1 Program in Neurobiology and Department of Pediatrics, Children's Memorial
Institute for Education and Research and The Feinberg School of Medicine,
Northwestern University, Chicago, IL 60614, USA
2 Department of Biology, University of Massachusetts, Amherst, MA 01003,
USA
** Author for correspondence (e-mail: j-kohtz{at}northwestern.edu)
Accepted 20 May 2004
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SUMMARY |
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Key words: Sonic hedgehog, Lipid modifications, Forebrain
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Introduction |
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Since its discovery as a key secreted regulatory protein, there have been
many studies investigating the mechanisms used by Shh to pattern the embryo.
One of the long-standing puzzles has been how Shh selectively acts in a
long-range or short-range manner. Early evidence that Shh may act through
long-range signaling in the vertebrate embryo resulted from comparisons of
Shh mRNA expression with that of its target genes
(Echelhard et al., 1993; Kraus
et al., 1993; Riddle et al.,
1993
; Roelink et al.,
1994
). In the chick spinal cord, expression analysis showed that
although Shh mRNA is limited to the ventral midline, direct targets
of Shh can be detected several cell diameters distant from Shh-expressing
cells (Marti et al., 1995a
;
Roelink et al., 1995
;
Ericson et al., 1995
). In
addition, concentration-dependent effects of the Shh protein suggested that
the level of exposure to Shh protein regulates regional diversity (Marti et
al., 1995; Roelink et al.,
1995
; Ericson et al.,
1995
; Ericson et al.,
1997
; Briscoe et al.,
2000
). Relative expression analysis combined with the ability of
Shh to directly activate distant targets in a concentration-dependent manner
did not rule out the possibilities that secondary relay mechanisms and/or cell
migration are involved instead of the morphogenetic activity of Shh.
Gritli-Linde et al. and Incardona et al. have shown that the Shh protein can
be detected at a distance from its source
(Gritli-Linde et al., 2001
;
Incardona et al., 2000
),
providing new evidence that long-range signaling may involve the direct action
of Shh protein. In a crucial experiment, Briscoe et al.
(Briscoe et al., 2001
) showed
that the long-range signaling effects of Shh depend upon the direct
interaction of Shh with its receptor Patched (Ptc)
(Marigo et al., 1996
;
Stone et al., 1996
) in the
chick spinal cord. Taken together, these experiments clearly show that Shh can
act as a morphogen in vertebrates, directly acting on targets far from its
source of secretion.
The crucial issue has now become, not whether Shh acts as a morphogen, but
how Shh acts as a morphogen. Several models for the mechanism of action of
morphogens have been proposed (reviewed by Telemann et al., 2001). Among these
are repeated cycles of endocytosis and secretion, cell growth, diffusion and
cytonemes (Ramirez-Weber and Kornberg,
1999). However, it is presently not clear which, if any, of these
mechanisms are used by Shh.
The unique nature of the two lipid moieties on Shh suggests that
investigation of these may lead to a better understanding of the long-range
signaling mechanisms used by Shh (reviewed by
Ingham, 2001). Previous work
showed that the addition of the C-terminal cholesterol requires an
autocatalytic cleavage event (Porter et
al., 1995
; Porter et al.,
1996a
) that results in the generation of a 19 kDa active
N-terminal fragment (Bumcrot et al.,
1995
; Marti et al.,
1995b
). Palmitoylation of this 19 kDa fragment generates a Shh
protein that contains two lipids, an N-terminal palmitate and a C-terminal
cholesterol (Pepinsky et al.,
1998
). The N-terminal lipid is required for activity in
Drosophila (Lee et al.,
2001
) and enhances differentiation activity in both a cell
line-based assay and forebrain neural explants
(Pepinsky et al., 1998
;
Kohtz et al., 2001
;
Taylor et al., 2001
). The
C-terminal cholesterol is responsible for tethering Shh to the surface of the
secreting cell (Porter et al.,
1996b
), but recent evidence suggests that the C-terminal
cholesterol is required for long-range signaling in the mouse limb
(Lewis et al., 2001
), and
punctate structures in the fly (Gallet et
al., 2003
). However, the mechanism by which the C-terminal
cholesterol mediates long-range signaling remains unknown. Zeng et al.
recently proposed a model suggesting that the C-terminal cholesterol is
necessary for the formation of multimers that hide the hydrophobic lipid
domains in a micelle structure mediating long-range signalling
(Zeng et al., 2001
). Taken
together, these data suggest that the N- and C-terminal lipids may play
different roles in Shh activity, tethering, multimerization and long-range
signaling. However, the individual contributions of the N- and C-terminal
lipids in these functions have yet to be defined.
In this study, Shh proteins containing different combinations of N- and C-terminal lipids were purified from stably-transfected neural cell lines and used to compare the relative roles of the N- and C-terminal lipids in ventral forebrain neuronal differentiation-inducing activity, membrane tethering, multimerization and expression profile in embryonic tissues. The data suggests that the N- and C-terminal lipids synergize during Shh multimerization and membrane tethering, but not during differentiation-inducing activity. The presence of the C-terminal lipid alone is sufficient both for multimerization and membrane tethering, whereas the N-terminal lipid alone is sufficient only for multimerization and not for membrane tethering. Contrastingly, the N-terminal lipid is required for differentiation-inducing activity, whereas the C-terminal lipid substantially reduces Shh differentiation-inducing activity. Purification of multimeric and monomeric forms of Shh proteins shows that multimerization is required but not sufficient for Shh differentiation-inducing activity. In addition, differences between Shh proteins in the embryonic brain and limb can be detected, supporting the hypothesis that expression of functionally distinct forms of Shh may be region specific. These data suggest that the N- and C-terminal lipids play both overlapping and distinct roles in Shh signaling, and that the key to understanding the morphogenic actions of Shh is a better understanding of the relative functions of its N- and C-terminal lipids.
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Materials and methods |
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Generation of stably transfected cell lines
The C17.2 cell line was grown according to Snyder et al.
(Snyder et al., 1992). Cells
were transfected with human Shh cDNAs capable of producing three different N-
and C-terminal lipid-containing Shh proteins (amino acids 1-197), including
the signal sequence. The S4 cell line (Liu
et al., 1998
), a C17.2 cell line containing wild-type Shh (wtShh)
was obtained from Dr A. Joyner (New York University). A point mutation
changing human SHH cysteine 24 to a serine was obtained from Dr E. Garber
(Biogen) (Pepinsky et al.,
1998
; Kohtz et al.,
2001
). Shh cDNAs (C24S-Shh, ShhN, C24S-ShhN) were
subcloned into the mammalian expression vector pZeo (Invitrogen) and
transfected into C17.2 cells. Selection for zeomycin (Invitrogen, 500
µg/ml)-resistant colonies was performed over a two-week period, after which
15 independent clones were recovered, as well as two groups of heterogeneous
cells for each Shh cDNA. Stably transfected cell lines were screened
by western analysis to obtain the clones that produced the highest levels of
Shh proteins.
Preparation of different N- and C-terminal lipid-containing Shh proteins
Culture supernatants from C17.2 stably transfected cell lines containing
soluble Shh proteins were collected over a 12-day period from 150 mm plates.
Cells were fed with fresh medium every 4 days. One to two liters of cell
culture supernatant was affinity purified on a 1 ml column (Pharmacia
AKTA-FPLC) containing 1 mg of purified 5E1 anti-Shh antibody
(Ericson et al., 1996) (a
monoclonal antibody against Shh, obtained from the Developmental Studies
Hybridoma Bank), cross-linked to CNBr-activated Sepharose (Pharmacia). The
supernatant was applied twice to the column, the column washed with TBS-0.5%
Tween 80, and proteins eluted in 100 mM glycine, pH 2.5. Shh proteins were
dialyzed against PBS/0.5 mM dithiothreitol, concentrated in Centricon filters
(Amicon), and quantified using uShhN as a standard by western blotting.
Determination of ratio of cell-associated and secreted Shh
In order to determine the ratio of cell-associated and secreted Shh,
stably-transfected cell lines were grown in 100 mM plates for 4 days. For the
secreted form: supernatants from each plate (10 ml) were immunoprecipitated
with 5E1 antibody that had been pre-incubated with protein G-agarose beads
(Roche). Secreted Shh bound to 5E1-protein-G agarose was recovered by
centrifugation and solubilized in Laemmli sample buffer. For the
cell-associated form: cells were scraped from the 100 mm plate, recovered by
centrifugation and solubilized in Laemmli sample buffer. Samples were then
western blotted using rabbit anti-Shh antibody as the probe.
Gel filtration of Shh proteins
Affinity-purified Shh proteins were applied to a Superdex 200 column (AKTA
FPLC) and 250 µl fractions were collected. The column was standardized
using the following range of proteins (Pharmacia): 669 kDa, 66 kDa, 43 kDa, 25
kDa and 13 kDa. Independently run purifications on the Superdex column
resulted in standards eluting into identical fractions. Fractions were
dialyzed against 100 mM ammonium carbonate (pH 7.5) and concentrated by
evaporation. One-fifth of each fraction was analyzed by western blotting and
probed with rabbit anti-Shh antibody. Multimeric and monomeric fractions were
dialyzed against PBS/0.5 mM DTT in order to assay for their relative
activities in the forebrain differentiation assay.
ShhN protein (Fig. 3A, lane 4) and wtShh protein (Fig. 3A, lane 1) were purified from the culture supernatant of stably transfected C17 cells, and separated by Superdex 200 gel filtration column (as shown in Fig. 5A,C). Fractions corresponding to M (fractions 37 and 38) and the ShhN monomeric form (59 and 61) were pooled, concentrated, and tested for their activity.
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Sightless 2 (GenBank Accession number AK003605): 5'-1(611-) ctt ggc aga gtg ggt ttg taa-3'; 3'-1(-1060) aca gtg ttg aca aca cca aac-5'; 5'-2(632-) cag gca cct ttg atc ttc aag-3'; and 3'-2(-1161) gat acc acg gtc gaa gtg cgt-5'.
Mouse dispatched (GenBank Accession number AY150698), 591 bp fragment: mdispatched 5'-(450) gac cca gag aaa ccc caa gaa-3' and mdispatched 3'-(1041) ttc cac gtg acc agc ctc tgt-5'.
Zebrafish injection of RNA and in situ hybridization
mRNA encoding different Shh lipid modifiable forms was transcribed in vitro
using T7 polymerase from the pT7TS plasmid linearized with BamHI.
Two- to four-cell stage zebrafish embryos were injected with 75 pg of mRNA
encoding ShhN, C24S-Shh and C24S-ShhN, Shh
(Ekker et al., 1995) and
ß-galactosidase. Injected embryos were fixed in 4% paraformaldehyde at
the 20 somite stage, and gene expression was assayed by in situ hybridization
using anti-sense digoxigenin, or fluorescein-labeled RNA probes to
nk2.2 (Barth and Wilson,
1995
), nk2.1b (Rohr
et al., 2001
), pax6
(Krauss et al., 1991
) and
emx1 (Morita et al.,
1995
). Double in situ labeling was performed described previously
(Jowett and Yan, 1996
), using
(1) an alkaline phosphataseconjugated anti-digoxigenin antibody (Roche) and
the NBT (Roche) reaction substrate, and (2) an alkaline phosphatase-conjugated
anti-fluorescein antibody (Roche) and the Fast Red (Roche) reaction substrate.
After color reactions were developed, embryos were fixed overnight and cleared
in 75% glycerol. Color reactions were timed and controlled for by expression
in comparison with uninjected controls. For lateral views, yolks and eyes were
removed. For the dorsal views, only yolks were removed. Mounted embryos were
photographed using a Zeiss compound microscope with DIC optics and images were
processed using Adobe Photoshop software.
Effects on gene expression were scored blind based on the degree of expansion of nk2.1b and nk2.2 expression and the degree of reduction of emx1 and pax6 expression. Embryos were placed into four different classes: (1) no effect, (2) mild ventralization, (3) moderate ventralization and (4) major ventralization (see Fig. 5).
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Results |
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The N- and C-terminal lipids synergize to increase membrane tethering
It has previously been shown that the C-terminal cholesterol tethers Shh to
the membrane of the secreting cell (Porter
et al., 1996a). However, the contribution of the N-terminal lipid
to membrane tethering has not been defined.
Fig. 2A shows that wtShh (N+C
lipid) exhibits the highest cell-associated:secreted protein ratio, whereas
C24S-ShhN (no lipid) exhibits the lowest cell-associated:secreted protein
ratio. Comparison of the ratios of cell-associated and secreted Shh proteins
in cell lines stably transfected with different lipid-containing Shh cDNA are
shown in Table 1. Cell
association and secretion are inversely related (as would be expected). That
the majority of cell-associated wtShh is membrane bound and not cytosolic was
previously determined by subcellular fractionation
(Kohtz et al., 2001
).
Ninety-eight percent of wtShh (N+C lipid) is cell associated. Fifty-four
percent of C24S-Shh (C lipid alone) remains cell associated, whereas only 1%
of ShhN (N lipid alone) remains cell associated. These data suggest that the
N- and C-terminal lipids synergize during membrane tethering. In addition, the
C-terminal lipid alone can tether Shh to the membrane, whereas the N-terminal
lipid cannot.
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The C-terminal lipid reduces the N-terminal lipid-mediated increase in early striatal neuronal differentiation-inducing activity
We recently reported that the Shh N-terminal lipid significantly enhances
the ability of Shh to induce the differentiation of rat ventral forebrain
neurons (Kohtz et al., 2001).
These experiments were performed using soluble recombinant proteins in vitro,
and virally produced proteins in vivo. In vivo, the inactivity of Shh lacking
the N-terminal lipid supported a requirement for the N-lipid in Shh signaling
in the forebrain. This inactivity also suggested that the C-terminal
cholesterol could not substitute for the N-terminal lipid to rescue activity
in the forebrain. Recent experiments suggest that the cholesterol group may
function in long-range signaling in the mouse limb
(Lewis et al., 2001
), and that
this may result from the formation of multimeric complexes
(Zeng et al., 2001
). We
observed that ectopic Dlx expression occurs at a distance from Shh virally
infected clusters of cells, consistent with the ability of wtShh (N+C lipid)
to signal in a long-range manner in the forebrain
(Kohtz et al., 2001
). These
results raise the issue of whether the inactivity of C24S-Shh to induce Dlx
expression in vivo results from its inability to signal once it reaches its
target cell, or from its inability to form multimeric complexes that would
enable it to signal in a long-range manner, or both.
Using in vitro neural explant differentiation assays, it is possible to
study this issue because cells within the explant are exposed to soluble Shh
and do not require Shh transportation. Thus, if C24S-Shh is inactive in vivo
primarily because it fails to participate in long-range signaling, but can
signal once it reaches the cell, then the C24S-Shh protein should be active in
vitro. If, however, C24S-Shh is inactive independent of its long-range or
short-range signaling properties, then it should be inactive in vitro and in
vivo. In order to distinguish between these possibilities, we purified
different N- and C-terminal lipid-containing Shh proteins from cell culture
supernatants of stably transfected C17 cell lines (see
Table 1 and
Fig. 3A, lanes 1-4). Purified
proteins were assayed for their differentiation-inducing activities at three
different concentrations (1 nM, 3 nM and 12 nM) in rat E11 telencephalic
explants (Fig. 3C-N).
Fig. 3B shows a diagram of the
dissection used to generate neural explants for the telencephalic
differentiation assay [modified from a method described previously
(Kohtz et al., 1998)].
Fig. 3C-N shows that the ShhN
protein has the highest differentiation-inducing activity, as indicated by the
number of Dlx and/or Islet1/2 expressing early striatal neurons
(Fig. 3E,I,M). ShhN is active
at concentrations as low as 1 nM (Fig.
3M). Shh proteins lacking the N-terminal lipid either failed to
induce Dlx or Islet1/2 expression (Fig.
3H,L,F,J,N), or did so at very low levels
(Fig. 3D). This supports the
hypothesis that C24S-Shh is inactive in vivo because it is unable to signal,
independent of its ability to be transported. Thus, the C-terminal lipid
cannot substitute for the activity-enhancing properties of the N-terminal
lipid either in vitro or in vivo.
These experiments also indicate that wtShh containing both N- and
C-terminal lipids is significantly less active than ShhN containing only the
N-terminal lipid. This suggests that the C-terminal lipid actually reduces the
differentiation-inducing activity of N-terminal lipid-containing Shh.
Fig. 3O quantifies the Dlx
expression data presented in Fig.
3C-N, and adds data obtained with in vitro N-terminal
fatty-acylated recombinant Shh protein (mShhN) for comparison
(Kohtz et al., 2001). At 1 and
3 nM, significant activity is only detected for ShhN. At 12 nM concentrations,
differentiation-inducing activity is detected for wtShh, ShhN and mShhN.
The question remains as to whether Shh can signal in its membrane-bound
form, or whether it requires secretion for activity. Although the majority of
wtShh is cell associated (Fig.
2), the ability to extract soluble wtShh and ShhN from membranes
using relatively low concentrations of Triton X-100
(Fig. 1B) suggested that it may
be possible to compare the differentiation-inducing activities of
membrane-extracted and secreted Shh proteins. However, affinity purification
of Triton X-100 membrane-extracted Shh proteins failed repeatedly (data not
shown). Triton X-100 (0.1%) membrane-extracted wtShh fails to bind a column
containing 5E1 (Fig. 3A, lanes
9-11), a function-blocking monoclonal anti-Shh antibody
(Ericson et al., 1996).
However, the same 5E1 column purifies secreted Shh forms
(Fig. 3A, lanes 1-4). These
data suggest that membrane extraction of wtShh alters its conformation so that
it is no longer recognized by the 5E1 monoclonal antibody.
Both N- and C-terminal lipids are required for the formation of the L multimer, but not the M multimer
Although a role for the C-lipid in multimerization has been proposed, the
role of the N-terminal lipid in this process remains unclear. In addition, the
relationship of multimerization to activity has not been defined. In order to
characterize large multimeric complexes of Shh, different N- and C-terminal
lipid-containing Shh proteins (Fig.
3A, lanes 1-4) were analyzed by Superdex 200 gel-filtration
(Pharmacia, AKTA-FPLC). Fractions collected after gel-filtration were western
blotted (Fig. 4A-D). Shh
proteins were primarily found in three peaks: fractions 24 and 25, >669 kDa
(L, large); fractions 30-39, 669-66 kDa (M, medium); and fractions 59-61
(monomers). Comparison of these profiles indicates that only wtShh forms the
largest multimeric Shh complex L (compare fraction 24 in
Fig. 4A-D). Loss of either
lipid eliminates the ability to form L (C24S-Shh or ShhN,
Fig. 4B and C, respectively),
and the loss of both lipids eliminates the ability to form L or M (C24S-ShhN,
Fig. 4D).
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The presence of either the N- or C-terminal lipid results in the formation
of medium sized multimeric complexes (Fig.
4A-C, fractions 36 and 39). Within M-containing fractions 36 and
39, SDS-PAGE stable multimers are present
(Fig. 4A,C). In the presence of
both N- and C-terminal lipids, SDS-PAGE stable multimers are detected at
30 kDa,
50 kDa and
90 kDa. The predominant SDS-PAGE stable
multimer migrates at approximately 30 kDa, and can be detected in the presence
of the N-terminal lipid either alone or together with the C-terminal lipid. In
the absence of both lipids, SDS-PAGE stable multimers are not detected
(Fig. 4D). The significance of
these SDS-PAGE stable multimers is presently unknown; however,
Fig. 1B shows that differences
in membrane extractability, as well as in relative ratios of SDS-PAGE stable
multimers, occurs in the embryonic brain and limb tissues.
ShhN M multimers induce early striatal neuronal differentiation
Our results show that the N- and C-terminal lipids synergize in the
formation of the L multimer. However, the C-terminal lipid reduces the
differentiation-inducing activity of Shh, suggesting that the relative roles
of the N- and C-terminal lipids in differentiation-inducing activity differ
from multimerization. One possible explanation for the C-terminal
lipid-mediated decrease in activity may be that multimerization occurs at the
expense of activity. Another is that some fraction of the monomeric form must
be present in order for Shh activity to occur.
In order to test directly the relationship between multimerization and differentiation-inducing activity, we compared the differentiation-inducing activities of the multimeric forms of ShhN, wtShh and C24S-Shh with monomeric ShhN. M multimeric ShhN is highly active at 6 nM (G), whereas M multimeric wtShh (E), M multimeric C24S-Shh (F) and monomeric ShhN (G) activities are not detected (Fig. 4). These data show that the presence of monomers is not required for activity. In addition, these data suggest that multimerization may be required for differentiation-inducing activity. However, in the absence of the N-lipid, multimerization is not sufficient for differentiation-inducing activity.
Shh containing the N- or C-terminal lipid ventralizes the zebrafish forebrain in vivo
We have previously determined that the N-lipid is required for ventralizing
the mouse forebrain in vivo (Kohtz et al.,
2001), at the same time showing that the C-lipid cannot substitute
for the N-lipid in this context. In this paper, we show that the inability of
the C-lipid to substitute for the N-lipid results from protein inactivity in
rat forebrain explants assays. In contrast to the mouse forebrain, in the
absence of the N-lipid, the C-terminal lipid-containing Shh is sufficient to
induce mouse digit duplications, but the C-lipid does not substitute for the
N-lipid in the fly wing (Lee et al.,
2001
). Taken together, these data suggest that the relative roles
of the N- and C-lipids are both context and species dependent. In order to
determine the degree of conservation of the relative roles of the N- and
C-lipids in the developing forebrain, we compared the activities of different
N- and C-terminal lipid-containing Shh proteins in the zebrafish forebrain
(Fig. 5). As previously shown,
the expression of the ventrally expressed Shh-responsive genes nk2.2
and nk2.1b was expanded dorsally in the zebrafish forebrain upon
injection of RNA encoding wild-type Shh
(Fig. 5)
(Karlstrom et al., 2003
;
Rohr et al., 2001
). Consistent
with ventralization of the CNS, the dorsally expressed genes emx1 and
pax6 were reduced in injected embryos
(Fig. 5)
(Macdonald et al., 1994
;
Morita et al., 1995
). The
ventralizing activities of constructs encoding the different N- and C-lipid
Shh proteins are classified as mild, moderate or major, based on (1) the
expansion of the ventral forebrain markers nk2.1b
(Fig. 5A,D,G) and
nk2.2 (Fig. 5B,E,H),
and (2) the reduction of the dorsal markers pax6 and emx1
(Fig. 5C,F,I). A comparison of
the percentage of the embryos exhibiting different ventralizing activities
reveals that N+C-terminal lipid-containing Shh and Shh containing N-terminal
lipid alone are similar in activity (Fig.
5M). Although Shh containing the C-lipid alone is less active than
N+C or N-lipid Shh, it retains significant ventralizing activity when compared
with Shh lacking both lipids (C24S-ShhN) or uninjected controls. These data
suggest that either the N- or the C-terminal lipid is necessary for zebrafish
ventralizing activity (Fig.
5M).
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Discussion |
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Significance of multimerization and synergism between the N- and C-terminal lipids
It is known that multimerization is involved in regulating the action of
diverse groups of proteins both in the extracellular and intracellular
compartments of the cell. In this study, we find that Shh forms different
sized multimers depending upon the presence of N- and/or C-terminal lipids.
The ability of wtShh, but not ShhN to participate in long-range signaling in
the limb (Lewis et al., 2001),
and the ability of the former, but not the latter, to participate in L
multimer formation, raises the possibility that L multimers may be crucial to
long-range signaling. In addition, although multimerization has been proposed
as a mechanism for long-range signaling
(Zeng et al., 2001
), we show
that it is required for soluble ShhN activity in vitro, and therefore for
activity in forebrain neural explant assays.
Our results confirm previous reports that the C-terminal lipid tethers Shh
protein to the surface of the secreting cell
(Porter et al., 1996b).
Comparison of the relative levels of cell-associated and secreted C-terminal
lipid, and N+C-terminal lipid-containing Shh proteins, suggests that the
N-terminal lipid increases cell association by 44% in the presence of the
C-terminal lipid. Thus, the N- and C-terminal lipids act synergistically to
maintain cell association. This synergistic interaction is slightly different
from that involved in multimerization. Formation of the L multimer requires
the presence of both N- and C-lipids. However, neither lipid alone is
sufficient for the formation of the L multimer. The formation of the M
multimer is not synergistic and occurs in the presence of either the N- or the
C-lipid alone. Finally, the antagonistic effect of the C-terminal lipid on the
N-terminal lipid-mediated enhancement of differentiation-inducing activity
clearly identifies a distinct role for the C-lipid.
Significance of the C-terminal lipid-mediated reduction in early striatal neuronal differentiation-inducing activity
The lack of activity of C24S-ShhN (no lipids) and the high activity of ShhN
(N-terminal lipid alone) confirms our previous data that the N-terminal lipid
enhances early striatal neuronal differentiation-inducing activity. However,
we obtained a surprising result in that the presence of the C-terminal lipid
reduces the differentiation-inducing activity of N-terminal lipid-containing
Shh. This differs from the findings of Zeng et al. who report that the C-lipid
increases Shh activity by 15-fold in a C3H10T1/2 cell line-based assay
(Zeng et al., 2001). Based on
their data, Zeng et al. propose that a highly active, large multimeric form of
Shh is secreted and functions in long-range signaling
(Zeng et al., 2001
). A
long-range signaling form that is more active than the short-range signaling
form suggests that some mechanism of inactivation must be present in order to
prevent activity during transport. This inactivation mechanism would
presumably not affect the short-range signaling form of Shh. The data in this
paper suggests that the long-range signaling form of Shh is likely to be less
active than the short-range signaling form. Therefore, some mechanism of
activation may be present once the long-range signaling form has reached its
target. It will be important to determine whether activation/inactivation
mechanisms are involved during this process.
Support for the fact that ShhN is more potent in vivo than wtShh has been
reported by Lewis et al. (Lewis et al.,
2001), who show that the short-range signaling activities in the
limb of N-Shh/Shhn mice are enhanced compared with wtShh/+. Our
data suggests that the increased short-range signaling activity of
N-Shh/Shhn results not only from greater activity, but also from
increased release from the membrane. Lewis et al. also report that expression
of ShhN in a wild-type background (N-Shh/+) results in expanded long-range Shh
signaling that is inconsistent with the loss of long-range activity in the
absence of the C-lipid (Lewis et al.,
2001
). Given our multimerization and activity data indicating that
both wtShh and ShhN are able to form multimers, it is possible that in the
N-Shh/+ mice, multimers consisting of both wtShh and ShhN are formed. Thus, it
is possible that such hybrid multimers would not only be able to be
transported, but also exhibit greater activity once at their target. Our data
showing that wtShh (N+C lipid) is less potent than ShhN (N-lipid) in
differentiation-inducing activity in vitro, together with the in vivo data of
Lewis et al. (Lewis et al.,
2001
), support the intriguing possibility that the C-terminal
lipid may function to keep Shh in an inactive state during long-range
signaling. We propose a model in which long-range signaling of Shh is mediated
by inactive N+C-terminal lipid-containing Shh multimers that are converted to
an active form upon reaching their long-range target cell (see
Fig. 6). Because of the small
fraction of L-multimeric wtShh, we were unable to purify sufficient amounts to
assay in the neural explant assay. Therefore, we cannot say whether the
L-multimer is active or inactive. This is reflected in the model, where a
question mark has been placed next to the L-multimer. It will be important to
determine whether the L-multimer undergoes an activation or inhibition
mechanism.
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In this paper, we clearly show that Shh proteins with different biochemical
properties are present in embryonic brain and limb tissues. Differences in
migration in SDS-PAGE, in the formation of SDS-PAGE-resistant multimers, and
in solubility strongly support the hypothesis that, within different tissues,
Shh proteins contain different N- and/or C-terminal lipids. The difference in
the proportions of Shh protein forms in the brain and limb may influence the
distance that Shh is transported across embryonic tissue, and/or may regulate
the type of extracellular matrix components through which Shh travels in
mesenchymal versus epithelial tissues. The relatively normal patterning
observed in hair, whiskers, teeth and lung development in mice lacking the
C-terminal lipid (Lewis et al.,
2001) contrasts with the severe defects in the limb and brain,
further supporting the idea that context determines the requirement for
different lipid-modified Shh proteins.
Another factor that may influence the ratio of different lipid-containing Shh proteins is the accessibility of the target tissue to secreted Shh. For instance, although the prechordal plate supplies Shh in the forebrain initially, as development proceeds, neural sources of Shh arise. Whether these neural sources arise in conjunction with a loss of the initial prechordal plate source remains to be determined. As the source of Shh changes from non-neural to neural tissue, the need for a long-range multimerized form of Shh may also change. Therefore, it is possible that the requirement for different ratios of lipid-containing Shh proteins may depend on the physical and structural relationship between any given source of Shh and its target. Clearly, our techniques for determining the ratios of Shh proteins do not distinguish between those Shh proteins that remain close to the source in secreting cells and those that travel further from the source. Understanding the precise locations of the different Shh forms may also be especially important in the limb, where the source of Shh expands with the outgrowth of the progress zone. This change may require a different complement of Shh proteins than those needed in regions where this relationship is static.
A major question raised by these experiments is do different N- and
C-terminal lipid-containing Shh proteins result from the removal of existing
lipids or the failure to add a lipid? The discovery of an enzyme responsible
for the addition of the N-terminal lipid to fly hh
(Lee et al., 2001;
Chamoun et al., 2001
;
Micchelli et al., 2002
) makes
it possible to study whether regulation of the addition of the N-terminal
lipid occurs during development. Although enzymes capable of removing a
thiol-linked N-terminal palmitate have been described
(Camp and Hofmann, 1993
;
Camp et al., 1994
), enzymes
capable of removing an amide-linked palmitate have not. Because the N-terminal
palmitate on Shh is thought to be amide-linked
(Pepinsky et al., 1998
), study
of the regulation of the removal of the N-terminal lipid will depend on the
discovery of such enzymes. As the data in this paper suggests, equally
important will be the study of whether regulation of the addition or removal
of the C-terminal lipid occurs.
![]() |
ACKNOWLEDGMENTS |
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![]() |
Footnotes |
---|
Present address: Program in Neurobiology and Behaviour, University of
Washington, Seattle, WA 98195, USA
Present address: Department of Medicine, University of California at San
Diego, La Jolla, CA 92093, USA
Present address: Vanderbilt School of Medicine, Nashville, TN 37232,
USA
¶ Present address: Genetics and Development, Columbia University, New York,
NY 10032, USA
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