Advanced Review
Morphogen transport: theoretical
and experimental controversies
Takuya Akiyama1 and Matthew C. Gibson1,2∗
According to morphogen gradient theory, extracellular ligands produced from a
localized source convey positional information to receiving cells by signaling in a
concentration-dependent manner. How do morphogens create concentration gradients to establish positional information in developing tissues? Surprisingly, the
answer to this central question remains largely unknown. During development, a
relatively small number of morphogens are reiteratively deployed to ensure normal
embryogenesis and organogenesis. Thus, the intracellular processing and extracellular transport of morphogens are tightly regulated in a tissue-specific manner. Over the past few decades, diverse experimental and theoretical approaches
have led to numerous conflicting models for gradient formation. In this review, we
summarize the experimental evidence for each model and discuss potential future
directions for studies of morphogen gradients. © 2015 Wiley Periodicals, Inc.
How to cite this article:
WIREs Dev Biol 2015. doi: 10.1002/wdev.167
INTRODUCTION
B
efore the dawn of modern developmental biology,
experimental embryologists postulated that diffusing factors governed body plan formation during
development and regeneration.1–8 This view gained its
crucial experimental support in 1924, when Spemann
and Mangold demonstrated that the dorsal blastpore
lip of an unpigmented salamander embryo has the
capacity to direct distinct cell fates in neighboring
cells following transplantation into a pigmented
host.2 Although Lewis had already performed nearly
identical manipulations of amphibian embryos,9
Spemann and Mangold’s use of pigmentation to
mark graft cells definitively proved the existence of
a diffusible substance emanating from the blastpore
lip with the capacity to organize surrounding tissue.
Importantly, these experiments were not able to
distinguish between a morphogen and a signal relay
mechanism, but strongly supported the existence of
diffusible factors during development. In the years
∗ Correspondence
to: MG2@stowers.org
1 Stowers
Institute for Medical Research, Kansas City, MO, USA
of Anatomy and Cell Biology, The University of
Kansas School of Medicine, Kansas City, KS, USA
2 Department
Conflict of interest: The authors have declared no conflicts of
interest for this article.
that followed, evidence to support the idea that gradients could play a critical role in patterning emerged in
the literature,3,5,8 although molecules that might form
such gradients remained unknown until Drosophila
bicoid was discovered in 1988.10,11 By that time, Alan
Turing had coined the term ‘morphogen’ to describe
a hypothetical diffusible molecule (in 1952),4 and the
French Flag model for morphogen gradients had been
introduced by Lewis Wolpert (in 1969).6 According
to this model, cells in a developmental field interpret
their relative position based on the concentration of
a morphogen in the local area and adopt distinct cell
fates by expressing different target genes.12–14
As described above, Driever and NüssleinVolhard discovered the first morphogen, Bicoid,
in the Drosophila embryo.10,11 bicoid mRNAs are
maternally deposited anteriorly in the oocyte and
then translated to create an intracellular concentration gradient in the syncytial embryo, establishing
anterior–posterior (A/P) patterning.10,11,15–20 Soon
after Bicoid was described, the Drosophila Bone morphogenetic protein2/4 (BMP2/4)-like ligand Decapentaplegic (DPP) was identified as a secreted morphogen
in the developing wing imaginal disc.21–23 To date,
numerous other growth factors, such as Transforming
growth factor-𝛽 (TGF-𝛽), Hedgehog (Hh), Wingless
(Wg)/Wnt, and fibroblast growth factor (FGF), have
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Accumulation
Spreading
(d)
D
V
(c)
Xenopus
embyo
A
Drosophila
wind disc
Drosophila
embyo
(b)
P
(e)
D
A
Vertebrate
limb bud
(a)
V
P
FIGURE 1 | Two models of morphogen gradient formation. (a)
Schematic illustrations of the accumulation and spreading models for
the formation of morphogen gradients. (b–e) Examples of morphogen
gradients established by accumulation (b and c) and spreading (d and
e). (b and c) BMP morphogen gradients in Drosophila (b) and Xenopus
(c) embyros. (d) DPP gradient in the developing Drosophila wing disc.
(e) Shh gradient in the developing vertebrate limb bud. D: dorsal, V:
ventral, A: anterior, P: posterior.
been identified as morphogens during both vertebrate
and invertebrate development.12–14,24–26
As the number of identified morphogens has
increased, two distinct mechanisms for gradient formation have been observed (Figure 1(a)). In the case
of ‘accumulation’, morphogens are widely expressed
and subsequently transported to a local area to establish a concentration gradient (Figure 1(a)–(c)). For
example, a graded distribution of BMP patterns the
dorsoventral (D/V) axis in both insects and vertebrates. In Drosophila, DPP is uniformly expressed
in the dorsal half of the embryo and transported
to the dorsal midline in order to direct dorsal cell
fates27,28 (Figure 1(b)). In Xenopus embryos, BMP4
is widely produced but is shuttled to the ventral pole
to direct cell fate specification25 (Figure 1(c)). Conversely, in other developmental contexts, such as the
Drosophila wing disc and vertebrate limb bud, morphogens spread through a cellular field to establish
a concentration gradient (Figure 1(a), (d), and (e)).
During Drosophila wing development, DPP protein
produced by central cells along the A/P boundary disperses laterally to generate a concentration gradient,
which regulates both wing growth and patterning in
a concentration-dependent manner13,14 (Figure 1(d)).
Similarly, Sonic Hh (Shh) creates a concentration gradient by spreading from the posterior side of the
developing vertebrate limb bud to specify future digit
regions26 (Figure 1(e)), or from the notochord to specify cell identities in the developing neural tube.29
Despite a long history and many recent
advances, precisely how morphogens generate robust
concentration gradients in different developmental
contexts remains both controversial and inconclusive.
For example, while accumulation-based mechanisms
for BMP signaling in embryos have achieved some
degree of consensus,25,27,28 numerous competing models have been proposed to explain DPP spreading in
the singular case of the Drosophila wing disc. Importantly, despite being a central model for morphogen
study, the DPP gradient in the wing disc is rarely
assayed with direct methods. Instead, tagged forms
of DPP are overexpressed at nonphysiological levels,
which could perhaps be one explanation for conflicting findings from different studies. Further, beyond
the difference between spreading and accumulation,
most secreted signals are also regulated through intracellular processing,28,30–32 controlled trafficking,33
post-translational modification,28,30–33 and extracellular modulation.12–14,24,27,28 In order to highlight
current challenges to the field, here we describe recent
advances in understanding the molecular regulation of
morphogen production and transport, and also highlight incisive yet controversial experimental evidence
for morphogen gradient formation.
INTRACELLULAR REGULATION
OF MORPHOGENS
The first critical step for the formation of a morphogen
gradient is production. Based on the ‘source and sink
model’,7 a balance between morphogen production
(source) and degradation (sink) critically impacts the
final form of the gradient. As expected, if the degradation rate of a morphogen is consistent, the gradient can
expand by increasing the amount of production and
vice versa (Figure 2(a)). In addition, misexpression of
a morphogen induces an ectopic concentration gradient in the developmental field, resulting in patterning
defects34 (Figure 2(b)). Thus, to ensure normal development, morphogen production needs to be tightly
regulated at different levels.
Proteolytic Processing
Morphogens in the TGF-𝛽/BMP family are initially translated as long precursors consisting
of a prodomain and a highly conserved ligand
domain,28,32,35,36 (Figure 2(c)). After translation, they
form a dimer and are subsequently cleaved to liberate
a bioactive ligand for signaling. Recent studies have
© 2015 Wiley Periodicals, Inc.
WIREs Developmental Biology
(a)
Morphogen transport
(b)
Extracellular
space
(c)
BMP/TGF-β
Hh
Cytoplasm
Cleavage
Wnt
Lipid modification
Lipid
modification
Cleavage
Dimerization
N
C
N
C
N
C
FIGURE 2 | Morphogen production and gradient formation. (a)
Source-Sink model of morphogen gradient formation. (b) Misexpression
of a morphogen (red dotted circle) results in ectopic gradient formation.
The border between the morphogen expressing and receiving cells is
indicated by the dotted line. (c) Differential regulation of TGF-𝛽/BMP,
Hh, and Wnt morphogen production. Lipid and cholesterol
modifications are indicated in red and blue, respectively. N and C
represent N- and C-terminal sides of the proteins.
shed light on how the less conserved prodomain
contributes to the context-dependent behavior of
the ligand to precisely regulate TGF-𝛽/BMP signaling activity.28,32 For instance, some prodomains
have been shown to regulate ligand production
through lysosomal function. The Danio rerio TGF-𝛽
proteins Cyclops (Cyc) and Squint (Sqt) are essential for mesoderm and endoderm formation in
zebrafish embryos, and possess different signaling
activities.37–40 When their mRNAs are injected into
a single cell of 128–256-cell stage embryos, Cyc and
Sqt exhibit short- and long-range signaling activities,
respectively, as monitored by target gene expression.38
Serial deletions of the Cyc prodomain reveal a lysosomal targeting region responsible for this short-range
signaling activity, and removal of this domain results
in expanded Cyc target expression.41 The in vivo
distributions of Cyc-GFP and Sqt-GFP confirm their
previously identified signaling ranges, supporting the
idea that ligand production can critically influence
extracellular ligand gradient formation.42
The majority of TGF-𝛽/BMP proteins carry multiple proconvertase (e.g., Furin) cleavage sites.28,32,36
Several lines of evidence suggest that differential
cleavage can elicit context-dependent behavior of
these proteins.28,32,36,43–49 For example, sequential
cleavage of BMP4 plays a critical role in the ligand
production.43 The BMP4 prodomain contains two
Furin cleavage sites adjacent to the ligand domain: an
upstream S2 site and a downstream S1 site. Mutating
the S1 site causes a loss of BMP4 ligand production
due to a defect of the S2 cleavage. This result indicates
a critical requirement of the S1 site for the subsequent
S2 cleavage. Conversely, when the S2 site is mutated,
the BMP4 ligand and prodomain form an intermediate complex that is rapidly targeted for lysosomal
degradation after the S1 cleavage, resulting in reduced
ligand production. Supporting this model, injection of
mRNA encoding an S2-mutant form of BMP4 into a
single blastomere of 32-cell stage Xenopus embryos
produces a shorter activity gradient than wild-type
BMP4.43 Interestingly, mice harboring an S2 site mutation exhibit defects in the germ line, suggesting a
tissue-specific requirement of this cleavage site during normal development.45 A similar tissue-dependent
regulation of Drosophila DPP has been observed.48
DPP acts as a short- and long-range morphogen in
the Drosophila embryonic midgut and the developing wing disc, respectively. Overexpression and rescue experiments with a dpp mutant allele carrying an
S2 site mutation indicate that the long-range activity of DPP in the developing wing disc requires both
S1 and S2 cleavages, while only the S1 cleavage is
sufficient to induce DPP signaling in the embryonic
midgut.48 Additionally, recent studies have identified
an alternative Furin cleavage site (NS) within the
prodomains of the Drosophila BMP5/6/7-like proteins
Glass bottom boat (GBB) and Screw (SCW).28,32,49–51
In the case of GBB, cleavages at both the NS and
S1 sites produce two totally different sizes of bioactive GBB ligand with distinct signaling properties.49
Whereas the NS cleavage of SCW produces a nonfunctional ligand, a mutation in the NS site of SCW
influences dimer formation, thereby affecting BMP signaling activity.28,32,50,51 The identification of an alternative cleavage site in the BMP prodomain opens a
new avenue to address the long-standing question of
how BMP signaling establishes different signaling outputs in a context-dependent manner. For instance,
Drosophila wing patterning requires long-range BMP
signaling activity, while short-range activity is essential for the germline stem cell maintenance in the
Drosophila ovary. Although conventional cleavage
of BMP family proteins generates 100–140 amino
acid bioactive ligands, cleavage of the Drosophila
BMP5/6/7-like protein GBB at the alternative site
produces a larger active GBB ligand (328 amino
acids).28,32,49,50 In addition, this cleavage is regulated
in a tissue-dependent manner, and the larger GBB ligand has stronger signaling activity and a longer range
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than the smaller conventional GBB ligand when they
overexpressed.49 These results suggest the possibility
that differential cleavage may be responsible for differential signaling outputs in each tissue. Furthermore,
this alternative cleavage site is evolutionarily conserved, and mutations in this site are associated with
several diseases.28,49 Thus, regulation of BMP signaling through alternative protein cleavage sites may be
a common mechanism throughout animals, and it will
be of great importance to examine the endogenous distribution of alternative BMP ligands in future studies.
Lastly, in addition to alternative proteolytic
processing, TGF-𝛽/BMP family proteins can signal
as either homo- or heterodimers, which can dramatically impact their signaling capability.27,28,52–60
For instance, switching between Activin and Inhibin
depends on dimerization of the 𝛼- and 𝛽-subunits
of Inhibin. Activin is composed of two 𝛽-subunits
and enhances follicle-stimulating hormone synthesis,
while Inhibin, consisting of 𝛼/𝛽 heterodimers, downregulates the same process.56–60 Furthermore, heterodimers of BMP ligands play critical roles during
the early embryogenesis (DPP/SCW) and posterior
crossvein formation (DPP/GBB) in Drosophila.27,28
Heterodimers have a stronger affinity for extracellular BMP binding proteins [Short gastrulation (Sog),
Twisted gastrulation (Tsg), and Crossveinless] than
homodimers, which is an essential feature for proper
BMP tissue distribution.52–55 Indeed, DPP itself fails
to be transported in scw or gbb mutant conditions in
both developmental systems.54,55 Combined, differential cleavages and dimerization play essential roles in
modulating the TGF-𝛽/BMP signaling pathway in disparate developmental contexts.
Post-translational Modifications
In both vertebrates and invertebrates, the production of active Hh ligands requires two essential
intracellular processes: autoproteolysis and lipid
modification.30,31,61–68 Hh family proteins are
synthesized as inactive precursors consisting of a
N-terminal ligand domain (Hh-N) and an autocatalytic C-terminal domain (Hh-C; Figure 2(c)).
Although Hh-C does not have any signaling activity, it contains an intein protein splicing domain
and a steroid recognition region which catalyzes
cholesterol-dependent autoproteolysis in the endoplasmic reticulum (ER) to generate a C-terminally
cholesterol modified Hh-N.64–68 Hh-C is then rapidly
degraded through ER-associated degradation68 and
the cholesterol-linked Hh-N is further modified by
palmitate via the membrane-bound O-acyltransferase
(MBOAT) family protein Rasp/Hhat.30,31,61–63
Misregulation of post-translational modification
differentially impacts Hh signaling. First, mutations
affecting human Shh autoproteolysis are associated
with holoprosencephaly.69–71 Similarly, defective autoproteolysis of Drosophila Hh leads to a loss of signaling activity in vivo.66 Second, Dispatched (Disp),
which regulates Hh-N secretion, requires cholesterol
modification.72,73 In disp1 mutant mice, Shh signaling
activity is significantly reduced in the ventral neural
tube, leading to a defect in spinal cord patterning.72
Likewise, when disp mutant clones are generated in
Hh expressing cells in the developing Drosophila wing
disc, Hh protein accumulates within the mutant cells,
resulting in a narrower Hh gradient.73 Interestingly,
despite its similarity to the Hh receptor Patched, Disp
has no function in Hh signal transduction.73 Third,
palmitoylation of mouse Shh by Hhat/Rasp is essential
for long-range signaling in both the neural tube and
limb bud.74 Drosophila embryos lacking both maternal and zygotic Rasp protein show a strong segmental polarity defect similar to hh mutants.75,76 In addition, Hh target gene expression is significantly reduced
when rasp mutant clones are induced in Hh producing cells in the developing wing disc.75–78 Lastly, these
lipid modifications are important for Hh oligomerization, which enhances signaling activity and is critical for the gradient formation by solubilizing Hh
proteins.74,79–82
A tight connection between post-translational
modification and intracellular trafficking also regulates Wg/Wnt production33,83–85 (Figure 2(c)). Newly
synthesized Wg/Wnt proteins are acylated and glycosylated in the ER and transported to the Golgi.
They subsequently employ the cargo receptor Wntless/Evenness interrupted (Wls/Evi) for trafficking
from the Golgi to the plasma membrane. Wg/Wnt proteins have two fatty acid modifications: a saturated
palmitic acid to a conserved N-terminal cysteine (e.g.,
C93 of Wg, and C77 of Wnt3a) and an unsaturated
palmitoleic acid to a conserved internal serine (e.g.,
S239 of Wg, and S209 of Wnt3a). Porcupine (Por,
an MBOAT family protein) acylates Wg/Wnt family
proteins, although whether the enzyme is responsible
for both lipid modifications remains unclear. por was
originally identified as a segment polarity gene in the
Drosophila.86 Cells lacking Por protein show intracellular accumulation of Wg both in the embryo and
wing imaginal discs.86,87
The precise requirement for each lipid modification in Wg/Wnt secretion and signaling is still
debated. The palmitoleic modification of a serine
residue in Wg/Wnt is important for the interaction
with Wls/Evi and secretion.87,88 S209A mutant Wnt3a
proteins do not physically interact with Wls/Evi in
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Morphogen transport
HEK293 cells.88 Likewise, S239 of Drosophila Wg is
essential for recognition of Wls/Evi in the wing disc.87
However, Tang et al. recently demonstrated that both
lipid modifications are required for Wls/Evi interaction and Wg secretion.89 In the Drosophila S2 cell
culture system, Wg[C93A, S239A] shows a dramatic
reduction in its interaction with Wls/Evi and exhibits
a secretion defect, while Wg[C93A] and Wg[S239A]
individually behave like wild-type Wg. Importantly,
it is notable that the Drosophila atypical Wg/Wnt,
WntD, which lacks the conserved internal serine, is
secreted normally.90 This finding suggests an existence
of a Wls/Evi-independent mechanism for Wg/Wnt
secretion.
Palmitic acid modifications of the cysteine
residues at C77 and C93 are essential for Wnt3a
and Wg signaling activity, respectively.91,92 In mouse
Wnt3a, mutation of C77 to A causes a loss of signaling
activity without affecting secretion.91 In Drosophila
wing discs, overexpression of Wg[C93A] results in
accumulation of the defective ligand in the ER and is
not associated with the misexpression phenotypes typical of wild-type Wg.92 However, in contrast, a recent
study in the same tissue shows that palmitic acid modification of C93 is not required for Wg activity.89 In
these experiments, overexpression of Wg[C93A] using
dpp-Gal4 induces the expression of a target gene in a
manner indistinguishable from wild-type Wg, while
overexpressed Wg[S239A] exhibits reduced signaling
activity. Murine Wnt3a[C77A] can also activate the
pathway in some contexts,93 suggesting that this modification is not absolutely necessary for the signaling
activity. Rather, the requirement for palmitoylation
may depend on the specific developmental context.
MORPHOGEN TRANSPORT
According to morphogen gradient theory, secreted ligands disperse into a morphogenetic field and establish a concentration gradient. To date, several distinct
models have been proposed for morphogen dispersal. Based on their characteristics, they are grouped
into several categories.12–14,24,94,95 ‘Active diffusion’
(e.g., planar transcytosis and cytonemes) suggests the
use of a cellular mechanism for gradient formation
(Figure 3). ‘Free diffusion’ and ‘restricted extracellular diffusion’ [e.g., heparan sulfate proteoglycan
(HSPG)-mediated transport] attribute gradient formation to a mechanism that does not require direct cellular energy (Figure 4). Intriguingly, although graded
information generated by morphogens is a common
developmental mechanism, it is increasingly clear that
developmental systems employ different means to the
same end.
(a)
(b)
Planar transcytosis
Cytoneme
Ectopic DPP
Dynamin mutant clone
(c)
FIGURE 3 | Planar transcytosis and cytonemes in gradient
formation. Models of transcytosis (a) and cytoneme (b) mediated
gradient formation and experimental evidence for each. (a) In planar
transcytosis, morphogens are transferred by a sequence of endocytic
and exocytic events (top). Morphogen transport is blocked when
endocytosis is inhibited with a dynamin mutant (bottom, see the text
for detail). (b) Cytonemes are proposed to transfer morphogens by a
contact-dependent mechanism (top). Ectopic morphogen expression
induces the formation of cytonemes, which orient toward the ectopic
morphogen source (middle). (c) Physical interaction between the
morphogen expressing cell and cytonemes projected from the
receiving cell.
Owing to research efforts on morphogen
transport during the past few decades, molecular
mechanisms underlying the accumulation of morphogens have been well characterized (see Figure 1(a)).
For instance, D/V patterning in the early Drosophila
embryo requires the BMP morphogen gradient, which
is mediated by the shuttling of two BMP ligands, DPP
and SCW (Figure 1(b)).27,28,53,54 DPP is expressed
uniformly in the dorsal half of the embryo and forms
a dimer with SCW. After secretion, Sog and Tsg
proteins bind to DPP/SWC heterodimers, inhibit their
receptor interaction, and transport them to the dorsal
midline, where these protein complexes encounter
the BMP-1 family metalloprotease Tolloid (Tld). Tld
cleaves Sog to release the DPP/SCW heterodimer to
create the BMP morphogen gradient at the dorsal
midline. As expected, mutations in these extracellular BMP transporters cause a loss of proper BMP
morphogen gradient formation, resulting in D/V patterning defects. In addition, during Drosophila pupal
development, a similar mechanism is employed for
posterior crossvein formation.27,28,52,55 Importantly,
these extracellular transporters are evolutionarily
conserved and BMP shuttling plays critical roles in
D/V patterning in vertebrate embryos, such as Xenopus25 (Figure 1(c)). By contrast, the molecular basis
for the spreading of morphogens remains unclear and
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(a)
(b)
HSPG-mediated transport
Before
FRAP
Free diffusion
Photobleach
HSPG mutant clone
Fast
Slow
FIGURE 4 | Models of free diffusion and HSPG-mediated transport.
Models of free diffusion (a) and HSPG-mediated transport (b) in
morphogen gradient formation and experimental evidence for each.
(a) Schematic illustration of the free diffusion (top). For free diffusion,
only a small fraction of morphogen diffuses freely in the extracellular
space. Spatial FRAP distinguishes between free diffusion and the other
models (bottom, see the text for detail). The efficiency of florescence
recovery between the two windows (entire region in the red dotted line
and central region in the green dotted line) is compared.
(b) Morphogens are transferred by extracellular HSPGs (top). Using
mutant clones, morphogens are not able to form a gradient across cells
lacking HPSGs (bottom).
controversial (see Figure 1(a)). Even in the case of
Drosophila DPP (Figure 1(d)), perhaps the leading
model for morphogen analysis, the mechanisms of
gradient formation are still hotly debated. Therefore,
to highlight the essential concepts and controversies,
in this section we mainly focus on the molecular
mechanisms for the spreading of morphogens through
a tissue.
Transcytosis
Morphogen dispersion by transcytosis utilizes endocytic components96–104 (Figure 3(a)). In this model,
morphogens spread through a field of cells by a
sequence of reiterated endocytic and exocytic events.
Briefly, morphogens on the cell surface are endocytosed in a Dynamin-dependent manner and are
subsequently exocytosed into the extracellular space.
According to the direction of the transport, two types
of transcytosis have been characterized: apico-basal
transcytosis and planar transcytosis.
In the developing Drosophila wing disc, Wg
is expressed in cells along the D/V boundary. The
secreted ligand then moves dorsally and ventrally to
pattern the D/V axis. Although Wg proteins are mainly
secreted apically, they generate a long-range gradient
basolaterally.103,104 This indicates the importance of
apico-basal transcytosis for gradient formation. When
endocytosis is blocked in the dorsal compartment of
the wing disc by expressing a dominant-negative form
of Rab5, extracellular Wg proteins accumulate on
both the apical and basal sides of the epithelium and
fail to generate the proper gradient.104 Overexpression
of DLP, a glycosylphosphatidylinositol (GPI) anchored
HSPG, enhances translocation of Wg proteins from
both apical and basal surfaces to the basolateral
domain.104
Transcytosis in the plane of the epithelium
could also play critical roles in morphogen gradient
formation.96–102 For example, the Wg gradient regulates segmental pattern formation (ventral denticle
belts) in the Drosophila embryo, and this gradient
requires planar transcytosis. Specifically, inhibiting
endocytosis by expressing a dominant-negative form
of Dynamin in the receiving cells results in a loss of
Wg movement into the cells and a segmental polarity defect.96–98 This transport mechanism has also
been proposed to underlie generation of the DPP morphogen gradient formation in the developing wing
disc99–102 (Figure 3(a)). Supporting this model, DPP
tagged with GFP (GFP-DPP) expressed in the stripe
of dpp expressing cells using the GAL4/UAS system
strongly accumulates around clones of cells lacking
Thickveins (Tkv; a BMP type I receptor), indicating
the importance of receptor-mediated DPP internalization for DPP transport.100 In addition, it is reported
that GFP-DPP does not pass through dynamin mutant
cell clones (shibirets1 ), resulting in the observation of
a ‘shadow’ behind the clones (Figure 3(a)). Moreover, the GFP-DPP diffusion coefficient determined
by fluorescence recovery after photobleaching (FRAP)
is reduced when shibire function is partially disrupted (0.06 μm2 /second versus 0.12 μm2 /second in
control).102
Combined, the results above suggest that DPP
is transferred from cell to cell by a combination
of receptor-mediated internalization and transcytosis. However, there are several arguments supporting alternative interpretations. First, Belenkaya et al.
repeated the shibire shadow experiment and report
that extracellular GFP-DPP is able to move across
the cells lacking Dynamin.105 A possible explanation for these conflicting results lies in the detailed
experimental conditions: the first study used 34∘ C to
eliminate Dynamin activity, while the latter handled
the shibirets experiments at 32∘ C.100,105 This difference is critical since culture of shibirets1 animals at
32∘ C only causes a partial loss of Dynamin activity.102
Another line of evidence against planar transcytosis is that cells lacking tkv are generally eliminated
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Morphogen transport
from wing epithelia due to the ectopic expression of
Brinker (Brk)106–108 and thus tkv mutant clones may
not reflect normal GFP-DPP behavior.106 Indeed, the
extracellular distribution of GFP-DPP is not affected
in tkv brk double mutant clones,106 indicating that
receptor-mediated endocytosis is not required for DPP
dispersion. Lastly, if Tkv-mediated endocytosis were
essential for DPP transcytosis, a reduction of Tkv function would be expected to result in a narrower morphogen gradient. However, tkv/+ wing discs exhibit an
expanded DPP/BMP activity gradient, while tkv overexpression in the posterior compartment reduces its
size.109 These results may emphasize the importance of
Tkv-mediated endocytosis for DPP degradation, but
not DPP movement. In sum, the evidence supporting
Tkv-mediated transcytosis as a mechanism for DPP
gradient formation remains inconclusive. Looking forward, it will be interesting to test whether altering Tkv
levels can influence the diffusion coefficient of DPP,
as does a partial loss of Dynamin activity. Ultimately,
crucial insight will come from further studies to probe
the endogenous regulation of ligand and receptor trafficking without wholesale disruption of the endocytic
machinery.
Cytonemes
Cytonemes, long cellular extensions, represent a
completely distinct model for the formation of
morphogen gradients (Figure 3(b)). In this case,
morphogens are proposed to be directly delivered
from the producing cells to the receiving cells via
a contact-dependent mechanism.110–119 Cytonemes
were initially identified through GAL4 enhancer trap
screening in Drosophila.115 While monitoring GAL4
expression patterns with a cytosolic GFP reporter,
one line exhibited very thin cellular projections emanating from the GFP-expressing cells. Based on their
characteristic features (cytoplasmic extension and
thread-like structure), these cellular projections were
termed cytonemes [cyto + neme (thread in Greek)].
Cytonemes are filopodial protrusions, which contain
actin filament bundles oriented with their plus ends
at the tips of filopodia. Moreover, cytonemes are
thought to be highly specialized for each signaling
pathway.112 They differ in size (length and thickness),
localization, contain distinct signaling receptors, and
respond differently to each signaling molecule.
Cytonemes were recently reported to play a critical role in Hh gradient formation in the Drosophila
wing disc.116 While originally reported as apical
structures, additional cytonemes are found on the
basal side of wing disc epithelium where they transport Hh proteins to form a gradient. Shortening the
cytonemes by inhibiting actin polymerization in the
Hh producing cells results in a narrower Hh gradient
compared with wild-type. This suggests that Hh transport via cytonemes/filipodia is critical for proper gradient formation. Furthermore, cytonemes are not able
to cross clones of cells lacking HSPGs, highlighting
the previously reported requirement of HSPGs for Hh
transport.120–122 However, the molecular mechanism
by which cytonemes utilize HSPGs on the receiving
cells remains elusive. Interestingly, a similar transport
mechanism has been reported for Shh transport in
the chick limb bud.119 Shh produced in the posterior region of the limb bud generates a concentration gradient required for proper digit patterning.26
The posterior Shh-expressing cells extend cytonemes
into the anterior region, and Shh proteins travel along
these cellular extensions to reach the receiving cells.119
These results emphasize the importance of this evolutionarily conserved mechanism for Hh transport.
However, molecular mechanisms by which the orientation of cytonemes is controlled and how Hh
transport via cytonemes creates a precise concentration gradient remain elusive.
Although it has been proposed that cytonemes
function in DPP transport in the Drosophila wing
disc, it is still unclear whether cytonemes influence
formation of the DPP morphogen gradient.110–115
In contrast with Hh cytonemes, DPP cytonemes
extend along the apical surface of the wing disc
epithelium, projecting toward a central domain
where DPP is expressed along the A/P compartment boundary.111,112,114,115 This orientation of the
cytonemes is disrupted when either DPP expression is reduced, or when uniform DPP expression is
induced by heat-shock.112,114 Additionally, ectopic
expression of DPP is able to induce the formation of
oriented cytonemes112 (Figure 3(b)). Physical contact
between the DPP expressing cells and the receiving
cells through cytonemes was reported based on application of the GFP Reconstitution Across Synaptic
Partner technique111 (Figure 3(c)). Expression of
membrane-localized CD4-GFP1-10 or CD4-GFP11
in dpp expressing and receiving cells, respectively,
produces a reconstituted GFP signal in dpp expressing
cells. Further, cytonemes contain Tkv-GFP receptor
puncta, which move in both anterograde and retrograde directions (5–7 μm/second).114 Altogether, these
results support the idea that cytonemes transport
DPP from its expressing cells to the receiving cells in a
contact-dependent manner through receptor-mediated
retrograde trafficking. However, as described above,
the role of cytonemes in DPP gradient formation is
still speculative. It will be critical to examine how
interfering with cytoneme function (e.g., inhibition
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Advanced Review
of actin polymerization) influences DPP morphogen
gradient formation in the future.
Free Diffusion
Free diffusion of morphogens is the simplest mechanism suggested to create concentration gradients, and
best represents the theoretical origins of the morphogen concept itself7,123–125 (Figure 4(a)). Intuitively,
if free-diffusing morphogens are relatively stable, their
extracellular distribution will become uniform across
the tissue, as does secreted GFP.100,125 It follows that
for diffusion to work as a mechanism of gradient formation, morphogen molecules would have to exhibit
both rapid dispersal and efficient elimination from the
extracellular space. Testing this idea, the global diffusion coefficient for several morphogens has been
measured by FRAP, revealing diffusion rates too slow
to support free diffusion.12,42,102,126 Arguably, however, this approach may not be suitable to investigate
morphogen transport.124 When morphogen transport
is dominated by other factors such as degradation
and extracellular trapping, FRAP cannot distinguish
200-fold differences in diffusion coefficients [0.1 μm2
(slow) versus 20 μm2 (fast)].124
Recently, FGF8 morphogen gradient formation
in zebrafish embryos was studied using fluorescence
correlation spectroscopy (FCS) to monitor local
diffusion coefficients.125 When mRNAs encoding
FGF8-GFP are injected into embryos, the majority
of FGF8-GFP moves rapidly relative to what has
been observed by FRAP (53 μm2 /second).125 These
findings suggest that overexpressed FGF8 proteins can
diffuse freely through the extracelluar space. Further,
manipulating endocytic activity in the receiving cells
alters the width of the FGF8 morphogen gradient.
The FGF8 gradient is expanded when endocytosis
is inhibited and vice versa. Altogether, these results
suggest that the FGF8 gradient is established through
free diffusion coupled with rapid degradation by a
receptor-mediated endocytosis.
Contrasting
with
the
transcytotic
or
cytoneme-based mechanisms described above, recent
evidence also supports a role for free diffusion in
creating the DPP gradient in the Drosophila wing
disc.124 DPP tagged with the photoconvertible protein
Dendra2 (Dendra2-DPP) diffuses rapidly away from
the endogenous dpp expression domain at the compartmental boundary (21 μm2 /second). In addition,
when Dendra2-DPP is pulse-labeled by photoconversion, no obvious dispersal is detected outside the area
of photoactivation, consistent with the idea that only
a small fraction of DPP (1–3% of total DPP) rapidly
diffuses in the extracellular space and majority of
DPP proteins are present within the cell or on the
cell surface. Furthermore, data from spatial FRAP
experiments support the free diffusion hypothesis
(Figure 4(a)). In brief, after photobleaching a large
area, the efficiency of flourescence recovery in the
central part of the bleached domain is compared
with that of the entire region. If morphogen transport is fast (free diffusion), fluorescence should be
recovered simultaneously in both areas. Conversely,
if morphogen transport is slow, recovery of the central region will be delayed compared to the whole
area. In experiments focused on Dendra2-DPP in the
wing disc, fluorescence recovery shows no obvious
difference between the two areas.124 However, other
spatial FRAP experiments support the transcytosis
model.102 One possible explanation for this discrepancy is that DPP was tagged with different fluorescent
proteins in each case. Further, each transgenic line
has different expression levels due to the positional
effect of the insertion site, which could influence
the rate of DPP diffusion. Indeed, GFP-DPP has a
slower local diffusion coefficient (10 μm2 /second)
than Dendra2-DPP.124
As described above, a fast local diffusion coefficient measured by FCS supports the free diffusion
model. However, the local diffusion rate alone is not
enough to distinguish between the free diffusion and
other transport models (e.g., HSPG-mediated transport) since neither degradation nor trapping by extracellular proteins necessarily affects local diffusion.
Furthermore, it has been shown that both DPP and
FGF8 morphogen gradients are critically affected by
extracellular HSPGs (see below).12,105,109,127 Therefore, it remains unclear whether free diffusion itself is
sufficient to establish a morphogen gradient.
HSPG-mediated Transport
Several lines of evidence have demonstrated that
HSPGs play critical roles in the formation of morphogen gradients12,105,109,120,127–130 (Figure 4(b)).
HSPGs consist of a protein core with HS chains
covalently attached, and are abundant both on the
cell surface and in the extracellular matrix. Based on
their protein structure, HSPGs are categorized into
three groups: syndecans, glypicans, and perlecans.120
Both syndecans and glypicans are localized on the
cell surface through a transmembrane or GPI anchor,
respectively, while perlecans are secreted into the
extracellular space.120 All three classes are evolutionarily conserved from worms to humans, and act as
either inhibitors or facilitators to fine tune morphogen
gradients.
HPSGs negatively regulate BMP4 morphogen
gradient formation during the embryonic development
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Morphogen transport
of Xenopus.128 The BMP4 ligand possesses a highly
conserved stretch of basic residues in the N-terminal
domain. When this region is eliminated, BMP4 is not
able to interact with heparin (a highly sulfated HS
chain) and the defective ligand migrates further than
its wild-type counterpart, suggesting that HSPGs trap
BMP4 proteins through this domain to limit their
diffusion. Consistent with this, wild-type BMP4 is
able to generate a wider gradient when embryos are
treated with Heparitinase to remove HS chains from
the HSPG protein cores. Additionally, injection of
exogenous heparin into zebrafish embryos increases
the global diffusion coefficient of FGF8-GFP by competing for binding with endogenous HSPGs compared
to the absence of exogenous heparin.12 These results
suggest that HPSGs trap FGF8 proteins on the cell
surface to restrict their dispersal. Finally, the movement of individual FGF2 molecules has been tracked
using photothermal heterodyne imaging in vitro.130
This technique allows for detection and tracking of
nanometer-sized objects in real time. Using FGF2
labeled with gold nanoparticles, HSPGs were shown
to regulate FGF2 diffusion in the pericellular matrix
by reversible interaction with HS chains.
HSPGs are also required for facilitating
morphogen gradient formation.105,109,120,127 For
instance, proper DPP morphogen gradient formation
in the developing Drosophila wing disc requires
HSPGs.105,109,127 Wing discs carrying a mutation
of the glypican dally exhibit a narrower DPP/BMP
activity (pMAD) gradient compared to controls.127 In
addition, extracellular GFP-DPP proteins are not able
to diffuse across HSPG mutant clones105 (Figure 4(b)).
These results indicate a critical requirement for HSPGs
in DPP transport. Supporting this view, DPP physically interacts with Dally and a mutant form of DPP
lacking this ability has a shorter half-life, resulting in
a failure of proper gradient formation.109 Moreover,
Dally antagonizes the effect of Tkv on DPP signaling.
Since Tkv regulates the levels of DPP protein by
receptor-mediated endocytosis, HSPGs may promote
gradient formation by inhibiting Tkv-dependent
degradation of DPP. Recent studies have identified
new secreted feedback regulators of DPP signaling:
Larval translucida (Ltl) acts as an antagonist, while
Pentagone (Pent) promotes signaling activity.131,132
Interestingly, these modulators both physically interact with HSPGs, but not DPP. While it is likely that
Pent and Ltl act together with HSPGs to modulate the
formation of the DPP morphogen gradient, the molecular mechanisms remain elusive. Adding an additional
layer of complexity, BMP signaling directly regulates
the expression of Dally, Pent, and Ltl.127,131,132 These
and yet unknown feedback loops are likely to play an
important role in shaping the final DPP morphogen
gradient.
CONCLUSIONS
‘It has been a great surprise and of considerable
importance to find that most embryonic fields seem to
involve distance of less than 100 cells, and often less
than 50’—Lewis Wolpert6
Inspired by this quote, Francis Crick proposed
that diffusion was a plausible mechanism for the
generation of morphogen gradients during early
development.7 Today, we still do not completely
understand whether and how veritable extracellular
morphogen gradients are established and maintained during development. Since the early theoretical
work of Turing, Wolpert and Crick,4,6,7 a number
of secreted signals have been identified and several
models for the formation of morphogen gradients
have been suggested.12–14,24–26 In some cases, different
research groups have proposed completely distinct
mechanisms to explain the formation of a single morphogen gradient, perhaps due to technical variations.
To date, only a few endogenous morphogen gradients
have been subjected to direct analysis. In a majority of
studies, secreted morphogens have been tagged with
fluorescent proteins and overexpressed in transgenic
animals or injected as mRNA into early embryos.
As described above, morphogen production is tightly
regulated at multiple levels, such as precursor cleavage and post-translational modifications, to create the
proper gradient. Therefore, it is possible (and perhaps
likely) that overexpression creates a nonphysiological
morphogen gradient and thus confounding results.
To minimize the influence of experimental variation
and the vagaries of transgene overexpression, it will
be crucial for future studies to visualize and study
endogenous morphogen gradients. Along these lines,
Alexandre et al. recently replaced the endogenous wg
locus with a membrane-tethered form in Drosophila.
Although the resulting homozygous mutant flies show
a developmental delay, they are viable and have normal adult morphology.133 In contrast to current Wg
morphogen theory, this unexpected result suggests
that Wg migration is dispensable for both patterning
and growth. Future studies using similar approaches
will undoubtedly continue to shed light on the molecular mechanisms of morphogen gradient formation.
Lastly, although majority of current morphogen
theories are based on production from a single
source, it is becoming clear that a single source
of morphogen is not always sufficient to generate
a proper gradient. For example, during Drosophila
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Advanced Review
wing disc development, Wnt4 and Wnt6 are expressed
in addition to Wg, and these mutants exhibit wing
phenotypes.134,135 Furthermore, another BMP ligand, GBB, is expressed uniformly in the developing Drosophila wing disc, and wing discs carrying a
mutation in gbb show a narrower DPP/BMP activity
gradient.136,137 These results indicate that both BMP
ligands, DPP and GBB, are required for proper gradient formation. Thus, to completely understand the
nature of morphogen gradients in the future, it will be
essential to reconsider models that are based on single
sources of morphogen for gradient formation.
ACKNOWLEDGMENTS
We thank A. Fritz for a critical reading of the manuscript, L. Gutchewsky for administrative support, and
members of the Gibson lab for discussions and advice. This work was supported by the Stowers Institute for
Medical Research.
REFERENCES
1. Morgan TH. Regeneration and liability to injury.
Science 1901, 14:235–248.
2. Spemann H, Mangold H. Induction of embryonic primordia by implantation of organizers from a different
species. Roux Arch Entw Mech 1924, 100:599–638.
3. Saunders JW Jr. The proximo-distal sequence of origin
of the parts of the chick wing and the role of the
ectoderm. J Exp Zool 1948, 108:363–403.
4. Turing AM. The chemical basis of morphogenesis. Philos Trans R Soc Lond Ser B Biol Sci 1952, 237:37–72.
5. Locke M. The cuticular pattern in an insect, Rhodnius
prolixus. J Exp Biol 1959, 36:459–477.
6. Wolpert L. Positional information and the spatial
pattern of cellular differentiation. J Theor Biol 1969,
25:1–47.
15. Driever W. The bicoid morphogen papers (II): account
from Wolfgang Driever. Cell 2004, 116:S7–S9 2 p
following S9.
16. Nusslein-Volhard CN. The bicoid morphogen papers
(I): account from CNV. Cell 2004, 116:S1–S5 2 p
following S9.
17. Driever W, Ma J, Nusslein-Volhard C, Ptashne M.
Rescue of bicoid mutant Drosophila embryos by
bicoid fusion proteins containing heterologous activating sequences. Nature 1989, 342:149–154.
18. Struhl G, Struhl K, Macdonald PM. The gradient morphogen bicoid is a concentration-dependent transcriptional activator. Cell 1989, 57:1259–1273.
19. Gibson MC. Bicoid by the numbers: quantifying a
morphogen gradient. Cell 2007, 130:14–16.
7. Crick F. Diffusion in embryogenesis. Nature 1970,
225:420–422.
20. Grimm O, Coppey M, Wieschaus E. Modelling the
bicoid gradient. Development 2010, 137:2253–2264.
8. Stumpf HF. Mechanism by which cells estimate their
location within the body. Nature 1966, 212:430–431.
21. Nellen D, Burke R, Struhl G, Basler K. Direct and
long-range action of a DPP morphogen gradient. Cell
1996, 85:357–368.
9. Lewis WH. Transplantation of the lips of the blastopore in Rana palustris. Am J Anat 1907, 7:137–143.
10. Driever W, Nusslein-Volhard C. The bicoid protein determines position in the Drosophila embryo
in a concentration-dependent manner. Cell 1988,
54:95–104.
11. Driever W, Nusslein-Volhard C. A gradient of bicoid
protein in Drosophila embryos. Cell 1988, 54:83–93.
12. Muller P, Rogers KW, Yu SR, Brand M, Schier
AF. Morphogen transport. Development 2013, 140:
1621–1638.
22. Lecuit T, Brook WJ, Ng M, Calleja M, Sun H,
Cohen SM. Two distinct mechanisms for long-range
patterning by Decapentaplegic in the Drosophila wing.
Nature 1996, 381:387–393.
23. Posakony LG, Raftery LA, Gelbart WM. Wing formation in Drosophila melanogaster requires decapentaplegic gene function along the anterior-posterior
compartment boundary. Mech Dev 1990, 33:69–82.
24. Muller P, Schier AF. Extracellular movement of signaling molecules. Dev Cell 2011, 21:145–158.
13. Restrepo S, Zartman JJ, Basler K. Coordination of
patterning and growth by the morphogen DPP. Curr
Biol 2014, 24:R245–R255.
25. Shilo BZ, Haskel-Ittah M, Ben-Zvi D, Schejter ED,
Barkai N. Creating gradients by morphogen shuttling.
Trends Genet 2013, 29:339–347.
14. Affolter M, Basler K. The Decapentaplegic morphogen
gradient: from pattern formation to growth regulation.
Nat Rev Genet 2007, 8:663–674.
26. McGlinn E, Tabin CJ. Mechanistic insight into how
Shh patterns the vertebrate limb. Curr Opin Genet Dev
2006, 16:426–432.
© 2015 Wiley Periodicals, Inc.
WIREs Developmental Biology
Morphogen transport
27. O’Connor MB, Umulis D, Othmer HG, Blair SS.
Shaping BMP morphogen gradients in the Drosophila
embryo and pupal wing. Development 2006,
133:183–193.
28. Wharton KA, Serpe M. Fine-tuned shuttles for bone
morphogenetic proteins. Curr Opin Genet Dev 2013,
23:374–384.
29. Cohen M, Briscoe J, Blassberg R. Morphogen interpretation: the transcriptional logic of neural tube patterning. Curr Opin Genet Dev 2013, 23:423–428.
30. Gradilla AC, Guerrero I. Hedgehog on the move: a
precise spatial control of Hedgehog dispersion shapes
the gradient. Curr Opin Genet Dev 2013, 23:363–373.
31. Briscoe J, Therond PP. The mechanisms of Hedgehog
signalling and its roles in development and disease. Nat
Rev Mol Cell Biol 2013, 14:416–429.
32. Constam DB. Regulation of TGFbeta and related
signals by precursor processing. Semin Cell Dev Biol
2014.
33. Clevers H, Nusse R. Wnt/beta-catenin signaling and
disease. Cell 2012, 149:1192–1205.
34. Zecca M, Basler K, Struhl G. Sequential organizing activities of engrailed, hedgehog and decapentaplegic in the Drosophila wing. Development 1995,
121:2265–2278.
35. Massague J. The transforming growth factor-beta
family. Annu Rev Cell Biol 1990, 6:597–641.
36. Bragdon B, Moseychuk O, Saldanha S, King D, Julian
J, Nohe A. Bone morphogenetic proteins: a critical
review. Cell Signal 2011, 23:609–620.
37. Feldman B, Gates MA, Egan ES, Dougan ST, Rennebeck G, Sirotkin HI, Schier AF, Talbot WS.
Zebrafish organizer development and germ-layer
formation require nodal-related signals. Nature 1998,
395:181–185.
38. Chen Y, Schier AF. The zebrafish Nodal signal
Squint functions as a morphogen. Nature 2001, 411:
607–610.
39. Schier AF. Nodal morphogens. Cold Spring Harb
Perspect Biol 2009, 1:a003459.
40. Langdon YG, Mullins MC. Maternal and zygotic
control of zebrafish dorsoventral axial patterning.
Annu Rev Genet 2011, 45:357–377.
41. Tian J, Andree B, Jones CM, Sampath K. The
pro-domain of the zebrafish nodal-related protein
cyclops regulates its signaling activities. Development
2008, 135:2649–2658.
42. Muller P, Rogers KW, Jordan BM, Lee JS, Robson D,
Ramanathan S, Schier AF. Differential diffusivity of
nodal and lefty underlies a reaction-diffusion patterning system. Science 2012, 336:721–724.
43. Cui Y, Hackenmiller R, Berg L, Jean F, Nakayama T,
Thomas G, Christian JL. The activity and signaling
range of mature BMP-4 is regulated by sequential
cleavage at two sites within the prodomain of the
precursor. Genes Dev 2001, 15:2797–2802.
44. Degnin C, Jean F, Thomas G, Christian JL. Cleavages within the prodomain direct intracellular trafficking and degradation of mature bone morphogenetic
protein-4. Mol Biol Cell 2004, 15:5012–5020.
45. Goldman DC, Hackenmiller R, Nakayama T, Sopory
S, Wong C, Kulessa H, Christian JL. Mutation of an
upstream cleavage site in the BMP4 prodomain leads
to tissue-specific loss of activity. Development 2006,
133:1933–1942.
46. Sopory S, Nelsen SM, Degnin C, Wong C, Christian JL.
Regulation of bone morphogenetic protein-4 activity
by sequence elements within the prodomain. J Biol
Chem 2006, 281:34021–34031.
47. Kunnapuu J, Bjorkgren I, Shimmi O. The Drosophila
DPP signal is produced by cleavage of its proprotein
at evolutionary diversified furin-recognition sites. Proc
Natl Acad Sci USA 2009, 106:8501–8506.
48. Sopory S, Kwon S, Wehrli M, Christian JL. Regulation of Dpp activity by tissue-specific cleavage of an
upstream site within the prodomain. Dev Biol 2010,
346:102–112.
49. Akiyama T, Marques G, Wharton KA. A large bioactive BMP ligand with distinct signaling properties is
produced by alternative proconvertase processing. Sci
Signal 2012, 5:ra28.
50. Fritsch C, Sawala A, Harris R, Maartens A, Sutcliffe
C, Ashe HL, Ray RP. Different requirements for
proteolytic processing of bone morphogenetic protein
5/6/7/8 ligands in Drosophila melanogaster. J Biol
Chem 2012, 287:5942–5953.
51. Kunnapuu J, Tauscher PM, Tiusanen N, Nguyen M,
Loytynoja A, Arora K, Shimmi O. Cleavage of the
Drosophila screw prodomain is critical for a dynamic
BMP morphogen gradient in embryogenesis. Dev Biol
2014, 389:149–159.
52. Shimmi O, Ralston A, Blair SS, O’Connor MB. The
crossveinless gene encodes a new member of the
Twisted gastrulation family of BMP-binding proteins
which, with Short gastrulation, promotes BMP signaling in the crossveins of the Drosophila wing. Dev Biol
2005, 282:70–83.
53. Shimmi O, Umulis D, Othmer H, O’Connor MB.
Facilitated transport of a Dpp/Scw heterodimer by
Sog/Tsg leads to robust patterning of the Drosophila
blastoderm embryo. Cell 2005, 120:873–886.
54. Wang YC, Ferguson EL. Spatial bistability of
Dpp-receptor
interactions
during
Drosophila
dorsal-ventral patterning. Nature 2005, 434:229–234.
55. Matsuda S, Shimmi O. Directional transport and
active retention of Dpp/BMP create wing vein patterns
in Drosophila. Dev Biol 2012, 366:153–162.
56. Robertson DM, Foulds LM, Leversha L, Morgan FJ,
Hearn MT, Burger HG, Wettenhall RE, de Kretser
DM. Isolation of inhibin from bovine follicular
© 2015 Wiley Periodicals, Inc.
wires.wiley.com/devbio
Advanced Review
fluid. Biochem Biophys Res Commun 1985, 126:
220–226.
57. Ling N, Ying SY, Ueno N, Esch F, Denoroy L,
Guillemin R. Isolation and partial characterization
of a Mr 32,000 protein with inhibin activity from
porcine follicular fluid. Proc Natl Acad Sci USA 1985,
82:7217–7221.
58. Ling N, Ying SY, Ueno N, Shimasaki S, Esch F,
Hotta M, Guillemin R. Pituitary FSH is released by a
heterodimer of the beta-subunits from the two forms
of inhibin. Nature 1986, 321:779–782.
59. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J. Purification and
characterization of an FSH releasing protein from
porcine ovarian follicular fluid. Nature 1986, 321:
776–779.
60. Santibanez JF, Quintanilla M, Bernabeu C.
TGF-beta/TGF-beta receptor system and its role
in physiological and pathological conditions. Clin Sci
(Lond) 2011, 121:233–251.
61. Steinhauer J, Treisman JE. Lipid-modified morphogens: functions of fats. Curr Opin Genet Dev
2009, 19:308–314.
62. Guerrero I, Chiang C. A conserved mechanism of
Hedgehog gradient formation by lipid modifications.
Trends Cell Biol 2007, 17:1–5.
63. Mann RK, Beachy PA. Novel lipid modifications of
secreted protein signals. Annu Rev Biochem 2004,
73:891–923.
64. Lee JJ, Ekker SC, von Kessler DP, Porter JA, Sun
BI, Beachy PA. Autoproteolysis in hedgehog protein
biogenesis. Science 1994, 266:1528–1537.
65. Hall TM, Porter JA, Young KE, Koonin EV, Beachy
PA, Leahy DJ. Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and
self-splicing proteins. Cell 1997, 91:85–97.
66. Porter JA, Young KE, Beachy PA. Cholesterol modification of hedgehog signaling proteins in animal development. Science 1996, 274:255–259.
67. Porter JA, Ekker SC, Park WJ, von Kessler DP,
Young KE, Chen CH, Ma Y, Woods AS, Cotter
RJ, Koonin EV, et al. Hedgehog patterning activity:
role of a lipophilic modification mediated by the
carboxy-terminal autoprocessing domain. Cell 1996,
86:21–34.
68. Chen X, Tukachinsky H, Huang CH, Jao C, Chu
YR, Tang HY, Mueller B, Schulman S, Rapoport TA,
Salic A. Processing and turnover of the Hedgehog
protein in the endoplasmic reticulum. J Cell Biol 2011,
192:825–838.
69. Traiffort E, Dubourg C, Faure H, Rognan D,
Odent S, Durou MR, David V, Ruat M. Functional characterization of sonic hedgehog mutations
associated with holoprosencephaly. J Biol Chem 2004,
279:42889–42897.
70. Maity T, Fuse N, Beachy PA. Molecular mechanisms of
Sonic hedgehog mutant effects in holoprosencephaly.
Proc Natl Acad Sci USA 2005, 102:17026–17031.
71. Roessler E, El-Jaick KB, Dubourg C, Velez JI, Solomon
BD, Pineda-Alvarez DE, Lacbawan F, Zhou N,
Ouspenskaia M, Paulussen A, et al. The mutational spectrum of holoprosencephaly-associated
changes within the SHH gene in humans predicts
loss-of-function through either key structural alterations of the ligand or its altered synthesis. Hum
Mutat 2009, 30:E921–E935.
72. Tian H, Jeong J, Harfe BD, Tabin CJ, McMahon AP.
Mouse Disp1 is required in sonic hedgehog-expressing
cells for paracrine activity of the cholesterol-modified
ligand. Development 2005, 132:133–142.
73. Burke R, Nellen D, Bellotto M, Hafen E, Senti
KA, Dickson BJ, Basler K. Dispatched, a novel
sterol-sensing domain protein dedicated to the release
of cholesterol-modified hedgehog from signaling cells.
Cell 1999, 99:803–815.
74. Chen MH, Li YJ, Kawakami T, Xu SM, Chuang
PT. Palmitoylation is required for the production of
a soluble multimeric hedgehog protein complex and
long-range signaling in vertebrates. Genes Dev 2004,
18:641–659.
75. Chamoun Z, Mann RK, Nellen D, von Kessler DP,
Bellotto M, Beachy PA, Basler K. Skinny hedgehog, an acyltransferase required for palmitoylation
and activity of the hedgehog signal. Science 2001,
293:2080–2084.
76. Micchelli CA, The I, Selva E, Mogila V, Perrimon
N. Rasp, a putative transmembrane acyltransferase, is
required for hedgehog signaling. Development 2002,
129:843–851.
77. Amanai K, Jiang J. Distinct roles of central missing and
dispatched in sending the hedgehog signal. Development 2001, 128:5119–5127.
78. Lee JD, Treisman JE. Sightless has homology to
transmembrane acyltransferases and is required to
generate active Hedgehog protein. Curr Biol 2001,
11:1147–1152.
79. Callejo A, Torroja C, Quijada L, Guerrero I. Hedgehog lipid modifications are required for Hedgehog
stabilization in the extracellular matrix. Development
2006, 133:471–483.
80. Gallet A, Ruel L, Staccini-Lavenant L, Therond PP.
Cholesterol modification is necessary for controlled
planar long-range activity of hedgehog in Drosophila
epithelia. Development 2006, 133:407–418.
81. Zeng X, Goetz JA, Suber LM, Scott WJ Jr, Schreiner
CM, Robbins DJ. A freely diffusible form of Sonic
hedgehog mediates long-range signalling. Nature
2001, 411:716–720.
82. Feng J, White B, Tyurina OV, Guner B, Larson T, Lee
HY, Karlstrom RO, Kohtz JD. Synergistic and
antagonistic roles of the Sonic hedgehog N-
© 2015 Wiley Periodicals, Inc.
WIREs Developmental Biology
and
C-terminal
131:4357–4370.
lipids.
Morphogen transport
Development
2004,
83. Gross JC, Boutros M. Secretion and extracellular space
travel of Wnt proteins. Curr Opin Genet Dev 2013,
23:385–390.
98. Moline MM, Southern C, Bejsovec A. Directionality
of wingless protein transport influences epidermal
patterning in the Drosophila embryo. Development
1999, 126:4375–4384.
84. Port F, Basler K. Wnt trafficking: new insights into
Wnt maturation, secretion and spreading. Traffic
2010, 11:1265–1271.
99. Gonzalez-Gaitan M, Jackle H. The range of
spalt-activating Dpp signalling is reduced in
endocytosis-defective Drosophila wing discs. Mech
Dev 1999, 87:143–151.
85. MacDonald BT, Tamai K, He X. Wnt/beta-catenin
signaling: components, mechanisms, and diseases.
Dev Cell 2009, 17:9–26.
100. Entchev EV, Schwabedissen A, Gonzalez-Gaitan M.
Gradient formation of the TGF-beta homolog Dpp.
Cell 2000, 103:981–991.
86. van den Heuvel M, Harryman-Samos C, Klingensmith
J, Perrimon N, Nusse R. Mutations in the segment
polarity genes wingless and porcupine impair secretion
of the wingless protein. EMBO J 1993, 12:5293–5302.
101. Kruse K, Pantazis P, Bollenbach T, Julicher F,
Gonzalez-Gaitan M. Dpp gradient formation by
dynamin-dependent endocytosis: receptor trafficking
and the diffusion model. Development 2004, 131:
4843–4856.
87. Herr P, Basler K. Porcupine-mediated lipidation is
required for Wnt recognition by Wls. Dev Biol 2012,
361:392–402.
88. Coombs GS, Yu J, Canning CA, Veltri CA, Covey
TM, Cheong JK, Utomo V, Banerjee N, Zhang
ZH, Jadulco RC, et al. WLS-dependent secretion of
WNT3A requires Ser209 acylation and vacuolar acidification. J Cell Sci 2010, 123:3357–3367.
89. Tang X, Wu Y, Belenkaya TY, Huang Q, Ray L, Qu J,
Lin X. Roles of N-glycosylation and lipidation in Wg
secretion and signaling. Dev Biol 2012, 364:32–41.
90. Ching W, Hang HC, Nusse R. Lipid-independent
secretion of a Drosophila Wnt protein. J Biol Chem
2008, 283:17092–17098.
91. Willert K, Brown JD, Danenberg E, Duncan AW,
Weissman IL, Reya T, Yates JR 3rd, Nusse R. Wnt
proteins are lipid-modified and can act as stem cell
growth factors. Nature 2003, 423:448–452.
92. Franch-Marro X, Wendler F, Griffith J, Maurice MM,
Vincent JP. In vivo role of lipid adducts on Wingless.
J Cell Sci 2008, 121:1587–1592.
93. Doubravska L, Krausova M, Gradl D, Vojtechova M,
Tumova L, Lukas J, Valenta T, Pospichalova V, Fafilek
B, Plachy J, et al. Fatty acid modification of Wnt1 and
Wnt3a at serine is prerequisite for lipidation at cysteine
and is essential for Wnt signalling. Cell Signal 2011,
23:837–848.
94. Rogers KW, Schier AF. Morphogen gradients: from
generation to interpretation. Annu Rev Cell Dev Biol
2011, 27:377–407.
95. Schwank G, Basler K. Regulation of organ growth by
morphogen gradients. Cold Spring Harb Perspect Biol
2010, 2:a001669.
96. Bejsovec A, Wieschaus E. Signaling activities of the
Drosophila wingless gene are separately mutable and
appear to be transduced at the cell surface. Genetics
1995, 139:309–320.
97. Dierick HA, Bejsovec A. Functional analysis of Wingless reveals a link between intercellular ligand transport and dorsal-cell-specific signaling. Development
1998, 125:4729–4738.
102. Kicheva A, Pantazis P, Bollenbach T, Kalaidzidis
Y, Bittig T, Julicher F, Gonzalez-Gaitan M. Kinetics of morphogen gradient formation. Science 2007,
315:521–525.
103. Gallet A, Staccini-Lavenant L, Therond PP. Cellular
trafficking of the glypican Dally-like is required for
full-strength Hedgehog signaling and wingless transcytosis. Dev Cell 2008, 14:712–725.
104. Marois E, Mahmoud A, Eaton S. The endocytic
pathway and formation of the Wingless morphogen
gradient. Development 2006, 133:307–317.
105. Belenkaya TY, Han C, Yan D, Opoka RJ, Khodoun M,
Liu H, Lin X. Drosophila Dpp morphogen movement
is independent of dynamin-mediated endocytosis but
regulated by the glypican members of heparan sulfate
proteoglycans. Cell 2004, 119:231–244.
106. Schwank G, Dalessi S, Yang SF, Yagi R, de Lachapelle
AM, Affolter M, Bergmann S, Basler K. Formation of
the long range Dpp morphogen gradient. PLoS Biol
2011, 9:e1001111.
107. Gibson MC, Perrimon N. Extrusion and death of
DPP/BMP-compromised epithelial cells in the developing Drosophila wing. Science 2005, 307:1785–1789.
108. Shen J, Dahmann C. Extrusion of cells with inappropriate Dpp signaling from Drosophila wing disc epithelia. Science 2005, 307:1789–1790.
109. Akiyama T, Kamimura K, Firkus C, Takeo S, Shimmi
O, Nakato H. Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface.
Dev Biol 2008, 313:408–419.
110. Kornberg TB, Roy S. Cytonemes as specialized signaling filopodia. Development 2014, 141:729–736.
111. Roy S, Huang H, Liu S, Kornberg TB.
Cytoneme-mediated contact-dependent transport
of the Drosophila decapentaplegic signaling protein.
Science 2014, 343:1244624.
112. Roy S, Hsiung F, Kornberg TB. Specificity of
Drosophila cytonemes for distinct signaling pathways.
Science 2011, 332:354–358.
© 2015 Wiley Periodicals, Inc.
wires.wiley.com/devbio
Advanced Review
113. Kornberg TB. Cytonemes extend their reach. EMBO J
2013, 32:1658–1659.
gradient forms by a source-sink mechanism with freely
diffusing molecules. Nature 2009, 461:533–536.
114. Hsiung F, Ramirez-Weber FA, Iwaki DD, Kornberg TB. Dependence of Drosophila wing imaginal
disc cytonemes on Decapentaplegic. Nature 2005,
437:560–563.
126. Gregor T, Wieschaus EF, McGregor AP, Bialek W,
Tank DW. Stability and nuclear dynamics of the bicoid
morphogen gradient. Cell 2007, 130:141–152.
115. Ramirez-Weber FA, Kornberg TB. Cytonemes: cellular
processes that project to the principal signaling center
in Drosophila imaginal discs. Cell 1999, 97:599–607.
116. Bischoff M, Gradilla AC, Seijo I, Andres G,
Rodriguez-Navas C, Gonzalez-Mendez L, Guerrero
I. Cytonemes are required for the establishment of a
normal Hedgehog morphogen gradient in Drosophila
epithelia. Nat Cell Biol 2013, 15:1269–1281.
117. Bilioni A, Sanchez-Hernandez D, Callejo A, Gradilla
AC, Ibanez C, Mollica E, Carmen Rodriguez-Navas
M, Simon E, Guerrero I. Balancing Hedgehog, a
retention and release equilibrium given by Dally, Ihog,
Boi and shifted/DmWif. Dev Biol 2013, 376:198–212.
118. Rojas-Rios P, Guerrero I, Gonzalez-Reyes A.
Cytoneme-mediated delivery of hedgehog regulates the expression of bone morphogenetic proteins
to maintain germline stem cells in Drosophila. PLoS
Biol 2012, 10:e1001298.
119. Sanders TA, Llagostera E, Barna M. Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature 2013, 497:628–632.
120. Yan D, Lin X. Shaping morphogen gradients by
proteoglycans. Cold Spring Harb Perspect Biol 2009,
1:a002493.
121. Takei Y, Ozawa Y, Sato M, Watanabe A, Tabata T.
Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans. Development 2004, 131:73–82.
122. Han C, Belenkaya TY, Wang B, Lin X. Drosophila
glypicans control the cell-to-cell movement of Hedgehog by a dynamin-independent process. Development
2004, 131:601–611.
123. Lander AD, Nie Q, Wan FY. Do morphogen gradients
arise by diffusion? Dev Cell 2002, 2:785–796.
124. Zhou S, Lo WC, Suhalim JL, Digman MA, Gratton E,
Nie Q, Lander AD. Free extracellular diffusion creates
the Dpp morphogen gradient of the Drosophila wing
disc. Curr Biol 2012, 22:668–675.
125. Yu SR, Burkhardt M, Nowak M, Ries J, Petrasek
Z, Scholpp S, Schwille P, Brand M. Fgf8 morphogen
127. Fujise M, Takeo S, Kamimura K, Matsuo T, Aigaki T,
Izumi S, Nakato H. Dally regulates Dpp morphogen
gradient formation in the Drosophila wing. Development 2003, 130:1515–1522.
128. Ohkawara B, Iemura S, ten Dijke P, Ueno N. Action
range of BMP is defined by its N-terminal basic amino
acid core. Curr Biol 2002, 12:205–209.
129. Tabata T, Takei Y. Morphogens, their identification
and regulation. Development 2004, 131:703–712.
130. Duchesne L, Octeau V, Bearon RN, Beckett A,
Prior IA, Lounis B, Fernig DG. Transport of fibroblast growth factor 2 in the pericellular matrix is
controlled by the spatial distribution of its binding sites in heparan sulfate. PLoS Biol 2012, 10:
e1001361.
131. Vuilleumier R, Springhorn A, Patterson L, Koidl S,
Hammerschmidt M, Affolter M, Pyrowolakis G. Control of Dpp morphogen signalling by a secreted feedback regulator. Nat Cell Biol 2010, 12:611–617.
132. Szuperak M, Salah S, Meyer EJ, Nagarajan U, Ikmi
A, Gibson MC. Feedback regulation of Drosophila
BMP signaling by the novel extracellular protein larval
translucida. Development 2011, 138:715–724.
133. Alexandre C, Baena-Lopez A, Vincent JP. Patterning
and growth control by membrane-tethered Wingless.
Nature 2014, 505:180–185.
134. Lim J, Norga KK, Chen Z, Choi KW. Control of planar
cell polarity by interaction of DWnt4 and four-jointed.
Genesis 2005, 42:150–161.
135. Doumpas N, Jekely G, Teleman AA. Wnt6 is required
for maxillary palp formation in Drosophila. BMC Biol
2013, 11:104.
136. Khalsa O, Yoon JW, Torres-Schumann S, Wharton
KA. TGF-beta/BMP superfamily members, Gbb-60A
and Dpp, cooperate to provide pattern information
and establish cell identity in the Drosophila wing.
Development 1998, 125:2723–2734.
137. Bangi E, Wharton K. Dpp and Gbb exhibit different
effective ranges in the establishment of the BMP activity gradient critical for Drosophila wing patterning.
Dev Biol 2006, 295:178–193.
© 2015 Wiley Periodicals, Inc.