Creating Gradients by Morphogen Shuttling

Review

Creating gradients by morphogen

shuttling

Ben-Zion Shilo, Michal Haskel-Ittah, Danny Ben-Zvi,

Eyal D. Schejter, and Naama Barkai

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

Morphogen gradients are used to pattern a ﬁeld of cells Because the responding cells monitor the level of the

according to variations in the concentration of a signal- morphogen and differentiate accordingly, the change in

ing molecule. Typically, the morphogen emanates from morphogen levels over space should be sufﬁciently steep,

a conﬁned group of cells. During early embryogenesis, such that distinct levels can be reproducibly sensed at

however, the ability to deﬁne a restricted source for different positions. In addition, the distribution of the

morphogen production is limited. Thus, various early morphogen should not ﬂuctuate markedly when the pro-

patterning systems rely on a broadly expressed morpho- duction rates of the morphogen and the components it

gen that generates an activation gradient within its triggers are altered by asymmetric segregation, genetic

expression domain. Computational and experimental perturbations such as heterozygosity to mutations, or

work has shed light on how a sharp and robust gradient changing environmental conditions. Collectively, these

can be established under those situations, leading to a features are termed ‘robustness’.

mechanism termed ‘morphogen shuttling’. This mecha- Two different conﬁgurations have been shown to pro-

nism relies on an extracellular shuttling molecule that vide a source for the localized release of a morphogen. One

forms an inert, highly diffusible complex with the mor- setup involves the juxtaposition of two distinct types of

phogen. Morphogen release from the complex following tissues, one of which produces the morphogen but does not

cleavage of the shuttling molecule by an extracellular respond to it, whereas the other tissue does [through

protease leads to the accumulation of free ligand at the tissue-speciﬁc expression of a downstream factor(s)]. Al-

center of its expression domain and a graded activation ternatively, a select group of cells that was deﬁned by an

of the developmental pathway that decreases signiﬁ- earlier signaling event could release the morphogen to the

cantly even within the morphogen-expression domain. extracellular milieu (Figure 1a,b). The ability to deﬁne the

interface between two distinct compartments, or to allocate

Morphogen gradients a deﬁned fate to a small group of cells that will serve as the

A central feature in the patterning of multicellular organ- source of the morphogen is feasible when patterning a

isms is the ability to provide a group of cells with informa- tissue at an advanced stage of development, when sufﬁ-

tion regarding their global position within the developing cient cues are in place to clearly demarcate these cells.

embryo or organ. This is achieved by morphogen gradients, Thus, in the Drosophila wing imaginal disc, production of

which are typically represented by secreted proteins that Hedgehog in the posterior compartment (which does not

trigger cellular signaling by activating speciﬁc receptors. respond to the ligand) and its restricted range of signaling

The term ‘morphogen’ was originally coined by Alan Turing within the adjacent anterior compartment will induce

[1], and elaborated on by Lewis Wolpert, who provided the production of the morphogen Dpp in only a few rows of

French ﬂag analogy to the outcome of morphogen-induced cells [4]. Once produced, Dpp diffuses and activates its

signaling [2]. In essence, each color within the ﬂag repre- receptors symmetrically [5–7], thus providing an example

sents a distinct domain that is characterized by the target of the successive utilization of the two mechanisms of local

genes it will express. Morphogens are used in a variety of morphogen release.

tissue settings to pattern a ﬁeld of cells according to their

concentration gradients. The classical model of patterning The need for morphogen shuttling

by morphogen gradients involves three cardinal aspects: (i) During early stages of embryogenesis, coarse symmetry-

release of the morphogen from a restricted source; (ii) breaking events provide the initial patterning cues. Mor-

graded distribution of the morphogen across a ﬁeld of cells; phogen-based mechanisms are then recruited for elabora-

and (iii) the ability of cells to respond to different concen- tion of the patterning scheme. However, because the initial

trations of the morphogen in discrete ways, such that asymmetry is only roughly deﬁned, the capacity to delin-

distinct target genes will be induced by different morpho- eate a signaling interface between two cell types or to

gen levels [3]. induce a narrow group of morphogen-producing cells in

the center of the region that will be patterned is limited.

Corresponding authors: Shilo, B.-Z. ([email protected]); Barkai, N. This means that patterning systems in early embryos are

([email protected]).

more likely to rely on broadly expressed morphogens.

Keywords: morphogen gradients; embryonic patterning; BMP; Chordin; Spa¨tzle; Toll;

shuttling. How then can a gradient be generated within a ﬁeld of

Review Trends in Genetics June 2013, Vol. 29, No. 6 (a) Morphogen at compartment interface (b) Morphogen at center of ﬁeld

Morphogen

Response to morphogen Morphogen producon Morphogen producon (no producon) (no response)

Response Morphogen

Expression of morphogen Expression of shuling and protease molecule

(ii)

Receptor

TRENDS in Genetics

Figure 1. Mechanisms for generating morphogen gradients. (a) Morphogen gradient at the compartment interface. A tissue that produces the morphogen, but does not

respond to it, is positioned next to a tissue that can respond to the morphogen, but does not produce it. Graded distribution of the morphogen is generated within the

responding tissue, leading to a graded activation pattern within this compartment. (b) Morphogen release from a restricted source. When a restricted group of cells

produces the morphogen within a field of responsive cells, the morphogen will spread symmetrically, leading to a corresponding graded activation pattern. (c) Morphogen

shuttling. (i) Shuttling of the morphogen is initiated by broad expression of the morphogen within the patterned region, flanked by domains expressing the shuttling

molecule. The morphogen and shuttling molecule can associate, to generate a biologically inactive and highly diffusible complex. (ii) Due to the activity of a protease within

the patterned region, the shuttling molecule in the complex will be cleaved and release the ligand. When cleavage takes place within the lateral region, the released ligand

will bind another shuttling molecule, due to the graded distribution of the free shuttling molecule within the patterned region. Conversely, cleavage at the center of the

patterned region will preferentially lead to binding of the free ligand to the receptor. This will give rise to physical concentration of the free ligand towards the center. A low

diffusion rate of the free ligand is essential to preserve the graded distribution of morphogen.

responding cells, if the morphogen-expression domain is that the ﬁnal gradient will be robust and reproducible from

not spatially restricted? Moreover, how can this gradient one embryo to another.

be sufﬁciently steep and robust?

Nevertheless, sharp and robust gradients exist during The concept of shuttling

early embryogenesis and enable later establishment of The proﬁle of signaling activation by the morphogen gra-

classical morphogen patterning. This seeming paradox dient determines the developmental responses of the cells

has inspired a combination of computational and experi- within the patterned region. Thus, it was logical to assume

mental approaches to elucidate the patterning scenarios at that the solution to graded patterning within a broad

work. A mechanistic solution to the generation of early region with uniform morphogen expression should be exe-

gradients has recently been obtained, deﬁning a process cuted within the extracellular milieu, where the morpho-

termed ‘morphogen shuttling’, which is the focus of this gen exerts its activity. Genetic studies of each morphogen

review. This mechanism was proposed based on its ability pathway have identiﬁed a limited number of molecules

to deﬁne sharp gradients under conditions where the that function extracellularly. A computational approach

morphogen is broadly expressed, as well as its relative that focused on these extracellular components was taken

insensitivity to the morphogen production rate and other to search for parameters that would not only allow the

ﬂuctuating biochemical rate constants, thereby ensuring extracellular components to form a sharp gradient, but also

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lead to a stable gradient in response to perturbations. This which will cleave the shuttling molecule and release the

analysis converged on a solution coined morphogen shut- morphogen. The chance of the morphogen encountering a

tling. free shuttling molecule decreases dramatically if released

The basic shuttling mechanism depends on two diffus- at the center of the patterning region. Free morphogen will

ible extracellular components. The ﬁrst is the morphogen therefore remain in this central region for a signiﬁcantly

itself, which is usually expressed in a uniform manner in longer time. The ﬁnal outcome will thus be a physical

the broad region that will eventually be patterned. The concentration of the free morphogen within the center of

second is a shuttling molecule, which can associate with the patterning region, driven only by diffusion, to generate

the morphogen and form a complex that is readily diffus- the required sharp morphogen activation gradient.

ible but biologically inert. In addition, there is a third key

component, an extracellular protease that speciﬁcally BMP shuttling in the Drosophila embryo: case in point

recognizes and cleaves this complex, destroying the shut- With the general concept of shuttling in mind, we now

tling molecule and releasing the active morphogen present a concrete case, which represents the ﬁrst instance

(Figure 1c). in which shuttling was proposed and elucidated: pattern-

The critical asymmetry cue in this system is the expres- ing of the dorsal region of the early Drosophila embryo,

sion domain of the shuttling molecule. This domain ﬂanks following the initial subdivision of the embryo along the

the region where the morphogen gradient will form, which dorsoventral axis into the mesoderm (ventral), neuroecto-

we term the ‘patterning region’. The region where the derm (lateral), and dorsal regions.

shuttling molecule is expressed could be broad, but the The dorsal region is patterned by a gradient of BMP

critical spatial component is its interface with the pattern- activity, which peaks at the dorsal-most part and decreases

ing region. The free shuttling molecule diffuses into the towards the lateral regions. This gradient can be easily

patterning region and forms a gradient. The shuttling visualized by an antibody to the phospho-Smad protein

molecule can also associate with the morphogen, forming (termed Mad in Drosophila), detecting the activated state

an inactive complex. The activity of the protease within the of the BMP pathway [8]. Two BMP ligands participate in

patterned region is uniform, in line with its expression this patterning as morphogens, Decapentaplegic (Dpp) and

pattern. Each time a morphogen is released from the Screw (Scw). dpp is expressed broadly and uniformly in the

complex, it will ‘sample’ its environment. When sur- dorsal region, whereas Scw is expressed throughout the

rounded by high levels of the free shuttling molecule, it embryo. The gradient of BMP activation is therefore de-

will form a new complex. Conversely, in regions with low ﬁned well within the expression domain of its activating

levels of the free shuttling molecule, the morphogen will ligands, posing the question of how it is generated. Both

preferentially associate with its speciﬁc cell surface recep- the extracellular BMP inhibitor, Short gastrulation (Sog),

tor. which binds both BMP ligands to generate inert complexes

A central requirement of the shuttling mechanism is and represents the shuttling molecule, and the Sog-cleav-

that the morphogen diffuses primarily when bound to the ing protease Tolloid (Tld) are also involved in this pattern-

shuttling molecule, rather than as a free molecule. Such ing system. Sog is expressed in the lateral regions ﬂanking

differential diffusibility could result, for example, from the dorsal domain to be patterned, whereas Tld is

rapid binding of the free morphogen to its receptor or to expressed uniformly in the dorsal domain [9,10].

components of the extracellular matrix. Regardless of the Conceptually, the shuttling molecule is predicted to

molecular implementation, the fact that morphogen diffu- have dual and opposite roles: not only inhibiting the ligand

sion requires binding to a shuttling molecule that is asym- when complexed to it, but also facilitating maximal acti-

metrically produced fundamentally impacts its dynamics: vation by the ligand as it releases it at the center of the

the morphogen is physically translocated to the center of patterned region. Experimental evidence for such a dual

the ﬁeld, establishing a sharp concentration gradient that role for Sog stemmed from analysis of the mutant pheno-

peaks at that position (Figure 1c). In other words, despite type, which is marked by the lack of dorsal-most fates. In

uniform production of the morphogen, higher levels are addition, Sog was shown to form a complex with the ligands

found extracellularly at the center of the patterned region [9]. These observations led to the suggestion that Sog

as a result of shuttling. This outcome generates a symmet- mediates the trafﬁcking and deposition of Dpp at the dorsal

rical activation gradient of the underlying signaling path- midline [11].

way, similar to the one formed in cases where morphogen Combining these observations regarding key extracel-

production is restricted to the center of the ﬁeld lular elements, an unbiased computational screen

(Figure 1b). searched for solutions that would establish a robust gradi-

To see how this gradient is built, note ﬁrst that, because ent that matched the observations. This led to the proposed

the shuttling molecule is produced at the edges of the shuttling mechanism [12]. In fact, it was the analysis of

patterning region and is subject to degradation by the this patterning circuit that ﬁrst highlighted the theoretical

protease, it will form a concentration gradient. Notably, advantages of shuttling: the sharpness of the resulting

the parameters that give efﬁcient shuttling require that gradient and its robustness to variations in most biochem-

protease activity leads to complete absence of the shuttling ical parameters [12] (Figure 2). Theory predicts that, in

molecule from the center of the patterning region. Consider both aspects, the shuttling mechanism is superior com-

now the dynamics of a morphogen molecule. When in pared with an alternative, inhibition-based mechanism

complex with the shuttling molecule, it diffuses relatively that could also be implemented by the same circuit design.

freely. The complex, however, may encounter the protease, Inhibition-based mechanisms will generally form shallow

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(a) Regionalized expression of Sog and Dpp Sog Dpp (acve)

(b) Sog binds Dpp Sog/Dpp (inacve)

(d) Dpp accumulates dorsally: shuling Dpp receptors

TRENDS in Genetics

Figure 2. Shuttling of BMP ligands in the dorsal ectoderm of Drosophila embryos. The first cohort of zygotic genes that are expressed along the dorsoventral axis of the

embryo includes the BMP ligand Dpp, the protease Tolloid (Tld), and the shuttling molecule Short gastrulation (Sog). The interface between the expression domains of Sog

and Dpp, and the specific biochemical features of these components, leads to a mechanism of morphogen shuttling, which culminates in a dorsal peak of Dpp

accumulation. (a) Expression of Dpp is restricted to the dorsal region (pink cells), whereas expression of Sog is confined to the flanking neuroectoderm (brown cells). Both

are secreted to the extracellular perivitelline space. For simplicity, the figure only displays Dpp. In reality, Dpp forms a heterodimer with Screw (Scw), which is expressed

around the entire circumference of the embryo. (b) The heterodimeric ligand and Sog form an inactive complex, which can diffuse readily within the extracellular fluid. (c)

The extracellular protease Tld, expressed in the dorsal region, preferentially cleaves Sog when in complex with the ligand. (d) When ligand is released in the dorsolateral

region, it will preferentially rebind Sog. Conversely, release in the dorsal-most region will result in favored binding of the receptor. This will lead to physical concentration of

the free ligand in the dorsal-most region (i.e., shuttling).

gradients and should be exquisitely sensitive to any change BMP molecules must be physically translocated dorsally,

in the gene dosage of the inhibitor or ligand. generating a concentration gradient (rather than a mere

The computational analyses of this BMP-patterning activity gradient). Second, free ligand molecules should

system deﬁned several aspects that would be essential not be readily diffusible, and diffuse primarily when com-

for the operation of the proposed shuttling mechanism. plexed with Sog. These two predictions have been tested

The ﬁrst requirement is that Sog be preferentially cleaved and manipulated experimentally. First, dpp was

when in complex with the ligand. This was demonstrated expressed perpendicular to its normal expression domain

biochemically in vitro [9]. Furthermore, when endogenous to assess the consequences of morphogen misexpression.

Sog was replaced with a modiﬁed Sog protein that was As predicted, patterning of the dorsal region was main-

recognized directly by Tld regardless of whether it was in tained, because the ectopic ligand was trafﬁcked to the

complex with the ligand, the resulting gradient of activa- dorsal side, guided by the normal distribution of Sog [12].

tion by Dpp was ﬂattened [13]. This result was predicted by Second, by marking Dpp molecules in the extracellular

the shuttling mechanism: if Tld also cleaves free Sog space of the embryo after they were secreted, it was

within the patterned region, the levels of free Sog decrease directly shown that Dpp was internalized in the dorsal

too rapidly (exponentially), thereby enabling free BMP region, following association with the receptor [14,15].

ligand accumulation over a wide region. Although it is not currently possible to follow the distri-

Two additional critical requirements of the shuttling bution of the different protein complexes in the embryo,

models involve the dynamics of the BMP ligand. First, the the physical translocation of the morphogen provides

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unambiguous experimental demonstration of the shut- that achieving an accurate activation pattern would also

tling mechanism. require a positive feedback response that facilitates an-

To monitor experimentally the lower diffusion of free choring of the free ligand that is released in the dorsal-

Dpp, expression of a perpendicular dpp stripe was again most area [15,19]. It is also instrumental to consider the

analyzed, this time in embryos that were mutant for sog. In precise topological coordinates of the embryo, which is

contrast to the dramatic translocation of Dpp in wild type more pointed at its ends rather than being cylindrical,

embryos, here Dpp and the target genes it induced to get a better ﬁt of the observed activation proﬁle to that

remained conﬁned to the stripe in which dpp was predicted by the original model [20].

expressed, demonstrating the low diffusion capacity of free In addition, the role of the extracellular matrix protein

ligand, as predicted by the model [12]. Collagen IV (Viking-Vkg) in facilitating BMP shuttling in

The dual and opposing roles of Sog account for the the early Drosophila embryo was recently reported [21,22].

robustness to ﬂuctuations in its levels. On the one hand, Dpp has a Collagen IV binding site, which will anchor the

Sog creates the ﬂux of ligand shuttling, by dispersing the heterodimeric ligand Dpp/Scw. Sog also has a binding site

complex with the ligand throughout the circumference of for Collagen IV. Vkg can therefore serve as a scaffold to

the embryo. On the other hand, only the regions that facilitate the efﬁcient formation of the complex between

exhibit the lowest levels of free Sog are permissive to the shuttling molecule and the ligand. To release the

the deposition of free ligand. Changes in Sog levels are complex, the accessory secreted protein Tsg binds Sog

thus counteracted by the opposing roles of the same pro- and, by attenuating the binding of Sog to Collagen IV,

tein, providing robustness [16]. However, it is important to allows for release of the shuttling complex and its subse-

note that robustness is not an absolute feature, and that quent diffusion into the patterning region. Following li-

some alteration in patterning can be observed upon gand shuttling, the binding of free Dpp to Collagen IV also

changes in the dosage of Sog [17]. helps to reduce the diffusion range of the free ligand.

Shuttling of BMP ligands also takes place during postem-

Reﬁning the shuttling mechanism bryonic stages of Drosophila development (Box 1).

The mechanism by which shuttling of the BMP ligands by

Sog generates a morphogen gradient was elaborated and Shuttling in other organisms: Sog determines the

extended by several groups taking different approaches. proﬁle of Dpp signaling

First, it was suggested that receptor-mediated morphogen The shuttling model and the experimental observations

degradation provides an effective way of limiting the dif- supporting it alter the conventional way of thinking about

fusion of free Dpp. In this context, Sog fulﬁlls another role, morphogen gradients. As the ligand is shuttled, its original

by protecting Dpp from receptor-mediated degradation, site of expression is irrelevant to the ﬁnal activation pat-

increasing its effective diffusion range [17]. tern it will produce, as was seen when dpp was ectopically

Because the distribution of free Sog is the critical deter- expressed [12,14,15]. It is the distribution of the free form

minant that dictates the deposition of free ligand by shut- of the shuttling molecule that will deﬁne the site of ligand

tling, it is interesting to consider the determinants that deposition and, hence, the spatial proﬁle of the morphogen

impinge on the generation of this pattern. The model gradient. Ectopic expression of the shuttling molecule was

predicts that the free form is in equilibrium with the indeed capable of deﬁning the morphogen gradient accord-

complex of Sog and Dpp. Because the probability of cleav- ingly [23].

ing the complex increases as it traverses a longer distance With these characteristics of the shuttling mechanism

towards the midline, there will be fewer complexes and a in mind, it is interesting to explore patterning by the BMP

continuous ﬂux of ligand carried by the shuttling molecule system in other organisms. For example, in the beetle

towards the midline. The involvement of Dpp and Tld in

the distribution of Sog was observed experimentally when

Box 1. Shuttling is not just for embryos

examining the gradient of Sog at the dorsal midline, out-

side the domain where it is produced. In mutants for dpp or At later stages of Drosophila development, the cardinal patterning

processes in the larval wing and leg discs, which are also triggered

tld, the nonautonomous distribution of Sog was indeed

by BMP gradients, do not utilize ligand shuttling. Rather, expression

elevated [18]. The antibodies used in this study could

of Dpp in a restricted group of cells leads to a graded distribution of

not, however, distinguish between free Sog and the com-

the ligand, which patterns the neighboring cells [5–7]. However,

plex of Sog with Dpp. utilization of Dpp shuttling has also been described in the pupal

wing veins [44,45]. Dpp is expressed in the future longitudinal wing

Although the primary shuttling model and initial

veins, which also display the most prominent pattern of the

experiments started with the simpliﬁed situation of three

phosphorylated (activated) downstream transcription factor pMad.

extracellular components, genetic studies have identiﬁed

However, pMad can also be detected in a crossvein that connects

two additional extracellular components that impinge on the longitudinal veins, although it does not express the ligand.

the gradient. Incorporation of these elements into the Interestingly, Sog is expressed on both sides of the future crossvein,

in a pattern that prefigures these veins. It was demonstrated that

experimental and theoretical framework demonstrated

Sog-mediated shuttling of Dpp leads to ligand deposition in the

that the heterodimeric Dpp/Scw form is the cardinal ligand

future crossvein. Although most of the ligand is anchored to the

molecule that is shuttled, and the extracellular molecule

longitudinal veins where it is produced, by association with the

Twisted gastrulation (Tsg) facilitates the formation of the receptor which is expressed at a high level by these cells, a small

complex between the heterodimeric ligand and Sog [14]. amount of free ligand is shuttled to induce the future crossvein. The

process requires cleavage of the complex by the Tld-related

Detailed computational analysis of the BMP patterning

protease that is expressed in this context.

gradient in the early Drosophila embryo also suggested

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Tribolium castenaum, dpp is normally expressed in a The mechanism that sets the position of maximal BMP

stripe along the anterior–posterior axis, but BMP activity signaling opposite the site of Chordin expression was

peaks at the dorsal side due to the orthogonal ventral further analyzed in sea urchin embryos. Elimination of

expression of Sog [24]. endogenous Chordin led to a broad activation pattern by

This observation was expanded by examining the rela- endogenous BMPs that was also prominent at sites that

tion between the sites of expression of the BMP ligand, the were furthest from the source of BMP expression [25].

shuttling molecule (Sog or its vertebrate homolog Chor- Thus, in this system, Chordin shapes the BMP gradient

din), and the resulting activation pattern in a broad range primarily by local inhibition of signaling, rather than by

of organisms, including cnidarians, arthropods, sea urch- facilitating the transport of ligand to the opposite pole.

ins, hemichordates, and chordates. The sites of ligand

expression vary, such that, in some cases, ligand expres- Self-organized shuttling

sion is ﬂanking to, or opposite, the shutting molecule, The prepattern that is generated by the expression proﬁle

whereas in others (e.g., sea urchin) the expression patterns of the shuttling molecule will dictate the ultimate site of

overlap. Yet, the resulting peak of patterning by BMP is the peak of morphogen accumulation for the BMP path-

always dictated by the Sog-homolog expression domain way. We can now ask whether the shuttling concept can be

and is generated in the region that is furthest from the used to generate a sharp morphogen gradient, even in

site of expression of this molecule [25]. In Xenopus embry- cases where there is no prepattern of the shuttling mole-

os, shuttling was veriﬁed and also shown to provide the cule.

ability to scale the morphogen gradient with embryo size A case in point is the earliest establishment of dorso-

(Box 2). ventral polarity in the Drosophila embryo. The initial

dorsoventral patterning of the early embryo is carried

out by a pathway whose transcripts are deposited into

Box 2. Shuttling in Xenopus: scaling pattern with size the egg during oogenesis. This pathway includes several

extracellular proteases that are produced in the early

The canonical shuttling model generates a sharp gradient of

embryo and secreted to the perivitelline space between

morphogen distribution that is robust to fluctuations in gene

dosage of the critical components. However, the absolute pattern the plasma and vitelline membranes. A cascade of protease

of morphogen distribution that is generated is predicted to be activation is triggered, in which activation of the ﬁnal

independent of the size of the embryos. In other words, the basic

protease termed ‘Easter’ (Ea) is restricted to the ventral

shuttling process does not provide ‘scaling’: the pattern of the

40% part of the perivitelline space, underlying the vitelline

morphogen gradient does not adjust to the size of the embryo or the

membrane region that was modiﬁed earlier during oogen-

organ. Scaling requires that the relative (rather than the absolute)

positions of the developing structures be fixed. It is not known esis by Pipe, a ventral sulfotransferase [26–28]. Activated

whether a scaling mechanism exists in Drosophila melanogaster Ea in turn cleaves the ligand Spa¨tzle (Spz), to generate a

embryos to adjust the pattern to variations in embryo size. In frog

form that will activate the Toll receptor on the embryonic

embryos, however, manipulations involving removal of cells

plasma membrane [29–31]. The ﬁnal step in response to

resulted in smaller but well-proportioned organisms, implying an

Toll signaling is the translocation of the transcription

underlying scaling mechanism [46].

Computational and experimental examination of patterning by factor Dorsal (Dl), which is a member of the NFkB family,

the BMP system in Xenopus embryos revealed not only the basic

into embryonic nuclei [32–34] (Figure 3).

shuttling mechanism, but also an additional twist that provides

According to the nuclear levels of Dl, which reﬂect the

scaling. Although patterning of the dorsoventral axis is largely

distribution of the activated ligand Spz within the perivi-

conserved between arthropods and chordates, the chordate axis is

inverted relative to that of arthropods, such that the shuttling telline space, distinct sets of zygotic genes will be induced

molecule Chordin (the Sog homolog) is expressed on the dorsal [35]. Interestingly, Dl nuclear localization forms a sharp

side, whereas the BMP activation gradient peaks on the ventral side

gradient, which is positioned well within the broad ventral

[47,48]. In addition to ventrally expressed BMP ligands, the system

domain initially deﬁned by pipe expression [36–38]. Be-

contains a BMP ligand termed ADMP, which displays features that

cause the Pipe domain is the cardinal cue that is provided

are diametrically opposed to the BMPs [49]. ADMP is expressed at

the dorsal side of the embryo and is repressed by BMP signaling. [39], how can a sharp gradient be generated within this

Yet, ADMP is an activating ligand that can be shuttled by Chordin domain?

and released on the ventral side, following cleavage of the complex

Although shuttling can be a powerful way of generating

by the Tld-homolog protease Xolloid.

a sharp gradient, it was not clear that it could operate in

The special features of ADMP provide Xenopus embryos with the

this context, because no shuttling molecule was identiﬁed,

ability to scale the BMP gradient along their dorsoventral axis in the

following manner. Both ADMP and other BMP ligands are shuttled and a prepattern of gene expression ﬂanking the Pipe

to the ventral region and contribute to the generation of an domain does not exist. Biochemical characterization of

activation gradient, which is focused on the ventral side. Production

Spz has demonstrated that it can exist in several distinct

of ADMP persists as long as the activation gradient does not reach

forms [40,41]. The full-length protein is inactive, because

the dorsal side of the embryo. As development proceeds and the

its N-terminal pro-domain prevents the C-terminal ligand

levels of ADMP rise, the activation gradient will eventually reach the

dorsal side of the embryo and turn off admp expression at some domain from binding the Toll receptor. Interestingly, upon

fixed level of signaling. In smaller embryos, shutting off admp

cleavage by Ea, the pro-domain and the ligand remain

expression is expected to occur sooner (and at a sharper gradient)

noncovalently associated, but the ligand can now be recog-

than in larger ones [43]. The profile of the gradient is adjusted to

nized by the receptor. Binding to the receptor releases the

embryo size by modulating the duration of production and, hence,

level of ADMP that is produced: more ADMP is produced in larger pro-domain.

embryos than in smaller ones. Shuttling of BMP in a Chordin- Computational analysis that took all these features into

dependent manner has been demonstrated experimentally [43].

account suggested that shuttling could operate, assuming

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(a) Spz cleavage by Ea

Spz precursor (inacve)

Ea cleavage

Vitelline membrane VM Pipe domain N-C Spz (acve) (b) N-C Spz diﬀuses laterally

Toll binding

C Spz (acve) and N Spz (free)

Reassociaon C Spz+N Spz VM (inacve)

(d) Preferenal ventral release of C Spz: Shuling DI nuclear gradient

Cleavage of N Spz C Spz (acve)

TRENDS in Genetics

Figure 3. Self-organized shuttling of Spa¨ tzle (Spz) generates the initial morphogen gradient in Drosophila embryos. The biochemical versatility of the Spz ligand allows it to

function in several distinct forms. It first appears as an inactive ligand. Following cleavage by Easter (Ea), the two parts remain noncovalentaly associated, and can bind the

Toll receptor. This leads to dissociation of the Spz pro-domain. The active ligand and pro-domain can reassociate to form an inactive complex. Possible cleavage of the pro-

domain within the complex will release the active ligand. Based on these properties, the following self-organized shuttling mechanism takes place. (a) Spz is cleaved by Ea

in the ventral domain that was defined by pipe expression in the follicle cells during oogenesis, to generate an active ligand that is still associated with the pro-domain. (b)

Some of the active ligand will diffuse laterally. (c) Binding of the cleaved ligand to Toll will activate the receptor and release the pro-domain. The ligand and pro-domain can

reassociate to form an inactive complex. A ventral flux of this complex will be generated due to saturation of receptors within the Pipe domain, as well as the ventrally

biased activity of a putative protease (purple scissors) that cleaves the pro-domain within this complex. (d) The release of free ligand by the protease will lead to preferential

accumulation of free ligand within the ventral-most region, to generate the sharp activation gradient of Toll, manifested in the gradient of Dl-nuclear localization.

Abbreviations: C Spz, ligand domain; N Spz, Spz pro-domain; N-C Spz, active Spz following cleavage by Ea; C Spz+N Spz, reassociated inactive complex; DI, Dorsal; VM,

ventral midline.

two additional features. First, the released pro-domain can displayed throughout the circumference of the embryo.

reassociate with free Spz ligand to form a distinct, inactive Once receptors at the ventral region become occupied by

complex. Second, the activity of a protease bearing Tld-like free ligand, the cleaved (but still associated) products can

features: ventrally biased and capable of cleaving the diffuse to the lateral region. Binding to the Toll receptor in

inactive complex, thereby destroying the pro-domain and all parts will release the pro-domain, and to some extent

releasing active free ligand [42] (Figure 3). also the active ligand, and allow their reassociation. Pro-

According to the model, the ﬁrst cleavage of the ligand duction of free pro-domain within the ventral region, which

by Ea is restricted to the ventral domain, whereas Toll is depends on binding of cleaved Spz to the receptor, will be

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Review Trends in Genetics June 2013, Vol. 29, No. 6

limited. Conversely, inhibitor production becomes more diffusion of ligands, free and in complex with the shuttling

prominent at the ﬂanking lateral regions. The initial ex- molecule, and to elucidate the distribution of receptors and

cess of inhibitor production in the lateral region initiates a extracellular elements and their precise role in shaping the

ventral ﬂux. Shuttling is further facilitated by the ventral- signaling gradient. The underlying signaling network may

ly biased protease, which degrades the inhibitory pro- also play a part in the observed robustness of the signaling

domain when in complex with the ligand. pathway.

When an active ligand molecule is released from the We expect the shuttling mechanism to represent the

inert complex in the ventrolateral region, it will rebind the exception rather than the rule in creating morphogen

pro-domain. Conversely, release in the ventral-most do- gradients because it requires speciﬁc prepatterning mech-

main will result in preferential binding to Toll, thus creat- anisms to provide the expression of the shuttling molecule

ing a mechanism of ligand shuttling. The ventrally at the borders of the patterned domain. Shuttling also

localized protease is required to bias the gradient of the relies on sufﬁciently rapid diffusion settings that will

free pro-domain, which will allow active ligand deposition readily distribute the shuttling complex. Such an environ-

to peak at the ventral midline. In conclusion, the versatility ment is indeed available in the early Drosophila embryo

of the Spz protein substitutes in this setting for missing within the perivitelline ﬂuid. At later developmental

features of a BMP-type system, allowing for implementa- stages, diffusion may be more restricted and conﬁned.

tion of a shuttling mechanism. For example, the sea urchin embryo represents a case

As attractive as the model appears, it is necessary to where the canonical elements of the BMP pathway are

verify experimentally the central predictions, represented used for local inhibition of the ligand, but are not utilized

by the structural and functional versatility of Spz. It was for shuttling. However, in the biological scenarios where

indeed demonstrated that the two fragments of Spz can shuttling does operate, it provides a powerful way to

reassociate and form an inactive complex. Most notably, it generate gradients that are sharp and robust to ﬂuctua-

was possible to redirect patterning in the embryo by an tions.

artificially generated flux of the pro-domain, thus confirm-

ing the basic requirement of the morphogen-shuttling Acknowledgments

We thank the reviewers for insightful comments that helped to shape this

mechanism [42]. The identiﬁcation of the predicted ven-

review. This work was supported by the European Research Council,

trally biased protease that would cleave the inactive com-

Israel Science Foundation, Minerva, and the Helen and Martin Kimmel

plex awaits further experiments. One option is that Ea,

Award for Innovative Investigations to N.B., who is the incumbent of the

which carries out the ﬁrst proteolytic cleavage of Spz in the Lorna Greenberg Scherzer Professorial Chair, and the Minerva Founda-

ventral domain, might also have a ‘moonlighting’ job as the tion to B-Z.S., who is an incumbent of the Hilda and Cecil Lewis

Professorial Chair in Molecular Genetics.

second ventrally biased protease.

Therefore, the shuttling mechanism can operate not

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