Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan
* Author for correspondence (e-mail: ttabata{at}ims.u-tokyo.ac.jp)
SUMMARY
During the course of development, cells of many tissues differentiate according to the positional information that is set by the concentration gradients of morphogens. Morphogens are signaling molecules that emanate from a restricted region of a tissue and spread away from their source to form a concentration gradient. As the fate of each cell in the field depends on the concentration of the morphogen signal, the gradient prefigures the pattern of development. In this article, we describe how morphogens and their functions have been identified and analyzed, focusing on model systems that have been extensively studied.
Introduction
The term morphogen is used rigorously to describe a particular type of
signaling molecule that acts on cells directly to induce distinct cellular
responses in a concentration-dependent manner. Although there is abundant
evidence for concentration-dependent activity of secreted signaling molecules,
evidence for their direct action on cells has been lacking in many cases and,
so far, only a few such molecules fulfill the criteria of morphogens.
Nevertheless, the roles of morphogens during the development of
Drosophila appendages have been extensively studied, and a few
examples of morphogens have recently been identified in vertebrate
development. These include members of the Hedgehog (Hh) family, for example,
Hh in Drosophila appendage development
(Mullor et al., 1997;
Strigini and Cohen, 1997
) and
Sonic hedgehog (Shh) in chick neural tube development
(Briscoe et al., 2001
); the Wnt
family member Wingless (Wg), which acts during Drosophila appendage
development (Neumann and Cohen,
1997
; Zecca et al.,
1996
); and some members of the TGFß family, including
Decapentaplegic (Dpp) in Drosophila appendage development
(Lecuit et al., 1996
;
Nellen et al., 1996
) and
Squint in Zebrafish early embryonic development
(Chen and Schier, 2001
).
Activin, another member of TGFß family, has been well studied in the
Xenopus model system, in which the exogenously supplied signaling
molecule activates target genes in a dose-dependent manner, and this has
already been well described in a comprehensive review
(Gurdon and Bourillot, 2001
).
In this primer, we will give a brief history of strategies adopted for
identifying secreted morphogens, taking the development of Drosophila
appendages as a model system, and we discuss how these strategies could be
applied to the study in vertebrates.
However, even for those morphogens that have been identified, we still do not understand crucial issues such as how morphogens are moved through a tissue, how a gradient is maintained, and how morphogens coordinate growth and patterning. In addition, the morphogen gradient must be invariable despite genetic or environmental fluctuations. Recent studies have revealed significant roles for cell surface molecules in shaping morphogen gradients, and these include morphogen receptors and heparan sulfate proteoglycans (HSPGs). Several reports have suggested the involvement of transcytosis, cytonemes and argosomes in morphogen transport, whereas numerical analyses have favored restricted diffusion as a mechanism for the formation of morphogen gradients. More cell biological and biochemical studies will be needed to evaluate the role of these activities and structures in gradient formation.
Morphogens in Drosophila wing development
Hh: a short range morphogen in wing development
The first step towards identifying and analyzing a morphogen is to
determine whether a signaling molecule fulfills the criteria required to
qualify as a morphogen, i.e. whether the molecule induces distinct cellular
responses in a concentration-dependent manner and whether it acts directly on
cells at a distance from its source. The Drosophila adult wing arises
from the larval imaginal wing disc. An imaginal disc is a two-sided sac
comprising a columnar cell layer (which gives rise to eye, antennae, wing or
leg) and overlying squamous epithelium, known as the peripodial membrane
(Fig. 1B). The wing imaginal
disc is subdivided into non-intermingling anterior (A) and posterior (P)
compartments along the anteroposterior axis. The identity of cells in the P
compartment is imparted by the expression of the gene engrailed
(en) (Guillen et al.,
1995; Simmonds et al.,
1995
; Tabata et al.,
1995
). Under the control of En, cells of the posterior compartment
synthesize Hh, which is secreted into the A compartment
(Tabata and Kornberg, 1994
)
(Fig. 1C). There, Hh induces
several target genes, including patched, en and dpp, and
patterns the central domain of the wing
(Fig. 2).
|
|
Dpp: a long range morphogen in wing development
One of the targets of Hh regulation, Dpp, then functions as a second
morphogen that patterns the wing beyond the central domain
(Lecuit et al., 1996;
Nellen et al., 1996
)
(Fig. 2A). Thus, the wing is
patterned by two different types of morphogens, and this has been well
illustrated in experiments that ectopically express these proteins: Hh induces
the mirror image duplication of the entire A compartment, whereas the
duplication caused by an ectopic source of Dpp lacks the central domain
(Zecca et al., 1995
)
(Fig. 2B). Dpp is expressed
along the border between the A and P compartments, and induces several target
genes including sal and omb (bifid - FlyBase), with
omb being expressed in a wider domain than sal (Figs
2,
3). Dpp distribution in the
columnar cell layer can be monitored using an antibody against Dpp
(Gibson et al., 2002
) or by
fusing Dpp with green fluorescent protein (Dpp-GFP)
(Entchev et al., 2000
;
Teleman and Cohen, 2000
)
(Fig. 3). The space between the
two cell layers of the imaginal disc is called the disc lumen. Dpp is also
detected uniformly in the disc lumen and is thought to be required mainly for
cell survival (Gibson et al.,
2002
).
|
|
|
Shh during limb bud development
Anterior/posterior polarity of the vertebrate limb is regulated by a
posteriorly localized signaling center called the zone of polarizing activity
(ZPA). Shh mirrors the properties of the ZPA; ectopic Shh activity induces
digit duplications, with higher concentrations specifying increasingly more
posterior digits (Riddle et al.,
1993; Yang et al.,
1997
) (Fig. 1D).
Furthermore, an ectopic source of Shh induces a mirror image duplication of
the limb (Fig. 1C). Shh induces
dose-dependent production of a Dpp ortholog, BMP2
(Yang et al., 1997
), and
misexpressed BMP2 can induce ectopic formation of the most anterior digit
(Duprez et al., 1996
). These
observations readily prompt us to see the analogy between patterning by Shh of
the chick limb bud and the role of Hh in Drosophila wing
(Fig. 1C,D). However, no
evidence for the direct action of Shh in regulating different target genes has
yet been demonstrated, suspending the conclusion that Shh functions as a
morphogen in the limb development.
Shh in neural tube development
During development of the chick neural tube, however, Shh does function as
a morphogen. Shh emanates from the notochord to induce formation of the floor
plate. Subsequent Shh expression in the floor plate generates a
ventral-dorsal activity gradient of Shh that promotes the specification of a
series of ventral cell types (Ericson et
al., 1997). Furthermore, ectopic expression of a mutated form of
the Shh receptor, Patched (Ptc), which does not bind Shh but does antagonize
its signaling, causes cell-autonomous ventral-to-dorsal switches in neural
progenitor identity (Briscoe et al.,
2001
) (Fig. 6),
clearly indicating that Shh functions by acting on cells directly.
|
Cell surface molecules and morphogen gradients
Concentration and activity gradients of morphogens
Although morphogens form concentration gradients, the concentration of the
protein is not necessarily directly proportional to the gradient of the
signaling activity in morphogen-receiving cells. Dpp signaling activity can be
visualized in situ in the Dpp-receiving cells of the wing imaginal disc. The
Dpp signal is transduced by phosphorylation of the transducer Mad (Mothers
against dpp) (Fig. 4A). The
phosphorylated form of Mad (p-Mad) can be used as an intracellular marker to
monitor Dpp morphogen activity. To this end, p-Mad levels are visualized using
an antibody that specifically recognizes p-Mad
(Tanimoto et al., 2000). In
the wing imaginal disc, the amount of p-Mad is high in cells near the AP
border, where Dpp concentration is high, but is severely reduced in cells
along the AP border that express dpp, where the level of Dpp is also
very high (Fig. 7). This
reduction in p-Mad levels along the AP border, despite the high concentration
of Dpp, is a result of the direct repressive action of Hh. Hh also directly
organizes patterning in the region where it attenuates Dpp signaling, and it
is possible that Hh downregulates Dpp signaling in this region to prevent it
from interfering with patterning by Hh.
|
The distribution of Tkv may also regulate Dpp activity, rather than just
Dpp concentration. Hh-dependent reduction of p-Mad levels at the AP border
occurs largely by repressing transcription of the tkv gene.
Conversely, the higher Tkv level in the P compartment than in the A
compartment is maintained by the activity of the transcription factor
Engrailed. Both the Hh and En activities that regulate tkv levels are
mediated by the gene mtv (master of thickveins), which
encodes a putative nuclear protein
(Funakoshi et al., 2001).
The ability of receptor levels to regulate the distribution of receptor
ligands is not restricted to the Dpp morphogen, but is also seen for Hh. The
Hh receptor Ptc is expressed in the A compartment at low levels and is
strongly induced by Hh at the AP border. Here, Hh induces a high level of Ptc
to limit the range of the Hh distribution gradient
(Chen and Struhl, 1996).
Heparan sulfate proteoglycans
Recently, several reports have suggested that heparan sulfate proteoglycans
(HSPGs) play a key role in morphogen transport and/or signaling. HSPGs are
abundant cell surface molecules and are a part of the extracellular matrix.
HSPGs consist of a protein core (such as syndecan and glypican) to which
heparan sulfate glycosaminoglycan (HS GAG) chains are attached. GAG chains are
long unbranched polymers consisting of many sulfated disaccharides. Genetic
screens for mutations that affect morphogen signaling pathways in
Drosophila have identified genes that have sequence homologies to
genes that encode vertebrate GAG biosynthetic enzymes. These putative enzymes
are encoded by sugarless (sgl), sulfateless
(sfl), and members of the Drosophila EXT gene family
consisting of tout-velu (ttv), brother of ttv
(botv) and sister of ttv (sotv; Ext2 -
FlyBase), which encode proteins with homology to UDP-glucose dehydrogenase,
N-deacetylase/N-sulfotransferase and HS copolymerase, respectively. Mutations
in sgl compromise signaling mediated by Wg
(Binari et al., 1997;
Hacker et al., 1997
;
Haerry et al., 1997
) and Dpp
(Haerry et al., 1997
).
Similarly, the sfl mutation affects Wg and Hh signaling
(Lin et al., 1999
;
The et al., 1999
), and in
somatic sfl mutant clones Wg protein levels are diminished
(Baeg et al., 2001
). ttv,
botv and sotv mutants affect Hh, Dpp and Wg signaling
(Bellaiche et al., 1998
;
The et al., 1999
;
Takei et al., 2004
). In
addition, Notum, a gene that encodes a member of the
/ß-hydrolase superfamily, influences Wg protein distribution by
destabilizing the HSPGs (Gerlitz and
Basler, 2002
; Giraldez et al.,
2002
). Lastly, dally is proposed to encode a HSPG protein
core, and is required for Wg and Dpp activity. Dally, and the related
Dally-like protein (Dlp), bind and stabilize Wg at the cell surface
(Baeg et al., 2001
;
Strigini and Cohen, 2000
), and
may provide a pool of Wg protein that can become available for receptor
binding upon its release from HSPGs. Both dally and tkv
expression are regulated by Hh and Engrailed. In addition, elevated levels of
Dally increase the sensitivity of cells to Dpp, and alterations in the levels
of Dally affect formation of both Dpp ligand and activity gradients
(Fujise et al., 2003
).
Together, these findings indicate that HSPGs are major regulators of morphogen
gradients.
Hh protein levels are significantly decreased in clones of cells mutant for
the EXT genes ttv, botv and sotv (for Hh;
Fig. 8C) when these clones are
generated in the Hh-expressing cells of the wing. This indicates that HSPGs
are required for stable retention of Hh on the cell surface. In wild-type
imaginal discs, Hh protein synthesized in the P compartment appears to flow
into the A compartment, with a moderate concentration gradient starting from
the middle of the posterior compartment
(Fig. 8A). However, when the
EXT mutant clone is created in the A compartment along the AP boundary, Hh
accumulates abnormally in the P compartment
(Fig. 8B). This indicates that,
because of a lack of appropriate HSPG, Hh fails to move into the mutant cells
and, as a consequence, accumulates in posterior cells instead. Dpp-GFP and Wg
also accumulate abnormally in the cells near EXT mutant clones, probably
because these proteins cannot move into the cells mutant for EXT genes
(Takei et al., 2004). These
observations indicate that the HSPG-dependent diffusion is a common mechanism
for the distribution of the three morphogens, Hh, Dpp and Wg.
|
We have witnessed significant progress in understanding the mechanisms by
which morphogen signals regulate pattern formation in various contexts of
development. Nevertheless, we still do not know the answer to the simple and
fundamental question of how morphogens are propagated through tissues.
Movement by free diffusion alone cannot explain the graded pattern of a
morphogen because a secreted GFP fusion protein composed of GFP and the
secretory transport domains of Dpp (i.e. lacking the mature Dpp peptide) fails
to form a gradient (Entchev et al.,
2000). We are beginning to see cell-biological findings that
postulate mechanisms by which the morphogen molecules might traffic through a
developing tissue. These mechanisms involve planar transcytosis, cytonemes and
argosomes observed in imaginal disc development. Theoretical studies, however,
favor restricted diffusion in extracellular spaces as a mechanism by which
morphogens traffic through tissues. The term `restricted diffusion' is used
here to distinguish it from more elaborated mechanisms such as transcytosis.
It is also different from `free diffusion' in that `restricted diffusion'
implies interaction between the morphogen molecules and cell surface molecules
such as the receptors and HSPGs. We will return to restricted diffusion at the
last part of this section.
Planar transcytosis
One way to explain morphogen transport is that morphogens are transported
through the tissue by repeated cycles of endocytosis and resecretion - known
as planer transcytosis. The requirement for endocytosis in Dpp function was
indicated by an experiment using Dpp-GFP and a mutation in the gene
shibire (shi), which encodes Dynamin, a GTPase required for
clathrin-dependent endocytosis. When a shi clone is made shortly
after a short burst of Dpp-GFP expression, Dpp-GFP-positive endosomes are not
present in the area behind the shi clone
(Entchev et al., 2000).
Furthermore, the activity of the small GTPase Rab5 is required for the fusion
between endocytotic vesicles and early endosomes, and is thought to be rate
limiting in the early endocytic pathway. When a dominant-negative mutant of
Rab5 is expressed in the wing imaginal disc of wild-type flies, target gene
expression is restricted to the cells adjacent to the Dpp source
(Entchev et al., 2000
). By
contrast, overexpression of Rab5 broadens the expression domain of target
cells. In addition, another small GTPase, Rab7, targets endocytic cargo from
the early to the late endosome, and then to the lysosome for degradation.
Expression of a dominant gain-of-function mutant of Rab7 decreases the levels
of GFP-Dpp that are internalized and reduces the range of Dpp signaling
(Entchev et al., 2000
).
Nevertheless, Dpp could also be propagated in part by its diffusion in the
extracellular space; the digestion of the extracellular proteins of the intact
wing disc with proteinase K demonstrates that most of the Dpp-GFP signal
appears to be in the extracellular space
(Teleman and Cohen, 2000).
Thus, more careful genetic and cell biological studies will be required to
determine how much of the Dpp trafficking can be ascribed to the endocytotic
mechanism, extracellular movement or other mechanisms.
In contrast to the role of planer transcytosis in the transport of Dpp, a
report has argued against transcytosis as a mechanism of Wg trafficking in
wing imaginal disc development (Strigini
and Cohen, 2000). By devising a new antibody-staining protocol to
detect extracellular Wg protein, the authors revealed that the Wg protein
makes a shallow extracellular gradient
(Strigini and Cohen, 2000
). Wg
does not localize to punctate structures in the shi mutant clones (as
is the case for Dpp), and, in contrast with Dpp, Wg is internalized by
wild-type cells behind the shi clone. This shows that Wg can move
across the shi mutant tissue and is internalized by the adjacent
wild-type cells. In fact, higher levels of extracellular Wg protein are
present in shi mutant clones than in wild-type cells.
(Strigini and Cohen, 2000
).
Recent reviews discuss the problems of morphogen transport that we have not
been able to detail in this article
(Teleman et al., 2001
;
Vincent and Dubois, 2002
).
Cytonemes
Cells at the periphery of the imaginal disc have been found to extend
actin-based long processes, called cytonemes, towards the AP border where Dpp
is expressed (Ramirez and Kornberg,
1999). By using cytonemes, even cells far from the source of
morphogen can make direct contact with cells that express the morphogen
(Fig. 9). Although they await
functional analysis before their role in gradient formation becomes clear,
imaginal disc cells also extend other types of processes. It was proposed that
these processes have roles in morphogen transport. As discussed earlier, the
wing imaginal disc consists of a columnar cell layer, which gives rise to the
wing, as well as an overlying peripodial epithelium
(Fig. 1B). Inhibition of Dpp
signaling only in the peripodial cells nevertheless disrupts growth and
patterning of the wing (Gibson et al.,
2002
), suggesting that mechanisms that govern the growth and
patterning of peripodial cells coordinate with those of columnar cells. The
peripodial cells extend long cellular processes that traverse the acellular
space between these layers and terminate on the surface of the columnar cells
(Gibson and Schubiger, 2000
).
These processes may function to transmit the signal between the two layers of
cells.
|
Restricted diffusion and mathematical studies
The striking observation that blockage of endocytosis in clones of cells
causes defects in Dpp transport (Entchev
et al., 2000) led to the proposal that transcytosis can propagate
Dpp through tissue, and seems to present the strongest argument against the
restricted diffusion mechanism (the extracellular transport of functional
morphogen interacting with cell surface molecules). A numerical analysis,
however, has since shown that the observation in that experiment could also be
explained by the restricted diffusion mechanism, if the internalization of the
receptors by endocytosis is accounted for. The blockage of endocytosis is
known to cause increased cell surface receptor levels. As gradient shape
depends on cell surface receptor concentration (whereby the gradient becomes
steeper in the tissue with more receptors), a numerical analysis predicts
gradients to fall through such clones steeply enough to see the shadows behind
the clones (Lander et al.,
2002
). This could be tested experimentally in shi mutant
clones when antibodies against the morphogen and receptor become available.
Furthermore, Lander et al. also suggest that, because Dpp gradient in the wing
disc is almost fully established within 7 hours of the onset of Dpp
expression, transcytosis would have to occur at implausibly fast rates
(Lander et al., 2002
).
If morphogens are transported by restricted diffusion, another numerical
analysis also predicts the significance of morphogen receptors in establishing
robust gradients. It has been proposed that morphogens are decayed rapidly
close to the source and more gradually further away from the source in order
to ensure their robustness and long-range action
(Eldar et al., 2003). These
authors proposed two models: (1) morphogen signaling represses receptor
expression, while the receptor stabilizes the free morphogen; and (2)
morphogen signaling activates receptor expression, while the receptor enhances
the degradation of the free morphogen. They suggested that the former model
represents the Wg system, and the latter the Hh system, as Wg downregulates
its own recepter levels whereas Hh upregulates them, and they also showed that
the Wg receptor Dfz2 stabilizes free Wg
(Eldar et al., 2003
). The
models will be tested by experimental characterization of the
receptor-dependent regulation of degradation of free morphogens.
Future perspectives
In 1924, Hans Spemann and Hilde Mangold carried out a spectacular
experiment in which they transplanted the dorsal rip from an early gastrula of
a newt into another early gastrula in the region that would become ventral
epidermis. This resulted in a complete mirror image duplication of the whole
body (Spemann and Mangold,
1924) (Fig. 10).
We now know that many signaling molecules and their antagonists are involved
in this process. When the whole process driven by the organizer is analyzed at
the molecular level, it is probable that we will see successive rounds of
regulation by both morphogens and signal relay mechanisms.
|
Much of the knowledge about morphogens has come from the studies of
imaginal disc development, mainly because of the simple structure of the disc
and the powerful tools of analysis now available, especially mosaic clones
(Blair, 2003). Advances in
similar techniques will reveal more morphogens in vertebrates and will allow
us to determine how their gradients are regulated.
ACKNOWLEDGMENTS
We thank A. Kuroiwa, H. Nakato, T. Ogura and C. Tabin for helpful discussions, and A. Kouzmenko for critical reading of the manuscript.
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