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First published online July 27, 2006
doi: 10.1242/10.1242/dev.02472
Review |
MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Author for correspondence (e-mail:
xfranch{at}nimr.mrc.ac.uk)
SUMMARY
Members of the Hedgehog (Hh) family of proteins are conserved morphogens that spread and modulate cell fates in target tissue. Mature Hh carries two lipid adducts, a palmitoyl group at the N terminus and cholesterol at the C terminus. Recent findings have addressed how these lipid modifications affect the function and transport of Hh in Drosophila. In contrast to the palmitoyl adduct, cholesterol appears not to be essential for signalling. However, the absence of the cholesterol adduct affects the spread of Hh within tissues. As we discuss here, the exact nature of this effect is controversial.
Introduction
A relatively small number of secreted proteins spread through tissues
during development and organise tissue patterning. Understandably, there has
been great interest in the mechanisms that modulate the range of action of
these developmental ligands. So far, three such secreted developmental
signals, Wingless/Wnt, Spitz (Drosophila Transforming Growth Factor
) and Hedgehog (Hh), are known to undergo lipid modification
(Jeong and McMahon, 2002
;
Miura et al., 2006;
Pepinsky et al., 1998;
Porter et al., 1996;
Willert et al., 2003). All
three are palmitoylated in the lumen of the endoplasmic reticulum (ER) during
biosynthesis. In addition, Hh also undergoes cholesterol modification
(Porter et al., 1996).
Although the palmitoylation of cytoplasmic proteins has been extensively
studied, the palmitoylation of proteins residing in the lumen of ER-derived
vesicles has only been recently recognised, and the enzymology of these
reactions is still poorly understood. In the cases of Spitz and Hh, the
reaction appears to be catalysed by the product of the gene skinny
hedgehog, which is also known as rasp or sightless
(Amanai and Jiang, 2001;
Chamoun et al., 2001;
Lee and Treisman, 2001;
Micchelli et al., 2002). By
contrast, more work is needed to confirm that Wingless palmitoylation is
catalysed by the gene product of porcupine, as has been suggested
(Kadowaki et al., 1996;
Willert et al., 2003).
Interest in the role of lipid adducts in morphogen signalling and activity
is heightened in the case of Hh because it undergoes a second lipid
modification. The N-terminal half of Hh becomes conjugated with cholesterol by
an autocatalytic reaction that simultaneously cleaves off the inactive
C-terminal polypeptide (Porter et al.,
1996). Thus, mature active Hh, which is about half the size of
full-length Hh, carries a palmitate group at the N terminus and a cholesterol
moiety at the C terminus. After biosynthesis, Hh must be released from
secreting cells. This requires the activity of a dedicated multipass-membrane
protein encoded by the dispatched (disp) gene
(Burke et al., 1999). There is
still some controversy as to the exact mode of Disp action. One view is that,
by virtue of its sterol-sensing domain, Disp allows the release of lipidated
Hh from the cell surface (Burke et al.,
1999). Alternatively, Disp has been suggested to guide Hh from the
basolateral membrane to the apical membrane of the cell, where the release of
Hh would take place (Gallet et al.,
2003), thus allowing its subsequent extracellular transport.
Lipid modification is believed to cause proteins to tightly associate with
cell membranes (Linder and Deschenes,
2004). Such an association is expected to cause lipidated ligands
to remain stuck at the surface of secreting cells, thus preventing their
subsequent transport. However, the transport of lipidated molecules, such as
Hh, does take place. So, how is membrane association compatible with
long-range transport and signalling? It has been suggested that specific
transport mechanisms have evolved to overcome this problem. For example, lipid
particles called argosomes have been proposed to extract lipidated ligands
from the cell surface and then to act as vehicles for long-range transport
(Eaton, 2006). According to
this view, cholesterol would be required for transport, as it would be needed
for the loading of a lipidated protein onto the transport vehicles.
Here, we review recent findings that address how lipid modifications of the
highly conserved ligand Hh affect its signalling activity and range in
Drosophila epithelia. One report
(Gallet et al., 2006) suggests
that cholesterol is indeed required for long-range Hh transport. Two other
reports (Callejo et al., 2006;
Dawber et al., 2005) propose
that, in the absence of a cholesterol adduct, the spread of Hh is increased,
although this is at the expense of its signalling activity near its source,
perhaps because of the dilution of unlipidated Hh in the extracellular space.
Below, we evaluate the evidence and then briefly relate it to the situation in
vertebrate embryos.
Tools and tissues for the study of Drosophila Hh
The wing imaginal disc system
One system of choice to study the range of extracellular ligands is the
wing imaginal disc of Drosophila. This structure, found in the
growing larva, consists of an epithelial sack that has a thick, columnar,
pseudostratified epithelium on one side and a squamous epithelium, called the
peripodial membrane, on the other (Fig.
1B). The space between the two epithelia is called the peripodial
space. It is the columnar epithelium in the central region of the disc (the
pouch) that gives rise to the wing proper, hence most attention has been
devoted to this tissue. Somewhat unusually for epithelia, the apical side of
this tissue faces the inside of the disc (the peripodial space)
(Pallavi and Shashidhara,
2005) (Fig. 1B). Hh
is produced from the posterior half of the disc [the posterior (P)
compartment], from where it spreads into the anterior (A) compartment
(Basler and Struhl, 1994;
Tabata and Kornberg, 1994)
(Fig. 1A, parts a,b). It forms
a gradient (over about 10 cell diameters) and activates a series of target
genes in a concentration-dependent manner
(Fig. 1A, parts c,d). Examples
of `high level' targets include engrailed (en) and
patched (ptc), while decapentaplegic
(dpp), cubitus interruptus (ci), collier
(col) and iroquois (iro) are low level targets
(Torroja et al., 2005). How
the range of Hh is regulated so that it activates its targets in the right
pattern is an issue of considerable interest.
Modulators of Hh distribution
Several proteins that are expressed in Hh-receiving tissues have been shown
to affect the range and distribution of Hh after its release from secreting
cells. One class of such proteins comprises the Heparan Sulfate Proteoglycans
(HSPGs) dally and dally-like (dlp)
(Bellaiche et al., 1998;
Bornemann et al., 2004;
Han et al., 2004a;
Han et al., 2004b;
Takei et al., 2004;
Takeo et al., 2005).
Dally-like is required for Hh signalling itself, perhaps by acting as a Hh
co-receptor (Desbordes and Sanson,
2003; Han et al.,
2004b; Lum et al.,
2003). Dlp and dally might also participate in
transport, although a cell biological understanding of how they achieve this
is not yet available (Desbordes and
Sanson, 2003; Glise et al.,
2005; Gorfinkiel et al.,
2005; Han et al.,
2004b; Lum et al.,
2003). Another protein that might be involved in the distribution
of Hh is the secreted molecule encoded by shifted. Shifted, which is
related to vertebrate Wnt inhibitory factors, such as WIF-1
(Hsieh et al., 1999), can
diffuse readily within imaginal discs
(Glise et al., 2005;
Gorfinkiel et al., 2005).
Genetic data have suggested that it could mediate the interaction between
Hedgehog and HSPGs (Glise et al.,
2005; Gorfinkiel et al.,
2005). Despite its homology to WIF-1, Shifted does not modulate
Wingless activity in flies (Gorfinkiel et
al., 2005). Another important molecule that modulates the range of
Hh is Patched (Ptc). This multipass-transmembrane protein, which is the
primary receptor for Hh, represses downstream signalling in the absence of Hh.
Upon Hh binding, this inhibition is relieved and downstream Hh targets are
activated. The ptc gene itself is a target of Hh signalling, and high
level expression of Ptc sequesters Hh by endocytosis, thereby limiting its
further transport (Chen and Struhl,
1996; Chen and Struhl,
1998; Torroja et al.,
2004). Thus an increased production of Hh leads to more of it
being sequestered. This negative-feedback loop ensures that the range of Hh is
always kept under control.
Experimental variants of Drosophila Hh
Three groups have sought to assess how lipid modifications affect the range
of Hh by expressing mutated forms of Hh that are expected to lack either or
both lipid adducts (Callejo et al.,
2006; Dawber et al.,
2005; Gallet et al.,
2006). The various forms of Hh used were expressed in flies using
the Gal4/UAS system, which allows the expression of so-called UAS transgenes
in specific patterns (Brand and Perrimon,
1993). Although extremely powerful, this system has the drawback
that the transgenes are usually expressed at a substantially higher level than
are endogenous genes. Several forms of Hh were expressed in this fashion:
Hhppalm;choles, HhNpalm and HhC85Scholes, as
discussed below. For ease, we use a unified nomenclature to describe the
various constructs used in these experiments, as the key mutations were
identical in all cases, even though they were made independently in the three
laboratories. The constructs differed in that some were tagged with GFP while
others were not. However, extensive evidence suggests that the GFP tag does
not affect the signalling or trafficking behaviour of Hh
(Callejo et al., 2006;
Torroja et al., 2005;
Gorfinkiel et al., 2005;
Burke et al., 1999;
Dawber et al., 2005). The
production of Hhppalm;choles results from expression of the
full-length form of Hh, which is processed during its biosynthesis to give
rise to mature active Hh (the N terminal-half of pro-Hh, bearing a lipid
adduct at each end; Fig. 2).
HhNpalm represents the N-terminal half of pro-Hh without the
cholesterol moiety (Fig. 2B).
In HhC85Scholes, the cysteine at position 85, which is the target
of palmitoylation in wild-type Hh, is mutated to a serine
(Fig. 2C). This form of Hh is
strongly impaired in signalling (both in vertebrates and Drosophila),
implying an essential role for the palmitoyl group in signal transduction
(Callejo et al., 2006;
Dawber et al., 2005;
Gallet et al., 2003;
Pepinsky et al., 1998). By
contrast, HhNpalm is signalling competent. It is assumed that the
lack of cholesterol does not preclude palmitoylation, otherwise it would also
be signalling incompetent (Burke et al.,
1999; Chamoun et al.,
2001). Because target gene expression provides an important
readout of the range of Hh signalling, we will not consider the
palmitate-deficient forms of Hh any longer and will focus on the effect of
cholesterol on the range of Hh in Drosophila.
|
Cholesterol and Hh multimerisation
As expected, cholesterol mediates the association of mature Hh with the
plasma membrane. This was shown in both Drosophila S2 cells and
mammalian cells (HEK293T) (Chamoun et al.,
2001; Chen et al.,
2004; Gallet et al.,
2006; Zeng et al.,
2001). Cells expressing Hhppalm;choles showed a strong
accumulation of Hh at the cell surface, whereas cells expressing
HhNpalm at a comparable level did not. Fractionation studies of the
supernatant of Hh-expressing cells showed that Hhppalm;choles
participates in high-molecular weight structures that are likely to represent
mutimeric complexes (Callejo et al.,
2006; Chen et al.,
2004; Gallet et al.,
2006; Zeng et al.,
2001). HhNpalm is not found in these structures,
suggesting that cholesterol mediates multimerisation, as has been shown before
for vertebrate Shh (Chen et al.,
2004; Goetz et al.,
2006; Zeng et al.,
2001). Gallet et al. attempted to assess the activity of the
different fractions obtained by size exclusion chromatography from the cell
culture supernatant. Although it is inherently difficult to ensure that
comparable amounts of Hedgehog are used for each fraction, the authors
provided good evidence that the multimeric fraction is more active than the
monomeric one. They concluded from these results that cholesterol modification
(and multimer formation) ensures high signalling activity. Because the
monomeric fraction retains some signalling activity, it appears that
cholesterol is not essential for signalling although it may contribute to its
maximal activity.
Subcellular localisation of Hh
Gallet et al. found that wild-type Hh is mostly present on the apical side
of the columnar cells in Drosophila imaginal discs
(Gallet et al., 2006). From
this observation, they suggest that, following biosynthesis, wild-type Hh is
released apically and then transported along the apical side of the columnar
epithelium. The cholesterol adduct could be important for this as they found
that (untagged) HhNpalm accumulates at a basal localisation.
Callejo et al. also believe that wild-type Hh is secreted apically
(Callejo et al., 2006).
However, they suggest that it then finds its way to the basolateral domain,
where it is transported to and then activates downstream signalling. They too
stained fixed imaginal discs, although they followed a staining protocol that
is designed to detect specifically extracellular GFP-tagged forms of Hh
produced from clones of cells. This was achieved by inducing, at random
locations, clones of cells that activate the expression of gal4. Gal4
in turn activates the relevant forms of Hh (this is colloquially called the
`Flp-Out' technique) (Harrison and
Perrimon, 1993; Struhl and
Basler, 1993). Callejo et al. found that, at the surface of
Hh-producing cells, all forms of Hh are found mainly at the apical surface,
consistent with apical secretion. In the receiving cells, wild-type Hh is
basolateral, whereas HhNpalm remains at the apical surface, as if
cholesterol was needed for the transfer of Hh from one side to the other. In
order to map where, along the apicobasal axis, various Hh variants are
internalised, they looked at Hh variant distribution following a transient
block of endocytosis. Consistent with their reported steady-state
distribution, they found that wild-type Hh accumulates basolaterally in
receiving cells, while HhNpalm seems to build up at an apical
location. Overall, Callejo et al. argue that cholesterol-modified
Hhppalm;choles diffuses along the basolateral surface, while
unmodified HhNpalm spreads apically.
Here, it is relevant to note that the HSPG dally-like, which
contributes to Hh transport (Han et al.,
2004a; Han et al.,
2004b; Takei et al.,
2004), is relatively more abundant at the basolateral surface of
the columnar epithelium (Kreuger et al.,
2004; Marois et al.,
2006). The differences between the observed distributions of Hh
variants may be due to the differences between the assays. In one case
(Gallet et al., 2006), the
steady-state distribution of total (intra- and extracellular) endogenous Hh
was measured, whereas, in the other
(Callejo et al., 2006), the
extracellular distribution of overexpressed ligand was assessed. Clearly,
total staining does not necessarily give an indication of the routes of
export/transport, nor does it tell us whether the ligands are inside or
outside of the cells. Extracellular staining is more specific, but it can give
rise to artefacts that are caused by a molecule's limited access to the apical
side of a wing disc cell (as this side is inside the disc). In addition to
looking at extracellular steady-state distribution, Callejo et al. also
stained discs following a block of endocytosis. The results of this experiment
seem to confirm that wild-type Hh is mainly present at the basolateral side in
receiving cells, although it should be kept in mind that a block of
endocytosis will necessarily affect the distribution of a large number of cell
surface molecules, some of which might be needed for Hh transport. Overall, we
believe that novel methods to assess the trafficking of Hh in imaginal discs
are needed to resolve the divergent views on the nature of the epithelial
surface where Hh spreads and signals. Better techniques will also be needed to
assess the distribution of the relevant Hh receptors. Presumably, they are
present on the epithelial surface where transport and signalling are taking
place. The HSPG co-receptor Dlp is thought to be mainly, although not
exclusively, present at the basolateral surface
(Kreuger et al., 2004;
Marois et al., 2006), whereas
Ptc has been reported to be mainly apical (Capdevilla et al., 1994;
Gallet et al., 2005). This
also needs confirmation.
|
). From these data, and from additional results obtained in
the Drosophila embryonic epidermis, Gallet et al. conclude that
cholesterol is required for long-range signalling, perhaps by targeting Hh to
the apical surface from where its long-range transport would be launched. This
shortened range of Hh in the absence of cholesterol is at odds with the
long-range Hh transport that is predicted from the finding that
HhNpalm expressed in the P wing disc compartment activates
dpp-lacZ expression throughout the A compartment
(Burke et al., 1999). In
support of this latter result, Callejo et al. report the widespread activation
of two low-level Hh targets, dpp-lacZ and iro, around clones
of cells overexpressing GFP-tagged HhNpalm
(Callejo et al., 2006). Dawber
et al. (Dawber et al., 2005)
also confirm this finding with an untagged HhNpalm variant. In
their study, they use two other low-level target genes, ci and
col, as a read-out for Hh signalling. The three reports agree that a
lack of cholesterol leads to a reduction in the activation of high-level Hh
targets, such as en and ptc, and a concomitantly shortened
range over which these target genes are activated. However, there is a clear
disagreement as to whether HhNpalm activates low-level Hh targets
over an extended or reduced range. How could this be explained?
|
). In their view, earlier studies failed to consider that Hh
produced from peripodial cells could behave differently from Hh made by
columnar cells (although Callejo et al. were aware of this concern). Indeed,
Flp-Out clones are, by nature, induced randomly, and do appear in the
peripodial epithelium at some frequency. These can easily be overlooked
because they are in a different focal plane from columnar clones. The presence
of one such clone could give the erroneous impression that columnar clones in
the same disc have a long-range effect. The model of Gallet et al. (see
Fig. 3A) suggests that, in the
columnar epithelium, Hh trafficks to the apical surface, where it would
normally be processed by Disp for release, and then subsequently transferred
to the transport machinery (Gallet et al.,
2006). Because of the absence of cholesterol, HhNpalm
would hardly reach the apical surface. Instead, it would be released in a
Disp-independent manner from the basolateral surface (which lies on the
exterior of the disc), where it would fail to access the apically located
long-range transport machinery and would eventually become diluted in the
haemolymph. According to Gallet et al., peripodial cells would secrete
HhNpalm equally towards both surfaces. A significant amount would
therefore reach the peripodial space, where it would be free to diffuse and
activate dpp expression in competent cells (throughout the A
compartment of the columnar epithelium;
Fig. 3A). Callejo et al. agree
that peripodially produced HhNpalm acts at a long range
(Callejo et al., 2006).
However, they find that this is also true for HhNpalm produced from
the columnar cells. This is based on the use of peripodial and
columnar-specific Gal4 drivers to express the various forms of Hh (although
the tissue specificity of the drivers was not rigorously verified). Overall,
the model of Callejo et al. (see Fig.
3B) is that HhNpalm;choles, which has high signalling
activity, is strongly retained at the basolateral surface of columnar cells
near the source of expression. It would therefore activate high-level target
genes over a short range. Because of the absence of cholesterol,
HhNpalm does not accumulate at the basolateral surface (owing to
the lack of an appropriate trafficking signal) and, because it has reduced
affinity for the cell membrane, it would be released on the apical side into
the peripodial space. Such a form of Hh would only activate low-level target
genes, albeit at a long range. In addition, Callejo et al. show that
cholesterol-deficient HhNpalm is internalised by cells throughout
the wing disc (consistent with long-range spread), both in a Ptc-independent
and Ptc-dependent manner (Callejo et al.,
2006). They suggest that two modes of Hh sequestration and
degradation could be at work. The Ptc-dependent mode would ensure a
self-limiting restriction of the range of highly active multimeric Hh, as
ptc expression is upregulated by high-level signalling. In parallel,
Ptc-independent internalisation of cholesterol-deficient HhNpalm
would function throughout the wing disc irrespective of Hh signalling. Because
such a system would not be activated by Hh signalling, it would not be
upregulated near the source and would thus allow a wider distribution of
Hh.
In short, although there is agreement that wild-type Hh originating from
the peripodial epithelium acts over a long range, there is a clear
disagreement on the actual range of cholesterol-deficient HhNpalm
when it is expressed in the columnar epithelium. Gallet et al. report that,
when produced by columnar cells, HhNpalm acts at a reduced range
(Gallet et al., 2006). By
contrast, Callejo et al. and Dawber et al. report that HhNpalm
produced by columnar cells acts over an extended range
(Callejo et al., 2006;
Dawber et al., 2005). There is
no obvious explanation for the difference. However, we note that the clones
shown by Gallet et al. tend to be small relative to the ones shown by Callejo
et al. and Dawber et al. Small clones have a large perimeter-to-surface ratio
and are therefore surrounded by a large number of wild-type cells, relative to
the amount of Hh produced. For such clones, Ptc-dependent internalisation is
expected to trap all the Hh produced and thus prevent further transport. When
the relative amount of Hh produced increases (because clones are bigger or Hh
is expressed at a higher level), the Ptc-dependent mechanism might become
saturated, allowing unsequestered Hh to travel further. Ptc-dependent
endocytosis might be saturated more readily by HhNpalm than it is
by wild-type Hh, as HhNpalm does not feed into the Ptc-mediated
negative-feedback loop and therefore does not enhance Ptc expression. This
would explain why large clones that express wild-type Hh would allow Hh to act
at a shorter range than does HhNpalm produced by similarly sized
clones. Differences between the experimental results could also arise from
differences between the detection protocols (such as the time allowed between
clone induction and staining), or differences between the constructs. The
exchange of the reagents and protocols used in the studies will ensure that
these data can be directly compared.
None of the three reports discussed above directly addresses whether
unlipidated Hh exists in nature or whether lipidated Hh alone can account for
the range of Hh signalling that is observed. One argument against the natural
existence of HhNpalm is that low-level target genes are not
activated in disp mutants (Burke
et al., 1999; Dawber et al.,
2005). Because Disp is not required for the release of
HhNpalm, one would expect dpp expression to be unaffected
by the absence of disp. It therefore seems that only
HhNpalm;choles is made and is capable of activating the full
complement of target genes.
The role of heparan sulfate proteoglycans
In the absence of tout-velu (ttv), which encodes an
enzyme that is required for the biosynthesis of HSPGs, Hh can no longer act at
a distance from its source (Bellaiche et
al., 1998). One likely interpretation of this finding is that
HSPGs contribute to the tethering of Hh to the plasma membrane of receiving
cells, thus preventing it from escaping the epithelium and allowing its
further transport or Ptc-dependent sequestration. Indeed, in the absence of
ttv activity, Hh can only be detected in cells adjoining its source.
By contrast, internalised HhNpalm can be detected throughout
ttv mutant clones, suggesting that cholesterol-deficient
HhNpalm does not require HSPGs for its membrane retention
(Callejo et al., 2006). This
result opens up the possibility that cholesterol could be important for the
interaction of Hh with HSPGs, maybe with the help of the secreted protein
Shifted (Glise et al., 2005;
Gorfinkiel et al., 2005). Such
interactions could be further tested and investigated in cell culture. As in
Drosophila, vertebrate glypicans have been suggested to act as
co-receptors for Shh in cell culture assays
(Carrasco et al., 2005).
Another component of the extracellular matrix, a secreted HSPG (perlecan) has
been proposed to act as a negative regulator of Shh signalling by controlling
the amount of Shh available for binding to Ptc
(Carrasco et al., 2005;
Datta et al., 2006).
Conclusion
These recent papers highlight the importance of cholesterol in modulating
the range and activity of Hh. However, readers are faced with conflicting
results and models. In this review, we have attempted to highlight the
differences in the hope that they can be resolved in the near future. First,
the groups of Guerrero and Therond differ in their views on apicobasal
localisation of Hh and its variants
(Callejo et al., 2006;
Gallet et al., 2006). This is
relevant because the different views lead to distinct models of the route
followed by Hh along the epithelium. There is also a clear disagreement on the
actual range of cholesterol-deficient HhNpalm. Gallet et al. report
that HhNpalm acts at a reduced range (when expressed in the
columnar epithelium) (Gallet et al.,
2006). By contrast, Callejo et al. and Dawber et al. report an
extended range for non-cholesterol modified Hh
(Callejo et al., 2006;
Dawber et al., 2005). These
different experimental results are probably attributable to differences in the
specific protocols, and further comparisons among the different groups will be
needed to resolve the differences. Hopefully, the resolution of these
differences will allow researchers to then integrate data from the study of
Shh in vertebrate systems. So far, data from vertebrates seem consistent with
the view proposed by Callejo et al.
(Callejo et al., 2006): ShhN
(no cholesterol) acts over a long-distance along the anteroposterior axis of
the mouse limb bud, suggesting that the cholesterol moiety restricts the
spread of Shh in this system (Li et al.,
2006). Significant further progress will require more than
resolving the differences outlined here. We believe that new approaches are
needed to answer questions about Hh trafficking in vivo. For example, better
pulse-chase experiments could be devised to track the various forms of Hh as
they undergo secretion transport and endocytosis. Ideally, these experiments
should be performed with a level of expression that is comparable to that of
the endogenous gene. Overall, it is hoped that a better understanding of the
mechanism that regulates the activity of Hh and Shh will lead to new ways of
controlling it in a therapeutic setting.
Footnotes
* These authors contributed equally to this work 
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