1 Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore
138673
2 Centre for Biomedical Genetics, Department of Biomedical Science, University
of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
3 Department of Biological Sciences, 14 Science Drive 4, National University of
Singapore, Singapore 117543
Author for correspondence (e-mail:
sudipto{at}imcb.a-star.edu.sg)
Accepted 2 December 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Hedgehog, Costal2, Suppressor of Fused, Zebrafish, Muscle
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There is evidence to indicate that among the primary events in this process
of Ci activation by the Hh signal is the neutralisation of the inhibitory
effects exerted by Cos2 on Ci, which results in the dissociation of the
Cos2-Fu-Ci-Su(fu) complex from the microtubules
(Ingham and McMahon, 2001;
Lum and Beachy, 2004
). Indeed,
the cytoplasmic tail of Smoothened (Smo), a seven-pass serpentine
transmembrane protein and component of the Hh receptor that is essential for
the intracellular transmission of the Hh signal, interacts with Cos2 and has
recently been implicated in the Hh-mediated abrogation of Cos2 activity during
the process of intracellular signal transduction
(Jia et al., 2003
;
Lum et al., 2003
;
Ogden et al., 2003
;
Ruel et al., 2003
).
In vertebrates, at least three distinct paralogous Gli proteins, the
orthologues of Drosophila Ci, regulate the transcriptional responses
to Hh, and their activities appear to be similarly modified by phosphorylation
and proteolytic cleavage (Ingham and
McMahon, 2001). Furthermore, the nuclear access of the activator
and repressor forms of the Gli proteins and their transcriptional regulatory
functions have been shown to be controlled by analogous antagonistic effects
of Su(fu) and Fu, raising the possibility that a similar or an identical
cytoplasmic complex of proteins could perform an essential function in
modulating the signalling pathway in Hh target cells of vertebrates as well as
Drosophila (Ingham and McMahon,
2001
). However, despite the remarkable overall evolutionary
conservation of the signalling mechanism, in the absence of any evidence yet
implicating the involvement a Cos2-like protein, the significance of such a
cytoplasmic complex in vertebrate Hh signalling has not been fully
appreciated.
We report the identification and functional characterisation of a kinesin-like protein in the zebrafish embryo that represents the first vertebrate orthologue of Cos2 and show that it plays a crucial and conserved role as an intracellular repressor of the Hh signalling pathway.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning of zebrafish cos2
We used the web-based CODEHOP (consensus-degenerate hybrid oligonucleotide
primer) programme
(http://blocks.fhcrc.org/codehop.html)
to design appropriate primers for degenerate PCR based on sequence information
derived from the Drosophila, mouse, Fugu and human homologues of
cos2: Cos2FORWARD, 5'-AGATCGACAACCTGCGACARGARAARGA-3';
Cos2REVERSE, 5'-CAGCTGCATGTTCCGCTCRTGYTCYTT-3'.
RACE reactions were performed using reagents from Clontech according to the
manufacturer's instructions. A full-length zebrafish cos2 cDNA was
constructed by piecing together 5' and 3' RACE products at their
overlapping sequence. This cDNA was subsequently fused in frame with GFP at
the 5' end in the pCS2 mRNA expression vector. mRNA expression construct
for shh was used as reported previously
(Blagden et al., 1997).
Approximately 2-3 nl of 0.5 mg/ml of synthetic mRNA of the different
constructs was injected into each fertilised egg.
Antisense cos2 MOs
The sequence of the antisense MOs are as follows: Cos2START,
5'-GCCGACTCCTTTTGGAGACATAGCT-3'; Cos2SPLICE,
5'-AAATACTCACAAATGCTGGCTTCCC-3'. cos2 genomic sequences
used for delineating the exon-intron boundaries were identified in the
zebrafish BAC/PAC sequence database (BAC zK265M8; LG7) at the Wellcome Trust
Sanger Centre. The MOs were used at a concentration of 1 mM each and 3-4 nl of
the MO solution was injected into each fertilised egg. As a combination of the
two MOs consistently gives a slightly stronger phenotype than when each is
used singly, we have provided data that we obtained on using this mixture. The
sequence and use of the Su(fu) MOs have been previously reported
(Wolff et al., 2003).
Consistent with published data from our laboratory as well as others, the
effect of the MOs was most prominent in somites in the anterior two-thirds of
the embryo, possibly owing to the degradation and dilution of the MOs over
time. As a test for the specificity of the cos2 morphant phenotype,
the effect of the MOs was titrated by co-injection of synthetic
gfp-cos2 mRNA. This construct carries the cos2
translational start sequence that is recognised by the Cos2START antisense MO,
immediately downstream of the GFP coding sequence, with a single mismatch
(CGCTATG instead of AGCTATG, cos2 start codon in bold;
see also Fig. 4) introduced to
ensure in-frame fusion of the cos2 ORF with that of gfp.
However, the intronic sequence that is recognised by the Cos2SPLICE MO is
completely absent from this chimaeric cDNA. These modifications possibly
account for the ability of this construct to effectively titrate the
inhibitory effects of the MOs. The MO injected syut4 and
smub641 embryos were identified by their curled down tails
and U-shaped posterior somites as described before
(Wolff et al., 2003
). The
numbers of embryos of a particular genotype that exhibited a specific
phenotype from among the total number of embryos of that genotype that were
analysed for each MO experiment have been expressed as `n' values.
Quantification of MP cell numbers in embryos with different levels of Hh
activity was carried out as described by Wolff et al.
(Wolff et al., 2003
).
|
Cell culture, co-immunoprecipitation and western blotting
Mammalian 293T cells were transfected with pCS2-GFP, pCS2-GFP-Cos2,
pCS2-Su(fu)-GFP and pcDNA-His-Gli1 either singly or in combination using the
Qiagen Superfect transfection kit. Lysates from untransfected 293T cells or
single transfections of pCS2-GFP-Cos2, pCS2-Su(fu)-GFP and pcDNA-His-Gli1,
each containing 40 µg of protein, were fractionated by SDS-PAGE. For
co-immunoprecipitation reactions, lysates from untransfected 293T cells,
pcDNA-His-Gli1 and pCS2-GFP single transfections, or co-transfections of
pCS2-GFP, pCS2-GFP-Cos2 or pCS2-Su(fu)-GFP, together with pcDNA-His-Gli1, each
containing
500 µg of protein, were incubated with purified mouse
anti-GFP antibodies (Sigma) overnight at 4°C. The immune complexes were
collected by incubation with protein A-sepharose beads (Oncogene) for 1-4
hours at 4°C, followed by centrifugation. The immunoprecipitates were then
washed twice with washing buffer and fractionated by SDS-PAGE. For single
transfections, membranes were probed with mouse monoclonal anti-His (Santa
Cruz Biotechnology) or anti-GFP antibodies, whereas for
co-immunoprecipitations, only the anti-His antibodies were used.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
In the epidermal cells of the Drosophila embryo, the distribution
of Cos2 has been shown to mirror the distribution of microtubules in the
cytoplasm, consistent with Cos2 being a microtubule binding protein
(Sisson et al., 1997). We
performed double-labelling studies with antibodies to ß-tubulin to assess
the relative distribution patterns of zebrafish Cos2 and microtubules within
the cytoplasm of the somitic cells, and found a substantial overlap of the GFP
signal with ß-tubulin, reminiscent of the pattern in Drosophila
embryos (Fig. 2G,H). In
addition, high-resolution confocal microscopy of mammalian 293T cells
transfected with a construct expressing the GFP-Cos2 fusion protein, showed
significant colocalisation of GFP with ß-tubulin
(Fig. 3A-C), further
substantiating the view that zebrafish Cos2, like its Drosophila
homologue, possibly functions by associating with microtubules.
|
In a series of earlier studies, we showed that distinct muscle fibre-types
superficial slow-twitch fibres (SSFs), muscle pioneer (MP) slow fibres
and medial fast-twitch muscle fibres (MFFs) differentiate in the
myotome of the zebrafish embryo in response to different levels and timing of
Hh activity emanating from the midline
(Lewis et al., 1999;
Wolff et al., 2003
;
Wolff et al., 2004
). These
unique muscle identities are specified by the combinatorial effects of the
Gli1 and Gli2 proteins, and serve as very sensitive cellular readouts of the
status of the activities of individual components of the Hh pathway that
modulate Gli function (Wolff et al.,
2003
). We used two different MOs designed against cos2
mRNA one targeted at the translational start and the other at the
splice junction between the first coding exon and the succeeding intronic
sequences (Fig. 4B; see also
Materials and methods). Injection of either one or a combination of these MOs
into wild-type embryos resulted in ectopic activation of the pathway, as
evidenced by an upregulation and expansion in the domain of patched1
(ptc1) expression in the muscle precursor cells
(Concordet et al., 1996
), which
encodes the ligand-binding component of the Hh receptor complex and, in
addition, is a direct and immediate transcriptional target of Hh activity in
responding cells (Fig. 4C,D).
Consistent with this expansion in the domain of ptc1, analysis of the
specification of muscle cell identities in such morphant (i.e. MO injected)
embryos revealed an increase in the population of all three Hh-dependent
muscle fibre types as a consequence of Cos2 inactivation the effects
on the MPs and MFFs being more pronounced than the SSFs
(Fig. 4E-H; Table 1). Such ectopic
induction of Hh-dependent muscle fates was suppressed in embryos co-injected
with the MOs, as well as synthetic mRNA encoding the GFP-Cos2 fusion protein
(Fig. 4I, see Materials and
methods for alterations in the MO recognition sequences in the
gfp-cos2 chimaeric cDNA), thereby confirming the specificity of the
morphant phenotype and suggesting that it could result from improper
translation and splicing of the endogenous cos2 transcript.
|
|
The activity of zebrafish Cos2 is epistatic to Shh and Smo
To explore this interpretation further and obtain additional evidence that
the effects of Hh-dependent cell fate determination on inhibition of Cos2
function arise specifically from a de-repression of the Hh pathway, we
injected the MOs into embryos compromised to varying degrees in their ability
to transduce Hh, either because of deletion of the shh gene
(Schauerte et al., 1998;
van Eeden et al., 1996
) or as
a consequence of mutations in the Smo protein that render it completely
non-functional (Chen et al.,
2001
; Varga et al.,
2001
). Although embryos that lack shh activity lack all
MP cells (Lewis et al., 1999
;
Schauerte et al., 1998
), a
muscle fibre that forms in response to the highest levels of Hh activity
(Wolff et al., 2003
), this
cell type was effectively restored in the mutant embryos injected with the
cos2 MOs (Fig. 6A-C).
Moreover, in smu mutant embryos, where no Hh-dependent muscle
cell-types are specified (Barresi et al.,
2000
; Wolff et al.,
2003
), loss of Cos2 activity resulted in the restoration of some
SSFs [cells that require the lowest levels of Hh
(Wolff et al., 2003
)],
indicating that inhibition of Cos2 is sufficient for triggering the activity
of Gliact, even under conditions where Smo-dependent signal
transduction is completely abolished (Fig.
6D-F).
|
Given this scenario, we analysed whether the phenotypic effects resulting
from the de-repression of Hh signalling as a consequence of the loss of Cos2
activity could be further potentiated by the simultaneous inhibition of the
activity of the zebrafish Su(fu) homologue. Indeed, embryos co-injected with
MOs directed against Su(fu) as well as cos2 exhibit high
levels of ectopic Hh signalling that represents a marked enhancement of the
effects observed when either the Cos2 or Su(fu) proteins are individually
inactivated with a striking increase in the numbers of all muscle cell fates
that depend upon Hh activity (Fig.
7A,B; Table 1) (see
also Wolff et al., 2003). We
also examined the effects of the concurrent loss of Cos2, as well as Su(fu),
in smu mutant embryos that lack all Hh signalling activity. Like
Cos2, inhibition of Su(fu) alone in the absence of Smo-mediated signalling,
results in a weak de-repression of the pathway as manifest by the
differentiation of a few SSFs in the myotome
(Fig. 7C). By contrast,
simultaneous loss of Cos2 and Su(fu) from smu embryos leads to the
differentiation of substantial numbers of SSFs and MFFs, as well as more
sporadic MPs cell types that are otherwise completely eliminated in
the absence of Smo activity (Fig.
7D-F; Table 1).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have noted that in contrast to situations where the Hh ligand is
mis-expressed, the effects of the loss of activity of zebrafish Cos2 in
instigating ectopic signalling is relatively mild. MO-mediated gene
inactivation dramatically reduces, but does not completely eliminate, the
translation of gene products (Nasevicius
and Ekker, 2000), and this could explain the restricted
de-repression of the Hh pathway that is observed in the cos2
morphants. In addition, the endogenous Cos2 protein could be highly stable
its sustained activity counteracting the effects of the MOs. Moreover,
we cannot rule out the existence of an additional paralogue(s) of Cos2 in the
zebrafish genome in which case, like the multiple Hh, Ptc and Gli proteins of
vertebrates, they could have overlapping functions in the regulation of the Hh
pathway. Although we are presently unable to distinguish between these
possibilities, we have nevertheless shown that the negative regulatory effect
of zebrafish Cos2 on Hh signalling is subject to dramatic enhancement when the
activity of the Su(fu) protein is also eliminated. Su(fu) is a known
antagonist of Gli function in flies as well as vertebrates and it plays a
dedicated role in restraining the nuclear access, as well as the
transcriptional activities of the Gli proteins. Although it is conceivable
that the augmentation of signalling levels in cos2; Su(fu)
double morphants again reflects the additive effects of inefficient MO
activity [because maximal activation of the Hh pathway in Drosophila
also requires the simultaneous loss of Cos2 and Su(fu) function], it seems
parsimonious to conclude that our results more likely point to a collaborative
effect of Cos2 and Su(fu) in regulating Gli activity that represents a
conserved event in the Hh signalling cascade.
There is now overwhelming support from investigations in
Drosophila for the activity of the C-terminal intracellular tail of
Smo in mediating the communication of Hh with the intracellular components of
the pathway through an association with the Cos2 protein
(Jia et al., 2003;
Lum et al., 2003
;
Ogden et al., 2003
;
Ruel et al., 2003
). Thus, Cos2
not only serves as a scaffold for the assembly and interaction of molecules
like Fu, Ci and Su(fu) with each other, but could also act to route the signal
from the Smo receptor at the membrane to these proteins in the cytoplasm. In
this connection we note that there are conflicting reports on the requirement
of the cytoplasmic tail of mammalian Smo for normal Hh signalling
(Jia et al., 2003
;
Murone et al., 1999
). In
addition, the stability as well as activity of Smo seems to be regulated by Hh
very differently in flies and mammals
(Kalderon, 2000
;
Taipale et al., 2002
).
Moreover, in contrast to Drosophila, our understanding of the precise
function of Fu, Su(fu) and the Gli proteins and their inter-molecular
interactions during the signalling process in vertebrates is rather
incomplete. Even more intriguingly, recent genetic as well as biochemical
screens for Hh pathway components in mammals and the zebrafish have led to the
isolation of a number of new constituents
(Bulgakov et al., 2004
;
Wolff et al., 2004
), the roles
of which have either not been elucidated or whose activities are not required
for regulating Hh signalling in flies, pointing to the possibility that a
substantial degree of diversification of the signalling mechanism has occurred
in different groups of animals.
Of particular interest in this regard is the observation that mutations in
intraflagellar transport (IFT) proteins, which includes a kinesin family
member unrelated to Drosophila Cos2, Kif3a, affect Hh signal
transduction in the mouse embryo (Huangfu
et al., 2003). However, in contrast to zebrafish Cos2, the
activities of these proteins appear to be required positively within the
signalling cascade. Currently, it is unclear how these IFT proteins interface
with the other more conserved intracellular players of the Hh pathway,
especially Su(fu), Fu and Gli. It is also possible that the IFT proteins have
assumed a committed role in Hh signalling exclusively in mammals because
mutations in their homologues seem not to result in any obvious defects in
Hh-dependent developmental processes in the zebrafish embryo
(Sun et al., 2004
;
Tsujikawa and Malicki, 2004
).
In light of all of these new but disparate findings, our discovery of an
obligate role of a Cos2 orthologue in regulating Hh signal transduction in the
zebrafish is noteworthy, as it closes a major gap in the mechanistic parallels
that exist between the signalling cascades that operate in flies and
vertebrates. In addition, it provides an essential framework for further
investigations directed at understanding the details of the interactions
between the Gli proteins, Cos2 and other members of the cytoplasmic complex,
and if and how these associations are modulated by the activity of Hh proteins
during development of the vertebrate embryo.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barresi, M. J., Stickney, H. L. and Devoto, S. H.
(2000). The zebrafish slow-muscle-omitted gene product is
required for Hedgehog signal transduction and the development of slow muscle
identity. Development
127,2189
-2199.
Blagden, C. S., Currie, P. D., Ingham, P. W. and Hughes, S.
M. (1997). Notochord induction of zebrafish slow muscle
mediated by Sonic hedgehog. Genes Dev.
11,2163
-2175.
Brand, M., Heisenberg, C. P., Warga, R. M., Pelegri, F.,
Karlstrom, R. O., Beuchle, D., Picker, A., Jiang, Y. J., Furutani-Seiki, M.,
van Eeden, F. J. et al. (1996). Mutations affecting
development of the midline and general body shape during zebrafish
embryogenesis. Development
123,129
-142.
Bulgakov, O. V., Eggenschwiler, J. T., Hong, D-H., Anderson, K.
V. and Li, T. (2004). FKBP8 is a negative regulator of mouse
sonic hedgehog signalling in neural tissues.
Development 131,2149
-2159.
Chen, W., Burgess, S. and Hopkins, N. (2001). Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity. Development 128,2385 -2396.[Medline]
Concordet, J. P., Lewis, K. E., Moore, J. W., Goodrich, L. V.,
Johnson, R. L., Scott, M. P. and Ingham, P. W. (1996).
Spatial regulation of a zebrafish patched homologue reflects the roles of
sonic hedgehog and protein kinase A in neural tube and somite patterning.
Development 122,2835
-2846.
Ding, Q., Fukami, S-i., Meng, X., Nishizaki, Y., Zhang, X., Sasaki, H., Dlugosz., A., Nakafuku, M. and Hui, C-c. (1999). Mouse Suppressor of fused is a negative regulator of Sonic hedgehog signalling and alters the subcellular distribution of Gli1. Curr. Biol. 9,1119 -1122.[CrossRef][Medline]
Du, S. J., Devoto, S. H., Westerfield, M. and Moon, R. T.
(1997). Positive and negative regulation of muscle cell identity
by members of the hedgehog and TGF-beta gene families. J. Cell
Biol. 139,145
-156.
Goldstein, L. S. (1993). With apologies to scheherazade: tails of 1001 kinesin motors. Annu. Rev. Genet. 27,319 -351.[CrossRef][Medline]
Huangfu, D., Liu, A., Rakeman, A. S., Murcia, N. S., Niswander, L. and Anderson, K. V. (2003). Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426,83 -87.[CrossRef][Medline]
Ingham, P. W. and McMahon, A. P. (2001).
Hedgehog signalling in animal development: paradigms and principles.
Genes Dev. 15,3059
-3087.
Jia, J., Tong, C. and Jiang, J. (2003).
Smoothened transduces Hedgehog signal by physically interacting with
Costal2/Fused complex through its C-terminal tail. Genes
Dev. 17,2709
-2720.
Kalderon, D. (2000). Transducing the hedgehog signal. Cell 103,371 -374.[Medline]
Karlstrom, R. O., Talbot, W. S. and Schier, A. F.
(1999). Comparative synteny cloning of zebrafish you-too:
mutations in the Hedgehog target gli2 affect ventral forebrain patterning.
Genes Dev. 13,388
-393.
Karlstrom, R. O., Tyurina, O. V., Kawakami, A., Nishioka, N.,
Talbot, W. S., Sasaki, H. and Schier, A. F. (2003). Genetic
analysis of zebrafish gli1 and gli2 reveals divergent requirements for gli
genes in vertebrate development. Development
130,1549
-1564.
Kogerman, P., Grimm, T., Kogerman, L., Krause, D., Undén, A. B., Sandstedt, B., Toftgård, R. and Zaphiropoulous, P. G. (1999). Mammalian Suppressor-of-Fused modulates nuclear-cytoplasmic shuttling of GLI-1. Nat. Cell Biol. 1,312 -319.[CrossRef][Medline]
Lewis, K. E., Currie, P. D., Roy, S., Schauerte, H., Haffter, P. and Ingham, P. W. (1999). Control of muscle cell-type specification in the zebrafish embryo by Hedgehog signalling. Dev. Biol. 216,469 -480.[CrossRef][Medline]
Lum, L. and Beachy, P. A. (2004). The Hedgehog
Response Network: Sensors, Switches, and Routers.
Science 304,1755
-1759.
Lum, L., Zhang, C., Oh, S., Mann, R. K., von Kessler, D. P., Taipale, J., Weis-Garcia, F., Gong, R., Wang, B. and Beachy, P. A. (2003). Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2. Mol. Cell. 12,1261 -1274.[CrossRef][Medline]
McMahon, A. P., Ingham, P. W. and Tabin, C. J. (2003). Developmental roles and clinical significance of hedgehog signalling. Curr. Top. Dev. Biol. 53, 1-114.[Medline]
Murone, M., Rosenthal, A. and de Sauvage, F. J. (1999). Sonic hedgehog signalling by the patched-smoothened receptor complex. Curr. Biol. 9, 76-84.[CrossRef][Medline]
Murone, M., Luoh, S-M., Stone, D., Li, W., Gurney, A., Armanini, M., Grey, C., Rosenthal, A., de Sauvage, F. J. (2000). Gli regulation by the opposing activities of Fused and Suppressor of Fused. Nat. Cell Biol. 2,310 -312.[CrossRef][Medline]
Nakano, Y., Kim, H. R., Kawakami, A., Roy, S., Schier, A. F. and Ingham, P. W. (2004). Inactivation of dispatched 1 by the chameleon mutation disrupts Hedgehog signalling in the zebrafish embryo. Dev. Biol. 269,381 -392.[CrossRef][Medline]
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Odenthal, J., van Eeden, F. J. M., Haffter, P., Ingham, P. W. and Nusslein-Volhard, C. (2000). Two distinct cell populations in the floor plate of the zebrafish are induced by different pathways. Dev. Biol. 219,350 -363.[CrossRef][Medline]
Ogden, S. K., Ascano, M., Jr, Stegman, M. A., Suber, L. M., Hooper, J. E. and Robbins, D. J. (2003). Identification of a functional interaction between the transmembrane protein Smoothened and the kinesin-related protein Costal2. Curr. Biol. 13,1998 -2003.[CrossRef][Medline]
Pearse, R. V. 2nd, Collier, L. S., Scott, M. P. and Tabin, C. J. (1999). Vertebrate homologues of Drosophila suppressor of fused interact with the gli family of transcriptional regulators. Dev. Biol. 212,323 -336.[CrossRef][Medline]
Robbins, D. J., Nybakken, K. E., Kobayashi, R., Sisson, J. C., Bishop, J. M. and Therond, P. P. (1997). Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2. Cell 90,225 -234.[Medline]
Ruel, L., Rodriguez, R., Gallet, A., Lavenant-Staccini, L. and Therond, P. P. (2003). Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog. Nat. Cell Biol. 5, 907-913.[CrossRef][Medline]
Schauerte, H. E., van Eeden, F. J., Fricke, C., Odenthal, J.,
Strahle, U. and Haffter, P. (1998). Sonic hedgehog is not
required for the induction of medial floor plate cells in the zebrafish.
Development 125,2983
-2993.
Sisson, J. C., Ho, K. S., Suyama, K. and Scott, M. P. (1997). Costal2, a novel kinesin-related protein in the Hedgehog signalling pathway. Cell 90,235 -245.[Medline]
Stone, D. M., Murone, M., Luoh, S-M., Ye, W., Armanini, M. P.,
Gurney, A., Phillips, H., Brush, J., Goddard, A., de Sauvage, F. J. and
Rosenthal, A. (1999). Characterisation of the human
Suppressor of fused, a negative regulator of the zinc-finger transcription
factor Gli. J. Cell Sci.
112,4437
-4448.
Sun, Z., Amsterdam, A., Pazour, G. J., Cole, D. G., Miller, M.
S. and Hopkins, N. (2004). A genetic screen in zebrafish
identifies cilia genes as a principal cause of cystic kidney.
Development 131,4085
-4093.
Taipale, J., Cooper, M. K., Maiti, T. and Beachy, P. A. (2002). Patched acts catalytically to suppress the activity of Smoothened. Nature 418,892 -897.[CrossRef][Medline]
Taylor, M. D., Liu, L., Raffel, C., Hui, C. C., Mainprize, T. G., Zhang, X., Agatep, R., Chiappa, S., Gao, L., Lowrance, A. et al. (2002). Mutations in SUFU predispose to medulloblastoma. Nat. Genet. 31,306 -310.[CrossRef][Medline]
Tsujikawa, M. and Malicki, J. (2004). Intraflagellar transport genes are essential for differentiation and survival of vertebrate sensory neurons. Neuron 42,703 -716.[CrossRef][Medline]
van Eeden, F. J., Granato, M., Schach, U., Brand, M.,
Furutani-Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C. P., Jiang,
Y. J., Kane, D. A. et al. (1996). Mutations affecting somite
formation and patterning in the zebrafish, Danio rerio.Development 123,153
-164.
Varga, Z. M., Amores, A., Lewis, K. E., Yan, Y. L., Postlethwait, J. H., Eisen, J. S. and Westerfield, M. (2001). Zebrafish smoothened functions in ventral neural tube specification and axon tract formation. Development 128,3497 -3509.[Medline]
Wang, G., Amanai, K., Wang, B. and Jiang, J.
(2000). Interactions with Costal2 and suppressor of fused
regulate nuclear translocation and activity of cubitus interruptus.
Genes Dev. 14,2893
-2905.
Wang, Q. T. and Holmgren, R. A. (1999). The
subcellular localisation and activity of Drosophila cubitus interruptus are
regulated at multiple levels. Development
126,5097
-5106.
Wolff, C., Roy, S. and Ingham, P. W. (2003). Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo. Curr. Biol. 13,1169 -1181.[CrossRef][Medline]
Wolff, C., Roy, S., Lewis, K. E., Schauerte, H., Joerg-Rauch,
G., Kirn, A., Weiler, C., Geisler, R., Haffter, P. and Ingham, P. W.
(2004). iguana encodes a novel zinc-finger protein with
coiled-coil domains essential for Hedgehog signal transduction in the
zebrafish embryo. Genes Dev.
18,1565
-1576.
Related articles in Development: