# Mechanisms of Morphogen Movement

Maura Strigini*

Department of Zoology, University of Cambridge, Downing Street,
Cambridge CB2 3EJ, United Kingdom

Received 11 January 2005; accepted 12 January 2005

**ABSTRACT:** Morphogens are defined as signaling molecules that are produced locally, yet act directly at a distance to pattern the surrounding field of cells in a concentration-dependent manner. In recent years many laboratories have devoted their attention to how morphogens actually reach distant cells. Several models have been proposed, including diffusion in the extracellular space and planar transcytosis. A combination of genetic, developmental, and cell-biological approaches have been taken to tackle this issue. I will present the models and discuss the types of experiments that have been designed to test them. It stands out that most of the work has been carried out in *Drosophila*. Morphogens contribute to

patterning of the vertebrate nervous system, and the same signaling molecules have recently been shown to play important, possibly instructive, roles in axon guidance. Little, if anything, is known about the movement of morphogens in the context of nervous system development. The long-standing tradition of biophysical studies on diffusion in the brain extracellular space, along with the sophisticated *in vitro* culture systems developed in neurobiology laboratories, may provide new tools and ideas to test these models in a new context. © 2005 Wiley Periodicals, Inc. J Neurobiol 64: 324–333, 2005

**Keywords:** morphogen; gradient; diffusion; transcytosis; trafficking

## INTRODUCTION: MORPHOGEN GRADIENTS

Positional information in a developing tissue is often imparted by a secreted signaling molecule that is produced in a restricted group of cells. In some cases this molecule can act only at short range on nearby cells, but initiates a series of secondary “relay signals” that sweep across the tissue and pattern it by eliciting different responses. In other cases the molecule distributes away from its source, forming a concentration gradient that signals directly to nearby and to distant cells. Patterning is achieved because the response of the receiving cells changes according to the local concentration of the signaling molecule. The latter scenario defines the signaling molecule as a morphogen (Wolpert, 1969). Members of the Wnt, TGF-β, Hedgehog, and FGF families of secreted signaling

molecules have been shown to act as morphogens both in vertebrate and in invertebrate model organisms. The morphogen concentration gradient typically translates into an activity gradient, which results in the differential activation of target genes in different cells as a function of their distance from the morphogen source. So- called high-threshold targets respond only to high levels of morphogen signaling, while low-threshold targets respond to low levels. Mechanisms of morphogen gradient interpretation have been reviewed by Gurdon and Bourillot (2001); see also the recent research article by Casali and Struhl (2004) and references therein. The activity gradient can also be monitored by looking at the level of activation of the signaling pathway elicited by the morphogen, for example using an antibody against the activated form of a pathway component. Work from several laboratories has shown that many factors impinge on the activity gradient, including complex interplays between morphogens and their receptors (reviewed in Strigini and Cohen, 1999; Cadigan, 2002), and between morphogens and their antagonists (for example Giraldez et al., 2002). As a result, the

<footnote>Correspondence to: M. Strigini (ms624@cam.ac.uk).

© 2005 Wiley Periodicals, Inc.
Published online in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/neu.20164</footnote>
morphogen concentration and the activity gradients may differ from each other to some extent. Still, the shape of the morphogen concentration gradient is obviously a key determinant of the corresponding activity gradient, and it is in turn a function of the rate of production, degradation, and movement of the morphogen across the tissue.

## The Source and the Sink

Theoretical models postulate a source and a sink for the morphogen (Wolpert, 1969). The cellular basis of the source is the group of cells that produce the morphogen. The genetic/developmental programs that define them and the temporal window in which the source remains active are known in several cases. Instead it is not clear to what extent the actual rate of production of active morphogen molecules (which may depend on post-transcriptional and post-translational modifications as well as on secretion) may be under developmental regulation (Pfeiffer et al., 2002 and references therein; see also Schlesinger et al., 2004 for the regulation of the secretion of Spitz, the short-range signaling EGF receptor ligand).

Degradation (the sink) is equally important in mod-
ulating the shape of the gradient, as suggested by
experiments in which intracellular trafficking
(reviewed in Gonzalez-Gaitan, 2003; Torroja et al.,
this issue) or the levels of morphogen receptors (Cadi-
gan, 2002) are altered. A particularly nice example is
the one uncovered by Dubois and colleagues (2001),
who were able to document the developmentally regu-
lated degradation of the Wingless morphogen in the
*Drosophila* embryo. Wingless (a protein of the Wnt
family) is produced in one row of cells in each seg-
ment in the fly epidermis. The range of Wingless sig-
naling is broader anteriorly than posteriorly. Dubois
et al. (2001) showed that restricted activation of the
EGF receptor pathway posterior to the Wingless
source leads to increased Wingless degradation and
thus to an asymmetric gradient.

Movement Away from the Source

A major issue regarding the mode of action of a
morphogen remains the one of how it reaches distant
cells. Two main mechanisms have been considered:
(i) diffusion in the extracellular matrix (ECM) and
(ii) planar transcytosis (Fig. 1). The latter involves
successive rounds of endocytosis and exocytosis, so
that the signaling molecule is internalized, trafficked
through the endosomal compartments, and then
recycled and passed on to neighboring cells. In this

way, part of the morphogen journey is spent in intra-
cellular vesicles. Additional models, briefly presented
later in this article, include dispersal of the morpho-
gen via (iii) cell movement, (iv) particles (argo-
somes), and (v) cellular processes (cytonemes).

This review will focus on diffusion and planar
transcytosis. I will discuss the type of questions and
experimental approaches that characterize this field.
Further details, examples, and opinions on what
remains a rather controversial topic can be found in
other recent publications, including Zhu and Scott,
2004, Gonzalez-Gaitan et al., 2003, Lander et al.,
2002, and Vincent and Dubois, 2002.

MODELS: DIFFUSION/EXTRACELLULAR
MOVEMENT VERSUS PLANAR
TRANSCYTOSIS

For a long time it has been assumed that secreted sig-
naling molecules, including long-range morphogens,
move away from the secreting cells through the tissue
by diffusion in the ECM (see for example McDowell
et al., 1997). Diffusion in the ECM is not the same as
diffusion in solution: first, the “geometry” of the ECM
imposes limitations on the paths a molecule can take
and how smoothly it can travel through it; second, the
secreted molecule interacts with many other molecules
that can, for example, (i) bind and immobilize it, (ii)
bind and target it to degradation, (iii) prevent its degra-
dation. The distinguishing feature of the diffusion
model is that the signaling molecule follows an exclu-
sively extracellular path to reach its target cells, where
it activates the relevant receptor(s). Accordingly, the
terms “facilitated diffusion,” “restricted diffusion,”
“extracellular spreading,” or “extracellular move-
ment” are now preferred to “diffusion”.

The planar transcytosis model argues that morpho-
gens cannot travel far in the ECM. Most secreted sig-
naling molecules, most notably Wnt molecules, tightly
bind heparin, heparan sulfate proteoglycans (HSPGs),
and possibly other cell membrane and ECM compo-
nents (reviewed in Lin and Perrimon, 2000; Perrimon
and Bernfield, 2001; Selleck, 2001). Accordingly, it is
often difficult to biochemically purify active molecules
from cell culture supernatants unless heparin is added
(Reichsman et al., 1996). It is therefore proposed that
their journey in the ECM may be continuously halted
by interactions with ECM components, and thus ren-
dered extremely inefficient. An intracellular, vesicle-
mediated mechanism of morphogen transport could
provide a way to escape the “stickiness” of the extrac-
ellular route: only the short distance between two
neighboring cells would be left to diffusion, while
<figure><img src="image_1.png" /></figure>

**Figure 1** Diffusion and transcytosis models and predictions after blocking endocytosis. Pools of secreted morphogen molecules are depicted as dots. Intracellular vesicles are depicted as circles surrounding the morphogen (secretory vesicles at the source are not drawn for simplicity). Endocytosis-defective cells are shaded. The diffusion model (top left) proposes that morphogen molecules are secreted from the source cells and reach all of their target cells via an extracellular route. Internalized molecules are sent for degradation. If endocytosis is blocked in some of the receiving cells (top right), extracellular morphogen may accumulate around these cells because of a local decrease in degradation, but its movement to more distant cells is not halted. The transcytosis model (bottom left) proposes that morphogen molecules are secreted by source cells, internalized by adjacent responding cells, and resecreted (recycled); subsequent rounds of endo- and exocytosis ensure that morphogen molecules can reach distant cells. If endocytosis is blocked in some of the receiving cells (bottom right), morphogen molecules cannot spread further away.

most of the journey would be spent inside cells. More specifically, the transcytosis model states that secreted morphogen molecules are internalized by immediately adjacent responding cells and then (at least in part) “recycled” to the cell surface, thus being passed on to other cells (Fig. 1). Essentially molecules do not move around cells but through them.

Cell biologists use the term “transcytosis” to describe the sorting or trafficking of proteins from the apical to the basolateral domains of the cell membrane in polarized epithelia (and vice versa). This occurs via endocytosis from the membrane domain facing one side of the epithelium, regulated endosomal trafficking, and exocytosis to the other side. Immunoglobulin A and its receptor are a very well studied paradigm (Mostov, 1994; Mostov et al., 2000). Transcytosis also plays a crucial role in the selective crossing of endothelia and the blood-brain barrier (Rippe et al., 2002). In the case of morphogens one does not refer to transcytosis across an epithelium, but to the intracellular vesicle-mediated transport of molecules from cell to cell. This model was first proposed for the patterning of the *Drosophila* embryonic epidermis, a monolayer epi-

thelium, where the Wingless morphogen moves along the plane of the epithelium (Gonzalez et al., 1991; Bejsovec and Wieschaus, 1995). Therefore, the term “planar transcytosis” was used (I will mostly use “transcytosis” for brevity). This model actually poses no requirement for the morphogen to be trafficked and “recycled” in any specific way with regard to the polarity of the cells and could be applied also to the movement of morphogens across multilayer epithelia and nonepithelial tissues.

## Visualizing the Gradients

The very first prerequisite to test experimentally these two scenarios is the visualization of the gradient. This can be achieved by antibody staining, and protocols have been developed to specifically detect extracellular versus total pools of molecules (Strigini and Cohen, 2000). In many cases though, the protein gradient cannot be easily detected, even if it can be inferred on the basis of a corresponding activity gradient. Tagged versions of the molecule of interest can then be delivered to the relevant tissue, for example in transgenic animals or upon implantation of beads
soaked in the molecule. Tagged molecules should be
functional and ideally should be able to replace the
endogenous molecule. Fluorescent derivatives are
particularly valuable for *in vivo* imaging (Pfeiffer
et al., 2000; Entchev et al., 2000; Teleman et al.,
2001; Torroja et al., 2004; Scholpp and Brand, 2004;
Williams et al., 2004).

Morphogen molecules can usually be detected
both inside and outside cells. Intracellular staining
appears punctate, consistent with vesicular accumula-
tion. Double labeling with vesicle markers suggests
that some of these vesicular structures are lysosomes,
and therefore marks molecules in their final steps to
degradation. The existence of this intracellular pool is
consistent with both models, because both are com-
patible with the eventual internalization and intracel-
lular degradation of morphogens (the sink). The chal-
lenge is then to show whether any of the remaining
pools of morphogen-containing vesicles will eventu-
ally fuse to the plasma membrane and release the
morphogen. A useful experimental approach that has
not been fully developed yet involves the *in vivo*,
quantitative observation (tracking) of vesicles con-
taining fluorescently labeled morphogens, including
photoactivatable versions, to directly follow the fate
of endocytosed molecules (see for example Pfeiffer
et al., 2000, 2002; Greco et al., 2001).

Modulating Intracellular Trafficking

In the past few years many tools and approaches bor-
rowed from cell biologists have been adapted by
developmental biologists to look into the trafficking
of the morphogens and to ask whether support for the
transcytosis model can be found. Given that the trans-
cytosis model states that the morphogen must be
internalized in order to spread, a first set of experi-
ments involves blocking endocytosis. To what extent
is the movement of the morphogen away from its
source impaired in this situation? The relevant experi-
ments were initially done in flies mutant for a temper-
ature-sensitive mutation in Dynamin, a GTPase
required for the internalization of vesicles during cla-
thrin-mediated endocytosis (van der Bliek and
Meyerowitz, 1991). Recently endocytosis has been
manipulated also by interfering with the activity of
Rab5, a small GTPase that promotes the initial step in
the trafficking of endocytic vesicles towards early
endosomes (Zerial and McBride, 2001). When Dyna-
min temperature-sensitive homozygous mutant flies
are kept for a certain length of time at the restrictive
temperature, endocytosis is blocked and morphogens
appear to accumulate at the source, consistent with

the hypothesis that endocytosis is required for normal
morphogen distribution. This was reported for Wing-
less in the embryo (Bejsovec and Wieschaus, 1995),
Wingless in the wing disc (Strigini and Cohen, 2000),
and Dpp (a TGF-β family member) in the wing disc
(Entchev et al., 2000). In order to bypass the compli-
cations of a possible requirement for Dynamin in
morphogen production/release from the source cells,
experiments were subsequently developed so that the
source of the morphogen would be wild-type for
Dynamin function, and only the responding tissue
would be mutant. When this was done while looking
at Dpp (in the *Drosophila* wing disc) and Wingless
(in the *Drosophila* embryo), the two molecules could
not be detected in cells at a distance from the source,
supporting again the transcytosis model (Moline
et al., 1999; Entchev et al., 2000). Recently, though,
Belenkaya and colleagues (2004) reported that
extracellular Dpp could be detected at a distance from
its source when they conducted this type of experi-
ment. Following a conceptually similar approach,
only small groups of responding cells were made
mutant for Dynamin (or Rab5) in an otherwise wild-
type tissue; this can be achieved by generating mutant
clones in *Drosophila* (Strigini and Cohen, 2000;
Entchev et al., 2000; Belenkaya et al., 2004; Han
et al., 2004b; Torroja et al., 2004), mosaic embryos in
zebrafish (Scholpp and Brand, 2004), or “mosaic
explants” in *Xenopus* (Williams et al., 2004). The
question then was whether the morphogen could be
detected in the mutant tissue and whether it was able
cross mutant patches of cells. If lower morphogen
levels could be detected inside the clone and even in
wild-type tissue just “behind” the mutant clone (i.e.,
on the side of the clone far away from the source;
Fig. 1), it would be taken as a strong indication that
endocytosis is required for morphogen movement.
Instead the results suggest that these morphogens can
cross the mutant cells, arguing that endocytosis may
not be required for movement. In an even more ele-
gant set of experiments, a group of cells defective in
endocytosis was challenged with a “transient wave”
of morphogen. This last set-up allows one to look at
the system before steady-state is reached and before
compensatory movement from surrounding wild-type
tissue may mask any effect. Entchev et al. (2000) and
Belenkaya et al. (2004) took this approach to look at
Dpp distribution in the fly wing disc, and came up
with different conclusions, an issue that is still con-
troversial.

Overall the requirement of endocytosis for move-
ment seems to be supported in some systems but
interestingly not in all. Recent work on the movement
of FGF in the zebrafish ectoderm strongly suggests
that endocytosis is not required (Scholpp and Brand, 2004). Similarly it has been argued that Hedgehog and Wingless spread in an endocytosis-independent manner in the wing imaginal disc (Han et al., 2004b; Torroja et al., 2004; Strigini and Cohen, 2000), and that so does the TGF-β family member Xnr2 in *Xenopus* (Williams et al., 2004). Work on Dpp in the fly wing supports a role for endocytosis in morphogen movement: in some situations Dpp cannot be detected in endocytosis defective tissue, nor can it cross it (Entchev et al., 2000), although Lin and colleagues reached different conclusions (Belenkaya et al., 2004; see also Eldar and Barkai, 2005 on the interpretation of this type of experiment). Finally, transcytosis may mediate movement of Wingless in the fly embryonic epidermis (Bejsovec and Wieschaus, 1995; Moline et al., 1999). It would be interesting to learn whether different morphogens move by different mechanisms, and whether the same molecule may use different modes of movement in different contexts. For the time being, whether the differences are real or simply reflect different experimental designs and interpretations remains a matter of hot debate.

Small GTPases of the Rab family localize to distinct subcellular organelles involved in vesicular traffic, where they mediate the tethering and fusion of cargo vesicles. As mentioned above Rab5 promotes the initial step in the trafficking of endocytic vesicles towards early endosomes. Rab7 and Rab11 are instead specific to the lysosomal and recycling compartments of endosomes, respectively (reviewed in Zerial and McBride, 2001). Modulating their activity can be used as a means to change the relative balance of traffic through the relevant compartment while monitoring morphogen movement, a promising experimental approach that has not been fully developed yet (Entchev et al., 2000).

Incidentally, the experiments discussed above also allow us to assess the requirement for endocytosis and endosomal trafficking in morphogen stability and signaling (discussed in this issue by Isabel Guerrero and colleagues; Torroja et al., 2005). Endocytosis contributes to clearance of most morphogens, most likely by allowing their internalization and subsequent intracellular degradation: endocytosis-defective groups of cells accumulate higher levels of extracellular morphogen (Strigini and Cohen, 2000; Entchev et al., 2000; Belenkaya et al., 2004; Scholpp and Brand, 2004; Han et al., 2004b; Torroja et al., 2004). In this sense it is clear that endocytosis does contribute to shaping concentration gradients, although it is not usually known to what extent ligand clearance is developmentally regulated (see also the section The Source and the Sink). It is also clear that endocytosis

modulates signaling, now an important focus in research. Endocytosis is positively required for Dpp signaling (Belenkaya et al., 2004), while it appears to be dispensable for Hedgehog signaling (Han et al., 2004b; Torroja et al., 2004). Endocytosis of an activated receptor can also down-regulate the extent and duration of signaling, as is the case for some of the signaling events downstream of the EGF receptor (Vieira et al., 1996). All this must be kept in mind when morphogen distribution cannot be monitored directly but is inferred from its activity: if endocytosis and trafficking are required for signaling, then loss of signaling cannot be taken as an indication of a drop in morphogen concentration.

## Test of Temperature Dependence

Transcytosis is an active, energy-requiring process. As such it should be blocked at low temperatures (4°C). On the other hand diffusion in the ECM is a passive process and should remain largely unaffected at 4°C. Following this reasoning, McDowell and colleagues (2001) monitored, by antibody staining, the movement of TGF-β1 released from beads in *Xenopus* explants kept at different temperatures. The range of spreading was similar at 23 and 4°C, suggesting that TGF-β1 moves by diffusion (McDowell et al., 2001). In order to apply the same criterion to other systems, the challenge is now to modulate the temperature within the tissue under investigation, and repeat the same experiment while looking at endogeneously produced morphogens.

## HSPGs: Facilitated Diffusion

Several lines of evidence indicate that HSPGs, consisting of a protein backbone carrying complex sulfated glycosaminoglycan chains (GAG), contribute to morphogen signaling. It has been proposed that they may do so by functioning as coreceptors and/or by regulating morphogen stability (reviewed by Lin and Perrimon, 2000; Perrimon and Bernfield, 2001; Sell-eck, 2001). Do they also contribute to their movement? It is possible to affect the levels and activity of HSPGs by using mutations in their protein core, mutations in the enzymes involved in the biosynthesis of their sugar chains, and by pharmacological treatments. Interfering with HSPG function has been reported to affect the distribution of Dpp, Hedgehog, and Wingless (Fujise et al., 2003; Baeg et al., 2004; Han et al., 2004a,b; Belenkaya et al., 2004, and references therein; reviewed in Tabata and Takei, 2004). The most convincing piece of data on the function of
HSPG in morphogen movement comes perhaps from the work of Lin and colleagues (Belenkaya et al., 2004). They showed that GFP-tagged Dpp cannot cross clones of cells carrying defective HSPG. Dpp levels were dramatically decreased both inside and behind the clones lacking *dally*, a GPI-linked glypi-can (an HSPG protein core); this dramatically con-trasts with what the authors observed when blocking Dynamin activity in clones of cells in similar (but not identical!) experimental setups (see the section “Modulating Intracellular Trafficking”). They sug-gest that HSPG could provide a molecular highway for morphogen movement, whereby morphogen mol-ecles are captured by the sugar chains of proteogly-cans and are displaced to the next ones down the con-centration gradient. This model is somewhat reminiscent of the “bucket brigade” model originally proposed, on a theoretical basis, by Kerszberg and Wolpert (Kerszberg and Wolpert, 1998; Kerszberg, 1999): morphogens would travel around cells, but were tightly associated with cell membranes because of their being effectively handed over from receptor to receptor.

Finally, it should be emphasized that the expres-sion, modification state, and activity of HSPG is developmentally regulated, often indeed under con-trol of morphogen signaling itself (see Kreuger et al., 2004 and Kirkpatrick et al., 2004 and references therein). This may contribute in part to the “robustness” of the gene network responsible for patterning (see the next section; Eldar et al., 2003). But this also means that, given that HSPGs appear to modulate the distribution and the signaling activities of morpho-gens, and that additional feedback loops exist, for example, between morphogens and their receptors, it has been difficult to experimentally tell apart the con-tributions of each component of the gene network to specific functions.

It has been argued that an active mode of transport for morphogens, like transcytosis, would offer the possibility to regulate the process. It is now evident that diffusion can be indirectly modulated. Collec-tively, the work on HSPG suggests there is more than just passive diffusion in the extracellular movement of morphogens.

## Computer Modeling

Given that the experimental setups used to test these models are rather complicated and may affect simul-taneously more than one process and parameter, computer modeling has now emerged as a valuable approach. Morphogen movement can be modeled and theoretical predictions generated, which can then be

confronted with experimental data. For instance, interfering with endocytosis affects the levels of receptors on the membrane. It has been argued that the resulting increase in high affinity receptors on the cell surface of an endocytosis-defective group of cells may be enough to block the spreading of morphogen through them. Computer modeling was used to recre-ate the case of the movement of the Dpp morphogen in the fly wing disc, postulating that it diffuses in the extracellular space and encounters endocytosis-defec-tive cells with elevated receptor levels at their surface (Lander et al., 2002). The prediction of the model is that the morphogen molecules will be trapped by the first encountered mutant cells. In other words, even in a “diffusion scenario” endocytosis may affect morphogen movement similarly to what is predicted by the transcytosis model and to what was reported upon experimental manipulation of endocytosis (Lander et al., 2002; Entchev et al., 2000). This model was recently challenged by Julicher, Gonzalez-Gaitan, and colleagues (Kruse et al., 2004). They elaborated on the diffusion model, accounting, for instance, for diffusion in two dimensions. They then came up with different predictions, which did not match the exist-ing experimental data on Dpp, arguing that a model based on diffusion alone cannot explain the observed roles of endocytosis. Discrepancies partly came from the choice of numerical parameters that were fed into the models, which in turn reflected the biochemical and biophysical properties of the system. Experimen-tal validation of such parameters is crucial, but will necessitate time. In the meantime estimates can only be made on the basis of published data on similar molecules. A continuous reciprocal feedback between theory and experiment must sustain this type of approach.

Morphogen gradients are established with a strik-ing degree of reproducibility under diverse environ-mental conditions and genetic backgrounds. For example, morphogen-dependent patterning is indis-tinguishable in individuals carrying two or one func-tional copies of the morphogen-encoding gene. “Robustness” is the capacity to buffer fluctuations in gene dosage or environmental conditions. Eldar, Bar-kai, and colleagues showed that, by theoretical analy-sis of mechanisms that facilitate robustness, it is pos-sible to unravel the machinery responsible for gener-ating the spatial distribution of morphogens (reviewed by Eldar et al., 2004). In particular, their novel approach, tightly linked to the experimental analysis of specific model systems in *Drosophila*, highlighted the importance of feedback loops leading to an enhanced degradation of the morphogen in regions of high morphogen signaling. In order to
achieve robust long-range signaling, morphogens should decay rapidly close to their source but at a significantly slower rate over most of the field. This could be accomplished if morphogens enhance their own degradation, leading to a higher degradation rate close to their source. If morphogen clearance is dependent on receptor-mediated endocytosis, enhanced degradation may be achieved through upregulation of receptor levels by morphogen signaling (as seems to be the case for Hedgehog in the developing wing; Han et al., 2004b; Torroja et al., 2004). Alternative plausible scenarios include the induction of proteases degrading the ligand, or of other components affecting the extracellular matrix (Eldar et al., 2003).

## OTHER MODELS: DISPERSAL VIA CELL MOVEMENT, ARGOSOMES, AND CYTONEMES

As mentioned in the section “Movement Away from the Source,” a number of other models have been proposed to explain the movement and long range signaling activity of morphogens, most notably dispersal of the morphogen via growth and cell displacement, in association with particles (argosomes), and by cellular processes (cytonemes).

It has been argued that morphogens could move away from their source along with cells that once acted as source but are then displaced as a consequence of tissue growth. Essentially the “dispersal via growth and cell displacement model” proposes that some of the synthesized signaling molecules may not be promptly secreted, but may stay in the producing cells, as these cells divide and are displaced during tissue growth. Although such cells stop producing “new” molecules as they move away from the source, they effectively keep on secreting “old” ones (i.e., molecules that have been retained in the secretory pathway). There is evidence that suggests that such a mechanism contributes in part to the dispersal of the Wingless morphogen in the *Drosophila* embryo (Pfeiffer et al., 2000). Another interesting case is the one of the FGF activity gradient that promotes growth and patterns the anterior-posterior axis of the early vertebrate embryo. Dubrulle and Pourquie (2004) showed that fgf8 mRNA is transcribed only in the growing posterior tip of the embryo. fgf8 mRNA is progressively degraded in the newly formed tissues, resulting in the formation of an mRNA gradient in the posterior part of the embryo. This fgf8 mRNA gradient is then translated into a gradient of FGF8 protein and downstream signaling activity.

The “argosome model” proposes that the signaling molecules remain tightly associated with the cell membrane upon secretion from the producing cells, and effectively move on to and through neighboring cells along with membrane vesicles that are shed from cells (called exovesicles or argosomes). Such a mechanism has been invoked to explain the long-range distribution of Wingless in the developing *Drosophila* wing, where Wingless appears to localize to such exovesicles (Greco et al., 2001).

The “cytoneme model” suggests that responding cells have long cellular protrusions that grow towards the source of the morphogen and bypass the need for the morphogen to spread towards the responding cells. The model was proposed by Ramirez-Weber and Kornberg (1999), who documented the existence of these structures, and suggested that they may be needed for the long-range activity of Dpp in the *Drosophila* wing disc. So far, no substantial evidence has been brought in support of this model.

## MORPHOGEN MOVEMENT IN THE NERVOUS SYSTEM

Members of the FGF, Hedgehog, Wnt, and TGF-β protein families are all expressed in the nervous system, where they are known to play important roles in patterning (reviewed by Jessel, 2000). In some cases they have been shown to act as bona-fide morphogens, that is, signaling from a localized source to distant target cells in a direct and concentration-dependent manner (see for example Briscoe et al., 2001). Recent work has revealed additional unexpected roles for these molecules in the development and regeneration of the nervous system, including the control of cell differentiation, cell migration, axon growth and guidance, synaptogenesis, proliferation, cell death, and tumorigenesis.

If we consider, for example, the role of these signaling molecules in axon growth and guidance, several cases have been described where they control the trajectory of growing axons in both vertebrate and invertebrate model systems (Colavita et al., 1998; Augsburger et al., 1999; Trousse et al., 2001; Yoshikawa et al., 2003; Charron et al., 2003; Butler and Dodd, 2003; Lyuksyutova et al., 2003; reviewed by Bovolenta in this special issue). Direct action at a distance has been shown in some of these new scenarios, and in at least one case, that of Sonic Hedgehog for midline crossing in the vertebrate spinal cord (Charron et al., 2003), the graded activity of the molecule,
emanating from a localized source, appears to be instructive in axon guidance.

Could we learn more about morphogen movement by looking at how these secreted molecules are distributed in the nervous system? Most of this work has been undertaken in vertebrate model systems and could therefore contribute to counterbalance the pre-dominance of *Drosophila* as a system to study morphogen movement. Vertebrate systems bring along a distinct set of experimental approaches, most notably well established *ex vivo* systems based on cultured explants. These are amenable to pharmacological treatments and classic manipulations, and can often be combined with genetic manipulations, including the transient expression of recombinant DNA and the use of transgenic animals as tissue and cell donors.

Finally, a long-standing school of biophysical studies on the diffusion of molecules in the brain and spinal cord exists. Most of these studies focus on the diffusion of small, “inert” molecules, and they aim at describing the volume and geometrical complexity of the extracellular space (ECS) (Nicholson and Sykova, 1998). Molecules of up to 70,000 Daltons have been shown to be able to diffuse through the ECS, although subject to significant hindrance. These studies typically measure the diffusion parameters of molecules released from a source that approximates a point, whose local concentration around the source can be monitored (e.g., using electrophysiology or optical imaging). Such studies have shown that the biophysical properties of the ECS may be different in different parts of the nervous system, and, more interestingly, may vary during development and as a consequence of electrical activity and injury (see for example Binder et al., 2004; Sykova, 2004).

More recently, analyses of biologically relevant secreted molecules such as neurotrophins and EGF have been reported (Stroh et al., 2003, 2004; Thorne et al., 2004). Although such studies often rely on experimental setups that do not coincide with the *in vivo* situations (most importantly working with an excess concentration of the signaling molecule), it may be possible to apply these techniques to study the dynamics and temperature-dependence of morphogen gradient formation, to help distinguish the relative contributions of diffusion and vesicle-mediated transport mechanisms.

I thank Michalis Averof, Eva Sykova, and Xinhua Lin for critical reading of the manuscript, as well as the anonymous referees for constructive criticism.

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