1 One DNA Way, Genentech Incorporated, South San Francisco, CA 94080, USA
2 1550 Fourth Street, Room 282, UCSF Mission Bay Campus, San Francisco, CA
94158, USA
3 University of Florida College of Medicine, Department of Molecular Genetics
and Microbiology, 1600 SW Archer Road, Gainesville, FL 32610-0266, USA
4 Department of Genetics, Harvard Medical School, NRB room 360, 77 Avenue Louis
Pasteur, Boston, MA 02115, USA
5 Department of Molecular and Cellular Biology, The Biolabs, Harvard University,
16 Divinity Avenue, Cambridge, MA 02138, USA
Author for correspondence (e-mail:
mcmahon{at}mcb.harvard.edu)
Accepted 4 November 2004
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SUMMARY |
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Key words: Dispatched1, Shh, Cholesterol, Paracrine, Autocrine
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Introduction |
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As extracellular signals, Hh proteins are synthesized in discrete subsets
of cells in many organs and act in short and long-range signaling processes.
The best characterized mammalian Hh-target field is the developing ventral
neural tube, where progenitors differentiate into several different cell types
in response to a morphogen gradient of Shh, issuing from two point sources:
the notochord and floorplate (Briscoe and
Ericson, 1999; Briscoe and
Ericson, 2001
; Jessell,
2000
; McMahon et al.,
2003
). The notochord is a ventral rod of mesoderm that underlies
the neural tube while the floorplate is a population of support cells at the
ventral midline of the neural tube that is induced by notochordal Shh.
Distinct ventral neural progenitors are induced at specific positions with
respect to the source, and apparent concentration, of Hh ligand.
All Hh proteins are synthesized as full-length precursors that undergo an
autocatalytic cleavage reaction. This removes the C-terminal catalytic domain
and attaches a cholesterol molecule to the C terminus of the N-terminal
signaling fragment (N-Shhp; `p' stands for `processed' in the N-Shh signaling
moiety) (Porter et al.,
1996a). The hydrophobic cholesterol moiety is thought to bind Hh
to the cell membrane. The hydrophobicity of the Hh molecule is further
increased by the addition of a palmitoyl group to a conserved cysteine residue
that is exposed at the N terminus after signal peptide cleavage
(Pepinsky et al., 1998
). These
modifications regulate Hh activity, oligomerization, range of action, potency
and might in many cases, shape a signaling gradient
(Burke et al., 1999
;
Chamoun et al., 2001
;
Chen et al., 2004
;
Chen and Struhl, 1996
;
Kohtz et al., 2001
;
Lee et al., 2001
;
Lewis et al., 2001
;
Zeng et al., 2001
). The
requirement for such modifications may depend on the context in which Hh
proteins act (Chen and Struhl,
1996
; Lewis et al.,
2001
). As cholesterol modification is unique to Hh-ligands, the
role of cholesterol has attracted considerable attention. Forms of Hh ligand
have been engineered that lack the autocatalytic cleavage site and C-terminal
cleavage domain, and therefore are not cholesterol-modified [Hh-N in
Drosophila (Chen and Struhl,
1996
) and NShh in mammals
(Lewis et al., 2001
)]. In both
instances, palmitoylation appears to be largely independent of cholesterol
addition (Chen et al., 2004
;
Gallet et al., 2003
).
Despite dual lipid modifications that normally retain protein on the cell
membrane, Drosophila Hh and mouse Shh and Ihh protein can be detected
at significant distances from their expression domains
(Chen and Struhl, 1996;
Gallet et al., 2003
;
Gritli-Linde et al., 2001
;
Lewis et al., 2001
;
Porter et al., 1996a
). This
long-distance action is an active process involving a transmembrane protein
Dispatched (Disp). Disp was first identified in Drosophila studies,
where its activity is required specifically within Hh-producing cells for
movement of cholesterol modified ligand into Hh target fields
(Burke et al., 1999
). By
contrast, Hh-N signaling is Disp independent
(Burke et al., 1999
).
The mouse has two Disp homologs, Disp1 and Disp2, but only Disp1 is able to
rescue a Drosophila disp mutant phenotype
(Ma et al., 2002). Genetic
studies in zebrafish suggest that Disp2 is not involved in Hh
signaling (Nakano et al.,
2004
), and mouse studies support this view (H.T. and A.P.M.,
unpublished). Three mutant alleles of mouse Disp1 have been
described: Disp1
8,
Disp1C829F and Disp1
2
(Caspary et al., 2002
;
Kawakami et al., 2002
;
Ma et al., 2002
;
Tian et al., 2004
). Analysis
of these alleles lead to similar general conclusion, that Disp1 is involved in
Hh signaling during early embryonic development. Among these mutant alleles,
the Disp1
8 deletion allele
(Kawakami et al., 2002
;
Ma et al., 2002
) and
Disp1C829F missense allele
(Caspary et al., 2002
) are
likely to represent null alleles of Disp1. Homozygous
Disp1
8 and Disp1C829F
mutants do not survive beyond E9.5, and exhibit gross morphological features
that are similar to Smoothened (Smo) mutants in which all Hh
signaling activity is abolished (Zhang et
al., 2001
). In Disp1C829F/C829F and
Disp1
8/
8 embryos, Hh
signaling is retained but only in midline cells of the notochord that both
produce Shh and respond to Shh signals (Ma
et al., 2002
). Disp1
2, by
contrast, encodes a hypomorphic allele
(Tian et al., 2004
).
Homozygous Disp1
2 mutants die at birth
with facial midline patterning defects, characteristic of attenuated Shh
signaling (Wallis and Muenke,
1999
). This hypomorphic allele has permitted us to demonstrate the
genetic interaction of Disp1 with specific components of the Hh
signaling pathway. By combining a Disp1
2
hypomorphic allele with Disp1C829F,
Shhnull and patched 1 (Ptch1null)
alleles, we created a set of graded facial midline and neural tube phenotypes
demonstrating that Disp1 gene dose regulates the level of Shh
signaling activity in vivo. Furthermore, rescue of the
Disp1
2/
2 mutant upon
removing one copy of Ptch1 suggested that Disp1 functions exclusively
in the Hh pathway (Tian et al.,
2004
).
Although these phenotypic analyses demonstrated a conserved requirement for Disp1 in the Hh signaling pathway and highlighted the importance of Disp1 dose for normal levels of Shh signaling, they did not address the specific cellular or molecular limits to Disp1 action in mammalian Hh signaling. To determine whether Disp1 activity is required in Shh-producing cells or in paracrine signaling in the target field, we used a ShhCre knock-in allele to remove Disp1 exon 2 specifically from Shh-producing cells. Our results indicate that Disp1 activity in these cells is essential for Hh signaling to the target field. Furthermore, using an allele that produces N-Shh, we demonstrate that Disp1 activity is required only for the paracrine action of the cholesterol modified form of Shh.
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Materials and methods |
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Generation of Disp12C and Disp1
2 alleles
To remove exon 2 of Disp1, a targeting vector was engineered in
which exon 2 was flanked by loxP sites. Exon 2 encodes the amino terminal
cytoplasmic domain and first transmembrane domain of Disp1
(Tian et al., 2004). After
homologous recombination at the Disp1 locus in AV3 ES cells, a
heterozygous ES cell line was injected into blastocysts of the C57BL6/J strain
to generate chimeras. These were bred with Swiss Webster mice to obtain
Disp1
2C/+ offspring. Chimeric males were
also bred with ß-actin-Cre females, Cre activity in the
preimplantation embryo allow the recovery of
Disp1
2/+ heterozygous offspring.
RNA in situ hybridization
Embryos were fixed in 4% paraformaldehyde at 4°C overnight. Whole-mount
and section in situ hybridization using digoxigenin-labeled RNA probes was
performed as described previously
(Schaeren-Wiemers and Gerfin-Moser,
1993; Wilkinson,
1992
).
Immunofluorescence
Immunofluorescence on embryonic sections was performed as described for
sections (Yamada et al.,
1991). Antibodies and dilutions were as follows: rabbit
Foxa2, 1:8000 (Ruiz i Altaba et al.,
1995
);
Nkx6.1, 1:3000
(Cai et al., 2000
);
Nkx2.2, 1:4000 (Briscoe et al.,
1999
);
Olig2, 1:5000
(Takebayashi et al., 2000
);
mouse
Nkx2.2, 1:50 (Ericson et al.,
1997a
);
Pax6, 1:20
(Ericson et al., 1997b
);
Pax7, 1:20 (Ericson et al.,
1996
); and
MNR2, 1:20 (DHSB).
Skeletal preparations
For skeletal preparations, 18.5 dpc embryos were processed as described
previously (Karp et al.,
2000).
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Results |
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To knock down Disp1 activity specifically within Shh-producing
cells, we used a Cre knock-in allele in which sequence encoding a GFP-Cre
fusion protein was inserted into the Shh locus
(ShhGFP-Cre, herein ShhCre)
(Harfe et al., 2004). This
allele no longer expresses a normal Shh transcript, and is a null allele for
Shh activity. When combined with a Cre reporter line R26R
(Soriano, 1999
),
ShhCre gave rise to lacZ expression that was
almost identical to Shh mRNA distribution at E10.5
(Fig. 1A,B), indicating that
the GFP-Cre fusion protein is functionally active within all Shh expression
domains. To further confirm the functional specificity of GFPCre fusion
protein, ShhCre was crossed with a Shh conditional allele
(Shh
2C)
(Lewis et al., 2001
) to remove
Shh in the Shh-producing cell.
|
Appropriate crosses were set up to generate mice that were either
Disp12/
2C, ShhCre/+ or
Disp1C829F/
2C, ShhCre/+. In both cases,
pups died within 1 day of birth. In the former, ShhCre
activity should generate Shh-expressing cells that are homozygous for the
Disp1
2/
2 allele. In the latter, the only
Disp1 activity comes from a single Disp1
2
hypomorphic allele. In both examples, Disp1 activity is decreased on a
background where Shh dose is lowered by the presence of the
ShhCre null allele. Ordinarily, reducing Shh levels by
removing one allele of Shh produces no discernible phenotype
(Chiang et al., 1996
;
St-Jacques et al., 1998
) but
reduction of Shh dose enhances the neural phenotype in Disp1
hypomorphic combinations (Tian et al.,
2004
) Importantly, as the production of Cre recombinase is linked
to Shh expression, some level of Shh signaling must take place prior
to Cre-mediated modification of Disp1 alleles. This is evident in
ShhCre/C embryos where a few Nkx6.1-producing cells are
observed (Fig. 1N), while no
Nkx6.1 are produced in Shh-/- embryos
(Fig. 1L,M).
In both genotypes (Disp12/
2C,
ShhCre/+ or Disp1C829F/
2C,
ShhCre/+), pups displayed facial midline defects similar to
Disp1 hypomorphic mutants (Disp1
2/
2,
Shh+/- and Disp1C829F/
2,
Shh+/-), in which Disp1 exon 2 was deleted in the
entire embryo, the severity of the phenotype increased as Disp1 activity
decreased (Fig. 2A-E). Importantly, no phenotype was observed in
Disp1
2/
2C, Shh+/- embryos (data
not shown). The midline facial defects were clearly visible at E10.5 by
comparing the ventral disposition of the medial and lateral nasal process and
proximal mandibular and maxillary arches that are highlighted by the
expression of Fgf8 (Fig.
2F-J). In Disp1
2/
2C,
ShhCre/+ embryos, the two nasal pits were positioned closer to
the midline, indicating a loss of facial midline structure. A more severe
phenotype was seen in Disp1C829F/
2C,
ShhCre/+ embryos as the two nasal pits start to fuse where
Shh-dependent medial nasal cells were also affected
(Fig. 2J). The severity of the
midline loss in conditional mutants was comparable with Disp1
hypomorphic mutants Disp1
2/
2,
Shh+/- and Disp1C829F/
2;
Shh+/- (compare Fig. 2G and
I with 2H and J).
Skeleton preparations of newborn pups revealed that the premaxilla was missing
from Disp1
2/
2C, ShhCre/+ and
Disp1C829F/
2C, ShhCre/+ mutant embryos,
just as in Disp1C829F/
2, Shh+/- and
Disp1
2/
2, Shh+/- embryos
(Fig. 2K-O). Although the
parietal bone was also lost in Disp1C829F/
2,
Shh+/- embryos, we only observed a delay in ossification of
parietal bones in Disp1C829F/
2C,
ShhCre/+ mutant (compare
Fig. 2N,O). As expected from
the analysis of Disp1
2/
2, Shh+/-
and Disp1
2/C829F, Shh+/- mutants,
expression of Ptch1, one of the principle transcriptional targets of
Hh signaling, was greatly downregulated in the frontal nasal process (FNP) of
the conditional mutants at E9.5 (Fig.
2P-T, arrows). This confirms that the loss of midline structures
of the frontal nasal process in the conditional mutants
Disp1
2/
2C, ShhCre/+ and
Disp1C829F/
2C, ShhCre/+ was due to the
attenuation of Shh signaling in this region. Furthermore, Shh
expression within the ventral forebrain, which is itself a target of a
mesendodermal derived Shh signal, was lost in all allelic combinations
(Fig. 2U-Y). This most probably
explains the initial defect in FNP development in the Disp1 mutant
background.
|
|
Signaling by N-Shh is independent of Disp1 activity
Next, we addressed the specific requirement for Disp1 for cholesterol
modified Shh ligand. We have previously reported on an N-Shh allele
generated by inserting a stop-codon into the endogenous Shh gene at the
position where normal cleavage and cholesterol addition occurs
(Lewis et al., 2001). The
protein produced is identical to that of the normal Shh signal and is expected
to undergo N-terminal palmitoylation (Chen
et al., 2004
). Unfortunately, this allele is dominant lethal,
highlighting the importance of cholesterol modification to normal Hh
regulation (Lewis et al.,
2001
). To overcome this dominant lethality and to enable us to
address N-Shh activity in a Disp1C829F/C829F mutant
background that lacks all Disp1 activity, we created a conditional N-Shh
(N-ShhC) allele (J.J. and A.P.M., unpublished). Mice
heterozygous for this allele are viable and fertile permitting genetic
intercrosses with the Disp1C829F allele. Cre-mediated
recombination leads to exclusive production of N-Shh from the endogenous
Shh locus, an outcome that is essentially identical to the original
non-conditional N-Shh allele. To initiate recombination throughout
the embryo, we used a Sox2Cre transgene to induce recombination in
the entire embryo (Hayashi et al.,
2002
). As the resulting N-Shh allele is under identical
cis-regulatory control to the wild-type Shh allele, N-Shh
expression was restricted to Shh-expressing cells (data not shown). Unlike
Disp1C829F/C829F mutants, which do not survive beyond 9.5
dpc and show gross defects in neural, somite, cardiac, vascular, facial and
limb development (Fig. 4A,B), Disp1C829F/C829F, NShhC/Shhn,
Sox2Cre embryos were alive at E10.5 but die at or around birth.
Morphologically, these embryos were indistinguishable from the
N-ShhC/Shhn, Sox2Cre mutants
(Fig. 4C,D), which also employ
N-Shh as the only available Shh ligand. At this stage, both
Disp1C829F/C829F, N-ShhC/Shhn,
Sox2Cre and N-ShhC/Shhn, Sox2Cre mutant
embryos are very similar to the wild type, except for midline defects in the
frontal nasal process. Thus, Disp1 does not appear to be required for N-Shh
activity.
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Discussion |
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In this study we have used a variety of genetic strategies to examine Disp1
action. In the previous studies, the precise cellular requirements for Disp1
were unclear. Here, we specifically attenuated Disp1 activity in
Shh-expressing cells; strikingly, the phenotypes closely resemble
those observed when Disp1 activity is reduced in the entire embryo
(Tian et al., 2004). That
these phenotypes are not identical is most likely a technical limitation of
our approach. In this, we used a conditional hypomorphic allele of
Disp1 where essential sequence encoded within exon 2 was flanked by
loxP sites and combined this allele with a ShhCre allele
to `knock down' Disp1 levels exclusively within Shh-expressing cells.
In this genotype, Cre-dependent recombination at the
Disp1
2C locus can occur only after Shh
transcription is initiated; hence, it is likely some Shh signaling occurs
while there is a sufficient level of Disp1 to lead to normal signaling
(embryos heterozygous for Disp1null/+ alleles are
phenotypically wild-type). Alternatively, as this approach is only expected to
modify Ihh signaling where Shh and Ihh expression overlap,
wild-type Ihh activity from non-Shh expressing cells may contribute to the
weaker phenotype in the conditional allele. The presence of a few
Nkx6.1+ cells in ShhCre/C embryos is consistent
with the former explanation.
With these provisos in mind, it is striking that the phenotypes of
attenuated Disp1 activity in the Shh expression domain and in the
whole embryo are so similar. Clearly, most if not all, paracrine Shh signaling
within the face, neural tube and limb is dependent on Disp1 function in the
ligand-producing cell, supporting a model in which Disp1 acts in signal
production and not target cell response. How Disp1 acts at the molecular and
cellular level is not clear. Studies in Drosophila report the
accumulation of Hh ligand in Disp1 mutant cells in the posterior
compartment of the imaginal disc (Burke et
al., 1999), suggesting that Disp1 may regulate the release of bulk
ligand from Hh-producing cells. Further work implicates Disp in apical
trafficking (Gallet et al.,
2003
) within Hh-producing epithelia, suggesting that the defective
release and ligand accumulation in Hh-producing cells may be secondary to
altered membrane trafficking. However, we failed to observe any obvious
accumulation of Shh in Shh-producing cells in Disp1-null mutant embryos (data
now shown). We also failed to observe any differences in the release of bulk
Shh protein into the medium when Shh was expressed in
Disp1C829F/C829F or wild-type fibroblasts
(Tian et al., 2004
). Whether
this represents a difference between polarized epithelia and fibroblasts is
unclear. Interestingly, reports of a highly active multimeric complex of lipid
modified Hh ligands (Zeng et al.,
2001
) raises the possibility that Disp1 may function not in
general release of ligand, but rather in the formation and/or release of an
active fraction that is composed of Hh oligomers. Clearly, there is enhanced
bioactivity in media conditioned by Shh-expressing fibroblast when Disp1 is
active in these cells (Ma et al.,
2002
).
Disp1 is only required for paracrine signaling activity of cholesterol modified forms of Shh
Cholesterol-modification of Hh ligands plays a key role in Hh signaling. In
Drosophila, removal of cholesterol from Hh increases the range of
ligand action, at least in part through a disruption of normal Ptch-dependent
feedback control that normally sequesters ligand
(Chen and Struhl, 1996).
Whereas Ptch1 mediated sequestration of N-Shh is also defective in the mouse,
cholesterol modification of N-Shhp is essential for long-range action in the
limb (Lewis et al., 2001
).
Thus, although there appears to be species or context-dependent differences in
the role of cholesterol in Hh signaling, in both flies and mice
cholesterol-modification of Hh ligands is essential for normal signaling
within a multicellular target field. In Drosophila, Disp is required
only for signaling by cholesterol-modified forms of Hh
(Burke et al., 1999
). Our work
in the mouse indicates a similar requirement for Disp1. Whereas,
Disp1-null embryos arrest at E9.5 dpc with multiple defects,
including an absence of ventral cell identities in the neural tube, expression
of a single allele of N-Shh is sufficient to rescue many of these
deficiencies. In the resulting neural tube, all Shh-dependent ventral cell
identities are represented leading to a phenotype identical to that of
N-Shh/Shhn embryos on a wild-type Disp1
background.
Interestingly, when we examined N-Shh localization in the floorplate and
notochord in N-Shh/Shhn embryos, we were unable to detect
any immunoreactivity (in contrast to embryos carrying a single wild-type
alleles of Shh). Thus, N-Shh appears to be rapidly lost from Shh
secreting cells, whereas NShhp is retained and accumulates within the cell, a
finding supported by earlier cell culture analyses
(Bumcrot et al., 1995;
Porter et al., 1996b
). These
results highlight the challenge faced in moving a cholesterol-tethered Shh
ligand from the initial Shh-producing cell into the target field and the vital
role Disp1 plays in this process. Recent studies have demonstrated that N-Shh
fails to generate a soluble multimeric protein complex, lending further
support to a link between Disp1 and oligomeric forms of Shh ligand
(Chen et al., 2004
;
Zeng et al., 2001
). That Disp1
shares a sterol sensing domain with Ptch1 and several proteins that regulate
cholesterol biosynthesis or trafficking
(Carstea et al., 1997
;
Hua et al., 1996
) suggests
cholesterol sensing by Disp1 plays some role in regulation of N-Shhp
export.
Finally, given that Hh ligands undergo a second lipid modification, an
N-terminal palmitoylation, how does this relate to Disp1 function? Early
studies first noted that when NShh was highly expressed in tissue cultures, a
reduced fraction of secreted ligand was palmitoylated compared with cells
expressing wild-type N-Shhp (Pepinsky et
al., 1998). However, more recent studies suggest that
palmitoylation is largely independent of cholesterol addition
(Chen et al., 2004
;
Gallet et al., 2003
).
Furthermore, loss of palmitoylation, but not cholesterol, results in a
dramatic reduction in Hh and Shh activity
(Chamoun et al., 2001
;
Chen et al., 2004
;
Lee et al., 2001
). However,
N-Shh retains biological activity in the neural tube (data herein) and limb
(Lewis et al., 2001
). Thus, it
is likely that the N-Shh allele we have generated gives rise to an
N-terminal palmitoylated ligand. If so, this lipid modification does not
appear to be sufficient for retention of Shh ligand in Shh-producing cells
from our data. Although both nonpalmitoylated and non-cholesterol tethered Shh
(N-Shh) ligands both fail to form oligomers
(Chen et al., 2004
), N-Shh
retains bioactivity while bioactivity is lost in non-palmitoylated Shh. One
possible explanation for these results is that the ready secretion of N-Shh
ligand may counteract the failure of oligomerization in paracrine signaling in
the embryo. By contrast, continued membrane retention and an absence of
oligomerization of non-palmitoylated cholesterol-modified Shh ligand may lead
to an absence of sufficient active signal within the target field to mediate
any paracrine signaling in the mouse embryo.
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ACKNOWLEDGMENTS |
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Footnotes |
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