Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA
Author for correspondence (e-mail:
chuang{at}cvrimail.ucsf.edu)
Accepted 30 September 2002
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SUMMARY |
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Key words: Dispatched, Hedgehog, Mouse, Protein transport
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INTRODUCTION |
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Extensively studies on Hh signaling in both invertebrates and vertebrates
have led to a prevailing model of Hh reception. Hh signal is transduced
through hedgehog binding to patched 1 (Ptch), a multipass transmembrane
protein (reviewed by Ingham and McMahon,
2001; Kalderon,
2000
). Genetic and molecular studies suggest that Ptch inhibits
the signaling activity of smoothened (Smo), a seven transmembrane protein that
shares sequence similarity with G-protein-coupled receptors (reviewed by
Ingham and McMahon, 2001
;
Kalderon, 2000
). Though the
molecular mechanism remains to be elucidated, Hh binding to Ptch appears to
relieve the Ptch-mediated repression of Smo. As a consequence, activated Smo
can initiate the signaling cascade, turning on transcription of key Hh
targets.
An attractive model to account for the activity of Hh is the generation of
a Hh protein concentration gradient, which provides positional information in
the morphogenetic field. The mechanism by which Hh protein moves across tens
of cell diameters is not obvious because of the fact that Hh protein is
membrane anchored through lipid modification. The Hh protein precursor
undergoes autoproteolysis to generate an N-terminal signaling fragment (Shh-N)
(Bumcrot et al., 1995;
Lee et al., 1994
), followed by
two types of post-translational modification. A cholesterol molecule is
covalently attached to the C terminus of Shh-N
(Porter et al., 1996a
;
Porter et al., 1996b
) and a
palmitoyl group is added to the N-terminus of Shh-N
(Pepinsky et al., 1998
) (the
resulting lipid-modified form of Shh-N will be denoted as Shh-Np). The role of
lipid modification in Hh signaling is not completely understood, but in vitro
studies have shown that Shh-Np becomes membrane anchored as a consequence of
lipid modification. It is conceivable that an important step in Hh signaling
is to release the membrane-anchored Hh from the Hh-producing cells to allow
for subsequent `movement' through the morphogenetic field. Interestingly,
movement of lipid-modified Hh in Drosophila depends on the activity
of tout velou (ttv) in Hh-receiving cells
(Bellaiche et al., 1998
).
ttv encodes a glycosaminoglycan transferase, suggesting TTV generates
a proteoglycan that may mediate the transfer of Hh protein between cells. The
role of ttv vertebrate homologs, the Ext genes
(Stickens et al., 1996
), in Hh
signaling has not yet been established.
Some insight into the process of Hh release from Hh-producing cells came
from the identification of the dispatched (disp) gene in
Drosophila that is predicted to encode a twelvepass transmembrane
protein and is required in Hh-producing cells to transport lipid-modified Hh
protein (Burke et al., 1999).
Drosophila mutants in the disp gene display phenotypes
reminiscent of hh mutants as Hh protein, instead of moving out of
Hh-producing cells, accumulates to a higher level in these cells
(Burke et al., 1999
). Disp
exhibits sequence similarity to an emerging family of multipass membrane
proteins, including Ptch, all of which contain a characteristic sterol-sensing
domain (SSD) (reviewed by Kuwabara and
Labouesse, 2002
). These observations suggest that Hh movement is
closely linked to lipid modification and likely employs novel cellular
mechanisms in releasing and transporting a membrane-anchored cell surface
protein. However, the biochemical mechanisms by which Disp facilitates Hh
movement remain unknown. To address the issue of Hh movement in vertebrates,
we report the identification of the mammalian dispatched gene and studies
aimed to understand its role in Hh protein transport during vertebrate
embryonic development.
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MATERIALS AND METHODS |
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Cloning of mouse dispatched (Disp) cDNA
A mouse EST clone (IMAGE 1430982) containing sequence similarity to the
Drosophila disp gene was used to screen a mouse embryonic cDNA
library and several partial Disp cDNAs were obtained. The 5'
end of the Disp cDNA was obtained by RT-PCR. A full-length
Disp cDNA (4721 bp) was acquired by ligating together restriction
fragments of partial cDNAs. The GenBank Accession Number for mouse
Disp cDNA is AY150577.
Generation of Disp null mice
Mouse Disp cDNA was used to screen a mouse 129/SvJ genomic
library. To construct a positive/negative targeting vector for removing exon 8
of the Disp gene (the resulting allele is designated Disp
E8), a 2.7 kb fragment containing sequences upstream of intron 7 was used
as the 5' region of homology (Fig.
3A). A 3.5 kb fragment containing sequences downstream of exon 8
was used as the 3' region of homology and was inserted upstream of the
MC1-tk-pA cassette (see Fig.
3A). A PGK-neo-pA cassette was inserted between the
5' and 3' homology regions and replaces the seventh intron and
eighth exon of the Disp gene (Fig.
3A). E14Tg2A.4 (E14) feeder-independent ES cells
(Nichols et al., 1990
) were
electroporated with a SalI-linearized targeting vector and selected
in G418 and FIAU as described (Joyner,
2000
). Heterozygous E14 ES cells were injected into blastocysts of
C57BL/6 strain mice to generate germline chimeras. Chimeric males were mated
with C57BL/6, 129/Sv, 129/Ola or Swiss-Webster females (to maintain the
Disp mutant allele in different genetic backgrounds) and heterozygous
animals were identified by Southern blotting of tail-tip DNA
(Fig. 3B).
|
Histology and in situ hybridization
Histological analysis, whole-mount in situ hybridization using
digoxigenin-labeled probes and section in situ hybridization using
33P-labeled riboprobes were performed as described
(Wilkinson and Nieto, 1993).
The mouse Disp in situ probe encompasses the last kb of the
Disp cDNA.
Western blotting
We collected wild-type, Disp E8+/- and
Disp
E8-/- embryos at 9.5 dpc (genotypes confirmed
by Southern blotting) for western blotting to detect the processing event of
Shh. In addition, we transfected COS7 cells, using Lipofectamine Plus reagent
(Invitrogen), with expression constructs, which encode either the full-length
Shh or the N-terminal fragment of Shh (Shh-N) without post-translational
modifications. Transfected cells were harvested 2 days after transfection. To
control for the specificity of Shh antibodies, we also collected Shh
mutant embryos at a stage similar to that of Disp
E8-/- embryos. COS7 cells or 9.5 dpc mouse embryos were lysed
in buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton
X-100 (v/v), 0.5 mM DTT, 1 mM PMSF and 2 µg/ml each of aprotinin, leupeptin
and pepstatin A. Insoluble materials were sedimented by centrifugation at
20,000 g for 30 minutes at 4°C. The supernatants were
transferred into a fresh tube and the samples were separated on 15% SDS-PAGE
and transferred onto PVDF membranes for immunoblotting
(Harlow and Lane, 1999
). The
membranes were blocked in 10% w/v fat-free milk powder in phosphate buffered
saline (PBS) containing 0.1% Tween 20 overnight and incubated with primary
antibody against Shh for 2 hours. The membranes were incubated with secondary
antibodies followed by chemiluminescent detection according to manufacturer's
instructions (ECL, Amersham Pharmacia Biotech).
Immunohistochemistry
We followed a protocol kindly provided by Dr Gritli-Linde
(Gritli-Linde et al., 2001)
with some minor modifications. The embryos were fixed overnight in Sainte
Marie's fixative (95% ethanol, 1% acetic acid) at 4°C. After washing the
embryos three times, 30 minutes each, in 95% ethanol, we proceeded to paraffin
embedding and sectioning at 5 µM. Tissues were dewaxed in xylene twice, 5
minutes each and rehydrated to water by taking through 100% ethanol twice, 5
minutes each, 95% ethanol twice, 5 minutes each and PBS once for 5 minutes.
The endogenous peroxidases were blocked by incubating the slides in 3%
H2O2 in methanol for 10 minutes in the dark at room
temperature. The slides were rinsed in PBS three times, 5 minutes each, and
the nonspecific staining was blocked by incubating the slides in PBS with 5%
sheep serum, 0.2% BSA and 0.1% Triton X-100 for 40 minutes at room
temperature. Slides were incubated overnight at 4°C with the primary
antibody (anti-Shh) diluted 1:500 in PBS with 0.2% BSA and 0.1% Triton X-100
in a humidified chamber. The signal was amplified using a Tyramide signal
amplification kit (TSA Biotin kit NEL700 or 700A from PerkinElmer). We
followed a modified version of the manufacturer's protocol outlined below. The
slides were rinsed three times, 5 minutes each, in TNT (0.1M Tris, pH 7.5,
0.15 M NaCl, 0.025% Tween 20). Slides were then incubated with goat
anti-rabbit biotinylated secondary antibody at 5 µg/ml (Vector
laboratories) in TNT solution containing 2% w/v fat-free milk powder for 45
minutes at room temperature in a humidified chamber. Slides were rinsed three
times, 5 minutes each, in TNT solution. The slides were incubated for 30
minutes in TNB buffer (0.1 M Tris, pH 7.5, 0.15 M NaCl, 0.5% blocking reagent)
in the dark at room temperature in a humidified chamber. The slides were
incubated for 30 minutes with SA-HRP diluted 1:100 in TNB buffer at room
temperature in the dark in a humidified chamber. The slides were rinsed three
times, 5 minutes each, in TNT solution and they were incubated in the
Biotinyl-tyramide amplification reagent diluted to a working concentration of
1:50 for exactly 9 minutes in the dark. The slides were rinsed three times, 5
minutes each, in TNT solution and incubated with SA-HRP diluted 1:100 in TNB
buffer for 30 minutes at room temperature in the dark and in a humidified
chamber. Next, The slides were rinsed three times, 5 minutes each and once for
2 minutes, in TNT solution and developed in DAB solution (Vector laboratories)
for 3-20 minutes. Finally, the slides were rinsed in PBS for 5 minutes and
counterstained using 0.5% Toluidine Blue with 10 mM sodium acetate (pH
4.6).
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RESULTS |
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The expression domains of Disp during mouse embryogenesis
overlap with those of Shh and Ihh
As a first step towards understanding the potential role that Disp
plays in Hh signaling, we examined the temporal and spatial expression
patterns of Disp in mouse embryos collected from 7.5 days post coitum
(dpc) to 18.5 dpc. Shh is first detected at late streak stages of
gastrulation (7.75 dpc) in the midline mesoderm arising from the node
(Echelard et al., 1993
)
(Fig. 2A). Weak Ihh
expression is also detected in the posterior part of the node at 7.75-8.0 dpc
(Zhang et al., 2001
). Genetic
analysis demonstrated that Shh and Ihh play partially
redundant roles in Hh signaling in the mouse node
(Zhang et al., 2001
). At 7.75
dpc, Disp is barely detectable by whole-mount in situ hybridization
(Fig. 2B) but a
4.7 kb
Disp transcript could be detected at this stage on a Northern blot
(data not shown). By late-headfold stage just prior to somite formation,
Shh expression is detected in the node and head process
(Echelard et al., 1993
)
(Fig. 2C). At this stage,
Disp is only very weakly expressed in cells immediately adjacent to
the midline mesoderm (arrowheads in Fig.
2D) as well as at junctions between neural and surface ectoderm
(arrows in Fig. 2D).
Subsequently, Shh expression is detected in several signaling
centers, including the notochord, floor plate and ZPA of the limb and in
several endoderm derivatives (Echelard et
al., 1993
). As somites form (
8 dpc) and the embryonic axis
extends caudally, the notochord, which represents the caudal extension of the
head process, also expresses Shh. Disp is initially weakly activated
in the notochord and its expression is upregulated by 9.5 dpc
(Fig. 2I,M). By 8.5 dpc, when
Shh is induced in the floor plate at the ventral midline,
Disp expression is only very faintly expressed in the floor plate at
this stage as well as at later stages (Fig.
2I,M and data not shown). At
9.5 dpc Shh is
activated in the ZPA of the forelimb
(Echelard et al., 1993
) and
Disp is broadly expressed throughout the limb mesenchyme as well in
the apical ectodermal ridge (AER) (Fig.
2H-J). Expression levels of Shh in the ZPA increase from
9.5 to 10.5 dpc (Echelard et al.,
1993
). At 10.5 dpc, Disp expression in both fore- and
hindlimb is still broad, but its expression is downregulated both in ZPA and
surrounding regions (arrow in Fig.
2L). Expression of Disp is also detected in the somite
and branchial arches (Fig.
2H,M,L).
|
At later stages of development, Ihh expression is detected in
developing chondrocytes. Ihh expression is first detected at 12.5 dpc
in chondrocytes in the center of cartilage condensation of long bones
(Bitgood and McMahon, 1995;
St-Jacques et al., 1999
). At
13.5 dpc, Ihh expression is downregulated in the more mature central
cells that are undergoing hypertrophy
(Bitgood and McMahon, 1995
;
St-Jacques et al., 1999
). At
this stage, the expression domain of Disp largely overlaps with that
of Ihh (data not shown). In addition, a strong Disp
expression domain was detected in the articular chondrocytes facing the joint
cavity (data not shown). At later stages, Ihh expression is
restricted to the prehypertrophic chondrocytes between the zones of
proliferating and hypertrophic chondrocytes
(Bitgood and McMahon, 1995
;
St-Jacques et al., 1999
)
(Fig. 2O); Disp
expression remains associated with Ihh expression in the
prehypertrophic condrocytes in addition to maintaining its strong expression
in the articular chondrocytes (Fig.
2P). Taken together, these findings suggest a potential role of
Disp in Hh signaling since its expression domains overlap with those
of both Shh and Ihh during early mouse embryogenesis.
Mouse embryos deficient in the Disp gene do not survive
beyond 9.5 dpc and resemble Smo mutant embryos
To better understand the role Disp plays in Hh signaling, we
generated a null allele of Disp using gene targeting in mice. The
Disp gene is located on mouse chromosome 1 and the genomic locus
consists of eight exons. The eighth exon encodes the last 1193 amino acids of
Disp protein, which include all twelve predicted transmembrane domains
(Fig. 3A). We targeted the
eighth exon to generate a null allele of the Disp gene (designated
Disp E8) (Fig.
3B). The gross morphology of homozygous Disp
E8
mutant embryos at 9.5 dpc (Fig.
4B) is remarkably similar to embryos deficient in the Smo
gene (Zhang et al., 2001
)
(Fig. 4C). Furthermore, similar
to Smo mutants, homozygous Disp
E8 mutants do not
survive beyond 9.5 dpc. By contrast, Disp
E8 heterozygous
embryos cannot be distinguished from their wild-type littermates (data not
shown). Disp
E8 mutants exhibit cyclopia and
holoprosencephaly. In addition, Disp
E8 mutants fail to
complete embryonic turning (Fig.
4B). The embryonic lethality observed in homozygous
Disp
E8 mutants is most probably due to defective heart
development. Disp
E8 mutants fail to undergo normal rightward
looping of the heart, which remains as a linear tube and is surrounded by a
bloated pericardial sac (Fig.
4B,F). All of these phenotypes have been reported in embryos
defective in Hh signaling (Chiang et al.,
1996
; Zhang et al.,
2001
).
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To confirm that the observed defects in Disp E8 mutants are
due to defective Hh signaling, we examined the expression of the Hh targets,
Ptch, Hip1 and Gli1
(Chuang and McMahon, 1999
;
Goodrich et al., 1996
;
Marigo et al., 1996
;
Platt et al., 1997
). In situ
hybridization was used to monitor their expression in Disp
E8
mutants in wholemounts and sections. Expression of Hip1 is known to
be completely dependent on Hh signaling
(Chuang and McMahon, 1999
)
while Ptch expression is initially Hh independent but is strongly
upregulated upon Hh signal transduction
(Goodrich et al., 1996
). In
Disp
E8 mutants at 9.5 dpc, Shh is expressed in the
notochord, the ZPA, the gut endoderm and the branchial arches
(Fig. 5B,J), but expression of
Hip1 (Fig. 5F) and
Gli1 (Fig. 5H) is
completely absent in Disp
E8 mutants. Expression of
Ptch is greatly reduced and only weak expression is detected in the
sclerotome of the somite, the ventral neural tube and the distal posterior
margin of the forelimb (Fig.
5D,L), which may reflect Hh-independent expression of
Ptch. Taken together, these findings indicate that Disp is
required for Hh signaling during mouse embryogenesis.
|
Disp mutants display multiple defects in LR axis
determination and in the development of the axial structure, ventral neural
tube, somite and limb, because of defective Hh signaling
To better understand the molecular mechanisms that underlie the defects
observed in Disp E8 mutant embryos, we performed a detailed
histological and marker analysis. Our analysis focused on LR axis
determination, the axial structures, the ventral neural tube, the somite and
the limb, as the role Hh signaling plays in patterning these structures has
been well characterized (Chiang et al.,
1996
; Lewis et al.,
2001
; Marti et al.,
1995b
; Riddle et al.,
1993
; Roelink et al.,
1995
; Zhang et al.,
2001
). In addition, formation of these structures involves both
short- and long-range Hh signaling.
Disp mutants are first distinguishable at the six- to seven-somite
stages (8.5 dpc) by the abnormal morphology of the forebrain, indicative
of loss of ventral midline fate, and by a delay in cardiac morphogenesis (data
not shown). The failure to complete embryonic turning and the absence of heart
looping in Disp
E8 mutants suggested that LR axis development
may be affected, as has been previously reported in Smo mutants
(Zhang et al., 2001
).
Pitx2, which encodes a bicoid-related homeobox protein, is expressed
in the left lateral plate mesoderm (LPM) from two- to three-somite (
8
dpc) to 10 somite (8.5 dpc) stages in wild-type embryos
(Piedra et al., 1998
;
Ryan et al., 1998
;
Yoshioka et al., 1998
).
Pitx2 expression is greatly reduced in the left LPM in two- to
six-somite Disp
E8 mutants, whereas expression of
Pitx2 in the head mesenchyme and yolk sac is unaltered (data not
shown). These results suggest that defective Shh and Ihh
signaling in the node affects the establishment of LR asymmetry
(Zhang et al., 2001
) in
Disp
E8 mutants.
Analysis of Shh mutant mice suggests that Shh is required
for the maintenance but not the formation of the notochord
(Chiang et al., 1996). If
Disp is required for Hh signaling, phenotypes resembling the axial
defects in Shh mutants should be observed in Disp
E8
mutants. Consistent with this hypothesis, expression of brachyury [which is
required for differentiation of the notochord and is normally expressed in the
primitive streak, the node and developing notochord
(Herrmann and Kispert, 1994
)]
becomes discontinuous in the rostral region of Disp
E8 mutant
embryos (arrow in Fig. 6B).
Though the origin of the floor plate is not completely understood, the floor
plate and notochord share similar expression profiles (including Shh
and Hnf3b) and there is good evidence to suggest that expression of
Shh in the notochord acts short-range to induce floor plate
(Le Douarin and Halpern, 2000
;
Placzek et al., 2000
). In
Disp
E8 mutant embryos, Shh
(Fig. 5B,J) and
Hnf3ß (Fig. 6D,F)
are not detected in the ventral midline of the neural tube, suggesting that
the floor plate fails to form. These results indicate that Disp is
required for Shh signaling in the axial midline.
|
Shh signaling from both the notochord and the floor plate plays a
key role in patterning the ventral neural tube in a dosedependent manner
(Chiang et al., 1996;
Roelink et al., 1995
). To
examine whether dorsoventral patterning of the neural tube is affected in
Disp
E8 mutants, we probed the expression of molecular markers
that define different dorsoventral positions in the early neural tube
(Briscoe and Ericson, 1999
;
Briscoe and Ericson, 2001
). In
the neural tube, Pax3 expression is normally restricted to the dorsal
half (alar plate) of the spinal cord from the tail to the diencephalons
(Fig. 6G) and Pax6 is
only weakly expressed in the alar plate and more strongly throughout the
ventral half (basal plate) of the neural tube, except at the ventral midline
(Fig. 6I). In Disp
E8 mutants at 9.5 dpc, Pax3 expression in the spinal cord
extends ventrally (Fig. 6H),
whereas Pax6 expression level is quite low (to a level characteristic
of normal alar plate expression) (Fig.
6J). Wnt1 (data not shown) and Wnt3a
(Fig. 6P) are expressed in the
roof plate in Disp
E8 mutants. These results indicate that the
ventral neural fate is not properly specified in the absence of Disp.
Consistent with this conclusion, expression of a set of homeodomain proteins
in neuroprogenitor cells (such as Dbx1, Dbx2, Nkx6.1 and
Nkx2.2) was not detected in Disp
E8 mutants (compare
Fig. 6K with 6L and data not
shown). Expression of these homeodomain genes is induced or repressed in
response to graded Shh signaling (reviewed by
Briscoe and Ericson, 1999
;
Briscoe and Ericson, 2001
).
Recent studies suggest that the resulting overlapping expression domains of
these genes specify different neuronal types, including interneurons and
motoneurons, at distinct positions of the ventral neural tube. Loss of the
homeodomain code resulted in absence of islet 1 expression, a marker for
motoneurons, in Disp
E8 mutants
(Fig. 6N), as well as loss of
En1, which is expressed in V1 interneurons (data not shown).
Many studies have shown that Shh signaling in the floor plate and
notochord induces expression of sclerotomal marker Pax1 and
suppresses the dorsal dermomyotomal marker Pax3
(Chiang et al., 1996;
Fan et al., 1995
;
Fan and Tessier-Lavigne,
1994
). In Disp
E8 mutants at 9.5 dpc,
Pax1 expression is not induced in the somite, suggesting that
sclerotomal differentiation does not occur
(Fig. 6R). By contrast,
Pax3 expression in the somite is expanded ventrally
(Fig. 6H,T). We then asked
whether dermomyotomal development is affected in the absence of Disp.
In wild-type embryos, the first myogenic bHLH gene to be expressed is
Myf5 at 8 dpc (Summerbell et al.,
2000
), followed by the activation of myogenin at 8.5 dpc
(Tajbakhsh et al., 1997
).
Myod1 expression is detected about 2 days later at 9.75 dpc
(Tajbakhsh et al., 1997
). In
Disp
E8 mutants at 9.5 dpc, Myf5 was detected at low
levels in the dermomyotome (Fig.
6V). Myogenin and Myod1 expressions are not detected at
these stages (Fig. 6X and data
not shown). These results suggest that dermomyotomal development is initiated
but does not proceed in Disp
E8 mutants.
Shh signaling from the ZPA specifies digit identity along the
anteroposterior (AP) axis of the limb
(Chiang et al., 1996;
Lewis et al., 2001
;
Riddle et al., 1993
;
Yang et al., 1997
). As
described above, though Shh expression in the ZPA appears to be
normal in the forelimb buds of Disp
E8 mutants at 9.5 dpc
(Fig. 5B), Hh targets are
either not induced (Hip1 and Gli1)
(Fig. 5F,H) or the expression
levels are greatly reduced (Ptch)
(Fig. 5D,L), suggesting that
proper AP patterning is disrupted. Consistent with this, Hand2
(dHand) expression, which normally shows broader,
Shh-dependent expression over almost half of the AP axis at this
stage (Charite et al., 2000
)
(indicated by the bracket in Fig.
6Y), is truncated in Disp
E8 mutants (arrow in
Fig. 6Z). Interestingly,
expression of Hoxd13, the most posteriorly restricted Hoxd family
member that is regulated by Shh signaling
(Zakany and Duboule, 1999
)
(Fig. 6AA), is only slightly
reduced in Disp
E8 mutants
(Fig. 6BB). Shh
signaling is known to induce Fgf4 expression in the apical ectodermal
ridge (AER), which regulates proximodistal (PD) outgrowth of the limb bud
(reviewed by Martin, 1998
)
(Fig. 6CC). Fgf4 also
functions to maintain Shh expression in the ZPA. In Disp
E8 mutants at 9.5 dpc, Fgf4 expression is not detected in the
AER (Fig. 6DD). This could be
due to retarded growth of the mutants as well as defective Hh signaling to
induce Fgf4 expression. By contrast, Fgf8 expression in the
AER of Disp
E8 mutants cannot be distinguished from that of
wild-type embryos (reviewed by Martin,
1998
) (Fig.
6EE,FF). Dorsoventral (DV) patterning of the limb appears to occur
normally in Disp
E8 mutants
(Parr and McMahon, 1995
) (data
not shown). Together, these findings indicate an absolute requirement of
Disp in multiple aspects of Hh signaling.
Shh protein is properly processed but the distribution of Shh protein
is restricted to its sites of synthesis in Disp mutants
Studies in Drosophila suggest that disp is involved in
facilitating the movement of the cholesterol-modified form of Hh and does not
affect Hh synthesis or processing (Burke et
al., 1999). As Shh expression appears to be normal in
Disp
E8 mutants, we asked whether processing of Shh to
generate a cholesterol-modified N-terminal fragment of Shh also occurs
normally in Disp
E8 mutants. On western blots, Shh antibodies
recognized the unprocessed (upper arrow in
Fig. 7) as well as the
processed form of Shh (Shh-Np) (lower arrow in
Fig. 7) in wild-type and
Disp
E8+/- embryos. Shh antibodies also recognized
Shh-N, which migrates slower than Shh-Np on an SDS-PAGE. By contrast, neither
the unprocessed form of Shh nor the processed Shh-Np or Shh-N could be
detected in lysate from Shh mutant embryos. In lysates from
Disp
E8-/- embryos, a band running at the same
position as Shh-Np was detected by Shh antibodies, suggesting that Shh
processing occurs in the absence of Disp. In addition, the ratio of
processed to unprocessed (a very small amount) (data not shown) form of Shh in
Disp
E8-/- embryos could not be distinguished from
that of their wild-type littermates. These results suggest that Shh processing
occurs normally in the absence of Disp.
|
To investigate whether the phenotype observed in Disp E8
mutants is due to defective Hh movement, we examined the distribution of Shh
protein in wild-type and Disp
E8-/- embryos. Using
the procedure described by Gritli-Linde et al., we found that in wild-type
mouse embryos at 9.5 dpc, Shh immunoreactivity is strong in the notochord and
extends outwards in a graded fashion (arrows in
Fig. 8A), upwards towards the
ventral neural tube along the extracellular matrix (arrowheads in
Fig. 8A) as previously shown
(Gritli-Linde et al., 2001
)
and downwards towards the branchial pouch (data not shown). Similar patterns
of Shh immunoreactivity extending from the notochord were observed on embryo
sections where the floor plate has not yet been induced
(Gritli-Linde et al., 2001
).
In Disp
E8 mutant embryos at this stage, Shh immunoreactivity
is confined to the notochord and no immunoreactivity is detected outside the
notochord (Fig. 8B). By
contrast, in Smo mutant embryos, Shh immunoreactivity is detected in
the notochord and extends in a graded fashion though at a lower level than
that in wild type (data not shown). Taken together, these results indicate
that while Disp
E8 and Smo mutants share similar
phenotypes, the underlying molecular defects are different. Hh transport
appears to be normal in Smo mutants but Hh protein is not capable of
transducing its signal in Hh-responding cells. By contrast, in the absence of
Disp, processed Hh protein fails to be transported out of
Hh-producing cells and Hh-responding cells never receive the Hh signal.
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DISCUSSION |
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Mouse dispatched in Hh signaling
Disp exhibits a dynamic expression pattern during mouse
embryogenesis. It is possible that regulation of Disp expression
involves Hh signaling. Expression of Disp in midline axial structures
is relatively weak, although analysis of Disp mutants strongly
suggests that Disp plays an essential role in midline Hh signaling.
In this case, it is not known whether Disp is required continuously
for proper signaling of Hh protein as initial expression levels of
Disp are low. In addition, Disp expression in the limb
becomes downregulated in locations where Shh is upregulated. It is
possible that Disp is not continuously required or a low level of
Disp expression is sufficient for Hh transport. It is interesting to
note that in many structures Disp is expressed at a lower level in
regions of Hh expression and at a higher level adjacent to regions of Hh
signaling. One possibility is that Disp could be involved in a
feedback mechanism to modulate Hh signaling. Alternatively, expression of
Disp outside Hh expression domains may imply a potential role in
processes not mediated by Hh signaling.
Our mutant analysis revealed the essential role Disp plays in Hh
signaling, including Shh and Ihh signaling. As the
phenotypes observed in Disp mutants and Smo mutants are
identical in our analysis, it is most likely that no Hh signal is transduced
in the absence of Disp, despite the prominent expression of Hh
protein. Hh signaling involves both short- and long-range signaling, and it is
somewhat surprising that in Disp mutants even short-range signaling
is defective. For example, induction of floor plate does not occur in
Disp mutants, and this process requires direct cell-cell contact of
ventral midline cells with the notochord and not long-range movement of Hh
protein (Le Douarin and Halpern,
2000; Placzek et al.,
2000
). It is possible that the Hh protein is not presented to the
cell surface in the absence of Disp, although the Hh protein is properly
processed in the secretory pathway of Hh-producing cells. Alternatively, Disp
may be required directly in short-range signaling once the Hh protein is
localized on the cell surface of Hh-producing cells. For example, Disp may be
involved in partitioning Shh into membrane microdomains essential for Hh
binding to Ptch or Disp may direct membrane to membrane transfer of Shh
between Hh-producing and Hh-responding cells.
As Disp mutants do not survive beyond 9.5 dpc, it has not been
possible to assess the role Disp plays in Ihh signaling in the developing
chondrocytes and gut endoderm (Bitgood and
McMahon, 1995; Ramalho-Santos
et al., 2000
; St-Jacques et
al., 1999
) as well as Dhh signaling in the developing testis and
peripheral nerves (Bitgood et al.,
1996
; Parmantier et al.,
1999
). It is also possible that Disp has Hh-independent functions,
because expression of Disp is detected in locations where none of the known Hh
proteins is expressed. Answers to these issues will require further genetic
and molecular studies.
A conserved mechanism of Hh transport in Hh-producing cells
Although the issue of lipid modification and its role in Hh movement in
Hh-responding cells is not yet completely resolved, the crucial step of moving
Hh protein out of Hh-producing cells appears to be evolutionarily conserved.
Molecular analysis of Drosophila disp revealed its essential role in
facilitating movement of the lipid-modified form of Hh protein in Hh-producing
cells (Burke et al., 1999). Our
studies demonstrate that the mouse ortholog of Dispatched also plays
a similar role in Hh transduction. Because Disp-deficient mice
phenocopy Smo mutants (Zhang et
al., 2001
), it is likely that Disp is involved in
transporting all three mammalian hedgehog proteins. These results suggest that
the molecular mechanism by which lipid-modified Hh is released from
Hh-producing cells is conserved. However, it is not known whether Disp is
dedicated to facilitate the movement of lipid-modified Hh proteins or it also
plays a role in transporting other lipid-modified proteins. The function of
Disp-related is not known, but the fact that its restricted
expression domain does not overlap with Hh expression (T'N. K. and P.-T. C.,
unpublished) suggests that Disp-related is unlikely to be involved in
the same process as Disp.
Potential molecular mechanisms by which Disp mediates Hh
movement
Generation of an active Hh signal is a highly regulated process. It
involves autoproteolytic cleavage, lipid modification and regulated transport.
Our studies show that Disp is not required for Hh protein synthesis or
processing but rather is involved in moving Hh protein from its sites of
synthesis. Mosaic analysis in Drosophila suggests that Disp is only
required in Hh-producing cells but not in Hh-receiving cells to facilitate Hh
movement, despite ubiquitous expression of disp mRNA
(Burke et al., 1999). It is not
known whether Disp also functions exclusively in Hh-producing cells for
vertebrate Hh signaling. Compared with disp, mouse Disp
exhibits a relatively restricted expression domain, although Disp protein
distribution has not been determined. How Disp functions to facilitate Hh
movement is also not known. Disp contains 12 predicted membrane-spanning
domains but its subcellular localization remains to be determined. It is
possible that Disp resides in the ER/Golgi to mediate the transport of Hh
protein in the secretory pathway. Proteins with SSDs have been implicated in
vesicular transport (Kuwabara and
Labouesse, 2002
) and Disp may be involved in a similar process to
direct the movement of Hh-containing vesicles to the plasma membrane.
Alternatively, Disp may function on the plasma membrane to promote the release
of Hh protein from Hh-producing cells. Interestingly, the topology of Disp
bears similarity to that of ion channels or transporters. Cellular and
biochemical studies will be required to uncover the molecular mechanisms by
which Disp facilitates transport of the lipid-modified form of Hh protein in
Hh-producing cells.
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ACKNOWLEDGMENTS |
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Footnotes |
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