Ift882-3ßgal). At E11.5, embryos carrying
both alleles (Ift88n/2-3ßgal) showed
phenotypes characteristic of
Ift882-3ßgal/2-3ßgal 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
Ift882-3ßgal/2-3ßgal 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).
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). 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.
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.
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 wild-type 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 conditional mutants, suggest that Ift88 function is required in the limb bud mesenchyme only to direct proper anteroposterior patterning.
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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).
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).
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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; St-Jacques 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).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 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 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 ß-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 ß-catenin, the ectopic cells observed in
Ihh;Gli3 mutants expressed markers of preosteoblasts. By contrast,
mice with conditional loss of ß-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 ß-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.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Department of Pediatrics, University of Iowa, Iowa City,
IA, USA
Present address: Whitehead Institute for Biomedical Research, Cambridge,
MA, USA
|
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