Mouse Kif7/Costal2 is a cilia-associated protein that
regulates Sonic hedgehog signaling
Karel F. Liem, Jr.a, Mu Hea,b, Polloneal Jymmiel R. Ocbinaa,c, and Kathryn V. Andersona,1
aDevelopmental Biology Program, Sloan–Kettering Institute, 1275 York Avenue, New York, NY 10065; andbBiochemistry, Cell and Molecular Biology
Program, andcNeuroscience Program, Weill Graduate School of Medical Sciences, Cornell University, 445 East 69th Street, New York, NY 10065
Contributed by Kathryn V. Anderson, June 24, 2009 (sent for review May 20, 2009)
Mammalian Sonic hedgehog (Shh) signaling is essential for em-
bryonic development and stem cell maintenance and has critical
roles in tumorigenesis. Although core components of the Shh
pathway are conserved in evolution, important aspects of mam-
malian Shh signaling are not shared with the Drosophila pathway.
Perhaps the most dramatic difference between the Drosophila and
mammalian pathways is that Shh signaling in the mouse requires
a microtubule-based organelle, the primary cilium. Proteins that
are required for the response to Shh are enriched in the cilium, but
it is not clear why the cilium provides an appropriate venue for
signal transduction. Here, we demonstrate that Kif7, a mammalian
homologue of Drosophila Costal2 (Cos2), is a cilia-associated pro-
tein that regulates signaling from the membrane protein Smooth-
identified in a reporter-based genetic screen, we show that, similar
to Drosophila and zebrafish Cos2, mouse Kif7 acts downstream of
Smo and upstream of Gli2 and has both negative and positive roles
in Shh signal transduction. Mouse Kif7 activity depends on the
presence of cilia and Kif7-eGFP localizes to base of the primary
cilium in the absence of Shh. Activation of the Shh pathway
promotes trafficking of Kif7-eGFP from the base to the tip of the
cilium, and localization to the tip of the cilium is disrupted in a
motor domain mutant. We conclude that Kif7 is a core regulator of
Shh signaling that may also act as a ciliary motor.
Gli2 ? intraflagellar transport ? smoothened ? neural tube ? ENU
way appear to diverge among animals. In particular, in mammals
it is not clear how the membrane protein Smoothened (Smo)
controls the activity of the Gli transcription factors that imple-
ment the pathway (1). In Drosophila, activation of the Hh
pathway leads to formation of a protein complex that includes
Costal2 (Cos2) on the cytoplasmic C-terminal tail of Smo, but
vertebrate Smo lacks the major binding site for Cos2 that is
crucial for complex formation (2). Kif7, a vertebrate homologue
of Cos2, is important for Hh signaling in zebrafish (3), but
cell-based assays have argued that Kif7 does not act in the
mammalian pathway (2). Fused, a serine/threonine kinase that
binds Drosophila Cos2, is important for Hh signaling in zebrafish
(4), but the single mouse Fused homologue is not essential for
Shh signaling and instead is important for the formation of
motile cilia (5–7). Suppressor of fused (Sufu) plays a minor role
in the Drosophila pathway that is only detected in the absence of
Fused, but is an essential regulator of the pathway in zebrafish
(4, 8), and is a very strong negative regulator of Shh signaling in
the mouse (9, 10).
The most surprising difference between the Drosophila and
mammalian pathways is that signaling from Smo to the Gli
transcription factors in the mouse requires the primary cilium.
ciliogenesis, including the intraflagellar transport (IFT) machin-
ery and basal body proteins that promote cilia formation and
maintenance, are essential for all responses to mammalian
Hedgehog (Hh) ligands in both early embryos and all other cell
lthough core components of the Hedgehog (Hh) pathway
are conserved in evolution, important aspects of the path-
types that have been tested (11–14). In contrast, IFT proteins are
not required for Hh signaling in Drosophila, and the role of cilia
in the zebrafish pathway is controversial (15, 16). In the mouse,
it appears that all of the core pathway proteins required for the
the Shh receptor, is localized in cilia in the absence of ligand and
moves out of cilia after exposure to ligand (17). In a similar time
course, the membrane protein Smo moves into cilia in response
Trafficking of Smo and Patched between vesicular and plasma
membrane compartments in response to ligand also takes place
in Drosophila, but not to any defined plasma membrane com-
partment (19), and Hedgehog-responding cells are not ciliated in
Drosophila. In the mouse, the Gli transcription factors that
implement Shh signals and the negative regulator Sufu are
localized to cilia tips both in the presence and absence of ligand
(20). Despite the clear connection between cilia and mammalian
Hh signaling, it is not yet clear why the cilium is the site of Hh
One proposed explanation for these differences between
Drosophila and mammals is that the IFT machinery that builds
cilia substitutes for the function of Drosophila Cos2, a kinesin-
related protein (1, 21, 22). Consistent with this hypothesis,
experiments in mammalian cells indicated that the mouse pro-
teins most similar to Cos2, Kif7 and Kif27, do not play a role in
mammalian Hh signaling (2). Here, we show that, in contrast to
this hypothesis, Kif7 is essential for mouse Shh signaling and that
Kif7 protein is associated with cilia and may act as a ciliary
motor. Thus, Kif7 and ciliary trafficking act in concert in
the connection between cilia and Hedgehog signaling.
Mutation in Kif7 Expands Ventral Fates in the Mouse Neural Tube. We
carried out a genetic screen to identify new recessive N-ethyl-
Shh signaling. Among its many functions in mammalian devel-
opment, the role of Shh in the specification of ventral cell types
in the developing neural tube is particularly well understood. For
example, spinal motor neurons are induced in response to an
intermediate level of Shh, and the number and position of motor
neurons within the ventral neural tube is a sensitive indicator of
Shh activity (23). We used an HB9-GFP transgenic reporter
mouse that expresses GFP in motor neurons (24) (Fig. 1A and
Fig. S1A) to identify mutations that altered Shh signaling. An
interesting mutation, matariki (maki), was identified that caused
an expanded motor neuron domain in the embryonic day 10.5
(E10.5) neural tube (Fig. 1A). Analysis of cross-sections con-
Author contributions: K.F.L., M.H., and K.V.A. designed research; K.F.L., M.H., and P.J.R.O.
performed research; K.F.L., M.H., P.J.R.O., and K.V.A. analyzed data; and K.F.L. and K.V.A.
wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
August 11, 2009 ?
vol. 106 ?
no. 32 ?
firmed that motor neurons were increased in number and
expanded dorsally (Fig. 1B). The floor plate, which is specified
by the highest level of Shh activity, appeared normal in the
mutant embryos (Fig. 1B and Fig. S2). Nkx2.2 is expressed in the
V3 interneuron progenitors that are specified by high levels of
Shh signaling adjacent to the floor plate. The Nkx2.2 domain was
types that are specified by lower levels of Shh activity also shifted
dorsally (Fig. S2). This neural patterning phenotype is consistent
with a moderate elevation of Shh pathway activity. maki mutants
died at the end of gestation, when other phenotypes character-
istic of elevated Shh signaling were apparent, including preaxial
polydactyly (Fig. 1C). To assess whether maki affected the Shh
pathway directly, we analyzed the expression of a direct target
gene of the Shh pathway, Ptch1. Ptch1-lacZ (25) was expressed
in a modestly expanded domain in the maki neural tube, which
confirmed that the mutation increased the activity of the Shh
pathway (Fig. 1D).
We used meiotic recombination mapping to localize the gene
responsible for the maki phenotype to a small interval on
chromosome 7 and found that the maki phenotype was associ-
ated with a missense mutation (L130P) in a conserved region of
the motor domain of Kif7 (Fig. S1 B and C) (see Experimental
Procedures), which encodes a kinesin homologous to Drosophila
Cos2. Because the motor domain of Drosophila Cos2 is required
for its microtubule-dependent motility and point mutations in
expression (26, 27), the L130P mutation is likely to cause loss of
Kif7 function. Both maki mutants and zebrafish Kif7 morphants
show mild ectopic activation of Shh pathway targets (3), sup-
porting the conclusion that the maki phenotype is caused by loss
of Kif7 activity. Thus, contrary to a previous report (2), Kif7/
Cos2 has a conserved role in mammalian Hh signaling.
Kif7 Acts as Both a Negative and Positive Regulator of Shh Signal
Transduction. Drosophila Cos2 is required to relay the signal from
the transmembrane protein Smo to the Ci transcription factor.
Cos2 acts as part of a protein complex that includes Fused and
binds to the C-terminal tail of Smo (28). Because Shh signaling
is not disrupted in mouse Fused mutants (5, 6) and the major
Cos2 binding domain is not present in vertebrate Smo (2), it was
important to define the step in the Shh pathway controlled by
Smo is required for embryos to survive past ?E9.0 and for
specification of all Shh-dependent ventral neural cell types (29).
Smo Kif7makidouble mutant embryos continued to develop until
motor neurons and some Nkx2.2? cells (Fig. 2A). Thus, loss of
Kif7 activated the pathway in the absence of Smo, indicating that
Kif7 causes ligand-independent activation of the Shh pathway at
a step downstream of Smo. Consistent with the hypothesis that
Kif7 acts at a step downstream of Smo, we found that, as in wild
type, Smo protein moves into cilia of Kif7makimouse embryo
fibroblasts in response to Shh.
The Gli2 and Gli3 transcription factors implement the re-
sponses to Shh in the neural tube (30–32). Gli2 is the principal
transcriptional activator of Shh target genes. Gli2 mutants lack
the most ventral cell type in the neural tube, the floor plate, and
have a reduced number of Nkx2.2? V3 progenitors (Fig. 2A)
(33). In Gli2 Kif7makidouble mutants, the motor neuron domain
was not expanded dorsally and the floor plate and Nkx2.2?
domains were absent (Fig. 2A), indicating that the dorsal
expansion of the motor neuron and Nkx2.2 domains seen in
Kif7makiwas due to increased activity of Gli2. The steady-state
level of Gli2 protein was higher in E10.5 Kif7makiembryo extracts
than in wild type (Fig. 2D), which may account, in part, for the
elevated Gli2 activity in the mutants.
In wild-type embryos, Gli3 is proteolytically processed to a
transcriptional repressor that keeps pathway targets off in the
absence of ligand. Although Gli3 single mutants do not have
defects in the pattern of motor neurons or V3 progenitors (34)
(Fig. 2A), Gli3 Kif7makidouble mutants showed a greater ex-
pansion of these ventral neural cell types than Kif7makisingle
mutants (Fig. 2A), indicating the Gli3 and Kif7 have indepen-
dent roles in preventing ligand-independent activation of the
pathway. We found, however, that the amount of processed Gli3
protein and the ratio of processed/full-length Gli3 were de-
creased in Kif7makimutant extracts (Fig. 2D), suggesting that a
decrease in the amount of transcriptional repressor could con-
maki than in wild-type E10.5 embryos. (B) Cross-sections through the brachial
domain of HB9-eGFP is expanded dorsally in maki embryos, whereas the
domain of Shh expression in the floor plate is the same in maki and wild type.
Dorsal, up. (C) Skeletal preparations of postnatal day 1 forelimbs show that
maki embryos have preaxial polydactyly; this defect is completely penetrant.
(D) Sections through the brachial neural tube of E9.5 embryos carrying a
Ptch1-lacZ transgene, stained for ?-galactosidase activity.
The maki phenotype. (A) The expression of HB9-eGFP is stronger in
www.pnas.org?cgi?doi?10.1073?pnas.0906944106Liem et al.
treatment with Smoothened agonist (SAG) (44). When as-
sayed 24 h after SAG addition, GFP was enriched at the cilia
tips of 80% of the cells (Fig. 4 D and E). The time course of
Kif7 movement to the cilia tip was comparable with that of
Smo localization to cilia: Smo began to be detected in cilia at
1 h after SAG activation (17), and one-third of the Kif7-eGFP-
expressing cells (4 of 12) had GFP at the tip 1 h after SAG
addition. As in the IMCD3 cells, the Kif7L130P-eGFP mutant
protein was detected at the base of the cilia of NIH 3T3 cells,
but did not move to the cilia tips after addition of SAG (0 of
18 cells with tip GFP after SAG treatment; 16 of 18 had GFP
at the cilia base). These findings indicate that activated Smo
promotes the movement of Kif7 from the base of the cilia to
the cilia tips and suggest that the relocalization of Kif7 to the
cilia tip requires a functional motor domain.
Our findings demonstrate that Kif7/Costal2 is a member of the
core Shh signal transduction pathway that is conserved in
Drosophila, zebrafish, and the mouse. Kif7 acts as a negative
regulator of the pathway: loss of Kif7 activity causes an expan-
sion of ventral neural cell types in the neural tube because of an
expanded domain of expression of Shh target genes. The double-
mutant analysis shows that Kif7 negatively regulates the pathway
by preventing inappropriate activation of the Gli2 transcription
factor in the absence of ligand. We also observe a decrease in the
amount of Gli3 repressor in Kif7makimutant embryos, which may
contribute to activation of the pathway in the mutants. However,
Gli3 repressor is present in Kif7 mutants, and the genetic analysis
shows that Kif7 and Gli3 cooperate in the restriction of Shh
activity and ventral fates in the neural tube.
In addition to its role as a negative regulator, genetic
experiments revealed that Kif7 also promotes Shh signaling.
The positive role of Kif7 was seen in 2 double mutants: the
neural tube of Kif7makiPtch1 double mutants is less strongly
ventralized than that of Ptch1 single mutants, and Kif7maki
Dync2h1 double mutants lack motor neurons, a stronger
dorsalization than seen in Dync2h1 single mutants. The dual
negative and positive roles suggest that Kif7-containing pro-
tein complexes may be reorganized in response to ligand, as is
the case for Cos2 (28). For example, Kif7 may act in one
complex to negatively regulate the pathway in the absence of
ligand and become incorporated into a distinct, positively
acting complex in response to Shh.
Our results contrast with experiments in which Kif7 knock-
down or overexpression had no detectable effect on Hh signaling
in NIH 3T3 cells (2). In that study, Sufu knockdown caused a
clear elevation of Gli-luciferase reporter assays, but no elevation
of reporter activity was observed when Kif7 activity was reduced.
Because Kif7 has both negative and positive roles in the pathway,
its removal in the embryo causes a net activation of the pathway
that is much less than that seen in Sufu mutant embryos. It is
therefore possible that the cell-based reporter assays were not
sensitive enough to detect changes in pathway activity that are
clear in the embryo.
Like Cos2 (27), Kif7 changes its subcellular localization in
response to pathway activation: Kif7-eGFP is enriched at the
base of the cilium in the absence of ligand and moves to the cilia
tip when the pathway is activated. We therefore propose that the
negative activity of Kif7 takes place at the base and its positive
role takes place at the tip. Because zebrafish Kif7 binds Gli
proteins directly (3), we suggest that, in the absence of pathway
activation, Kif7 at the base of the cilium negatively regulates the
pathway by targeting Gli2 away from the cilium or promoting
Gli2 turnover at the basal body, where proteasomes are enriched
(45). In response to activation of Smo, Kif7 is loaded onto
axonemal microtubules and moves to the cilia tip. At the cilia tip,
Kif7 may interact with the Gli proteins and Sufu that are
enriched in that compartment (20). Cos2 is also required for
responses to high levels of Drosophila Hh, and this has been
attributed to antagonism of Sufu (37). Similarly, Kif7 at the cilia
tip may antagonize Sufu to promote activation of Gli proteins.
Although Cos2 has been shown to have motor activity, the
Cos2 motor lacks conserved motifs important for binding of
ATP and microtubules and moves 4- to 10-fold more slowly
than a conventional kinesin (27). It has therefore been pro-
posed that Cos2 acts primarily as a scaffold protein for
cytoplasmic signaling complexes (46, 47). In contrast, the Kif7
motor domain includes all of the sequence motifs necessary for
ATP and microtubule binding, and the maki mutation dem-
onstrates that the motor domain is essential for its function in
the Shh pathway and its ability to move to the cilia tip. These
findings raise the possibility that the ancestral Kif7/Costal2
required efficient motor activity and that the lineage that led
to Drosophila might have lost both full motor activity of
Cos2/Kif7 and the association of Hedgehog signaling with the
primary cilium in parallel.
The data suggest that, once activated, Smo promotes trans-
location of Kif7 into the ciliary axoneme, and Kif7 may act as an
anterograde IFT motor to transport itself and perhaps other
cargo to the cilia tip. These results suggest that Kif7 is an
essential component of the Shh pathway and may also act as an
IFT motor. Such a dual function would be sufficient to explain
the coupling of mammalian Shh signaling to the primary cilium.
Mouse Strains and Phenotypic Analysis. ENU mutagenesis and the genetic
screen were carried out as described in ref. 48, except that mutagenized
C57BL6/J males and F1males were mated with females homozygous for an
HB9-eGFP reporter in the FVB genetic background, made by using a
patterning were performed as described in ref. 49. Mutant strains were as
follows: Ptch1tm1Mps(25), Smobnb(50), Ift172wim(11), Gli2 (33), and Gli3Xt
(51); genotyping was performed as described. Alcian blue and Alizarin red
were used to stain cartilage and bone, as described in ref. 52. For Western
blot analysis, whole E10.5 embryos were lysed in RIPA buffer (0.15 mM
NaCl/0.05 mM Tris?HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/
0.1% SDS) and processed using standard methods. Gli3 antibodies were the
kind gift of B. Wang (Weill Cornell Medical College, New York), and Gli2
antibodies were the kind gift of J. T. Eggenschwiler (Princeton University,
Identification of the maki Mutation. The maki mutation was induced in a
C57BL6/J chromosome and all outcrosses were to the FVB strain (Fig. S1A). We
were therefore able to map the maki mutation to a segment of chromosome 7
in DNA from mutant embryos on genome-wide SNP panel (53). Further genetic
mapping localized the gene between 2 SNPs, rs37700257 (85.4 Mb) and
rs32302579 (86.95 Mb) (Ensembl build M37; www.ensembl.org). Contrary to a
previous report (2), we found that Kif7 is widely expressed in the midgestation
embryo, so we were able to sequence RT-PCR products amplified from E10.5
mutant and wild-type cDNAs. The Kif7makimutation is a T-to-C missense substi-
tution that disrupts an AluI restriction site present in the wild-type sequence,
creating a restriction fragment length polymorphism (RFLP) that was used for
genotyping. PCR amplification from genomic DNA (primers, 5?-GGTGGTGGTG-
GTGGATACTT and 5?-GCAGATCTCGGAACTCTTCCT) generated a 214-bp PCR
but cleaves wild type to give 132- and 82-bp fragments.
Kif7-eGFP Expression and Localization. C-terminally tagged Kif7wt-eGFP and
Kif7maki-eGFP constructs were generated by subcloning full-length Kif7 and
Kif7makicDNAs into the pEGFPN3 mammalian expression vector (Clontech). To
generate NIH 3T3 cells stably expressing Kif7-eGFP or Kif7maki-eGFP, cells were
After 48 h of transfection, cells were selected in 500 ?g/mL G418. Primary
antibodies to mouse acetylated ?-tubulin (1:1000; Sigma) and secondary anti-
NIH 3T3 cell culture and immunofluorescent staining were performed as de-
scribed in ref. 17. To assay Kif7-eGFP localization in response to Shh pathway
activation, NIH 3T3 cells were grown to confluence and serum starved with the
Liem et al.PNAS ?
August 11, 2009 ?
vol. 106 ?
no. 32 ?
addition of 100 nM SAG for 24 h before fixation and staining; the eGFP-tagged
fusion protein was detected either by fluorescence or with mouse anti-GFP
upright Leica TCS SP2 AOBS laser-scanning microscope. Images were taken with
a 63? objective and 2? zoom. Confocal datasets were processed by using the
Volocity software package (Improvision).
that described similar phenotypes of targeted null mutations in Kif7.
ACKNOWLEDGMENTS. We thank Ivo Lieberam and T. M. Jessell (Columbia
University, New York), Jonathan Eggenschwiler, Baolin Wang, Alexandra
Joyner (Sloan-kettering Institute, New York), and Matthew Scott (Stanford
University, Stanford CA) for constructs, mouse strains, and reagents; the
Memorial Sloan-Kettering Molecular Cytology and Mouse Genetics Core fa-
cilities for technical assistance; Tamara Caspary and Christine Larkins for
technical advice; and Sarah Goetz for comments on the manuscript. The work
was supported by grants from Project ALS and National Institutes of Health
Grant NS044385. Monoclonal antibodies to neural patterning markers were
from the Developmental Studies Hybridoma Bank.
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