1 Department of Biological Science, Graduate School of Science, University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 RIKEN Center for Developmental Biology, Kobe, 650-0047, Japan
3 Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871,
Japan
4 Biology Department, University of Massachusetts, Amherst, MA 01003-9297,
USA
* Author for correspondence (e-mail: atkawaka{at}biol.s.u-tokyo.ac.jp)
Accepted 30 December 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Neural tube patterning, Muscle cell specification, Hedgehog signaling, Cyclopamine, cAMP-dependent kinase A, Forskolin, Midline mutant, Adaxial cells, dzip1, PEST sequence, Zebrafish
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because Hh signaling plays such a central role in development and disease,
the Hh signaling pathway has been investigated in considerable detail. Genetic
and in vitro studies in Drosophila have revealed that Hh signals are
transduced by binding of Hh ligands to the Patched (Ptc) cell-surface
receptor, resulting in the activation of the transmembrane protein Smoothened
(Smo). In Drosophila, the intracellular regulation of Hh signaling is
mediated by post-translational modifications of Cubitus interruptus (Ci), a
zinc-finger-containing transcription factor of the Gli family that can be both
an activator and a repressor of Hh target genes
(Methot and Basler, 2001). In
the absence of Hh signal, proteolytic cleavage converts Ci to a
transcriptional repressor (Aza-Blanc et
al., 1997
; Wang and Holmgren,
1999
). In the presence of Hh signals, cleavage of Ci is inhibited
and a full-length activator isoform predominates. In vertebrates, at least
three Gli genes, Gli1, Gli2 and Gli3, mediate the
transcriptional response to Hh signals
(Hui et al., 1994
;
Ruiz i Altaba, 1998
). The
functions of these different Gli genes have been analyzed in mouse,
Xenopus, zebrafish and cultured cells (reviewed by
Ingham and McMahon, 2001
;
Koebernick and Pieler, 2002
;
Ruiz i Altaba et al., 2002
).
Although Gli1 is dispensable for normal mouse development
(Park et al., 2000
;
Bai et al., 2002
), analysis of
Gli1/; Gli2/+
mutants (Park et al., 2000
)
and the rescue of Gli2 mutant by Gli1 (Bai
and Joyner, 2001
) suggest that Gli1 has an activator function.
Consistently, gli1 mutations in zebrafish lead to a loss of Hh target
gene expression (Karlstrom et al.,
2003
). As yet there is no evidence that Gli1 activity is regulated
by protein processing, it appears to be solely an activator of the Hh response
(Epstein et al., 1996
;
Marigo et al., 1996
;
Hynes et al., 1997
;
Lee et al., 1997
;
Dai et al., 1999
). By
contrast, Gli2 appears to be both an activator and repressor of Hh target
genes, depending on the tissue being examined. Mouse Gli2 mutations
are perinatal lethal and result in the downregulation of Hh target genes
(Ding et al., 1998
;
Matise et al., 1998
). The
C-terminal region of Gli2 appears to be essential for the activation function,
as the C-terminally truncated Gli2 proteins do not activate Hh target genes
(Karlstrom et al., 1999
;
Ruiz i Altaba, 1999a
;
Sasaki et al., 1999
;
Karlstrom et al., 2003
).
Several studies have shown that Gli2 can also repress the expression of Hh
target genes (von Mering and Basler,
1999
; Sasaki et al.,
1999
; Aza-Blanc et al.,
2000
; Karlstrom et al.,
2003
). Similarly, several lines of evidence suggest that Gli3 can
act both as an activator and as a repressor of Hh signaling
(Masuya et al., 1995
;
Dai et al., 1999
;
Ruiz i Altaba, 1999a
;
Sasaki et al., 1999
;
Shin et al., 1999
;
Aza-Blanc et al., 2000
;
Tole et al., 2000
;
Litingtung and Chiang, 2000
;
Wang et al., 2000
;
Persson et al., 2002
). Despite
suggestions that proteolytic processing may regulate Gli2 and Gli3 function,
the in vivo cleavage of these proteins has not been directly demonstrated.
Thus, in both vertebrates and invertebrates, Hh signaling controls the
expression of target genes by modulating the activity of the downstream Gli/Ci
transcription factors. Studies in Drosophila have identified a large
number of proteins that are involved in the regulation of this Gli/Ci activity
(reviewed by McMahon, 2000;
Ingham and McMahon, 2001
;
Nybakken and Perrimon, 2002
).
Within target cells, Ci forms a protein complex with Fused kinase (Fu)
(Monnier et al., 1998
;
Stegman et al., 2000
) and
Costal2 (Cos2) (Robbins et al.,
1997
; Sisson et al.,
1997
) that is tethered to microtubules. Activated Smo signals to
this cytoplasmic protein complex, resulting in the release of Ci and active
transport into the nucleus where it can activate Hh target genes
(Ohlmeyer and Kalderon, 1998
;
Ding et al., 1999
;
Kogerman et al., 1999
;
Methot and Basler, 2000
;
Murone et al., 2000
;
Wang et al., 2000
). The
transport of Ci to the nucleus is mediated by another protein, Suppressor of
Fused [Su(Fu)]. In addition to such a positive regulation in response to Hh
ligands, a negative pathway that includes cAMP-dependent protein kinase (PKA)
also regulates Hh signaling (Li et al.,
1995
; Pan and Rubin,
1995
; Lepage et al.,
1995
; Hammerschmidt et al.,
1996
). In the absence of Hh ligands, PKA directly phosphorylates
Ci to promote its proteolytic cleavage, generating the repressor isoform
(Chen et al., 1998
;
Chen et al., 1999
;
Price and Kalderon, 1999
).
Studies in vertebrates have shown that these intracellular components of the
Hh signaling pathway have been largely conserved through evolution
(Hammerschmidt et al., 1996
;
Pearse et al., 1999
;
Kogerman et al., 1999
;
Ding et al., 1999
;
Murone et al., 2000
).
Genetic studies of Hh signaling in zebrafish complement the analyses in fly
and other vertebrate species, and provide an approach to look into the
regulation of Hh signaling in vertebrates. In vertebrates, Sonic hedgehog
(Shh) is expressed in the notochord and floor plate
(Echelard et al., 1993;
Krauss et al., 1993
;
Roelink et al., 1994
;
Ekker et al., 1995
), and is
essential for the induction of floor plate, motoneurons and a class of ventral
interneurons in the neural tube (Chiang et
al., 1996
; Ericson et al.,
1996
). Shh signaling is also required for the induction of muscle
and sclerotome cell types in somites (reviewed by
Bumcrot and McMahon, 1995
). A
large number of zebrafish mutations, collectively called the midline mutants,
have been identified that lead to ventral neural tube defects, absence of an
optic chiasm and defects in slow muscle fiber formation
(Brand et al., 1996
;
Chen et al., 1996
;
Karlstrom et al., 1996
;
van Eeden et al., 1996b
). Many
of these midline mutants have now been shown to encode components of the Hh
signal cascade. shh is disrupted in sonic-you (syu)
mutants (Schauerte et al.,
1998
), smo is disrupted in slow-muscle-omitted
(smu) (Chen et al.,
2001
; Varga et al.,
2001
), gli2 is disrupted in you-too
(yot) (Karlstrom et al.,
1999
), gli1 is disrupted in detour
(dtr) (Karlstrom et al.,
2003
) and dispatched1 is disrupted in chameleon
(con) (Nakano et al.,
2004
). As several molecularly uncharacterized midline mutants
share many phenotypes with these known Hh pathway mutants, it is likely that
they encode additional components of Hh signaling.
In this study, we show that igu mutations lead to reduced Hh target gene expression in the neural tube. Surprisingly, these same mutations cause the ectopic activation of Hh target genes in somites. Our analyses reveal that Igu function is required for the full activation of Hh signaling in response to Hh ligands. We also show that the igu mutations, directly or indirectly, affect the negative regulation of Hh signaling that is required for silencing the Hh target gene expression in the absence of Hh ligands. Positional cloning of the igu gene revealed that the gene encodes Dzip1, a novel component of the Hh signaling pathway. We show that Igu/Dzip1 acts as a necessary permissive factor for the proper regulation of Hh signaling.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
RNA injection
For RNA injections, PCR fragments of igu-coding sequences from
wild-type and mutant alleles were cloned into pCS2+ vector. Capped mRNAs were
synthesized in vitro using the mMessage Machine kit (Ambion) according to the
manufacturer's instruction and injected into embryos (1- to 4-cell stage) that
were obtained from igu+/ incrosses.
Cyclopamine and forskolin treatments
Cyclopamine and forskolin were dissolved in 100% dimethyl sulfoxide (DMSO)
at 10 mM and 60.9 mM, respectively. Embryos were dechorinated with pronase at
50% epiboly and placed in 6 ml of embryo medium
(Westerfield, 1993) containing
50 µM of cyclopamine or the desired concentrations of forskolin. Surviving
embryos were fixed and processed for in situ hybridization.
In situ hybridization and genotyping
Whole-mount in situ hybridization was performed as described
(Schier et al., 1997). Embryos
were photographed in 80% glycerol, and genomic DNA was recovered from each
embryo by proteinase K treatment (1 mg/ml) in 50 µl of lysis buffer (10 mM
Tris-HCl pH 8, 50 mM KC1, 0.3% Tween 20, 0.3% NP40, 1 mM EDTA) for 5 hours to
overnight at 55°C. For genotyping, 5 µl of genomic DNA was used for
each PCR reaction. Mutant embryos were identified using tightly linked genetic
markers. Primers used for genotyping were unp172F
(5'-TCATGACGAAGCAGTTTGGA-3') and unp172R
(5'-CAGGTGTCGTTTTCAGGGTTA-3') for iguts294e,
z1660 for yotty119, and z14475 for
dtrts269.
Transfection and antibody staining
PCR amplified wild-type and mutant (ts294e allele)
igu/dzip1-coding sequences were subcloned into pcDNA4-HisMax TOPO
(Invitrogen). Transfection into NIH3T3 or HEK293T cells was performed
according to the manufacturer's instructions (Fugene, Roche). Cells were fixed
at 2 days, permeabilized with methanol and stained with Omni-probe antibody
(Santa Cruz Biotechnology) in combination with anti-rabbit FITC (Jackson
ImmunoResearch). Lysosomes were visualized with anti-Lamp1 antibody
(Hughes and August, 1981) in
NIH3T3 cells. We also made FLAG-tagged constructs and obtained a similar
result (data not shown).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To verify that defects in nk2.2 and en1 expression are
correlated with the changes in Hh signaling, we examined the expression of
patched1 (ptc1), a sensitive indicator of Hh signaling
(Concordet et al., 1996;
Goodrich et al., 1996
). In
agreement with the observed en1 phenotypes, ptc1 expression
is reduced in somites of syu/shh, dtr/gli1, yot/gli2 and con
mutants. More specifically, ptc1 expression is severely reduced in
con mutants both in the neural tube and in somites
(Fig. 1F6,F7), whereas it is
less severely reduced in the neural tube in syu/shh, dtr/gli1 and
yot/gli2 mutants (Fig.
1C6-E6,C7-E7). Intriguingly, in yot/gli2 mutant embryos,
the medial-ventral regions of somites retain ptc1
(Fig. 1E7). This region
corresponds to developing sclerotomal tissues
(Morin-Kensicki and Eisen,
1997
), which is also induced by Hh signals
(Fan and Tessier-Lavigne,
1994
). In marked contrast to the other Hh pathway mutants,
igu mutants show expanded ptc1 expression in somites
(Fig. 1B4-B7), indicating that
Hh signaling is ectopically activated in somites. The ectopic ptc1
expression is evident as early as 12 hpf
(Fig. 2I). By contrast,
ptc1 expression in the ventral neural tube appears to be unchanged or
slightly decreased compared with wild-type embryos
(Fig. 1A7,B7). Thus,
igu mutants show ectopic activation of Hh target genes in somites
despite the reduction of the Hh target genes ptc1 and nk2.2
in the neural tube. Such an aberrant regulation of Hh target gene expression
suggests that the igu gene product may play an important role in both
the positive and negative regulation of Hh signaling in different tissues.
|
|
|
igu mutations impair both the proper activation and repression of Hh target genes
Previous studies have established that PKA negatively regulates Hh
signaling by promoting the proteolytic processing of Ci/Gli proteins and their
conversion to repressor isoforms (Ohlmeyer
and Kalderon, 1998; Pan and
Rubin, 1995
; Lepage et al.,
1995
; Hammerschmidt et al.,
1996
). This processing is crucial for silencing target gene
expression in the absence of Hh ligands. To explore the possible relationship
between Igu and the negative regulation of Hh signaling, we manipulated the
PKA activity in igu embryos. First, we inhibited PKA activity by
overexpressing the dominant-negative regulatory subunit of PKA (dnPKA). This
inhibition of PKA activity in wild-type embryos leads to the ectopic
activation of Hh target genes in broad regions of the neural tube and somites
(Fig. 4A-C) (Hammerschmidt et al., 1996
).
However, the expression of dnPKA in igu embryos did not induce
expression of Hh-responsive genes (Fig.
4D-F; Table 1). As
dnPKA is thought to increase full-length Gli activators by disturbing the
processing of Gli proteins into repressor isoforms, our results suggest that
igu mutations impair the positive regulation of Gli proteins into a
fully active state. This is consistent with results from compound mutants
(Fig. 3) and results from
shh mRNA injections in which Shh overexpression did not affect the
igu mutant phenotype either in somites or in the neural tube
(Table 1; data not shown).
|
|
This predicted protein structure suggested that Igu/Dzip1 may localize in
the cytoplasm and/or nucleus. To characterize the subcellular localization of
Igu/Dzip1, we expressed wild type and mutant igu/dzip1 in cultured
cells. Wild type Igu/Dzip1 is present in the cytoplasm, and is found at
especially high levels in large vesicles
(Fig. 7A). These vesicles
correspond to lysosomes and/or endosomes based on the co-localization with the
lysosomal protein Lamp1 (Hughes and
August, 1981) (Fig.
7B,C). By contrast, the mutant Igu/Dzip1 protein, which lacks the
PEST sequences but retains the NLS, is strongly enriched in nuclei
(Fig. 7D-F). Because a single
zinc-finger domain does not bind to DNA, it is likely that Igu/Dzip1 is
involved in protein-protein interactions. Indeed, human DZIP1 has been
suggested to interact with DAZ, a protein required for spermatogenesis
(Moore et al., 2003
).
Therefore, our results raise the possibility that Igu/Dzip1 might regulate the
stability or the nuclear translocation of other proteins, including Gli
proteins or other components of the Hh signaling pathway.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our examination of compound mutant phenotypes also revealed that the activating ability of Gli proteins is generally reduced in igu mutants, despite the apparent upregulation of en1 and ptc1 expression in somites. This reduced Gli activator function explains the loss of Hh signaling in the neural tube of igu mutants and suggests that Igu/Dzip1 function is required for the full activation of Gli proteins in response to Hh signals.
Both positive and negative regulation of Hh signaling is impaired in igu mutants
Previous studies have shown that Hh signaling is tightly regulated in the
embryo, with different intracellular mechanisms positively and negatively
regulating Hh signal transduction. Hh signaling is positively regulated
through the functions of Fu, Cos2 and Su(Fu) and the nuclear trafficking of
Gli/Ci proteins (see Fig. 9) (Monnier et al., 1998;
Ding et al., 1999
;
Kogerman et al., 1999
;
Methot and Basler, 2000
;
Murone et al., 2000
;
Stegman, et al., 2000
;
Wang et al., 2000
). Negative
regulation is thought to occur via PKA-mediated processing of Gli/Ci proteins
into repressor isoforms in the absence of Hh ligands
(Lepage et al., 1995
;
Ohlmeyer and Kalderon, 1998
;
Pan and Rubin, 1995
;
Hammerschmidt et al., 1996
;
Jiang and Struhl, 1998
).
|
We also showed that igu mutations reduce the ability of PKA to
repress Hh target gene expression. igu mutants were resistant to
forskolin, which blocks Hh signaling by activating PKA. This reduced
sensitivity to forskolin treatment cannot be simply explained by defects in
the positive regulation of Gli proteins. In Drosophila, it has been
shown that mutations in PKA lead to the accumulation of Ci and the
upregulation of Hh signaling (Ohlmeyer and
Kalderon, 1998). Likewise, the upregulation of target genes in
igu mutants may partly be due to a defect in the negative regulation
of Gli proteins. Taken together, our analyses suggest that both the positive
and negative regulation of Hh signaling is impaired in igu mutants
(Fig. 9).
Igu protein structure and function
We showed that the igu gene encodes a single zinc-finger protein,
Dzip1. Although igu/dzip1 is conserved among vertebrate species such
as human, mouse, rat, chicken and frog, we could not identify a homologous
gene in Drosophila. Considering the high degree of conservation in
the Hh signaling pathway during evolution, a functionally equivalent protein
may present in invertebrates, though the sequence might be highly diverged.
Intriguingly, human postaxial polydactyly type A2 (PAPA2), a congenital defect
that is caused by the ectopic activation of Hh signaling
(Villavicencio et al., 2000),
maps to a region containing human DZIP1 at 13q32. DZIP1 might be the gene
disrupted in PAPA2.
Single zinc-finger domain proteins are not thought to bind DNA, instead
they are implicated in mediating protein-protein interactions. The presence of
PEST domains in the Dzip1 protein sequence suggests that the protein may be a
target for rapid degradation and is consistent with our observation that
Igu/Dzip1 accumulates in lysosomes in cultured cells. These facts suggest the
possibility that Igu/Dzip1 is involved in mediating the rapid turnover of
interacting proteins, perhaps components of the Hh signaling cascade. However,
the Igu/Dzip1 protein may not simply be a cytoplasmic protein, as it has a
nuclear localization signal (NLS) within the N-terminal conserved region.
Indeed, the truncated Igu/Dzip1 proteins encoded by igu are enriched
in nuclei in cultured cells, suggesting that the NLS is also functional. This
finding raises the possibility that wild type Igu/Dzip1 proteins shuttle
between the cytoplasm and the nucleus, and they could thus affect the nuclear
import of Hh pathway proteins. Although Gli proteins are possible candidates
for Igu/Dzip1 interacting partners, our preliminary data suggest that Igu
proteins do not physically interact with Gli proteins in vitro, and they do
not alter the transcriptional activation of Gli proteins in a luciferase
reporter assay (H.S. and A.K., unpublished). Igu proteins might interact with
other components of the Hh signaling pathway such as Fu, Cos2 or Su(Fu).
Intriguingly, Su(Fu) is involved in the nuclear trafficking of Gli proteins
and also contains a PEST sequence (Pham et
al., 1995; Pearse et al.,
1999
). Moreover, it was recently shown that reducing Su(Fu)
function in zebrafish using morpholino oligonucleotides resulted in the
upregulation of En expression in somites
(Wolff et al., 2003
). From
these data, we speculate that Igu/Dzip1 might be involved in the regulation of
the Su(Fu), Fu and Cos2 cytoplasmic protein complex that is known to regulate
Gli protein activities.
Our results also revealed that both wild-type and mutant versions of Igu/Dzip1 proteins do not have a dominant function when overexpressed in wild-type embryos. This, combined with our mutant analysis, indicates that Igu/Dzip1 function is a permissive factor required for the regulation of Hh signaling, and that mutant Igu/Dzip1 proteins do not function dominantly to interfere with the regulation of Hh signaling. These results support the idea that Igu/Dzip1 function is directly required for the negative regulation of Gli proteins. However, we cannot exclude the possibility that Igu mutant proteins have a new function that is masked in injected embryos by the presence of the wild-type protein. In fact, nuclear localization of mutant Igu/Dzip1 proteins in vitro could suggest a gained function for the mutant Igu/Dzip1 proteins. Although we attempted to generate a loss-of-function phenotype by blocking Igu/Dzip1 translation with antisense morpholino oligonucleotides, these morpholinos produced no phenotype in wild-type embryos, and did not alter Hh target gene expression in igu mutants. Considering the weak and ubiquitous expression of the igu gene and the permissive nature of Igu/Dzip1, it is possible that small amounts of Igu/Dzip1 proteins are sufficient for normal development, and that morpholinos are therefore unable to block expression sufficiently to produce a phenotype. Further biochemical analysis of Igu/Dzip1 will be needed to fully elucidate its function.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aza-Blanc, P., Ramirez-Weber, F.-A., Laget, M.-P., Schwartz, C. and Kornberg, T. B. (1997). Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 89,1043 -1053.[Medline]
Aza-Blanc, P., Lin, H. Y., Ruiz i Altaba, A. and Kornberg, T.
B. (2000). Expression of the vertebrate Gli proteins in
Drosophila reveals a distribution of activator and repressor activities.
Development 127,4293
-4301.
Bai, C. B. and Joyner, A. L. (2001). Gli1 can rescue the in vivo function of Gli2. Development 128,5161 -5172.[Medline]
Bai, C. B., Auerbach, W., Lee, J. S., Stephen, D. and Joyner, A. L. (2002). Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway. Development 129,4753 -4761.[Medline]
Barth, K. A. and Wilson, S. W. (1995).
Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate
hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic
forebrain. Development
121,1755
-1768.
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.
Bumcrot, D. A. and McMahon, A. P. (1995). Somite differentiation. Sonic signals somites. Curr. Biol. 5,612 -614.[Medline]
Chen, J. N., Haffter, P., Odenthal, J., Vogelsang, E., Brand,
M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Hammerschmidt,
M., Heisenberg, C. P. et al. (1996). Mutations affecting the
cardiovascular system and other internal organs in zebrafish.
Development 123,293
-302.
Chen, Y., Gallaher, N., Goodman, R. and Smolik, S.
(1998). Protein kinase A directly regulates the activity and
proteolysis of Cubitus interruptus. Proc. Natl. Acad. Sci.
USA 95,2349
-2354.
Chen, Y., Cardinaux, J., Goodman, R. and Smolik, S.
(1999). Mutants of cubitus interruptus that are independent of
PKA regulation are independent of hedgehog signaling.
Development 126,3607
-3616.
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]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][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.
Currie, P. D. and Ingham, P. W. (1996). Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish. Nature 382,452 -455.[CrossRef][Medline]
Dai, P., Akimaru, H., Tanaka, Y., Maekawa, T., Nakafuku, M. and Ishii, S. (1999). Sonic hedgehog-induced activation of the Gli1 promoter is mediated by GLI3. J. Biol. Chem. 19,8143 -8152.[CrossRef]
Ding, Q., Motoyama, J., Gasca, S., Rong, M., Sasaki, H.,
Rossant, J. and Hui, C.-C. (1998). Diminished Sonic
hedgehog signaling and lack of floor plate differentiation in Gli2 mutant
mice. Development 125,2533
-2543.
Ding, Q., Fukami, S., Meng, X., Nishizaki, Y., Zhang, X., Sasaki, H., Dlugosz, A., Nakafuku, M. and Hui, C. (1999). Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Gli1. Curr. Biol. 9,1119 -1122.[CrossRef][Medline]
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75,1417 -1430.[Medline]
Ekker, S. C., Ungar, A. R., Greenstein, P., von Kessler, D. P., Porter, J. A., Moon, R. T. and Beachy, P. A. (1995). Patterning activities of vertebrate Hedgehog proteins in the developing eye and brain. Curr. Biol. 5, 944-955.[Medline]
Epstein, D. J., Marti, E., Scott, M. P. and McMahon, A. P.
(1996). Antagonizing cAMP-dependent protein kinase A in the
dorsal CNS activates a conserved sonic hedgehog signaling pathway.
Development 122,2885
-2894.
Ericson, J., Morton, S., Kawakami, A., Roelink, H. and Jessell, T. M. (1996). Two critical periods of sonic hedgehog signaling required for the specification of motor neuron identity. Cell 87,661 -673.[Medline]
Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. M. and Briscoe, J. (1997). Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90,169 -180.[Medline]
Fan, C. M. and Tessier-Lavigne, M. (1994). Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell 79,1175 -1186.[Medline]
Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A. and Scott, M. P. (1996). Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by hedgehog. Genes Dev. 10,301 -312.[Abstract]
Goodrich, L. V. and Scott, M. P. (1998). Hedgehog and patched in neural development and disease. Neuron 21,1243 -1257.[Medline]
Hammerschmidt, M., Bitgood, M. J. and McMahon, A. P. (1996). Protein kinase A is a common negative regulator of hedgehog signaling in the vertebrate embryo. Genes Dev. 10,647 -658.[Abstract]
Hughes, E. N. and August, J. T. (1981).
Characterization of plasma membrane proteins identified by monoclonal
antibodies. J. Biol. Chem.
256,664
-671.
Hui, C. C., Slusarski, D., Platt, K. A., Holmgren, R. and Joyner, A. L. (1994). Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm and mesoderm-derived tissues suggests multiple roles during postimplantation development. Dev. Biol. 62,402 -413.
Hynes, M., Stone, D. M., Dowd, M., Pitts-Meek, S., Goddard, A., Gurney, A. and Rosenthal, A. (1997). Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene gli-1. Neuron 19, 15-26.[Medline]
Incardona, J. P., Gaffield, W., Kapur, R. P. and Roelink, H.
(1998). The teratogenic Veratrum alkaloid cyclopamine inhibits
sonic hedgehog signal transduction. Development
125,3553
-3562.
Ingham, P. W. and McMahon, A. P. (2001).
Hedgehog signaling in animal development: paradigms and principles.
Genes Dev. 15,3059
-3087.
Jiang, J. and Struhl, G. (1998). Regulation of the Hedgehog and Wingless signaling pathways by the F-box/WD40-repeat protein Slimb. Nature 391,493 -496.[CrossRef][Medline]
Karlstrom, R. O., Trowe, T., Klostermann, S., Baier, H., Brand,
M., Crawford, A. D., Grunewald, B., Haffter, P., Hoffmann, H., Meyer,
S. U. et al. (1996). Zebrafish mutations affecting
retinotectal axon pathfinding. Development
123,427
-438.
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., Schier, A. F. (2003).
Genetic analysis of zebrafish gli1 and gli2 reveals divergent requirements for
gli genes in vertebrate development. Development
130,1549
-1564.
Knapik, E. W., Goodman, A., Ekker, M., Chevrette, M., Delgado, J., Neuhauss, S., Shimoda, N., Driever, W., Fishman, M. C. and Jacob, H. J. (1998). A microsatellite genetic linkage map for zebrafish (Danio rerio). Nat. Genet. 18,338 -343.[Medline]
Koebernick, K. and Pieler, T. (2002). Gli-type zinc finger proteins as bipotential transducers of Hedgehog signaling. Differentiation 70,69 -76.[CrossRef][Medline]
Kogerman, P., Grimm, T., Kogerman, L., Krause, D., Unden, A. B., Sandstedt, B., Toftgard, R., and Zaphiropoulos, P. G. (1999). Mammalian suppressor-of-fused modulates nuclear cytoplasmic shuttling of Gli-1. Nat. Cell Biol. 1, 312-319.[CrossRef][Medline]
Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75,1431 -1444.[Medline]
Lee, J., Platt, K. A., Censullo, P. and Ruiz i Altaba, A.
(1997). Gli1 is a target of sonic hedgehog that induces ventral
neural tube development. Development
124,2537
-2552.
Lepage, T., Cohen, S. M., Diaz-Benjumea, F. J. and Parkhurst, S. M. (1995). Signal transduction by cAMP-dependent protein kinase A in Drosophila limb patterning. Nature 373,711 -715.[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 signaling. Dev. Biol. 216,469 -480.[CrossRef][Medline]
Li, W., Ohlmeyer, J. T., Lane, M. E. and Kalderon, D. (1995). Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 80,553 -562.[Medline]
Litingtung, Y. and Chiang, C. (2000). Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and Gli3. Nat. Neurosci. 3, 979-985.[CrossRef][Medline]
Macdonald, R., Barth, K. A., Xu, Q., Holder, N., Mikkola, I. and
Wilson, S. W. (1995). Midline signaling is required
for Pax gene regulation and patterning of the eyes.
Development 121,3267
-3278.
Marigo, V., Johnson, R. L., Vortkamp, A. and Tabin, C. J. (1996). Sonic hedgehog differentially regulates expression of gli and gli3 during limb development. Dev. Biol. 180,273 -283.[CrossRef][Medline]
Masuya, H., Sagai, T., Wakana, S., Moriwaki, K. and Shiroishi, T. (1995). A duplicated zone of polarizing activity in polydactylous mouse mutants. Genes Dev. 9,1645 -1653.[Abstract]
Matise, M., Epstein, D., Park, H., Platt, K. and Joyner, A.
(1998). Gli2 is required for the induction of floor plate and
adjacent cells, but not most ventral neurons in the mouse central nervous
system. Development 125,2759
-2770.
McMahon, A. P. (2000). More surprises in the hedgehog signaling pathway. Cell 100,185 -188.[Medline]
McMahon, A., Ingham, P. and Tabin, C. (2003). The developmental roles and clinical significance of Hedeghog signaling. Curr. Top. Dev. Biol. 53, 1-114.[Medline]
Methot, N. and Basler, K. (2000). Suppressor of
fused opposes hedgehog signal transduction by impeding nuclear accumulation of
the activator form of Cubitus interruptus. Development
127,4001
-4010.
Methot, N. and Basler, K. (2001). An absolute
requirement for Cubitus interruptus in Hedgehog signaling.
Development 128,733
-742.
Morin-Kensicki, E. M. and Eisen, J. S. (1997).
Sclerotome development and peripheral nervous system segmentation in embryonic
zebrafish. Development
124,159
-167.
Monnier, V., Dussillol, F., Alves, G., Lamour-Isnard, C. and Plessis, A. (1998). Molecular interactions between three members of the Drosophila Hedgehog signaling pathway: Suppressor of fused links the ser-thr kinase Fused and the transcription factor Cubitus interruptus. Curr. Biol. 8, 583-586.[Medline]
Moore, F. L., Jaruzelska, J., Fox, M. S., Urano, J., Firpo, M.
T., Turek, P. J., Dorfman, D. M. and Pera, R. A. (2003).
Human Pumilio-2 is expressed in embryonic stem cells and germ cells and
interacts with DAZ (Deleted in AZoospermia) and DAZ-like proteins.
Proc. Natl. Acad. Sci. USA
100,538
-543.
Murone, M., Luoh, S. M., Stone, D., Li, W., Gurney, A., Armanini, M., Grey, C., Rosenthal, A. and 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. and Ingham, P. W. (2004). Inactivation of dispatched 1 by the chameleon mutation disrupts Hedgehog signaling in the zebrafish embryo. Dev. Biol. (in press).
Nybakken, K. and Perrimon, N. (2002). Hedgehog signal transduction: recent findings Curr. Opin. Genet. Dev. 12,503 -511.[CrossRef][Medline]
Odenthal, J., van Eeden, F. J., 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]
Ohlmeyer, J. and Kalderon, D. (1998). Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator. Nature 396,749 -753.[CrossRef][Medline]
Pan, D. and Rubin, G. M. (1995). cAMP-dependent protein kinase and hedgehog act antagonistically in regulating decapentaplegic transcription in Drosophila imaginal discs. Cell 80,543 -552.[Medline]
Park, H. L., Bai, C., Platt, K. A., Matise, M. P., Beeghly, A.,
Hui, C. C., Nakashima, M. and Joyner, A. L. (2000).
Mouse Gli1 mutants are viable but have defects in SHH signaling in combination
with a Gli2 mutation. Development
127,1593
-1605.
Pearse, R. V., II, Collier, L. S., Scott, M. P. and Tabin, C. J. (1999). Vertebrate homologs of Drosophila Suppressor of fused interact with the Gli family of transcriptional regulators. Dev. Biol. 212,323 -336.[CrossRef][Medline]
Persson, M., Stamataki, D., te Welscher, P., Andersson, E.,
Bose, J., Ruther, U., Ericson, J. and Briscoe, J.
(2002). Dorsal-ventral patterning of the spinal cord requires
Gli3 transcriptional repressor activity. Genes Dev.
16,2865
-2878.
Pham, A., Therond, P., Alves, G., Tournier, F. B., Busson, D.,
Lamour-Isnard, C., Bouchon, B. L., Preat, T. and Tricoire, H.
(1995). The Suppressor of fused gene encodes a novel PEST protein
involved in Drosophila segment polarity establishment.
Genetics 140,587
-598.
Postlethwait, J. H. and Talbot, W. S. (1997). Zebrafish genomics: from mutants to genes. Trends Genet. 13,183 -190.[CrossRef][Medline]
Price, M. and Kalderon, D. (1999). Proteolysis
of cubitus interruptus in Drosophila requires phosphorylation by
protein kinase A. Development
126,4331
-4339.
Radhakrishna, U., Wild, A., Grzeschik, K.-H. and Antonarakis, S. E. (1997). Mutation in Gli3 in postaxial polydactyly type A. Nat. Genet. 17,269 -271.[Medline]
Rechsteiner, M. and Rogers, S. W. (1996). PEST sequences and regulation by proteolysis. Trends Biochem. Sci. 21,267 -271.[CrossRef][Medline]
Robbins, D., Nybakken, K., Kobayashi, R., Sisson, J., Bishop, J. and Therond, P. (1997). Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein Costal2. Cell 90,225 -234.[Medline]
Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., Ruiz i Altaba, A., Tanabe, Y., Placzek, M., Edlund, T., Jessell, T. M. et al. (1994). Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell 76,761 -775.[Medline]
Roy, S., Wolff, C. and Ingham, P. W. (2001).
The u-boot mutation identifies a Hedgehog-regulated myogenic switch for
fiber-type diversification in the zebrafish embryo. Genes
Dev. 15,1563
-1576.
Ruiz i Altaba, A. (1998). Combinatorial Gli
function in floor plate and neuronal inductions by Sonic hedgehog.
Development 125,2203
-2212.
Ruiz i Altaba, A. (1999a). Gli proteins encode
context-dependent positive and negative functions: implications for
development and disease. Development
126,3205
-3216.
Ruiz i Altaba, A. (1999b). Gli proteins and hedgehog signalingdevelopment and cancer. Trends Genet. 15,418 -425.[CrossRef][Medline]
Ruiz i Altaba, A., Sanchez, P. and Dahmane, N. (2002). Gli and hedgehog in cancer: tumours, embryos and stem cells. Nat. Rev. Cancer 2, 361-372.[CrossRef][Medline]
Sasaki, H., Nishizaki, Y., Hui, C., Nakafuku, M. and Kondoh,
H. (1999). Regulation of Gli2 and Gli3 activities by an
amino-terminal repression domain: implication of Gli2 and Gli3 as primary
mediators of Shh signaling. Development
126,3915
-3924.
Sbrogna, J. L., Barresi, M. J. F. and Karlstrom, R. O. (2003). Multiple roles for Hedgehog signaling in zebrafish pituitary development. Dev. Biol. 254, 19-35.[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.
Schier, A. F., Neuhauss, S. C. F., Helde, K. A., Talbot, W. S.
and Driever, W. (1997). The one-eyed pinhead gene
functions in mesoderm and endoderm formation in zebrafish and interacts with
no tail. Development
124,327
-342.
Seamon, K. B. and Daly, J. W. (1981). Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J. Cyclic Nucl. Res. 7,201 -224.
Shin, S. H., Kogerman, P., Lindstrom, E., Toftgard, R. and
Biesecker, L. G. (1999). GLI3 mutations in human
disorders mimic Drosophila cubitus interruptus protein functions and
localization. Proc. Natl. Acad. Sci. USA
96,2880
-2884.
Sisson, J. C., Ho, K., S., Suyama, K. and Scott, M. P. (1997). Costal2, a novel kinesin-related protein in the hedgehog signaling pathway. Cell 90,235 -245.[Medline]
Stegman, M., Vallance, J., Elangovan, G., Sosinski, J., Cheng,
Y. and Robbins, D. (2000). Identification of a
tetrameric hedgehog signaling complex. J. Biol. Chem.
275,21809
-21812.
Taipale, J., Chen, J. K., Cooper, M. K., Wang, B., Mann, R. K., Milenkovic, L., Scott, M. P. and Beachy, P. A. (2000). Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406,1005 -1009.[CrossRef][Medline]
Tole, S., Ragsdale, C. W. and Grove, E. A. (2000). Dorsoventral patterning of the telencephalon is disrupted in the mouse mutant extra-toes(J). Dev. Biol. 217,254 -265.[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. (1996a). Genetic analysis of
fin formation in the zebrafish, Danio rerio.
Development 123,255
-262.
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. (1996b). 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]
Villavicencio, E. H, Walterhouse, D. O. and Iannaccone, P. M. (2000). The sonic hedgehog-patched-gli pathway in human development and disease. Am. J. Hum. Genet. 67,1047 -1054.[Medline]
von Mering, C. and Basler, K. (1999). Distinct and regulated activities of human Gli proteins in Drosophila. Curr. Biol. 9,1319 -1322.[CrossRef][Medline]
Wallis, D. and Muenke, M. (2000). Mutations in holoprosencephaly. Hum. Mutat. 16, 99-108.[CrossRef][Medline]
Wang, B., Fallon, J. F. and Beachy, P. A. (2000). Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100,423 -434.[Medline]
Wang, Q. T. and Holmgren, R. A. (1999). The
subcellular localization and activity of Drosophila cubitus
interruptus are regulated at multiple levels.
Development 126,5097
-5106.
Weinberg, E. S., Allende, M. L., Kelly, C. S., Abdelhamid, A.,
Murakami, T., Andermann, P., Doerre, O. G., Grunwald, D. J. and
Riggleman, B. (1996). Developmental regulation of zebrafish
MyoD in wild-type, no tail and spadetail embryos.
Development 122,271
-280.
Westerfield, M. (1993). The Zebrafish Book. Eugene: University of Oregon Press.
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]
Related articles in Development: