1 Department of Biology, University of Massachusetts, Amherst, MA 01003,
USA
2 Developmental Genetics Program, Skirball Institute of Biomolecular Medicine
and Department of Cell Biology, New York University School of Medicine, New
York, NY 10016, USA
3 Department of Biological Science, University of Tokyo, Tokyo, Japan
4 Laboratory for Embryonic Induction, Center for Developmental Biology, RIKEN,
Kobe, 650-0047 Japan
5 Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871,
Japan
6 Department of Developmental Biology, Stanford University School of Medicine,
Stanford, CA 94305, USA
* Author for correspondence (e-mail: karlstrom{at}bio.umass.edu)
Accepted 14 November 2002
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SUMMARY |
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Key words: Forebrain patterning, Hedgehog signaling, Adaxial cells, floor plate, cyclopamine, Morpholino
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INTRODUCTION |
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In vertebrates, additional complexity in Gli function is caused by the
presence of at least three gli genes, gli1, gli2, and
gli3. The functions of the different gli genes have been
analyzed using mouse mutants and mis- and overexpression in Xenopus,
Drosophila and cultured cells (reviewed by
Ingham and McMahon, 2001;
Koebernick and Pieler, 2002
;
Ruiz i Altaba et al., 2002
).
While the in vivo relevance of some of these studies remains to be
established, current evidence suggests the following roles for Gli proteins.
Gli1 appears to be an activator of Hh target genes, but in contrast to Ci,
Gli1 activity is not regulated post-translationally but transcriptionally by
Hh-mediated gene activation (Epstein et
al., 1996
; Marigo et al.,
1996a
; Hynes et al.,
1997
; Lee et al.,
1997
; Dai et al.,
1999
). Both N- and C-terminal domains of Gli1 are necessary for
its activation function (Ding et al.,
1999
; Ruiz i Altaba,
1999
). Despite its apparent activator function, Gli1 is not
essential for normal mouse development
(Park et al., 2000
;
Bai and Joyner, 2001
;
Bai et al., 2002
). In contrast,
mouse Gli2 mutations are perinatal lethal and result in the
down-regulation of Hh target genes (Ding
et al., 1998
; Matise et al.,
1998
), supporting the idea that Gli2 is a Hh-dependent activator.
The C-terminal region of Gli2 appears to be essential for its activation
function because C-terminally truncated Gli2 inhibits Hh target genes
(Ruiz i Altaba, 1999
;
Sasaki et al., 1999
). Since a
C-terminally truncated form of Gli2 might be generated by proteolytic
processing, it has been suggested that Gli2 also has repressor activity
(Ruiz i Altaba, 1999
;
Sasaki et al., 1999
;
von Mering and Basler, 1999
;
Aza-Blanc et al., 2000
).
Similarly, Gli3 appears to be processed to a C-terminally truncated repressor
of Hh target genes (Ruiz i Altaba,
1999
; Sasaki et al.,
1999
; Shin et al.,
1999
; Aza-Blanc et al.,
2000
; Wang et al.,
2000
). Accordingly, Gli3 mouse mutants display ectopic
activation of Hh targets (Masuya et al.,
1995
; Ruiz i Altaba,
1998
; Litingtung and Chiang,
2000
; Tole et al.,
2000
). Hh signaling is thought to repress Gli3
transcription and Gli3 processing (Marigo
et al., 1996a
; Ruiz i Altaba,
1998
; Dai et al.,
1999
; von Mering and Basler,
1999
; Aza-Blanc et al.,
2000
; Wang et al.,
2000
). The full-length form of Gli3 has been postulated to act as
an activator of Hh targets (Dai et al.,
1999
; Sasaki et al.,
1999
; Borycki et al.,
2000
; Litingtung and Chiang,
2000
), but direct in vivo evidence is currently not available to
support this hypothesis.
Misexpression and cell culture studies give insights into potential Gli
functions, but the exact requirement for vertebrate Hedgehog signaling and
Gli genes has been studied in most detail during neural patterning in
mouse mutants. Sonic hedgehog 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, motor neurons and most classes of
ventral interneurons in the spinal cord
(Chiang et al., 1996
;
Ericson et al., 1996
). Gli2 is
required to mediate some aspects of Hh signaling in the ventral neural tube.
Whereas motor neurons and most interneurons develop normally in Gli2
mutants, the floor plate does not form
(Ding et al., 1998
;
Matise et al., 1998
). In
contrast, Gli1 mutant mice have an apparently normal spinal cord,
indicating that Gli1 is not essential for interpreting Hh signals in the
ventral CNS (Park et al.,
2000
). Double mutant analysis suggests, however, that Gli1 can
contribute to Hh signaling since
Gli1/;Gli2/+ mice
show ventral patterning defects not found in Gli2/+
mice (Park et al., 2000
).
Moreover, expression of low levels of Gli1 in place of Gli2 can rescue
Gli2 mutants (Bai and Joyner,
2001
). Taken together, these results support the idea that Gli1
and Gli2 are positive mediators of Hh signaling. In contrast, Gli3 appears to
be involved in the repression of Hh targets in the dorsal CNS
(Litingtung and Chiang, 2000
;
Tole et al., 2000
).
While mutant data indicate that Gli1 and Gli2 are activators and Gli3 is a
repressor of Hh targets, seemingly contradictory results are surprisingly
common in the analysis of Gli function. For instance, mis-expression studies
in Xenopus have led to the suggestion that Gli1 specifies floor plate
development in the neural tube while Gli2 restricts floor plate specification,
but induces motoneuron development and patterns the mesoderm
(Lee et al., 1997;
Marine et al., 1997
;
Ruiz i Altaba, 1998
;
Ruiz i Altaba, 1999
;
Mullor et al., 2001
). These
proposals contradict the observations that mouse Gli2 mutants lack
floor plate, but do not display defects in early mesoderm patterning, and that
Gli1 is not required for ventral patterning
(Ding et al., 1998
;
Matise et al., 1998
;
Park et al., 2000
). These
results might reflect the shortcomings of misexpression approaches or
complications due to redundancy, but they might also be indicative of
context-dependent differences in Gli function. For instance, depending on cell
type or species, the requirements and activities of Gli genes might
differ.
Genetic studies of Hh signaling in zebrafish complement mutant analysis in
the mouse and provide an approach to test the conservation and divergence of
Gli function in vertebrates. Loss of zebrafish Hh signaling leads to ventral
spinal cord defects, deficiencies in ventral forebrain specification, absence
of an optic chiasm due to retinal axon guidance defects, absence of slow
muscle fiber types, malformations of the dorsal aorta, ventral curvature of
the body and defects in pectoral fin development
(Brand et al., 1996;
Chen et al., 1996
;
Karlstrom et al., 1996
;
van Eeden et al., 1996b
;
van Eeden et al., 1996a
;
Schauerte et al., 1998
;
Karlstrom et al., 1999
;
Lewis et al., 1999
;
Barresi et al., 2000
;
Odenthal et al., 2000
;
Chen et al., 2001
;
Varga et al., 2001
). Forward
genetic screens have identified mutations that cause all or some of these
phenotypes and affect components of the Hh signaling cascade. These include
sonic-you (syu), which disrupts shh
(Schauerte et al., 1998
),
slow-muscle-omitted (smu), which inactivates
smoothened (smo) (Chen
et al., 2001
; Varga et al.,
2001
) and you-too (yot), which encodes
C-terminally truncated forms of Gli2
(Karlstrom et al., 1999
).
Moreover, several molecularly uncharacterized mutants have a subset of
hh loss-of-function phenotypes, suggesting that they might encode
additional components or mediators of Hh signaling. For instance, the
detour (dtr) mutant was originally isolated because of
errors in retinal axon guidance (Karlstrom
et al., 1996
) and ventral curvature of the body
(Brand et al., 1996
). Axons
that normally cross the midline of the diencephalon fail to do so in
dtr mutants, and no optic chiasm forms
(Karlstrom et al., 1996
). In
addition, lateral floor plate cells are absent, suggesting defects in Hh
signaling similar to those seen in syu/shh, smu/smo and
yot/gli2 (Odenthal et al.,
2000
). Cranial motor neurons also fail to differentiate in
dtr mutant embryos (Chandrasekhar
et al., 1999
). Unlike syu/shh, smu/smo and yot/gli2,
dtr does not appear to affect somite patterning, differentiation of slow
muscle fibers, or formation of the dorsal aorta. Here we identify the
dtr locus as gli1 and analyze the roles of gli1 and
gli2 during zebrafish development. Our results reveal contrasting
requirements for gli genes in mouse and zebrafish and suggest that
gli1 is an essential activator of Hh-regulated genes, whereas
gli2 has minor roles in activating or repressing Hh targets.
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MATERIALS AND METHODS |
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Genetic mapping and linkage analysis
We determined the position of dtr on the zebrafish genetic map
using centromere linkage analysis (Johnson
et al., 1996; Postlethwait and
Talbot, 1997
). Gynogenetic diploid embryos were obtained from
heterozygous females by early pressure treatment of eggs fertilized with
inactivated sperm. Mutant and wild-type progeny were identified by visual
inspection on day 1 or day 2. DNA prepared from individuals or from pools of
eight mutant or wild-type individuals was assayed by PCR using polymorphic
markers (simple sequence length polymorphisms)
(Knapik et al., 1998
). This
identified a genetic marker (z3581) on LG6 that was linked to dtr.
Finer mapping, using embryos obtained from pairwise matings of heterozygous
dtr parents in a WIK background, identified two other closely linked
markers (z4910, z4950). The detailed genetic map in the region of the
gli1 locus can be viewed online using the zebrafish information
network (ZFIN) at
http://zfin.org.
Cloning the zebrafish gli genes
Genomic clones were obtained by screening a gridded genomic bacterial
artificial chromosome (BAC) library (Genome Systems) using radiolabeled probes
for a mouse Gli2 cDNA at low stringency hybridization conditions. BAC
DNA was prepared for positive clones and the BAC ends were sequenced using T7
and SP6 vector primers. SP6 end sequence of clone 152g22 showed homology to
mouse Gli1. PCR primers based on sequence from the T7 end of clone
152g22 amplified a simple sequence length polymorphism (SSLP) detectable upon
electrophoresis through 2% agarose gels. This SSLP was used to map the BAC end
to LG6 and detect linkage to the dtr locus (0 recombinants in 83
meioses). A partial cDNA clone encoding gli1 was isolated from a 15-
to 19-hour embryonic cDNA library (generously provided by Bruce Appel and
Judith Eisen, University of Oregon, Eugene) using a radio-labeled PCR probe
generated to sequence from the SP6 end of BAC 152g22. 5' and 3'
RACE reactions (Invitrogen) identified cDNA fragments encoding the 3'
and 5' portions of zebrafish gli1. These fragments were cloned
into the pTOPO vector (Invitrogen) and their sequences assembled into the full
gli1 coding region (GenBank accession no. AY173030).
Sequencing mutant alleles
RT-PCR and cycle sequencing were used to sequence the three ENU-induced
dtr alleles. RNA was isolated from the following pools of 40 embryos:
(1) dtrts269wild-type siblings; (2)
dtrts269 mutants; (3)
dtrte370mutants; and (4)
dtrtm276mutants. First-strand cDNA was made using
Superscript reverse transcriptase (GIBCO). Fragments (500-1000 bp) were
amplified from first strand cDNA by PCR using primers based on the deduced
gli1 cDNA sequence. DNA fragments were then gel purified and cycle
sequenced (Stratagene Cyclist). Sequences were compared between pools and to
the gli1 cDNA sequence. The fragments containing the dtr
point mutations were also subcloned using the TA cloning system (Invitrogen).
DNA from two separately isolated clones was purified, and the mutant sequence
was verified.
PCR genotyping dtr/gli1 and yot/gli2 fish
Embryos or fin clippings were placed in 50 µl lysis buffer (10 mM Tris
pH 7.5, 50 mM KCl, 0.3% Tween 20, 0.3% NP40, 1 mM EDTA) and incubated for 10
minutes at 98°C. Tissue was then digested by adding Proteinase K (Roche)
to 2 mg/ml and incubating 2 hours to overnight at 55°C. Proteinase K was
then inactivated by incubation at 98°C for 10 minutes. For genotyping
dtrts26 fish, a mutant-specific reverse primer
designed for the dtrts269allele (ts269Mu.rv:
5'-TGGGATCATGTTGCCCA) was used with a forward primer (dtr8.fw:
5'-GTCTAAAGGCTAAATATGCAGC) to amplify a mutant-specific 560 bp product
from homozygous mutants and heterozygotes. A wild-type reverse primer
(ts269Wt.rv: 5'-TGGGATCATGTTGCCCG) served as an amplification control.
To genotype yotty17fish, two primers flanking the
mutation site (yot33.fw: 5'-CCACCTAGCATATCAGAGAAC, yot28.rv:
5'-CTTGCTCACCGATATTCTGAC) were used to amplify a 589 bp product which
was then digested using the NlaIV restriction enzyme. The
yotty17mutation eliminates a NlaIV
restriction site in the amplified region, resulting in the appearance of a
mutant-specific 363 bp band that can be visualized on an agarose gel.
In situ hybridization and antibody labeling
In situ labeling was performed as described previously
(Schier et al., 1997). A 1.4
kb gli1 probe was synthesized using the 3' RACE containing
plasmid (dtr3'RACE.pCRII) linearized with BamHI using the T7
promoter. Other probes used were zebrafish gli2
(Karlstrom et al., 1999
),
lim3 (Glasgow et al.,
1997
), myoD (Weinberg
et al., 1996
), nk2.2
(Barth and Wilson, 1995
),
shh (Krauss et al.,
1993
), ptc1
(Concordet et al., 1996
) and
pax6 (Krauss et al.,
1991
).
mRNA and morpholino antisense oligonucleotide injections
Embryos were pressure injected with 500 pl-1 nl of solution at the 1- to
4-cell stage. Embryos were injected in their chorions and held in agarose
troughs (Westerfield, 1993).
Injected, control injected and uninjected embryos were grown to
80%
epiboly at 28°C, then shifted to 22°C and grown to the 20-somite
stage, fixed in 4% paraformaldehyde and processed for in situ hybridization.
For morpholino antisense oligonucleotide (MO) injections, embryos were
injected with from 1-15 ng of MO diluted in 1x Danio solution
(Westerfield, 1993
).
zfgli1 (5'-CCGACACACCCGCTACACCCACAGT) and zfgli2 MO
(5'-GGATGATGTAAAGTTCGTCAGTTGC), and a random control MO
(5'-CCTCTTACCTCAGTTACAATTTATA) were synthesized by Gene Tools (Eugene,
OR) and kept as 25 mg/ml stocks in 1x Danio solution. Specificity of
these MOs is demonstrated by (1) the suppression of the yot/gli2
repressor phenotype by the gli2 MO and (2) phenocopy of the
dtr phenotype by the gli1 MO in wild-type embryos. Synthetic
mRNA was made using the Message Machine kit (Ambion) and diluted in water to 1
mg/ml. shh mRNA was synthesized from a pT7TS plasmid containing
shh (Ekker et al.,
1995
). Control, ß-gal-encoding mRNA was synthesized from a
pT7TS plasmid containing the lacZ gene.
Cell culture analysis of transcriptional activity
The rat neural stem cell line MNS70
(Nakagawa et al., 1996) was
co-transfected with different plasmid constructs containing a gli
gene in the pcDNA3.1-His cloning vector (Invitrogen) in combination with a
reporter plasmid containing luciferase inserted downstream of 8xGli
binding sites (Sasaki et al.,
1997
). Full-length gli1 and gli2 inserts were
subcloned into the pcDNA vector from pBluescript (Stratagene). Mutant
constructs were made by swapping the appropriate, mutation-containing DNA
fragment, which was generated by RT-PCR from cDNA made from mutant embryos.
One day before transfection, MNS70 cells were plated onto poly-D-lysine coated
six-well plates at the concentration of 2x105cells per well.
Four hours before transfection, cells were re-fed with fresh medium. 1 µg
(total) of plasmid DNA (0.4 µg of effector [0.2 µg each of two effectors
indicated in figure], 0.5 µg of reporter and 0.1 µg of reference
[SV-b-gal]) was transfected to a well by mixing with 6 µl of Fugene 6
transfection reagent (Roche) according to the manufacture's protocol. Cell
lysates were prepared 48 hours after transfection and assayed for luciferase
and ß-galactosidase activities as previously described
(Sasaki et al., 1997
). For
western analysis, epitope-tagged proteins were detected using an Omni-probe
antibody (Santa Cruz Biotechnology).
Cyclopamine treatments
2-4 cell embryos were treated with 100 µM cyclopamine (Toronto Chemical)
(Incardona et al., 1998) by
adding 10 µl of a 10 mM stock solution (in 95% ethanol) to 1 ml of egg
water (0.3 g/l Instant Ocean Salt, 1 mg/l Methylene Blue). Control embryos
were treated simultaneously with an equal volume (10 µl) of 95% ethanol
(cyclopamine carrier) in 1 ml egg water. Treatments were carried out in
12-well plates (40 embryos/well) at 28.5°C. Embryos were grown to the
4-somite stage, dechorionated using 0.2 mg/ml (final) pronase (Sigma) in egg
water, fixed with 4% paraformaldehyde, dehydrated in methanol, and processed
for in situ hybridization.
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RESULTS |
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Minor roles for full-length Gli2 in the activation of Hh target
genes
Whereas the C-terminal truncation alleles of gli2 provide
information about the effect of dominant repressors on Hh signaling in vivo,
they do not address the requirement for Gli2 during embryonic development.
Therefore, we characterized the phenotypes generated by injecting
gli2 MOs into wild-type embryos. Surprisingly, knock down of Gli2 in
wild-type embryos did not lead to significant defects in most structures
affected by Hh signaling. In particular, ventral CNS (ptc1, nk2.2,
fkd4) and somite (myoD) markers were expressed normally
(Fig. 8A,D,G). In some embryos,
ptc1 and fkd4 expression was slightly expanded
(Fig. 8A and data not
shown).
Previous studies have suggested that full-length Gli2 is a Hh-dependent
activator of Hh target genes (Ding et al.,
1998; Matise et al.,
1998
; Ruiz i Altaba,
1998
; Ruiz i Altaba,
1999
; Aza-Blanc et al.,
2000
; Bai and Joyner,
2001
), and that the C terminus of Gli2 is required for this
activity (Ruiz i Altaba, 1999
;
Sasaki et al., 1999
).
gli2 MO injection into wild-type embryos might still allow for some,
albeit reduced, generation of full-length Gli2. We therefore analyzed in more
detail embryos that produce no full-length Gli2 and express reduced levels of
C-terminally truncated Gli2 by injecting gli2 MO into
yot/gli2 mutants. Intriguingly, Hh targets in the nervous system such
as ptc1 (Fig. 8C),
nk2.2 (Fig. 8F) and
fkd4 (not shown) are robustly expressed. These results suggest that
full-length Gli2 is not required for Hh signaling in the zebrafish spinal
cord.
The limited requirement for full-length Gli2 might be due to redundancy with other gli genes. To test if Gli2 and Gli1 have overlapping roles, we injected gli2 MOs into dtr/gli1 mutants (Fig. 8). Like dtr/gli1 mutants, these embryos display defects in nk2.2 expression in the brain and floor plate (Fig. 8K). Interestingly, a tegmental patch of nk2.2 expression that remains in dtr/gli1 mutants is eliminated by injection of gli2 MOs, suggesting Gli2 may act as an activator of Hh signaling in this region (Fig. 8K). In addition, myoD expression in adaxial cells is slightly but consistently reduced in gli2 MO; dtr/gli1 embryos (Fig. 8M), revealing overlapping roles of Gli1 and Gli2. Taken together, these data suggest that Gli2 plays a minor role in activating Hh target genes and is partially redundant with Gli1.
Gli2 acts as a repressor of telencephalic nk2.1b
expression
Previous studies (Ruiz i Altaba,
1998; Sasaki et al.,
1999
; von Mering and Basler,
1999
; Aza-Blanc et al.,
2000
) and our cell culture and in vivo data (Figs
4 and
8) indicate that Gli2 can act
as a repressor of Hh target genes. In support of this, we found that in
gli2 MO-injected embryos, expression of nk2.1b was expanded
dorsally in the telencephalon and ventrally in the ventral diencephalon
(Fig. 9A,B). This contrasts
with the dramatic reduction in nk2.1b expression seen upon loss of Hh
signaling in smu/smo mutants (Fig.
9E). The expansion of nk2.1b expression caused by loss of
Gli2 function is Gli1-independent, since gli2 MO injection into
dtr/gli1 mutants leads to an expansion of nk2.1b in the
ventral telencephalon (Fig.
9H). This suggests that one role of Hh signaling might be to
overcome Gli2-mediated repression of nk2.1b. In this scenario,
blocking Gli2 function should partially suppress the loss of nk2.1b
in smu/smo mutants. Indeed, injection of gli2 MO into
smu/smo mutants partially restored nk2.1b expression in the
ventral telencephalon (Fig.
9F). These results suggest that Gli2 acts as a Hh-independent
repressor of some Hh target genes.
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DISCUSSION |
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Our analyses reveal that gli1 is disrupted in dtr mutants
and indicate that dtrte370 and
dtrts269 encode strong or complete loss-of-function
versions of Gli1. The dtrte370 and
dtrts269 alleles lack a C-terminal activation domain and
are inactive in cell culture, consistent with results obtained upon
overexpression of C-terminally truncated Gli1 in frog
(Ruiz i Altaba, 1999). In
vivo, dtrts269 mutants are impaired in the upregulation of
nk2.2 expression in response to ectopic Hh signaling in most regions
of the CNS. In contrast to truncated zebrafish Gli2, truncated zebrafish Gli1
does not appear to act as a dominant repressor of Hh signaling;
dtr/+ embryos do not display any obvious phenotypes
and truncated Gli1 does not interfere with gene activation by wild-type Gli1
in cell culture. Moreover, gli1 MO injection phenocopies
dtrte370 and dtrts269 mutants. Taken
together, these results suggest that these mutants and gli1 MO
embryos lack all or most Gli1 activity.
The third point mutation (dtrtm276) affects a conserved
tyrosine residue in the DNA binding region of Gli1 known to contact target DNA
(Pavletich and Pabo, 1993). On
its own, this protein does not activate reporter gene expression in cultured
cells, consistent with a potential defect in DNA binding. Interestingly,
however, dtrtm276 activates transcription in the presence
of wild-type Gli1. It is conceivable that the mutant protein forms a complex
with the wild-type protein, thus being recruited to DNA and providing a
transcriptional activation domain.
Together with previous studies (Brand et
al., 1996; Karlstrom et al.,
1996
; Chandrasekhar et al.,
1999
; Odenthal et al.,
2000
), our results reveal that loss of gli1 function
leads to ventral CNS patterning defects in zebrafish (summarized in
Table 3). dtr/gli1
mutants lack the lateral floor plate and show reduced expression of markers
for anterior pituitary and ventral diencephalon. These neural patterning
defects are similar to, but weaker than those seen in smu/smo mutants
or cyclopamine-treated embryos. For example, smu/smo mutant embryos
show a more severe loss of ventral diencephalon and strong to complete
reduction of ptc1, nk2.2, and nk2.1b expression
(Chen et al., 2001
;
Varga et al., 2001
). In
addition, dtr/gli1 mutants appear normal with respect to somite
development, pectoral fin formation and dorsal aorta differentiation, whereas
smu/smo mutants show severe defects in these structures. These data
indicate that gli1 is necessary for ventral CNS patterning, but that
it is required in only a subset of cells responding to Hh signals.
|
The phenotypic similarity between gli2 MO; dtr/gli1 and dtr/gli1 mutants also suggests a limited role for Gli2. Some overlapping functions of Gli1 and Gli2 are indicated by the reduction in myoD expression in somitic mesoderm and nk2.2 in the tegmentum in gli2MO; dtr/gli1 embryos. Overlapping roles of gli1 and gli2 are also evident in the loss of engrailed-expressing muscle cells upon reduction of both Gli1 and Gli2 (C. Wolff, S. Roy and P. Ingham, personal communication). These results suggest that Gli2 contributes as a positive mediator of Hh signaling to the activation of some Hh target genes. In contrast, telencephalic nk2.1b is expanded in gli2 MO embryos and expressed at reduced levels in smu/smo mutants. Blocking both Gli2 and Smo partially suppresses the smu/smo phenotype, indicating that Hh signaling relieves Gli2-mediated repression of nk2.1b. Importantly, neither expression nor expansion of nk2.1b are Gli1 dependent, indicating that Hh signaling might directly inhibit Gli2-mediated repression of nk2.1b. Taken together, these results suggest that zebrafish Gli2 can act as a Hh-dependent activator.
C-terminal truncations of Gli2 block Hedgehog signaling
Our results suggest that the C-terminally truncated Gli2 proteins encoded
by yot/gli2 alleles encode dominant repressors of Hh signaling. In
vitro, the truncated forms of Gli2 block Gli1-mediated transcriptional
activation, resembling the activity of C-terminally truncated mouse and frog
Gli2 proteins (Ruiz i Altaba,
1999; Sasaki et al.,
1999
). In vivo, yot/gli2 mutations reduce Hh signaling
(Karlstrom et al., 1999
).
Expression of Hh target genes such as ptc1 and nk2.2 is
reduced and several structures that depend on Hh signaling (lateral floor
plate, horizontal myoseptum, pectoral fins, dorsal aorta) do not form.
Injection of gli2 MO into yot/gli2 embryos rescues most of
the mutant phenotypes, demonstrating the antimorphic nature of the
yot/gli2 alleles. In addition, yot/gli2 heterozygotes have
subtle defects in somite patterning (van
Eeden et al., 1996b
; Karlstrom
et al., 1999
). These results suggest that zebrafish
yot/gli2 mutations turn Gli2 into a constitutive repressor of
Hh-regulated genes. Precedence for this scenario has been provided by human
GLI3 mutations that result in C-terminally truncated repressor forms
of GLI3 (Kang et al., 1997
;
Radhakrishna et al., 1997
;
Shin et al., 1999
) and by the
fact that truncated Gli proteins can act as dominant repressors in cell
culture (Sasaki et al., 1999
)
or when ectopically expressed in embryos
(Ruiz i Altaba, 1999
).
Interestingly, embryos that are heterozygous for both dtr/gli1 and yot/gli2 have a phenotype that is intermediate between the two homozygous mutant phenotypes (Fig. 1). This result indicates that truncated Gli2 blocks Gli1-mediated activation of Hh targets and uncovers roles for gli1 during somite development not revealed in dtr/gli1 mutants. Gli1 cannot be the only factor antagonized by yot, since yot/gli2 mutants have a more severe phenotype than dtr/gli1 mutants.
The finding that truncated Gli2 acts as an in vivo repressor of Hh target
genes has potential medical implications. Previous studies have shown that
decreased Hh signaling can result in congenital defects such as
holoprosencephaly (reviewed by Wallis and
Muenke, 2000). Our results in zebrafish suggest that C-terminal
truncations of Gli2 are candidates for the molecular basis of some cases of
holoprosencephaly. In addition, C-terminally truncated Gli2 could be employed
to repress the ectopic expression of Hh target genes in human cancers such as
Basal Cell Carcinoma or medulloblastoma (reviewed by
Ruiz i Altaba et al.,
2002
).
Species-specific roles of Gli genes
Vertebrate Gli function has been studied predominantly in Xenopus
using gain-of-function approaches and in mouse using loss-of-function
strategies. Our loss-of-function study in zebrafish suggests that gli
genes might not have identical roles in all vertebrates.
Comparison to Xenopus
Based on mis- and overexpression studies, multiple roles for Xenopus
Gli genes have been proposed. Gli1 has been considered to activate floor
plate and motor neuron differentiation in the spinal cord and induce ventral
cell types in the forebrain (Lee et al.,
1997; Ruiz i Altaba,
1998
; Ruiz i Altaba,
1999
). Our results reveal an essential role for zebrafish Gli1
during lateral floor plate induction (Fig.
5) (Odenthal et al.,
2000
), but do not indicate a requirement in motor neuron induction
(Brand et al., 1996
) or
telencephalic nk2.1b forebrain expression
(Fig. 8). Xenopus Gli2
has been proposed to restrict floor plate development, repress nk2.1b
expression in the forebrain, promote motor neuron formation and pattern
mesoderm (Marine et al., 1997
;
Ruiz i Altaba, 1998
;
Ruiz i Altaba, 1999
;
Brewster et al., 2000
;
Mullor et al., 2001
). Our
studies reveal only a minor and variable role for zebrafish Gli2 in the
repression of floor plate markers. Although our results provide evidence for
an essential role of zebrafish Gli2 in nk2.1b repression, this
activity of Gli2 is not simply achieved by repressing Gli1, as proposed in
Xenopus. In addition, we have found no evidence for a requirement of
Gli2 in motor neuron induction or early mesoderm patterning. The apparent
differences between zebrafish and Xenopus gli gene function might be
due to species-specific roles. Alternatively, they might reflect the
difficulty of comparing results gained in studies that test the requirement
for gene function using loss-of-function approaches with studies that assign
potential gene functions using gain-of-function strategies. Further
clarification of the potential differences in zebrafish and Xenopus
Gli function will require loss-of-function approaches in frog and
gain-of-function studies in zebrafish.
Comparison to mouse
Our analyses in zebrafish suggest surprisingly divergent requirements for
Gli1 and Gli2 in zebrafish and mouse. Genetic studies in mouse have shown that
Gli1 is dispensable for development, whereas Gli2 is a major mediator of Hh
signaling during neural development
(Matise et al., 1998;
Park et al., 2000
;
Bai and Joyner, 2001
). Two
lines of evidence suggest that mouse Gli2 acts predominantly as a
transcriptional activator of Hh target genes. First, replacing Gli2
with Gli1 in a knock-in approach results in normal development
(Bai and Joyner, 2001
). Second,
Shh;Gli2 double mutants have the same phenotype as Shh
mutants (Bai and Joyner, 2001
).
These results suggest that Shh signaling requires Gli2 to activate
Hh-regulated genes and does not de-repress Hh target genes by counteracting a
putative Gli2 repressor form. In clear contrast to these conclusions,
zebrafish Gli1 is an essential activator of Hh target genes during neural
development, while Gli2 appears to have only minor activator roles and acts as
a repressor of the Hh target gene nk2.1b in the telencephalon. It is
unlikely that these differences are simply the result of allele variations. In
the case of Gli1, strong (dtrts269;
dtrte370; gli1 MO) or even partial
(dtrtm276) loss of Gli1 function results in nervous system
defects not seen in mouse Gli1 null alleles. In the case of Gli2,
loss of a putative activator form of Gli2 or partial reduction of Gli2
activity does not result in the CNS phenotypes attributed to the loss of an
activator form of Gli2 in mouse.
The differences between orthologous gli genes are surprising in
light of the overall conservation of sequence, expression, regulation and
transcriptional activity in cell culture. Both overlapping functions of
gli genes and subtle differences in Gli activity or expression might
underlie the divergent requirements. In the case of gli2, it is
possible that another gli gene compensates for reduction in Gli2
activity. For instance, Gli2 and Gli3 have partially overlapping roles in
mouse foregut, tooth and skeletal development
(Mo et al., 1997;
Motoyama et al., 1998
). It is
thus possible that another Gli protein masks the role of Gli2 in zebrafish
development. gli2 MO injection into dtr/gli1 mutants leads
to only a minor enhancement of the dtr/gli1 mutant phenotype,
suggesting that a gli gene other than gli1 might compensate
for reduction in Gli2 activity.
We speculate that one of the major roles of Gli1 is to act as an amplifier
of vertebrate Hh signaling. In this model, Gli1 activity is required in
zebrafish, but not in mouse, because Hh target genes are insufficiently
activated by initial Hh signaling in zebrafish. This model is based on the
kinetics of gli gene activation. It has been shown that Gli1
is a transcriptional target of Hh signaling
(Epstein et al., 1996;
Marigo et al., 1996a
;
Lee et al., 1997
;
Dai et al., 1999
) and thus
acts as a delayed activator of Hh targets. In contrast, Gli2 and Gli3 protein
activity can be post-translationally regulated
(Ruiz i Altaba, 1999
;
Sasaki et al., 1999
;
von Mering and Basler, 1999
;
Aza-Blanc et al., 2000
;
Wang et al., 2000
;
Bai et al., 2002
) and in the
case of Gli3 has been shown to be directly modulated by Hh signaling
(von Mering and Basler, 1999
;
Aza-Blanc et al., 2000
;
Wang et al., 2000
). Hence, Hh
signaling is thought to be initially mediated by Gli2 and Gli3, leading to the
activation of downstream genes such as Gli1 and Ptc1
(Ingham and McMahon, 2001
).
Subsequently, Hh signaling can be maintained or amplified by Gli1. In some
contexts, this amplification might be essential for full activation of Hh
target genes. This model suggests that in the zebrafish CNS, the initial
activation of Hh target genes by Gli2, Gli3 or other Gli proteins might be
quite weak or short lived, requiring further enhancement by Gli1. In contrast,
in the mouse CNS, Hh-mediated modulation of Gli2 and Gli3 activity is
sufficient for Hh target gene activation. Interestingly, in
Gli1/;Gli2/+
mice, reduction of the levels of Gli2 leads to a requirement for Gli1 in Hh
target gene activation (Park et al.,
2000
; Bai et al.,
2002
). According to the Gli1 amplifier model, Gli1 becomes
essential because initial Hh-mediated signaling by Gli2 is weaker in
Gli2/+ than wild-type embryos. In this scenario,
Gli2/+ mouse embryos resemble zebrafish wild-type
embryos, requiring Gli1 for full Hh target gene activation. It is conceivable
that direct mediators of Hh signaling are less potent or expressed at lower
levels in zebrafish than mouse or negative regulators might be more active or
more highly expressed in zebrafish than mouse. In both cases, Gli1-mediated
amplification would be required to allow full Hh target gene activation in
zebrafish.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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