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First published online September 28, 2005
doi: 10.1242/10.1242/dev.02068
Review |
1 Section of Molecular Cell and Developmental Biology, and Institute for
Cellular and Molecular Biology, University of Texas, Austin, TX 78712,
USA
2 Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School,
Piscataway, NJ 08854, USA
3 Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School,
Piscataway, NJ 08854, USA
e-mail: wallingford{at}mail.utexas.edu and habasra{at}umdnj.edu
SUMMARY
The Dishevelled protein regulates many developmental processes in animals ranging from Hydra to humans. Here, we discuss the various known signaling activities of this enigmatic protein and focus on the biological processes that Dishevelled controls. Through its many signaling activities, Dishevelled plays important roles in the embryo and the adult, ranging from cell-fate specification and cell polarity to social behavior. Dishevelled also has important roles in the governance of polarized cell divisions, in the directed migration of individual cells, and in cardiac development and neuronal structure and function.
Introduction
In its original definition, the word `dishevelled' applied specifically to
one's hair, its definition being `without a hat' or, more commonly,
`un-coiffed'. Fittingly, the dishevelled gene was first identified
and so named because in flies bearing this mutation the body and wing hairs
fail to orient properly (Fahmy and Fahmy,
1959
). (It may be of interest to some scientists, as it was to one
of the authors, that dishevelled likely derives from the Spanish and French
words for `bald'.)
The dishevelled gene encodes a much-studied signal transduction protein that governs numerous biological processes. Nonetheless, our understanding of this protein has remained, well, dishevelled despite the identification of the first allele 50 years ago. It is clear that dishevelled encodes a protein that is an essential component of both the canonical WNT and the planar cell polarity (PCP) signaling cascades, and also signals via the WNT/Ca2+ pathway (Box 1, Fig. 1). The protein governs biological processes as distinct as cell polarity and cell fate specification, and even social behavior.
Much has been written about WNT signaling pathways, so this review will only briefly summarize our understanding of Dishevelled signaling activities and will instead concentrate on the biological processes that Dishevelled controls.
Dishevelled: an overview
The first alleles of the dishevelled (dsh) gene were
identified in Drosophila mutants
(Fahmy and Fahmy, 1959), and
were characterized by disruptions in hair and bristle polarity. The locus
attracted little attention until the 1980s, when it was discovered that this
gene played a key role in segment polarity in the early embryo
(Perrimon and Mahowald,
1987). Genetic experiments in early Drosophila embryos
first placed Dsh in the canonical Wingless/WNT signaling pathway governing
segment polarity (Couso et al.,
1994; Noordermeer et al.,
1994; Riggleman et al.,
1990; Siegfried et al.,
1994). Subsequent experiments demonstrated that
dishevelled was also involved in the Frizzled-dependent signaling
cascade governing PCP in the wing, legs and abdomen
(Krasnow et al., 1995;
Theisen et al., 1994). The
cloning of the Drosophila dsh gene unveiled a novel protein
(Klingensmith et al., 1994;
Theisen et al., 1994), and
additional experiments in the fly demonstrated that Dishevelled is positioned
at the branchpoint between the canonical WNT and PCP signaling pathways
(Axelrod et al., 1998;
Boutros et al., 1998).
The first vertebrate homolog of dishevelled, murine Dvl1,
was identified shortly thereafter, as was the Xenopus homolog
Xdsh, two additional mouse genes (Dvl2 and Dvl3)
and three human DVL genes (Klingensmith et
al., 1996; Pizzuti et al.,
1996; Semenov and Snyder,
1997; Sokol et al.,
1995; Sussman et al.,
1994; Tsang et al.,
1996). The vertebrate proteins are highly homologous to
Drosophila Dishevelled, and experiments in Xenopus
demonstrated that XDSH is an important part of the canonical WNT signaling
cascade that controls early patterning in vertebrates
(Dominguez et al., 1995;
Sokol et al., 1995). In fact,
interspecific experiments have demonstrated that Dishevelled function is
highly conserved between flies, mice and frogs
(Klingensmith et al., 1996;
Rothbächer et al.,
1995). More recently, vertebrate Dishevelled, like the fly
counterpart, has been shown to also signal via a PCP cascade, controlling cell
polarity during convergent extension cell movements that drive gastrulation
(Heisenberg et al., 2000;
Li et al., 1999b;
Tada and Smith, 2000;
Wallingford et al.,
2000).
The structure of Dishevelled proteins
Dishevelled proteins are 500 to 600 amino acids in length and are modular.
Each contains three highly conserved domains
(Box 1, Fig. 2), and whereas the
overall structure of Dishevelled has not been defined, structural descriptions
of each of the three major domains have been reported
(Box 1). In addition to these
commonly discussed domains of Dishevelled, several additional conserved
regions deserve attention (Box
1, Fig. 2). For
example, there is a basic region and scattered serine/threonine-rich stretches
between the DIX and PDZ domains, and there is a proline-rich region with a SH3
protein-binding motif downstream of the PDZ
(Penton et al., 2002;
Rothbächer et al.,
2000). A comparison of Dishevelled protein sequences from
hydrazoan to human reveals the presence of several invariant residues
downstream of the DEP domain, and the extreme C terminus is very highly
conserved across species (Fig.
3). The significance of these conserved C-terminal residues for
Dishevelled function remains unexplored.
|
A fascinating aspect of Dishevelled is that this protein forms a branchpoint that links several widely deployed signaling pathways (Fig. 1). In this section, we discuss the proteins in the canonical WNT and PCP signaling pathways that function upstream and downstream of Dishevelled. As these signaling cascades have been extensively reviewed elsewhere, we will only attempt a thumbnail sketch of each pathway.
Upstream of Dishevelled: WNTs, receptors and co-receptors
With the sequencing of the human genome, nineteen WNT ligands have been
identified (for reviews, see He et al.,
2004; Logan and Nusse,
2004). The receptors for the WNT ligand were identified as members
of the seven-pass Frizzled (FZ) gene family (see
Huang and Klein, 2004).
Members of the low-density lipoprotein-related receptor proteins (LRP),
including Drosophila Arrow and vertebrate LRP5 and LRP6, function as
co-receptors for WNTs (Pinson et al.,
2000; Tamai et al.,
2000; Wehrli et al.,
2000).
As the LRP co-receptors only impinge on canonical signaling, it is possible
that co-receptors may exist that channel WNT signals into the non-canonical
arm and several proteins, including NRH1 [p75(NTR)-related transmembrane
protein] (Chung et al., 2005;
Sasai et al., 2004), protein
tyrosine kinase 7 (PTK7) (Lu et al.,
2004b), receptor tyrosine kinase-like orphan receptor 2 (ROR2)
(Hikasa et al., 2002) and
glypican (Topczewski et al.,
2001), can modulate non-canonical WNT signaling. However, only
ROR2 has been shown to bind to WNT (Hikasa
et al., 2002), and as yet no evidence exists that these factors
exist in a WNT/Frizzled complex to imply bonafide co-receptor status for
non-canonical signaling.
`Activation' of Dishevelled
Downstream of the WNT/FZ/LRP complex, signaling is transduced to
Dishevelled, but it is hard to define the nature of `activated' Dishevelled.
Undoubtedly, Dishevelled becomes phosphorylated in response to WNT signaling,
but the role of this phosphorylation remains unclear
(Lee et al., 1999;
Rothbächer et al., 2000;
Willert et al., 1997;
Yanagawa et al., 1995).
Several kinases have been reported to phosphorylate Dishevelled, including
Casein kinase 1
, Casein kinase 2 and PAR1
(Cong et al., 2004a;
Ossipova et al., 2005;
Sun et al., 2001;
Willert et al., 1997). The
mechanisms by which the signal is transduced to `activate' Dishevelled remains
mysterious and incompletely understood, although a recent report has
demonstrated that FZ can directly bind to the PDZ domain of Dishevelled,
albeit with weak affinity (Wong et al.,
2003). The DEP domain of Dishevelled also appears to be involved
in signal reception, as a deletion construct of mouse DVL1 containing only the
DEP domain can inhibit the effects of overexpressed WNT, but not those of
overexpressed wild-type DVL (Wong et al.,
2000). Moreover, WNT signaling stimulates the DEP-mediated
translocation of Dishevelled to the cell membrane
(Axelrod et al., 1998;
Boutros et al., 2000). In
terms of downstream readouts, the mode of Dishevelled action is quite
different depending upon which pathway is being discussed.
Canonical WNT signaling
At least two models have been advanced to explain the effects of
Dishevelled on canonical WNT signaling
(Fig. 1A) (for reviews, see
Logan and Nusse, 2004;
Seto and Bellen, 2004;
Tolwinski and Wieschaus,
2004). In the first model, Dishevelled functions epistatically
downstream of the Frizzled/LRP complex. To bolster this model, studies have
revealed that the overexpression of Dishevelled can activate ß-catenin
signaling in Drosophila Arrow mutants
(Wehrli et al., 2000), and
that a constitutively active dFZ2-Arrow fusion cannot transduce signaling in a
Dishevelled mutant background (Tolwinski
et al., 2003). In this model, a likely mechanism by which
Dishevelled functions is via the inhibition of Axin function (see below).
Dishevelled can bind Axin and inhibit its activity
(Fagotto et al., 1999;
Kishida et al., 1999;
Li et al., 1999a;
Smalley et al., 1999). Axin
interacts with LRP5 (Mao et al.,
2001) in a WNT-stimulated manner and is a potent negative
regulator of WNT signaling (Zeng et al.,
1997). It is possible that the interaction of Dishevelled with
Axin is sufficient to inhibit the function of Axin, either through its
sequestration or by the induction of its degradation. It is also noteworthy
that both Axin and Dishevelled have been shown to cycle in and out of the
nucleus, although the role of this nuclear shuttling remains unclear
(Cliffe et al., 2003;
Cong and Varmus, 2004;
Habas and Dawid, 2005;
Itoh et al., 2005).
Furthermore, Frizzled overexpression can recruit Dishevelled to the membrane
(Boutros et al., 2000;
Rothbächer et al., 2000;
Steitz et al., 1996), where
Axin and Dishevelled have been found to be co-localized
(Fagotto et al., 1999;
Smalley et al., 1999). It is
thus tempting to speculate that such interactions facilitate the binding of
Dishevelled to Axin, thus activating canonical signaling.
| Box 1. The conserved elements of Dishevelled protein
At the N terminus of Dishevelled is a DIX (Dishevelled/Axin) domain that is
largely
For `canonical' WNT signaling, the DIX and PDZ domains of DSH are clearly
used, and some contribution may also be made by the DEP domain
(Axelrod et al., 1998
|
|
;
Schweizer and Varmus, 2003).
Another study has found that WNT-induced phosphorylation of Dishevelled can be
achieved independently of LRPs
(Gonzalez-Sancho et al.,
2004)
In the absence of WNT stimulation, ß-catenin is targeted for
degradation through the proteosomal pathway
(Aberle et al., 1997;
Liu et al., 2002). Whatever
emerges as the mechanism by which Dishevelled functions, it appears safe to
say that, upon WNT stimulation, Dishevelled acts to block the phosphorylation
of ß-catenin (Amit et al.,
2002; Dominguez et al.,
1995; van Noort et al.,
2002). This in turn results in the cytoplasmic accumulation of
ß-catenin, which then traffics into the nucleus, where it complexes with
members of the LEF/TCF family of transcription factors and induces the
transcription of WNT-target genes (Bienz
and Clevers, 2003; Liu et al.,
2002; Yanagawa et al.,
2002; Yost et al.,
1996).
Planar cell polarity signaling
The `non-canonical' WNT, or PCP, pathway signals downstream to the actin
cytoskeleton and appears to be independent of transcription
(Fig. 1B). A WNT signal, or
another extracellular PCP signal, is received by a Frizzled receptor.
Frizzled, and several other PCP effectors, including Strabismus, Diego and
Prickle, then work together to govern the asymmetric accumulation of a complex
of proteins at the plasma membrane that includes Dishevelled (for reviews, see
Klein and Mlodzik, 2005;
Veeman et al., 2003;
Wallingford et al., 2002).
Indeed, one recent study showed that Diego and Prickle competitively bind to
Dishevelled to regulate its function in the PCP pathway
(Jenny et al., 2005).
At the level of Dishevelled, two independent and parallel pathways lead
downstream to the activation of the small GTPases RHO and RAC
(Eaton et al., 1996;
Fanto et al., 2000;
Habas et al., 2003;
Strutt et al., 1997;
Tahinci and Symes, 2003). The
first pathway signals to RHO, and occurs through the molecule DAAM1
(Dishevelled associated activator of morphogenesis 1)
(Habas et al., 2001). This RHO
pathway leads to the activation of the RHO-associated kinase ROCK, which
mediates cytoskeletal re-organization (Kim
and Han, 2005; Marlow et al.,
2002; Veeman et al.,
2003; Winter et al.,
2001). A second pathway activates RAC, which in turns stimulates
JNK activity (Boutros et al.,
1998; Habas et al.,
2003; Li et al.,
1999b; Yamanaka et al.,
2002).
|
|
;
Tahinci and Symes, 2003). But
it is also important to recognize that Dishevelled may speak to the actin
cytoskeleton in a much more direct way. Dishevelled binds to DAAM1, a
formin-homology protein (Habas et al.,
2001), and formins are now recognized as being direct nucleators
of linear actin cables (Sagot et al.,
2002; Watanabe and Higashida,
2004). It seems likely that Dishevelled may employ multiple lines
of communication to the actin cytoskeleton.
WNT/Ca2+ and beyond
Finally, other non-canonical pathways are now emerging. Foremost among them
is the WNT/Ca2+ pathway, which may actually influence the function
of both the canonical and PCP pathways
(Fig. 1C)
(Miller et al., 1999a;
Sheldahl et al., 2003). In
addition to the canonical and non-canonical WNT pathways, studies indicate
that Dishevelled may also have `supracanonical' activities. For example,
Dishevelled governs microtubule stability in neurons via Glycogen synthase
kinase 3 (GSK3) and Axin, but not via other WNT pathway components
(Krylova et al., 2000).
Dishevelled also regulates cell migration in the Drosophila ovary in
collaboration with WNT and Frizzled, but not with other components of the
canonical or non-canonical pathways (Cohen
et al., 2002).
Pathway specificity
Tremendous effort has gone into comprehending how Dishevelled channels a WNT signal into a distinct pathway. Two streams of thought have emerged, which are by no means mutually exclusive. The first involves the choice of effector molecules and the second, subcellular localization.
Binding partners
In over two decades of research, at least 29 Dishevelled-binding proteins
have been identified (Table 1)
(see also Wharton, 2003). How
does one make sense of such a vast number of partners and how could they
generate specificity? The WNT signal utilizes distinct domains of Dishevelled
to branch signaling into the separate downstream pathways
(Axelrod et al., 1998;
Boutros et al., 1998). So, one
convenient approach to synthesizing this information is to discuss the
partners on the basis of the domains to which they bind.
|
;
Julius et al., 2000;
Kishida et al., 1999;
Rothbächer et al., 2000;
Smalley et al., 1999). When
overexpressed, the DIX domain has been shown to act as a potent
dominant-negative construct that effectively inhibits canonical WNT signaling
downstream of the WNT/FZ/LRP complex in Drosophila embryos and
Xenopus animal cap explants
(Axelrod et al., 1998;
Tamai et al., 2000). The PDZ
domain of Dishevelled is the region most often found to interact with
Dishevelled-binding proteins (note, most such studies actually use the PDZ,
and also some flanking, sequnce). This central PDZ domain functions in both
the canonical and non-canonical pathways. Through its interactions with the
Dishevelled-binding partners, the PDZ domain has been proposed to function as
the switch between the downstream pathways, depending on the binding partners
that engage with it. Factors such as DAAM1, Strabismus, Prickle, Diego, and
PAR1 can bind to the PDZ domain, and each functions in PCP signaling.
Conversely, Casein kinase, GSK3, GBP/FRAT (GSK3 Binding Protein/Frequently
Rearranged in T-Cell Lymphoma), Frodo, Dapper, Naked cuticle (NKD), Protein
phosphatase 2, IDAX (Inhibitor of Dishevelled and Axin) and Daple each
associate with the PDZ domain and function in canonical signaling.
The DEP domain functions in non-canonical signaling. This domain associates
with, and mediates the activation of, the small GTPase RAC, which in turn
leads to the activation of JUN kinase
(Boutros et al., 1998;
Habas et al., 2003;
Li et al., 1999b). The
requirement for the DEP domain in PCP is probably related to the fact that
this region associates with RAC (Habas et
al., 2003). The DEP domain also plays a central role in the
cytoplasmic-to-membrane translocation of Dishevelled in response to WNT
signaling or Frizzled overexpression
(Axelrod et al., 1998;
Krasnow et al., 1995;
Pan et al., 2004;
Rothbächer et al.,
2000), but the mechanism by which this occurs and the protein
factors required remain unknown.
Subcellular localization
Another mechanism by which Dishevelled specificity could be achieved is via
its discrete subcellular localization. In Drosophila, some studies
indicate that cytoplasmic Dishevelled is involved in canonical WNT signaling,
whereas membrane-localized Dishevelled is important for PCP signaling
(Axelrod et al., 1998).
Conversely, the activation of canonical WNT signaling is also associated with
the membrane translocation of Dishevelled
(Boutros et al., 2000;
Yanagawa et al., 1995).
Moreover, a recent study has shown that the localization of Dishevelled to
apical versus basolateral membrane, rather than to membrane versus cytoplasm,
influences pathway specificity (Wu et
al., 2004). Indeed, this study showed that the pool of available
Dishevelled is limiting, such that activation of one pathway sequesters
Dishevelled in a particular location, leaving it unavailable to activate the
other pathway (Wu et al.,
2004). This finding may explain results that show that the
activation of a non-canonical WNT pathway can downregulate canonical
signaling, and vice-versa (Axelrod et al.,
1998; Torres et al.,
1996; Wu et al.,
2004).
Clearly, binding partners and subcellular localization will be
interdependent. In Drosophila wing epithelia, both pathway
specificity and subcellular localization of Dishevelled can be directed by the
cytoplasmic portions of distinct Frizzled receptors
(Boutros et al., 2000;
Wu et al., 2004). Frizzled
receptors that are localized apically recruit Dishevelled to the apical plasma
membrane and specifically transduce PCP signals, whereas canonical WNT signals
use Frizzled receptors that are localized basolaterally and recruit
Dishevelled to the basolateral membrane
(Strigini and Cohen, 2000;
Wu et al., 2004). It is
likely that the mechanisms of Dishevelled pathway specificity will be
cell-type and context dependent, and it will be interesting in the future to
see whether a related mechanism of signal discrimination is at work in
mesenchymal cells. For example, cells in Xenopus or zebrafish
gastrula mesoderm have no defined apical or basolateral regions, yet still
respond to both canonical and non-canonical WNT signals during gastrulation
(see below).
In fact, the role of the subcellular localization of Dishevelled in
vertebrates remains poorly understood. The membrane translocation of
Dishevelled is a commonly reported consequence of the activation of canonical
WNT signaling in vertebrate cultured cells and in Xenopus animal caps
(Boutros et al., 2000;
Choi and Han, 2005;
Steitz et al., 1996;
Umbhauer et al., 2000;
Yang-Snyder et al., 1996). By
contrast, exposure of embryonic mouse kidney mesenchyme to WNT1 results in the
accumulation of Dishevelled in and around the nucleus
(Torres and Nelson, 2000).
Additionally, the association of Dishevelled with punctate intracellular
vesicles was found to be required for canonical WNT signaling in Chinese
hamster ovary (CHO) culture cells
(Capelluto et al., 2002) and
in Xenopus animal caps (Choi and
Han, 2005). However, in HEK293 cells (a human embryonic kidney
cell line), such punctate localization of Dishevelled correlates with
decreased canonical WNT signaling (Cong et
al., 2004a). Finally, in the mesoderm of gastrulating
Xenopus embryos, Dishevelled is localized to the plasma membrane in
cells undergoing convergent extension, but not in other cells
(Wallingford et al.,
2000).
Three very recent studies have further highlighted the interplay between
Dishevelled's binding partners and its subcellular localization during
vertebrate convergent extension. One study revealed that the phosphorylation
of Dishevelled by different isoforms of PAR1 in Xenopus resulted in
the transduction of either canonical WNT or PCP signals
(Ossipova et al., 2005).
Notably, phosphorylation by the PCP-specific isoform of PAR1 was associated
with the translocation of Dishevelled to the cell membrane. When such
phosphorylation was blocked, Dishevelled failed to accumulate at the membrane
and PCP signaling was disrupted (Ossipova
et al., 2005). Consistent with this result, another study used
engineered constructs to sequester Dishevelled at specific sites in the cell,
so demonstrating that the membrane localization of Dishevelled is essential
for PCP signaling (Park et al.,
2005). By contrast, the particular subcellular location of
Dishevelled did not influence the activation of canonical WNT signaling.
Instead, constructs that caused Dishevelled to be sequestered to either the
cell membrane or to the mitochondrial membrane were more active in canonical
signaling than was an equivalent amount of wild-type Dishevelled
(Park et al., 2005). The
final study showed that inversin, a vertebrate ortholog of the
Drosophila PCP effector Diego, activates PCP signaling and
simultaneously downregulates canonical WNT signaling by targeting Dishevelled
for proteosome-mediated degradation. Intriguingly, these authors show that
membrane-targeted Dishevelled is protected from such degradation
(Simons et al., 2005).
The biology of Dishevelled signaling
In this section, we focus on the biological processes that are governed by the many signaling capabilities of Dishevelled. We start with a summary of classical settings in which Dishevelled function has been studied, and proceed to more recently described functions.
Drosophila segment polarity
Dishevelled was first identified in Drosophila as a viable mutant
that had obvious defects in the orientation of the bristles on the wing and
thorax of the fly (Fahmy and Fahmy,
1959). Dishevelled was also identified in later genetic screens in
which removal of the maternal/zygotic product phenocopied Wingless and
Armadillo/ß-Catenin mutants, indicating that Dishevelled has an early
critical function in larval patterning in Drosophila
(Fig. 3A,B)
(Nusslein-Volhard and Wieschaus,
1980; Perrimon and Mahowald,
1987). This role and Dishevelled's placement in the Wingless/WNT
pathway was demonstrated by additional studies in the fly embryo
(Couso et al., 1994;
Noordermeer et al., 1994;
Riggleman et al., 1990;
Siegfried et al., 1994) and
wing (e.g. Heslip et al.,
1997).
Vertebrate dorsoventral axis patterning
The Xenopus model system has been pivotal in uncovering the
mechanism of WNT/Dishevelled signaling in early pattern formation during
vertebrate embryogenesis (Fig.
3C,D) (Harland and Gerhart,
1997). An endogenous complex, which translocates to the future
dorsalizing center of the early frog embryo, does indeed contain Dishevelled
(Miller et al., 1999b). The
translocation of Dishevelled and other factors likely restricts ß-Catenin
activation to the future dorsal region
(Larabell et al., 1997).
Recently, Xwnt11 was shown to be expressed maternally and to
translocate dorsally during cortical rotation, probably completing the picture
of how the canonical WNT pathway specifies dorsal fates
(Tao et al., 2005).
A word of caution must be inserted here. No complete loss-of-function study
of Xenopus Dishevelled has been reported. Therefore, its requirement
for early patterning remains a thorny issue. Indeed, mutant mice lacking both
dvl1 and dvl2 have revealed a requirement for Dishevelled
during neural fold closure and cardiac development, but not during early axial
patterning (Hamblet et al.,
2002; Lijam et al.,
1997). Nonetheless, a Dishevelled-mediated system of early axis
patterning appears to be very ancient and is implicated not only in
Xenopus, but also in sea urchins and even hydrazoans
(Hobmayer et al., 2000;
Weitzel et al., 2004).
Drosophila wing hair polarity
The hairs and bristles that cover the body and wings of insects are highly
polarized; all point posteriorly on the body and distally on the wing. This
issue attracted attention as early as 1940
(Wigglesworth, 1940), and
subsequently became a topic of much broader interest (for reviews, see
Adler, 1992;
Lawrence, 1973).
Early studies suggested that polarity information is provided by a
supracellular gradient of spatial information (non-cell autonomous), and that
a cell-autonomous mechanism also acts to maintain cell polarity (see
Wigglesworth, 1959). Evidence
that Dishevelled is a crucial player in this latter process came from the
observation that disruptions of its function disturbed the establishment of
planar polarity. The dsh1 allele, which harbors a mutation
in the DEP domain, disrupts the polarity of wing hairs, abdominal bristles,
and bracts on the legs (Theisen et al.,
1994). Although mutation of the Frizzled receptor results in
non-cell-autonomous defects in planar polarity
(Vinson and Adler, 1987),
Dishevelled affects planar polarity in a purely cell-autonomous manner
(Klingensmith et al., 1994;
Theisen et al., 1994).
In the case of wing hairs, planar polarity is established by the outgrowth
of an actin-rich prehair exclusively from the distal vertex of each cell
(Fig. 4A,B,E)
(Wong and Adler, 1993).
Directed actin polymerization at the distal vertex governs hair outgrowth. In
Dishevelled mutant flies, the abnormal positioning of prehairs presages the
disrupted polarity of the hairs themselves
(Wong and Adler, 1993). This
fundamental finding suggested that Dishevelled controls actin polymerization.
Importantly, Dishevelled protein accumulates asymmetrically at the site of
prehair initiation (Axelrod,
2001).
|
Drosophila ommatidal polarity
Dishevelled is also required for planar polarity in the eye, where polarity
is manifested by groups of cells, rather than by individual ones. To add
another layer of complexity, each ommatidium is itself asymmetric. As such,
polarity in the eye is manifested by the chirality or handedness of each
ommatidial cluster, and also by the orientation of the overall cluster.
Mutants have revealed that Dishevelled is necessary for both normal chirality
and normal overall orientation (Theisen
et al., 1994). Despite early suggestions to the contrary, several
additional genetic studies have demonstrated that Dishevelled signals through
the PCP cascade to control these aspects of ommatidial planar polarity
(Boutros et al., 1998;
Zheng et al., 1995).
Interestingly, the chirality of ommatidia depends upon a fate choice in the
R3/R4 pair of photoreceptor cells, which in turn depends upon the modulation
of Notch signaling downstream of a Dishevelled-mediated PCP signal
(Cooper and Bray, 1999;
Fanto and Mlodzik, 1999;
Strutt et al., 2002). In
fact, there has been at least one report of a physical association between
Dishevelled and Notch (Axelrod et al.,
1996). This is an area of future interest, as PCP signaling in the
eye is fundamentally different from that in the wing. It also remains unclear
whether such a PCP/Notch interaction occurs during vertebrate development.
Vertebrate gastrulation
The first vertebrate process in which Dishevelled was found to govern
planar cell polarity was convergent extension, a morphogenetic tissue movement
involving simultaneous lengthening and narrowing of a tissue
(Fig. 4C). This process
involves the planar polarization of a sheet of cells, where polarity is
manifested by stable lamellipodia that form specifically on the mediolateral
faces of the cells, but not on anteroposterior faces
(Fig. 4C,D,F). These
mediolaterally oriented lamellipodia exert tension on neighboring cells,
resulting in cell interdigitation (Fig.
4C,D,F) (Keller,
2002; Wallingford et al.,
2002).
Dishevelled was implicated in convergent extension
(Sokol, 1996), and later
studies revealed that Dishevelled functioned in a PCP-like pathway to govern
convergent extension in both frogs and fish
(Heisenberg et al., 2000;
Tada and Smith, 2000;
Wallingford et al., 2000).
Time-lapse imaging of cells revealed that Dishevelled has a conserved role in
controlling cell polarity: disrupting XDSH function randomizes the normally
polarized lamellipodial protrusions that drive convergent extension
(Wallingford et al., 2000).
As is the case for actin-rich prehairs in the fly wing, XDSH accumulates in
the actin-rich lamellipodia of cells undergoing convergent extension
(Kinoshita et al., 2003).
The importance of convergent extension for gastrulation is unlikely to be
limited to fish and frogs
(Solnica-Krezel, 2005).
Indeed, the disruption of Dishevelled function inhibits convergent extension
in the ascidian, a non-vertebrate chordate
(Keys et al., 2002). Likewise,
the importance of Dishevelled function during gastrulation is not limited to
convergent extension. Studies indicate that Dishevelled signaling via the PCP
cascade may govern several other cell behaviors during Xenopus and
zebrafish morphogenesis (e.g. Ewald et
al., 2004; Marsden and
DeSimone, 2001; Matsui et al.,
2005). Dishevelled may also signal via the WNT/Ca2+
pathway to govern additional aspects of amphibian gastrulation
(Winklbauer et al., 2001).
Future studies should aim to find the unifying features of the cellular events
that are governed by this protein.
Neural tube closure
Convergent extension not only drives gastrulation, but also occurs during
neural tube closure in vertebrate animals, including amphibians, chick and
mice (Jacobson and Gordon,
1976; Keller et al.,
1992; Lawson et al.,
2001; Sulik et al.,
1994; Van Straaten et al.,
1996). Dishevelled is essential for the convergent extension of
neural tissue (Wallingford and Harland,
2001). Moreover, disrupting Dishevelled function in
Xenopus has demonstrated that midline convergent extension is a
critical aspect of neural tube closure; it narrows the distance between the
forming neural folds and facilitates neural fold apposition and fusion
(Wallingford and Harland,
2002). This mechanism is conserved across vertebrates, as
disrupting mouse dvl1 and dvl2 genes also causes neural tube
closure defects (Hamblet et al.,
2002). Importantly, the phenotype of DVL mutant mice is
reminiscent of a severe human neural tube closure defect called
craniorachischisis (Kirillova et al.,
2000; Saraga-Babic et al.,
1993).
Directed migration of individual cells
Since the initial finding that a vertebrate cognate of the PCP cascade
governs cell polarity in vertebrates, many PCP genes identified in
Drosophila have been examined in vertebrates and have been found to
influence convergent extension (Mlodzik,
2002; Wallingford et al.,
2002). Despite the similarities, there is a crucial difference in
the functioning of the PCP cascade in Drosophila and in vertebrate
convergent extension. In flies, cross-talk between neighboring cells via PCP
signaling components allows feedback amplification that reinforces the
polarity decision (Fig. 4A,B)
(Tree et al., 2002). During
convergent extension, the vertebrate PCP pathway coordinates movement in a
population of cells that are constantly changing neighbors; therefore, no
long-term reinforcement of polarity from a particular neighboring cell is
possible (Fig. 4C,D).
The highly dynamic nature of vertebrate PCP signaling is highlighted by the
finding that a loss of Dishevelled function disrupts not only cell polarity
during convergent extension but also lamellipodial stability
(Wallingford et al., 2000).
Indeed, several recent studies demonstrate that Dishevelled controls dynamic
cell protrusions not only during polarized cell movements of large tissue
sheets, but also during directed migration of individual cells.
Foremost among these is the finding that Dishevelled-dependent PCP
signaling governs the directed migration, but not the specification, of
Xenopus neural crest cells (De
Calisto et al., 2005). Additional studies suggest that such a
function for Dishevelled is widely used. For example, Dishevelled is essential
for the directed migration of CHO cells during wound healing
(Endo et al., 2005), and PCP
signaling (and thus very likely Dishevelled) governs the migration of
cardiomyocytes during the development of the outflow tracts of the heart
(Phillips et al., 2005) (see
also Hamblet et al.,
2002).
Importantly, time-lapse studies in a variety of cell types hint at the
mechanism by which Dishevelled contributes to directed cell migration. During
convergent extension, cells lacking Dishevelled function are not only
de-polarized, but their lamellipodial protrusions become highly unstable
(Wallingford et al., 2000).
Likewise, Dishevelled is required to stabilize leading-edge lamellipodia in
migrating neural crest cells (De Calisto
et al., 2005). A similar effect was recently reported in
post-embryonic cells; disruption of DVL2 function with siRNA in bovine
arterial endothelial cells caused lamellipodia to undergo rapid extension and
retraction (Wechezak and Coan,
2005). Finally, a strong correlation between Dishevelled function
and the stability of cell protrusions has been observed in cultured Saos-2
cells (a human osteogenic sarcoma-derived cell line)
(Wiggan and Hamel, 2002).
This role is not limited to vertebrates. In the Drosophila ovary,
Dishevelled is required for proper cell migration during ovariolar
morphogenesis (Cohen et al.,
2002). In this case, WNT, Frizzled and Dishevelled are involved,
but other components of the PCP or canonical WNT pathways are not. As in
vertebrates, this defect in cell migration does not result from defective
polarity, but rather from the inability of cells to move productively
(Cohen et al., 2002). This
phenotype in the Drosophila ovary correlates with a failure to
accumulate focal adhesion kinase (Cohen et
al., 2002), raising the possibility that cross-talk occurs between
Dishevelled and the adhesion machinery of a cell. Notably, adhesion to
fibronectin has been found to cause Dishevelled to translocate to the cell
membrane (Marsden and DeSimone,
2001), and Dishevelled also associates with Ephrins and Eph
receptors (Tanaka et al.,
2003).
So far, the studies of Dishevelled function in individual cell migration
demonstrate a role in stabilizing protrusions. Future studies should determine
whether or not Dishevelled also plays a role in orienting the leading edge of
migratory cells. It will also be of interest to determine whether other PCP
genes share this functionality with Dishevelled, and to elucidate the ways in
which this signaling cascade has changed to accommodate the dynamic nature of
motile cells. In light of these findings, it is interesting to note that, from
studies of planar polarization of the insect denticle following wounding,
Nubler-Jung and colleagues presciently commented many years ago that `cell
migration and denticle formation may thus share similar orienting mechanisms'
(Nubler-Jung et al., 1987).
It will be important now to learn just how similar these mechanisms really
are.
Polarized cell division
Polarized cell divisions contribute to morphogenesis in a wide variety of
systems, and Dishevelled controls division polarity in several cell types.
What is peculiar is that Dishevelled may direct oriented cell divisions
through multiple, highly divergent mechanisms.
Dishevelled was first implicated in governing the orientation of
cytokinesis in Drosophila sensory organ precursor cells. These cells
divide in the plane of the epithelium, oriented along the anteroposterior
axis, but this polarity is randomized when Dishevelled is disrupted
(Gho and Schweisguth, 1998).
Because the PCP-specific dsh1 allele or mutations in other
PCP genes (e.g. flamingo) disrupt this polarity, it is likely that
Dishevelled signals via a PCP cascade to control these polarized cell
divisions (Bellaiche et al.,
2001; Gho and Schweisguth,
1998; Lu et al.,
1999).
This aspect of Dishevelled function is conserved in vertebrates; a recent
study found that dominant-negative Dishevelled constructs disrupt polarized
cell divisions in the gastrulating zebrafish. Here, again, Dishevelled acts in
a PCP-like signaling cascade, as manipulation of the PCP effector
Strabismus/Vangl2 also randomizes these normally polarized divisions
(Gong et al., 2004).
Dishevelled also controls a variety of polarized cell divisions in the
developing Caenorhabditis elegans embryo. Curiously, the orientation
of these mitoses requires Frizzled, Dishevelled and GSK-3, but not other
components of the WNT or PCP pathways
(Chang et al., 2005;
Schlesinger et al., 1999;
Walston et al., 2004).
Cell division orientation is coordinated by microtubules in the mitotic
spindles and asters (e.g. Bellaiche et al.,
2001; Kaltschmidt et al.,
2000). It is therefore intriguing that Dishevelled can regulate
microtubule stability via GSK3 (Ciani et
al., 2004; Krylova et al.,
2000). Indeed, GSK-3 has been shown to be an essential mediator of
normal mitotic spindle dynamics
(Wakefield et al., 2003).
This aspect of Dishevelled function is only just emerging, but should be of
particular interest, as it may represent yet another branch of Dishevelled
signaling.
Cardiac development
Following the establishment of the body axis, Dishevelled signaling is
re-used during organogenesis. For example, several studies implicate WNT
signaling in the control of cardiac development
(Olson and Schneider, 2003),
and all three major branches of Dishevelled signaling appear to be
involved.
The canonical WNT/ß-catenin pathway can negatively regulate
cardiogenesis at an early stage by suppressing the differentiation of the
cardiomyocyte precursors derived from the mesoderm
(Lickert et al., 2002;
Tzahor and Lassar, 2001). By
contrast, a DVL2-dependent WNT signal also triggers a cascade of signals
through ß-catenin, PITX2 and Cyclin D2 to promote cell proliferation
within the cardiac outflow tract (Kioussi
et al., 2002). Interestingly, the outflow tract defects in
Pitx2 knockout mice resemble those in Dvl2 knockout mice
(Hamblet et al., 2002).
This last result suggests that the only role for DVL2 in heart development
may be to signal through ß-catenin and activate gene expression. However,
it is likely that DVL2 also transduces crucial PCP signals during outflow
tract development, as the PCP effector Strabismus/Vangl2 is required for the
proper migration of cardiomyocytes during the development of the outflow
tracts of the heart (Phillips et al.,
2005).
Finally, studies have shown that, in Xenopus, WNT11 controls early
differentiation and morphogenesis in the heart
(Pandur et al., 2002). In
this setting, WNT11 acts through the DEP and PDZ domains of Dishevelled to
activate PKC and then JNK (Garriock et
al., 2005; Pandur et al.,
2002), suggesting that the WNT/Ca2+ pathway may be
involved. Additional studies will be necessary to pare out which pathways are
influencing which aspects of cardiac development.
Vertebrate neuronal development and social behavior
Mice lacking DVL1 exhibit defects in social behavior, such as in sleeping
behavior, nest building and whisker trimming
(Lijam et al., 1997;
Long et al., 2004), although
the mechanism underlying this phenotype remains elusive. The canonical WNT
pathway plays a role in the development and patterning of the brain. In fact,
it has been reported that Dishevelled can convert naive ectoderm into neural
tissue (Sokol et al., 1995),
an effect that is likely to be mediated by the ability of canonical WNT
signaling to repress BMP transcription in the ectoderm
(Baker et al., 1999). A more
established role for canonical WNT signaling is in neural patterning, where
WNT signals posteriorize neural ectoderm, generating anteroposterior pattern
in the central nervous system (McGrew et
al., 1997; McGrew et al.,
1995). Indeed, Dishevelled has the ability to mediate this
transformation (Itoh and Sokol,
1997).
Dishevelled also appears to be important for later events in neural
development, governing the morphology, differentiation and function of neurons
in vertebrates (e.g. Fan et al.,
2004; Kishida et al.,
2004; Luo et al.,
2002; Schulte et al.,
2005). In particular, two studies highlight Dishevelled's ability
to wear more than one hat. First, it has been shown that the atypical receptor
tyrosine kinase RYK can act as a WNT co-receptor
(Lu et al., 2004a). RYK forms
a complex with Frizzled and WNT, and also with Dishevelled, to transduce a
canonical WNT signal that is important for normal neurite outgrowth from
dorsal root ganglia, and for the guidance of axons in the craniofacial motor
nerves and the ophthalmic nerves in the mouse
(Lu et al., 2004a).
In the second study, DVL1 was found to control the complexity of dendritic
arbors in hippocampal neurons (Rosso et
al., 2005). In this capacity, DVL1 signals via a PCP cascade,
collaborating with WNT7b, RAC and JNK. Strikingly, in these neurons, DVL1 was
found to co-localize with microtubules in the axons and with actin in cellular
protrusions at the leading edge of extending neurons
(Rosso et al., 2005). This
latter association may be related to the effects of Dishevelled on
lamellipodial stability (e.g. De Calisto
et al., 2005; Wallingford et
al., 2000; Wechezak and Coan,
2005), whereas the former may be related to the ability of
Dishevelled to regulate microtubule stability (e.g.
Ciani et al., 2004;
Krylova et al., 2000). Further
studies of the function of Dishevelled in neuronal development and function
should be both illuminating and important.
Conclusion
Dishevelled signaling is an important regulator of a wide variety of signaling pathways and thus governs many important developmental processes. Some of these roles are very highly conserved across species, whereas others are not. In some cases, similar biological processes appear to require Dishevelled, but they need it to signal along divergent pathways. As we move forward, care should be taken. We should endeavor to find unifying features that will help us to understand this protein. But we should also be ready for the surprises that may be lurking in the next experiment.
ACKNOWLEDGMENTS
We thank J. Klingensmith, P. Lawrence, I. Quigley and the anonymous reviewers for help while writing this manuscript, and Jeff Axelrod and Keith Wharton for contributing images. R.H. is supported by a Basil O'Connor Starter Scholar Award, a Scientist Development Grant from the American Heart Association, and a Seed Grant from the Foundation of UMDNJ. J.B.W. is supported by a Burroughs Wellcome Fund Career Award and the NIH.
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