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: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
August 11, 2009 ?
vol. 106 ?
no. 32 ?