Department of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
* Author for correspondence (e-mail: amcmahon{at}mcb.harvard.edu)
Accepted 7 May 2004
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SUMMARY |
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Key words: Dispatched, Sonic hedgehog, Patched, Hypomorph, Mouse, Morphogenesis
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Introduction |
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Given the importance of these genes in regulating many aspects of embryonic
development, it is not surprising that aberrant Hh signaling activity
underlies a number of human abnormalities and diseases. Those include notably
holoprosencephaly (HPE) (Belloni et al.,
1996; Nanni et al.,
1999
), a midline patterning defect characterized by incomplete
separation of ventral forebrain into distinct cerebral hemispheres,
polydactyly (Masuya et al.,
1995
), brachydactyly type A-1
(Gao et al., 2001
), nerve
sheath degeneration (Johnson et al.,
1996
) and a variety of cancers
(McGarvey et al., 1998
).
All Hh proteins are synthesized as full-length precursors that undergo
autocatalytic cleavage to generate an N-terminal signaling fragment Hh-Np (`p'
stands for processed) (reviewed by Ingham
and McMahon, 2001). The mature Hh-Np ligand is dually
lipid-modified with a C-terminal cholesterol and an N-terminal palmitate
(Pepinsky et al., 1998
;
Porter et al., 1996
). Both
lipids are essential for tight membrane association and for full signaling
strength of Hh molecules as demonstrated by in-vivo and in-vitro experiments
(Chamoun et al., 2001
;
Taylor et al., 2001
).
Despite the tight membrane association of the Hh ligand, an Hh signaling
response can occur at considerable distances, as much as 150 to 200 µm in
the vertebrate limb bud (Lewis et al.,
2001). The first insights into how a membrane-associated Hh ligand
travels from Hh-producing cells to its target field began to emerge with the
identification of dispatched (disp) in Drosophila
(Burke et al., 1999
). Loss of
Disp function results in a mutant phenotype that is similar to the loss of Hh;
however, in disp mutants, Hh protein accumulates in the Hh-secreting
cell, and a weak activation of target genes is detected but only in a single
row of cells immediately adjacent to the Hh-secreting cell. Thus, the bulk of
Hh-Np produced in the secreting cell does not traffic into the target field.
Further, genetic studies suggest that Disp functions exclusively in the Hh
pathway, and its activity is required only within the Hh-secreting cell,
specifically for releasing cholesterol-modified Hh-Np. Signaling by Hh-N,
which lacks a cholesterol linkage, does not require Disp activity
(Burke et al., 1999
).
Disp, like the Hh receptor Ptch, is a multipass transmembrane protein that
shares a putative sterol-sensing domain (SSD). This phylogenetically conserved
SSD domain has been identified in key regulatory genes in cholesterol
homeostasis and lipoprotein signaling. Mutations in the SSD generally result
in loss of protein activity at least in part by altering the normal
localization of the protein in distinct membrane compartments
(Zhang et al., 2001a). By
analogy to the function of other SSD proteins in trafficking processes and
studies of disp mutants in Drosophila, Disp may regulate Hh
trafficking within the synthesizing cells
(Burke et al., 1999
;
Gallet et al., 2003
).
Recently, three groups have reported on two mammalian Disp homologs,
Disp1/A and Disp2/B (Caspary et al.,
2002; Kawakami et al.,
2002
; Ma et al.,
2002
); herein we adopt the nomenclature Disp1 and Disp2 to be most
consistent with the convention for mammalian gene families. Of the two murine
Disp homologs, Disp1 is expressed weakly but broadly at early stages
of embryonic development, overlapping with reported Shh and
Ihh expression domains (Kawakami
et al., 2002
). Two laboratories independently generated mouse
Disp1 null alleles, where exon 8
(Disp1
8), which encodes most of the
transmembrane spanning regions and C-terminal domain of Disp1, was removed
(Kawakami et al., 2002
;
Ma et al., 2002
). Notably,
homozygous Disp1
8 mutants do not survive
beyond embryonic day (E) 9.5 and exhibit gross morphological features that are
nearly identical to smoothened mutant embryos the model for complete
loss of Hh signaling activity (Zhang et
al., 2001b
). A third mutant with a point mutation in the second of
two cysteine-rich putative extracellular domains
(Disp1C829F), generated in an N-ethyl-N-nitrosourea (ENU)
chemical mutagenesis screen, gives rise to a similar mutant phenotype
(Caspary et al., 2002
). Thus,
Disp1C829F does not appear to produce functional Disp1
protein and is most likely a null allele. Interestingly, in Disp1
mutants, an Hh signaling response is retained in midline cells of the
notochord that both express Shh and respond to Shh signals
(Ma et al., 2002
). But the
absence of signaling in target fields that lie adjacent to the Hh-expressing
cells leads to a failure of L-R axis determination and defective patterning of
the ventral neural tube and somite.
As a result of the early embryonic lethality exhibited by Disp1
null mutants (between E8.5 and 9.5), the investigation of Disp1 function in
Hh-dependent signaling events at later developmental stages, the focus of most
studies to date, is compromised. Further, as Hh signaling involves the
transduction of a graded signal through multiple concentration thresholds,
examining the tissue response in the context of modulated Hh release may
provide novel insights into Hh action. To this end, new alleles of
Disp1, in particular hypomorphic alleles, may be important. Here, we
have taken advantage of a hypomorphic allele,
Disp12. Our findings are consistent with a
model in which Disp1 controls the levels of available Hh signal in the embryo.
Complementary cell culture studies suggest that secretion and cell-surface
accumulation of Shh are both Disp1-independent processes.
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Materials and methods |
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Mice
Disp1C829F/+ mice were kindly provided by Kathryn
Anderson (Caspary et al.,
2002). The Ptch1null and
Shhnull alleles have been described previously
(Goodrich et al., 1996
;
St-Jacques et al., 1998
).
Mutations were studied on mixed genetic background.
Generation of Disp12/
2 mutant
To remove exon 2 of Disp1, a targeting vector was engineered in which exon
2 was flanked by loxP sites. After homologous recombination at the Disp1 locus
in AV3 embryonic stem (ES) cells, a heterozygous ES cell line was injected
into blastocysts of the C57BL6/J strain to generate chimeras. Chimeric males
were bred with ß-actin-Cre females to obtain
Disp12/+ 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
).
Immunohistochemistry
Rabbit polyclonal antibodies DspN and DspC were raised against bacterially
expressed glutathione-S-transferase fusion proteins containing amino acids
1-100 and 1370-1521 of Disp1 respectively. The antiserums were affinity
purified as described elsewhere (Bumcrot et
al., 1995). For all antisera, inoculation of New Zealand White
rabbits, as well as test and production bleeding, were carried out at
Convance, Inc. Immunohistochemistry was performed as described for sections
(Yamada et al., 1991
) and
whole mounts (Lewis et al.,
2001
). Antibody and dilutions were as follows: rabbit
Shh,
1:100 (Marti et al., 1995
);
HNF-3ß, 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
);
Chox10, 1:5000 (Briscoe 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
);
En,1:30 (Developmental Studies Hybridoma Bank; DSHB);
Evx, 1:100
(Briscoe et al., 2000
).
Lim1/2, 1:100 (DSHB);
MNR2, 1:20 (DSHB).
Histology and skeletal preparations
For histological analysis, embryos were fixed in Bouin's fixative at
4°C overnight, then embedded in paraffin, sectioned at 8 µm, and
stained with hematoxylin and eosin. For skeletal preparations, 18.5 days post
coitum (dpc) embryos were processed as described previously
(Karp et al., 2000).
Cell culture-based assay
Disp1C829F/C829F mutant fibroblasts were isolated
directly from E9.5 embryos and maintained at high density in AV3 medium
(Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum,
1xnucleotides mix, 2mM L-Glutamine, 0.01 mM MEM non essential amino
acids and 0.05 mg/ml pen and strep, all from GIBCO). Mutant cell lines were
confirmed by PCR genotyping. Fibroblasts were transfected using lipofectamine
plus reagent (Invitrogen), following the manufacturer's recommendation. Cells
were analyzed 48 hours post-transfection. To immuno-precipitate N-Shhp in the
culture medium, we added 30 µl anti-Shh monoclonal antibody 5E1 (DSHB) to
1.5 ml of medium from Shh expressing cells for two hours at 4°C. The
antibody and bound proteins were then recovered by overnight incubation with
Protein A agarose beads at 4°C. The beads were washed and proteins
recovered in sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) sample buffer. Biotinylation of cell-surface protein was carried
out essentially as described (Denef et
al., 2000).
Western blotting
The proteins recovered from Protein A-agarose and streptavidin-agarose
beads were separated on a 4-15% SDS-PAGE and transferred to polyvinylidene
difluoride (PVDF) membranes for immunoblotting. The membranes were blocked in
5% skimmed milk in PBS with 0.1% Tween 20 (PBST) for 1 hour and incubated with
primary antibody against N-Shh (AB80, rabbit polyclonal antibody, 1:1000
dilution) for 1 hour. The membrane was washed three times with PBST, and
incubated with secondary antibody (HRP conjugated Donkey anti-rabbit IgG
1:3000 dilution) for 45 minutes, followed by chemiluminescent detection
according to the manufacturer's suggestion (Pierce).
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Results |
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Disp12/
2 mutants died
at birth due to breathing difficulties. All mutants showed mild midline facial
defects in which the nose, upper jaw and philtrum were hypoplastic and the
eyes were positioned close to the midline (compare
Fig. 1A,B,F,G). Examination of
the nasal cavity showed a narrowed or closed nasopharyngeal airway with a
resulting fusion of the paired, medial-lateral vomeronasal organs (data not
shown, and Fig. S3D,E at
http://dev.biologists.org/supplemental/).
Histological analysis further revealed midline defects: a loss of the primary
palate, upper incisors and pituitary hypoplasia (see Fig. S3). Analysis of
Fgf8 expression, which demarcates the lateral and medial frontal
nasal process and maxillary and mandibular epithelium, indicated that midline
tissue between these expression domains was absent by E10.5
(Fig. 1K,L). All these defects
are characteristic of holoprosencephaly (HPE), which in specific human cases
results from reduced Shh signaling in which only one allele is active
(reviewed by Wallis and Muenke,
1999
).
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If the midline facial defects reflected reduced levels of available Shh
signal, we reasoned that lowering the levels of Ptch1 may rescue
Disp12/
2 mice, as Ptch1
sequestration of Hh is known to reduce Hh protein levels in a responding
target field (Chen and Struhl,
1996
). Indeed,
Disp1
2/
2,Ptch1+/
mutant pups were viable, fertile and indistinguishable from wild-type
littermates (Fig. 1D,I,N). By
contrast, reducing Shh levels further might be expected to enhance the midline
phenotype. In agreement with this prediction,
Disp1
2/C829F, Shh+/
mutants exhibited an extreme proboscis-like nasal process, the result of a
fusion of the lateral frontal nasal processes, a phenotype observed in the
most severe forms of HPE and Shh null mutants
(Fig. 1E,J,O).
The induction of Shh and its targets during early forebrain development
Several lines of evidence suggest that the facial anomalies in HPE result
from earlier patterning defects in the anterior neural plate (reviewed by
Muenke and Beachy, 2000).
During early forebrain development, Shh is expressed throughout the
axial midline mesoderm, underlying the presumptive brain, and its activity is
required to induce secondary sites of Shh expression in the ventral
neural plate (reviewed by McMahon et al.,
2003
). In
Disp1
2/
2 mutants at the
10-somite stage (E8.5), expression of Shh in midline mesoderm was
comparable to that of wild-type embryos
(Fig. 2A,B, arrow). However,
only a weak induction of Shh was observed at the ventral midline of
the midbrain, whereas expression in wild-type embryos extended rostrally into
the forebrain and caudally into the hindbrain
(Fig. 2A,B, arrowhead).
Further, the Shh-dependent transcription factors, Nkx2.1 and
Nkx2.2 (Pabst et al.,
2000
), which show nested expression domains in the forebrain, were
markedly downregulated in
Disp1
2/
2 mutants
(Fig. 2E,F,I,J). Together,
these observations suggest that attenuating Disp1 activity results in markedly
reduced Shh-signaling activity during the early stages of forebrain
patterning. As a result, target gene activation is more ventrally restricted,
suggesting that the strength and range of Shh signaling are both diminished.
This is supported by analysis of the expression of the general Hh target gene
Ptch1, transcription of which provides a direct readout of the range
and strength of Hh signaling. In both E8.5 and E9.5
Disp1
2/
2 mutants,
Ptch1 expression was absent in the rostral forebrain and optic stalk
and the overall levels of remaining Ptch1 expression were reduced
(Fig. 2M,N and data not shown).
When Disp1 or Shh activity was further reduced in
Disp1
2/C829F and
Disp1
2/C829F, Shh+/
mutants, respectively, induction of Shh in the midbrain was almost absent, no
activation of Nkx2.1 or Nkx2.2 were observed in the
brain-forming regions of the anterior neural tube and only weak Ptch1
expression was seen in midbrain and more caudal regions of the neural plate
(Fig. 2C,D,G,H,K,L,O,P).
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|
To investigate ventral patterning defects in Disp1 mutants, we
looked at the expression of Class I and Class II homeobox proteins that
distinguish distinct classes of neural progenitors (reviewed by
Briscoe and Ericson, 2001;
Jessell, 2000
). Class I genes
(Dbx1, Dbx2, Pax6, Pax3 and Irx3) are repressed while Class
II genes (Nkx2.2, Nkx6.1 and Olig2) are activated at
different concentration thresholds of Shh. The ventral boundaries of Class I
and dorsal boundaries of Class II genes demarcate neural progenitor domains
within the ventral half of the neural tube. As with the forebrain, we observed
a progressive (ventral to dorsal) loss of ventral progenitor population that
correlated with the increasing severity of the Disp1 and Shh
mutant alleles. When
Disp1
2/
2 hypomorphic
embryos were examined, the floor plate markers (HNF3ß and
Shh) were not induced at the medial ventral neural plate
(Fig. 4A,E). The midline was
occupied by Nkx2.2+ pV3 progenitors
(Disp1
2/
2, 24±6,
n=5), whose numbers were only 40% of wild-type control (wt,
60±6, n=5, P<0.01)
(Fig. 4B,F,U,V). When Disp1
levels were further reduced in
Disp1
2/C829F embryos, we observed a
further reduction in pV3 (Disp1
2/C829F,
7±3 n=5) progenitors (Fig.
4I,J,U,V). An additional reduction of Shh-signaling strength in
Disp1
2/C829F, Shh+/ mutant
led to the complete loss of the pV3 progenitors
(Fig. 4M,N,U,V). The ventral
midline was now occupied by a reduced population of pMN cells (wt,
71±6, Disp1
2/C829F,
Shh+/, 5±2, n=5, P<0.01)
(Fig. 4N,V). The domain
occupied by Nkx6.1-producing cells (wt, 175±15, n=5) was also
reduced in the hypomorphic mutants
Disp1
2/
2 (125±10),
Disp1
2/C829F (60±5), and
Disp1
2/C829F, Shh+/
(63±5, n=5, P<0.01)
(Fig. 4C,G,K,O,W). As Nkx6.1
demarcates pV3, pMN and pV2 progenitors, the results suggest that even in the
most severe mutants, Shh-signaling levels are sufficient to induce pV2
progenitors, and pV2 progenitors expand ventrally on reduction of
Disp1 activity. In general, we observed a graded increase in severity
of the observed phenotypes with increasing severity of the allelic
combinations (floor plate>pV3>pMN>pV2), that mirrors the in-vitro
dependence of a given progenitor population on an increasing concentration of
Shh for induction (FP>pV3>pMN>pV2)
(Roelink et al., 1995
;
Ericson et al., 1997a
).
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The severe reduction of the pMN neural progenitor population translated to
a significant decrease in MNR-positive motoneuron precursors that were
abnormally positioned, occupying the ventral midline in all mutants (wt,
145±15, Disp12/
2,
98±14, Disp1
2/C829F, 80±10,
Disp1
2/C829F, Shh+/,
73±6, n=5, P<0.01)
(Fig. 5A,E,I,M).
Chox10+ V2, En1+ V1 and Evx1/2+ V0
interneuron precursors were present in approximately normal numbers, but there
was a higher degree of mixing between these populations, and a ventral
progression of their domains that correlated with the severity of the allelic
combinations (Fig. 5B,F,J,N). As expected, we also observed an accompanying ventral shift in Lim1/2
expression, a regulatory factor that is present within V0, V1 and V2
precursors, and D5 dorsal precursors that derive from dorsal pD5
progenitors.
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Skeletal patterning defects in Disp1 mutants
hedgehog signaling plays several distinct roles in skeletal patterning.
Initially, Shh signaling from midline (notochord and floor plate) induces
sclerotome formation in the adjacent ventral somite
(Fan et al., 1995). Sclerotome
precursors then generate the axial skeleton. Both the axial and appendicular
skeleton requires Ihh signaling to regulate proliferation and differentiation
of skeletal progenitors (reviewed by
McMahon et al., 2003
). By
contrast to Disp1 null mutants that fail to express ventral somite
markers prior to developmental arrest
(Caspary et al., 2002
;
Kawakami et al., 2002
;
Ma et al., 2002
),
Disp1
2/C829F embryos form a relatively
normal axial and appendicular skeleton, the only difference from wild-type
being a slight delay in chondrocyte differentiation at cervical levels (data
not shown). However, various deficiencies were apparent in the head skeleton
(Fig. 6A,B,C,D,E,F). As
expected from the loss of facial progenitors between the frontal nasal
processes that accompanies the midline defects in the
Disp1
2/C829F ventral forebrain,
Disp1
2/C829F embryos displayed a dramatic
loss of premaxillary frontal nasal components, including the primary palate
and upper incisor, which are all derived from the neural crest (Fig. S3). In
addition, we observed a complete absence of the parietal bone, whereas the
basisphenoid and occipital bones (supra-, ex-, basi-) are either misshapen or
reduced in size (Fig. 6E,F).
Thus, the data suggest that the anterior paraxial mesoderm of the cephalic
skeleton from which these bones derive is relatively more sensitive to
Disp1 function than more caudal somitic regions.
|
Disp1 is involved in Ihh signaling
Disp1 null mutants arrest early with L-R patterning defects that
resemble those observed in both Smo and Shh/Ihh compound
mutants (Chiang et al., 1996;
St-Jacques et al., 1999
;
Zhang et al., 2001b
). Thus, it
has been proposed that Disp1 plays a similar essential role in both Shh and
Ihh signaling (Kawakami et al.,
2002
; Ma et al.,
2002
). To investigate this possibility, we compared the phenotype
of Shh/ and
Disp1
2/C829F,
Shh/ mutants at E9.5. Whereas Shh/
embryos undergo heart looping, albeit randomized due to the loss of a midline
barrier (Fig. 7A,B)
(Meyers and Martin, 1999
),
Disp1
2/C829F, Shh/
embryos fail to undergo heart looping. Interestingly, these mutants do undergo
embryonic turning. So in this mutant combination, heart looping and turning,
both of which are distinct aspects of L-R asymmetry, are genetically
separable. Thus, the Disp1
2/C829F alleles
enhance the Shh null phenotype, consistent with an additional role
for Disp1 in Ihh signaling. Further evidence to support this conclusion comes
from analysis of the expression of Dbx1. Normal Dbx1
expression in p0 progenitors is dependent on hedgehog signaling
(Wijgerde et al., 2002
).
Further, the presence of some ventral Dbx1 expressing cells in
Shh mutants suggests a role for Ihh signaling in maintaining residual
expression (Wijgerde et al.,
2002
) (Fig. 7D,E).
However, when Disp1 activity was decreased in
Disp1
2/C829F, Shh/
embryos, all Dbx1 expression was lost, suggesting that residual Ihh
signaling is reduced to sub-threshold levels
(Fig. 7F). Given these results,
it is therefore surprising that by contrast to the marked skeletal defects
that result from perturbation of Ihh signaling
(St-Jacques et al., 1999
),
Disp1
2/C829F, Ihh+/ mutant
embryos, although slightly smaller than wild-type littermates at birth,
display a wild-type organization of the endochondral skeleton (data not
shown).
|
|
Next, we examined the cell-surface accumulation of Shh protein in Disp1C829F/C829F mutant fibroblasts. All cell-surface proteins were labeled with biotin and precipitated with streptavidin conjugated beads and separated by SDS-PAGE. The absence of ER protein BIP, Golgi protein GM130, as well as the full-length unprocessed Shh precursor in the pull-down fraction indicated that biotin labeling was specific for cell-surface proteins (data not shown). We co-transfected a transmembrane anchored Shh, Shh-CD4 (Mr 86 kDa), as a control for normalization of transfection efficiency (Fig. 8Db). Shh-CD4 can be labeled and pulled down effectively by streptavidin beads, in a similar way to N-Shhp. We detected only processed forms of N-Shhp on the cell surface, further evidence that the pulled-down components were from intact cells. After normalization for Shh-CD4 levels, Shh-Np accumulation on the cell surface, as well as in the cell lysate, showed no difference in the presence or absence of Disp1 in Disp1C829F/C829F mutant fibroblasts. Again, the presence of full-length Disp1 in this experiment was confirmed by Western blot analysis (data not shown).
Ma et al. (Ma et al., 2002)
have reported that Disp activity in Drosophila S2 cells increases the
extracellular export of a Shh::Renilla luciferase fusion protein. We
investigated production and secretion of Shh::luciferase in the presence or
absence of Disp1 in mouse fibroblasts. However, we observed almost no
normal processing of the Shh::luciferase precursor, which therefore precluded
any further analysis (data not shown). In agreement with the observations of
Ma et al. (Ma et al., 2002
),
co-culture of a Shh reporter cell line with fibroblast cells expressing Shh in
the presence or absence of Disp1 led to a modest (1.25 fold)
Disp1-dependent increase in signaling activity in reporter cells
(data not shown).
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Discussion |
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Disp1 in head and axial skeletal development
Attenuated Hh signaling, for example where one allele of Shh is inactive,
has been linked to midline facial defects that characterize HPE (for a review,
see Muenke and Beachy, 2000).
In comparison with other Hh-dependent signaling events, facial development
appears to be consistently more sensitive to attenuation of Hh-ligand
concentration or Hh-signaling strength. The loss of midline facial structures
and the associated ventral displacement of the eyes are all thought to result
from an initial perturbation in midline patterning of the ventral forebrain.
In the present study, we observed a relatively mild HPE phenotype in
Disp1
2/
2 mutants that
increased in severity in Disp1
2/C829F
embryos. When Shh dosage was decreased,
Disp1
2/C829F, Shh+/
embryos exhibited a further enhanced HPE with a proboscis nose that closely
resembled the most severe facial phenotype of Shh/
mutant embryos (Chiang et al.,
1996
). A simple explanation for the observed interaction between
Disp1 and Shh is that Disp1 regulates Shh signaling and that
Shh-signaling activity is sensitive to Disp1 levels. The observed facial
phenotypes probably arise secondary to defective ventral forebrain patterning
and the associated reduction in ventral forebrain-derived Shh. As
Disp1 and Shh activity were progressively reduced, we
observed an increasingly more severe failure in the induction of
Nkx2.1- and Nkx2.2-expressing ventral forebrain cell
identities that are dependent on mesendoderm-derived Shh signaling, despite
normal Shh expression in the mesendoderm
(Muhr et al., 1997
;
Pabst et al., 2000
). Since Shh
induces its own expression in the ventral forebrain, and ventral
forebrain-derived Shh is essential for facial patterning
(Hu and Helms, 1999
), reduced
Shh expression from this site probably plays a substantial role in
the generation of the observed facial phenotypes.
In addition to the loss of midline neural crest-derived structures, the
parietal bone was absent and several other bones that arise from the cephalic
paraxial mesoderm were reduced or misshapen in
Disp12/C829F mutants. To date, there is
little information on patterning of the cephalic mesoderm. Our evidence
suggests that Shh signaling is likely to play an important role, and the
reduced expression of two highly related Fox family members in this region,
FoxD1 and FoxD2, implicates these factors in the patterning
process. The lack of an obvious head skeletal phenotype in either single
FoxD mutant may reflect redundant roles for these closely related
forkhead genes (Hatini et al.,
1996
; Kume et al.,
2000
).
Surprisingly, although a considerable body of evidence indicates that Shh
signaling plays a central role in the initial step of axial skeleton
development and sclerotome induction in the paraxial mesoderm of the trunk,
somites (reviewed by McMahon et al.,
2003) and vertebrae form normally in the most extreme mutant
combinations examined (Disp1
2/C829F,
Shh+/). That Disp1 is required for sclerotome induction is
evident from analysis of Disp1 null mutants
(Caspary et al., 2002
;
Kawakami et al., 2002
;
Ma et al., 2002
). Thus, it is
likely that low levels of Shh signaling suffice for sclerotomal patterning; a
view consistent with in-vitro inductive assays of muscle and sclerotome
patterning in the zebrafish (Hammerschmidt
et al., 1996
).
Disp1 and spinal cord patterning
Arguably the best-characterized Hh target field with respect to the issue
of dose-dependent Hh signaling is the ventral spinal cord. As expected from
current models, the first cell identity that is expected to be lost as Shh
signaling is reduced is the midline floor plate. In vitro, floor plate
induction requires the highest Shh concentration threshold
(Ericson et al., 1997a). In
vivo, notochord transplants indicate that floor plate induction by the
notochord is contact-dependent (Placzek et
al., 1990
), consistent with an inductive event that may utilize
concentration thresholds in vivo that are only possible on the cell surface.
Disp1
2/
2 mutants fail to
undergo floor plate induction; they also exhibit reduced numbers of pV3
interneuron progenitors, the cell identity with the next highest concentration
requirement for Shh in vitro (Ericson et
al., 1997a
). By contrast, all other ventral cell identities are
present at approximately wild-type levels, a phenotype that closely resembles
loss of Gli2, a transcription effector of Shh signaling in the
ventral neural tube (Ding et al.,
1998
; Matise et al.,
1998
). pV3 progenitors are entirely lost when Shh signaling is
further attenuated in Disp1
2/C829F,
Shh+/ mutants, and pMN progenitor populations, that require the
next highest level of Shh input in vitro, are markedly reduced. In summary,
both the morphological and molecular analysis link Disp1 to the Shh
pathway, and the range of phenotypes we observe are completely consistent with
the view that Disp1 dosage regulates the level of hedgehog signaling
in target tissues.
Disp1 action
In principle, Disp1 may regulate hedgehog signaling in either the producing
or responding cell. Drosophila studies indicate that Disp is required
only in Hh-producing cells (Burke et al.,
1999). In the mouse, the persistence of Shh signaling in the
notochord of Disp1C829F/C829F mutants, in which cells both
produce and respond to ligands, suggest that in this autoregulatory loop,
Disp1 is not essential for transducing an Shh signal where autocrine signaling
is likely to be occurring (Caspary et al.,
2002
; Ma et al.,
2002
).
Previous studies in Drosophila and mouse cells have demonstrated
that translation and processing of hedgehog ligands are unchanged in
disp or Disp1 mutants
(Burke et al., 1999;
Kawakami et al., 2002
;
Ma et al., 2002
). Disp1 must
regulate Shh availability by some other mechanism. Several possible roles can
be imagined: (1) Disp1 may be involved in intracellular trafficking of Hh to
the cell surface; (2) it may regulate direct release of Hh protein into the
extracellular space; or (3) it may facilitate the effective surface
presentation of Hh to adjacent receiving cells. To distinguish between these
possibilities, we derived fibroblast cultures from
Disp1C829F/C829F embryos and gauged the levels of cell
surface-accumulated and medium-accumulated Hh protein.
First, accumulation of Shh on the surface of fibroblasts in culture was not
affected by the absence of Disp1. Whether fibroblasts and epithelial cells
differ in this regard is unclear. For example, a recent in-vivo study suggests
that disp is involved in regulating apical-specific accumulation of
Hh in the Drosophila epidermis, an issue that cannot be examined in a
non-epithelial fibroblast line (Gallet et
al., 2003). We also failed to observe any role for Disp1 in
secretion of Shh into the medium in fibroblast cell culture. Thus, the Shh
secretion we observed in vitro is Disp1 independent; the fact that there is
little or no response in Hh target fields of Disp1null
embryos (Caspary et al., 2002
;
Kawakami et al., 2002
;
Ma et al., 2002
) suggests that
this form of secretion is not likely to play a significant role in vivo.
However, the secreted N-Shhp from Disp1 null fibroblasts is biologically
active, as demonstrated by its ability to activate alkaline phosphatase
activity in C3H10T1/2 cells (data not shown). Interestingly, the existence of
a Disp-independent release (default release) of Hh protein has been
demonstrated in the Drosophila epidermis, where Hh retains the
ability to travel to adjacent receiving cells and elicit downstream signaling
events in cells posterior to the Hh-expressing domain in disp mutants
(Gallet et al., 2003
). Thus,
our studies argue against a simple model in which Disp1 only regulates release
of Shh into the extracellular space and in so doing, signals in the target
field. For example, Disp1 may be involved in efficiently presenting Hh ligand
to a specific mediator, which then relays Hh ligand to the target field.
By contrast to our findings, a role for Disp1 in Shh secretion from cells
has been suggested from other lines of experiment. Ma et al.
(Ma et al., 2002) reported
that when Shh-expressing Disp1/ mutant fibroblasts were
mixed with an Shh-responsive cell line, overexpressing Disp1 in these cells
leads to 1.6-fold increase in Hh-dependent response, suggestive of increased
ligand levels. We observed a similar low level of increase in the same assay
(1.25-fold, data not shown). Given that our results indicate that there is no
change in the bulk level of secreted Shh protein in the presence or absence of
Disp1 in vitro, the slight increase in activity may reflect a minor component
of this fraction, for example, in a more active oligomeric complex
(Zeng et al., 2001
). Elevated
Disp1 levels in Drosophila S2 cells were reported to enhance
accumulation of a Shh::Renilla luciferase fusion protein in the medium
(Ma et al., 2002
). However, we
were unable to verify this result in mouse fibroblasts, because this fusion
protein fails to undergo efficient processing. At present, the specific action
of Disp1 remains unclear.
Does Disp1 function exclusively in the Hh pathway in vertebrates, as
appears to be true for its Drosophila counterpart
(Burke et al., 1999)? A key
piece of evidence in support of this view comes from analysis of
Disp1
2/
2,
Ptch1+/ mutants, in which reducing Ptch1 dosage
completely rescues the
Disp1
2/
2 phenotype. Ptch1
plays two key roles in hedgehog signaling (reviewed by
Ingham and McMahon, 2001
):
inhibiting Smoothened activation of hedgehog targets and sequestering Hh
ligand in negative feedback control. Thus, either sensitizing the initial Hh
induction, or increasing available ligand by attenuating the feedback loop, is
sufficient to compensate for decreased Disp1 activity and the resulting
decrease in Shh signaling.
Does Disp1 play an equivalent role in signaling by other mammalian hedgehog
ligands? As Dhh phenotypes are most apparent in neonatal and adult
mice and Disp12/C829F mutants die at
birth, we focused our studies on Ihh. The strong resemblance of
Disp1 null mutants to Shh/Ihh compound mutants, which also
arrest at E9.5, favors the view that Disp1 mediates both Shh and Ihh signaling
(Caspary et al., 2002
;
Kawakami et al., 2002
;
Ma et al., 2002
). In agreement
with a Disp1 role in Ihh signaling in the early somite embryo, Dbx1
expression in the ventral neural tube of Shh/ embryos,
which probably depends on residual Ihh signaling
(Zhang et al., 2001b
), was
completely lost when Disp1 activity was reduced in
Disp1
2/C829F, Shh/
embryos. Later in development, Ihh plays a key role in co-coordinating
proliferation and differentiation of chondrocytes and in osteoblast
development in the long bones; as a result long bones of Ihh mutants
are one-fifth of their normal length at birth and have no bone
(Karp et al., 2000
;
Long et al., 2001
;
Pathi et al., 1999
;
St-Jacques et al., 1999
;
Vortkamp et al., 1996
).
Surprisingly, long bone development in
Disp1
2/C829F, Ihh+/ was
normal at birth. This result suggests two possibilities. First, Ihh signaling
in the bone is independent of Disp1 activity. The second possibility is that
Ihh signaling is Disp1-dependent in the bone, but relatively low levels of
signaling suffice, as in the induction of the dorsalmost ventral progenitor
populations in the neural tube and sclerotome of the somites, where Shh is the
key signaling factor. Distinguishing between these possibilities will require
a specific removal of Disp1 activity from the developing
skeleton.
In summary, our genetic interaction studies connect Disp1 specifically to the Hh pathway. Furthermore, our studies reveal a spectrum of responses to the attenuation of Hh-signaling strength in various Hh-dependent target fields, which might relate to tissue-specific (epithelial versus mesenchymal) differences, or differing requirements for active ligand by responding cells.
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ACKNOWLEDGMENTS |
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Footnotes |
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