Developmental Cell

Review

TGF-b Superfamily Signaling

in Embryonic Development and Homeostasis
Mary Y. Wu1 and Caroline S. Hill1,*
1Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK

*Correspondence: [email protected]
DOI 10.1016/j.devcel.2009.02.012

TGF-b superfamily signaling pathways emerged with the evolution of multicellular animals, suggesting that
these pathways contribute to the increased diversity and complexity required for the development and
homeostasis of these organisms. In this review we begin by exploring some key developmental and disease
processes requiring TGF-b ligands to underscore the fundamental importance of these pathways before
delving into the molecular mechanism of signal transduction, focusing on recent findings. Finally, we discuss
how these ligands act as morphogens, how their activity and signaling range is regulated, and how they
interact with other signaling pathways to achieve their specific and varied functional roles.

Introduction is often determined by localized maternal factors, sperm entry,

The transforming growth factor b (TGF-b) superfamily of growth or both, zygotic activation of Nodal and BMPs initiates complex
factors, which contains over 30 members including TGF-bs, circulatory loops of signaling to define and pattern the first
bone morphogenetic proteins (BMPs), growth and differentiation embryonic axis.
factors (GDFs), Activins, and Nodal, is vital for the development In Drosophila embryos, the BMP orthologs Decapentaplegic
and homeostasis of metazoans (Feng and Derynck, 2005). The (Dpp) and Screw (Scw) are required for dorsal-ventral (D/V)
ligands and their downstream pathway components are axis specification and patterning. Prior to cellularization of the
extremely well conserved during evolution, and they regulate blastoderm, dpp is expressed in the dorsalmost 40% of the
diverse cellular functions such as growth, adhesion, migration, embryo, limited ventrally by a maternal NF-kB ortholog, Dorsal
apoptosis, and differentiation. Moreover, their actions are modu- (Dl) (Morisato and Anderson, 1995). After cellularization, a
lated in time and space during embryonic development, leading gradient of Dpp signaling is established with highest activity
to a diversity of cellular responses that is staggering. The best at the dorsal midline, lower activity at the lateral regions, and
understood signal transduction pathway utilized by these growth no activity ventrally (O’Connor et al., 2006). Ubiquitously
factors is seemingly simple and linear. Ligand dimers bind and expressed Scw synergizes with Dpp to attain the highest levels
activate heteromeric complexes of type I and type II transmem- of BMP signaling (O’Connor et al., 2006). Cells along this D/V
brane receptors. Activated receptors then phosphorylate the axis respond to different levels of Dpp signaling and undergo
intracellular mediators (Smads), which form complexes with different programs of differentiation. Highest levels of Dpp
each other and other proteins to modulate transcription of target specify the amnioserosa, lower levels specify dorsal ectoderm,
genes in the nucleus. However, in order to perform their varied and lack of Dpp signaling allows the formation of neural ecto-
roles throughout animal life, there exists a level of complexity derm (O’Connor et al., 2006).
we are only just beginning to understand. In this review we begin While vertebrate and arthropod embryos share a similar body
by describing some key events in the development of both inver- plan, their D/V axes are inverted. In arthropods, neural tissue
tebrates and vertebrates that require TGF-b superfamily arises from ventral lateral ectoderm, while in vertebrates, it is
signaling, and some human diseases that result from deregu- derived from dorsal ectoderm. Despite this difference, molecular
lated signaling. We then look at the pathway in greater molecular requirements for defining and patterning the D/V axis in verte-
detail, concentrating on recent published studies. Finally, we tie brates and Drosophila are highly conserved (Little and Mullins,
together the functions of these ligands with the mechanisms by 2006). In Xenopus and zebrafish, an early zygotic manifestation
which they signal to explain how the TGF-b superfamily signaling of the D/V axis is the dorsal organizer, which was originally iden-
pathways achieve their complex in vivo roles. tified in a salamander gastrula embryo and is sufficient to induce
ectopic dorsal tissue (Spemann and Mangold, 1924). It is known
Functions of TGF-b Superfamily Members in Embryonic as the Spemann organizer in Xenopus and the shield in zebrafish.
Development and Disease In both systems formation of the dorsal organizer requires Nodal
Early Development, Axis Formation, and Patterning signaling (De Robertis and Kuroda, 2004; Feldman et al., 1998)
Members of the Nodal/Activin and BMP subfamilies are key (Figure 1A). Studies have revealed that many of the factors
players in the generation of axes and in the subsequent secreted from the organizer are BMP antagonists (De Robertis
patterning of tissues across these axes during embryogenesis. and Kuroda, 2004). From the onset of zygotic transcription to
They are morphogens that form concentration gradients and the end of gastrulation, Bmp4 and Bmp7 expression become
signal in a dose-dependent manner, thereby providing positional gradually limited to the ventral side of embryos in Xenopus,
information to a field of cells and initiating diverse downstream with Bmp2b (swirl) and Bmp7 (snailhouse) undergoing similar
molecular programs. Whereas the first asymmetry in the embryo temporal and spatial regulation in zebrafish (Little and Mullins,

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Figure 1. Disrupting TGF-b Family Signaling Produces Specific and Striking Effects during Early Development
(A) Overexpression (OE) of the Nodal ligand Xnr1 ventrally induces an ectopic organizer, resulting in a second axis, indicated by the white arrowheads.
(B) A ventral to dorsal gradient of BMP signaling patterns tissues in the early embryo. Bmp2b mutant fish are completely dorsalized, lacking ventrally derived
structures. Increasing expression of Xenopus BMP4 rescues the phenotype, and excess BMP signaling gradually ventralizes embryos.
(C) Inhibiting Nodal signaling in zebrafish with the type I ALK4/5/7-specific inhibitor SB-431542 results in gradual loss of mesodermal and endodermal tissue and
induces cyclopia and loss of anterior dorsal cell types. The arrow and the arrowhead indicate the eyes and notochord, respectively.
(D) Overexpression of Xnr1 in Xenopus embryos results in randomization of the L/R axis, which manifests as reversed heart and gut looping. v, ventricle.
(E) Overexpression of BMP antagonists Sog and Tsg in the Drosophila wing inhibits BMP signaling, resulting in lack of vein formation and a decrease in
proliferation.
(F) Loss of BMP2 and BMP4 in the mouse affects posterior digit development during limb morphogenesis as seen by Sox9 chondrogenic staining of digits.
The images were reproduced courtesy of Kishimoto et al. (1997) (B); Sun et al. (2006) (C); Sampath et al. (1997) (D); Shimmi and O’Connor (2003) (E); and
Bandyopadhyay et al. (2006) (F).

2006). Higher BMP activity then induces other secreted factors visceral endoderm (DVE), part of the extraembryonic tissue.
on the ventral side (De Robertis and Kuroda, 2004). In addition, Second, in response to Nodal, some cells of the visceral endo-
another BMP family member, Admp, is expressed in the Spe- derm (VE) secrete Nodal antagonists, and inhibition of Nodal in
mann organizer, being inhibited by high BMP levels on the the vicinity of these cells prevents Nodal-induced proliferation
ventral side (Reversade and De Robertis, 2005). The concerted in a portion of the VE, hence providing asymmetry and positional
actions of these molecules set up a ventral to dorsal gradient cues for determining anterior versus posterior. Third, Nodal
of BMP, capable of patterning tissues (Little and Mullins, 2006) induces cells of the DVE to migrate toward the anterior
(Figure 1B). In the vertebrate ectoderm, high BMP activity (becoming the anterior VE [AVE]), thus setting up the A/P axis.
induces epidermis and low activity specifies neural tissue. Germ-Layer Specification, Patterning, and Gastrulation
Neural crest cells are specified at the border between these In addition to its roles in axis specification, Nodal is also required
tissues in cells exhibiting intermediate BMP signaling (Sauka- in vertebrates for the induction of the three germ layers: endo-
Spengler and Bronner-Fraser, 2008). derm, mesoderm, and indirectly, ectoderm. It first induces mes-
In contrast, the first zygotically induced axis in mice is the endoderm, and then different levels of Nodal signaling are
anterior-posterior (A/P) axis, but again, Nodal is a key player. required for further patterning and refinement of domains, with
Nodal is initially expressed throughout the epiblast, the radially high levels inducing endoderm and lower levels inducing meso-
symmetrical cup-shaped embryo proper, and its involvement derm (Zorn and Wells, 2007). In mice, Nodal antagonism by
can be summarized in three steps (Schier, 2003; Yamamoto secreted factors from the AVE, along with complex positive feed-
et al., 2004). First, Nodal is required for the induction of the distal back circuits from the extraembryonic ectoderm (ExE), induces

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Review

a gradual localization of Nodal expression to the proximal poste- superfamily ligand functions is beyond the scope of this review,
rior region of the epiblast during A/P axis formation, thus setting so we will just highlight a few instructive examples.
up the Nodal gradient required for induction of endoderm and In Drosophila, Dpp signaling is vital for the morphogenesis and
mesoderm (Zorn and Wells, 2007). In Xenopus, the maternal development of the imaginal discs, which give rise to the external
vegetally localized Nodal-related ligand Vg1 (Birsoy et al., appendages and organs of the adult fly (O’Connor et al., 2006).
2006), in conjunction with zygotically expressed Nodal ligands, For example, in the early wing disc a Dpp gradient patterns the
creates the required Nodal gradient in the vegetal pole, which A/P axis, providing accurate positional cues for longitudinal
is high in the dorsal vegetal region and fades ventrally and vein (LV) formation (O’Connor et al., 2006) (Figure 1E). This initial
toward the animal pole (Schier, 2003). In zebrafish, a similar expression of dpp at the compartment boundary is also required
enrichment of Nodal exists dorsally, proposed to result from for proliferation of cells in the imaginal disc. Later during early
the accumulation of Squint (sqt) transcripts on the dorsal side pupal development, dpp expression is refined to areas where
of the embryo at the four-cell stage (Gore et al., 2005). In fish, LVs have been specified. This maintains LV fate and contributes
a mutant lacking both the short and long-range ligands, Cyclops to the induction of cross veins, which additionally requires
(Cyc) and Sqt respectively, develops with no endoderm and another Drosophila BMP ligand, Glass bottom boat (Gbb)
almost no mesoderm (Feldman et al., 1998), and a similar pheno- (O’Connor et al., 2006).
type is seen in embryos treated with an inhibitor of Nodal recep- In vertebrates, one or more TGF-b superfamily ligands play
tors (Sun et al., 2006) (Figure 1C). roles in the morphogenesis of most organs, and defects in
The third germ layer, ectoderm, is often considered a default signaling in this context can lead to serious human diseases.
tissue type, since tissues removed from the effects of Nodal Anti-müllerian hormone (AMH) is a highly specialized member
signaling become ectoderm (Zhang et al., 1998). However, in of the TGF-b family restricted in its expression both spatially
the embryo, active inhibition of Nodal signaling is required and temporally. This is in contrast to other family members previ-
(Schier, 2003). Moreover, normal gastrulation cannot occur ously discussed, such as BMP4 and Nodal, which are expressed
without the appropriate specification of the three germ layers, in many different tissues to elicit diverse responses. AMH is
and loss-of-function mutations in Nodal signaling lead to severe required for the regression of müllerian ducts in male fetuses
gastrulation and primitive streak defects (Zorn and Wells, 2007). and deficiencies in AMH or the AMH receptor (AMHR) result in
Left-Right Asymmetry Persistent Müllerian Duct Syndrome (PMDS), where fetuses
The left-right (L/R) axis is specified after the A/P and D/V axes that are genetically male develop with rudimentary female
and is important for the future position of the organs and the organs. Usually, functional testes develop but do not descend,
directional looping of tubules in the body. Here again, Nodal and obstructions or other defects which occur during the forma-
plays a crucial role (Figure 1D). Nodal signaling during L/R spec- tion of the secretory ducts can render individuals infertile (Josso
ification is dynamic, both spatially and temporally. In mice, Nodal et al., 2006). AMH is also required for follicular development in
is initially expressed symmetrically at E7.0 at the lateral edges of females. Its expression is used as a marker for follicular reserve
the node. By E7.5, its expression shifts, with higher levels found in adult women, and excessive AMH can cause polycystic ovary
in the left perinodal region (Marques et al., 2004). Shortly after at syndrome (Wang et al., 2007).
E8.0, Nodal expression is found in the left lateral plate mesoderm TGF-b family members are well known for their ability to
(LPM). Genetic ablation of Nodal expression in the node has induce epithelial-mesenchymal transition (EMT) (Yang and Wein-
demonstrated that Nodal is required for its own asymmetrical berg, 2008). This process allows polarized cells of an epithelial
expression later in the LPM, but how the signal is transduced sheet to delaminate, assume a spindle-like mesenchymal shape,
to manifest the later event is unclear (Brennan et al., 2002). In migrate from their site of origin, and invade surrounding tissue
Xenopus and zebrafish, where there are multiple Nodal ligands, (Yang and Weinberg, 2008). EMT is essential for a variety of
Xnr1 and Southpaw, respectively, are expressed asymmetrically developmental processes, a prominent example being the inva-
in the LPM (Long et al., 2003; Sampath et al., 1997). sion of the heart cushion by endocardial cells from the atrioven-
BMP signaling is also involved in L/R patterning. Evidence tricular (AV) canals, which eventually gives rise to heart valves
from mouse and chick reveal different roles. In mouse, BMP4 (Mercado-Pimentel and Runyan, 2007). In mice, TGF-b1, 2,
represses Nodal expression in the right LPM (Mine et al., and 3 are expressed in the developing heart in temporally distinct
2008). In the chick, BMP2 induces Nodal expression on the left phases. While mice null for each of the three TGF-b ligands have
side (Schlange et al., 2002). Experiments in zebrafish suggest been generated, only the TGF-b2 null mouse exhibited EMT-
that active BMP signaling can do both, but these are separate specific phenotypes in the heart. However, in chicken AV
events, regulated temporally. In this case, BMP4 is required at explants, use of neutralizing antibodies has demonstrated that
early stages to suppress Nodal in the right LPM, but later, it is TGF-b2 is required for initiation of EMT, while TGF-b3 affects
required for left-side-specific gene expression (Chocron et al., invasion/migration in a sequential manner, consistent with the
2007). order of expression of these ligands. Since the expression of
Organogenesis and Developmental Disease the three TGF-bs is also temporally controlled in the mouse
So far, we have discussed a few key players, mainly Nodal and heart, they may all contribute to EMT, but at different steps of
several BMPs, that are required for early embryogenesis. These this process.
ligands continue to be deployed for later developmental BMPs and GDFs make up the majority of the TGF-b superfamily
processes, along with other superfamily members that have members, and BMP2, 4, and 7, as well as GDF5 and 6, have all
extensive and specialized roles in tissue morphogenesis and been implicated in limb development (Bandyopadhyay et al.,
homeostasis. A comprehensive and detailed listing of all TGF-b 2006; Settle et al., 2003) (Figure 1F). The BMPs are redeployed

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at various stages, including apical ectodermal ridge (AER) forma- changes in signaling can also increase myofibroblast and
tion, AER regression, cartilage and bone differentiation, and inter- smooth muscle cell proliferation, leading to abnormal repair of
digital webbing regression, and their temporal coexpression can vessels (Eickelberg and Morty, 2007; Morrell, 2006).
play synergistic as well as antagonistic roles depending on the Myofibroblasts are key players in wound-healing and tissue
stage (Robert, 2007). In addition, TGF-b2 and 3 are also required repair. They respond to cues from TGF-b in order to carry out
for inducing programmed cell death during interdigital webbing this function. Therefore, disturbances in TGF-b signaling can
regression (Dunker et al., 2002). Mutations in TGF-b superfamily lead to fibrotic diseases, which are a result of excessive scarring
signaling components, such as the ligands GDF5 and CDMP1, due to increased extracellular matrix (ECM) deposition by over-
the type I BMP receptor BMPR1B, and the antagonist Noggin, active myofibroblasts. This abnormal scarring can eventually
can lead to severe defects resulting in shortening of limbs and interfere with organ function and lead to organ failure. Excessive
loss of joints and/or digits as seen in diseases such as brachydac- TGF-b signaling has been linked to fibrotic diseases and is
tyly (Lehmann et al., 2006, 2007; Seemann et al., 2005) and chon- thought to function by promoting the production of ECM compo-
drodysplasia (Faiyaz-Ul-Haque et al., 2008; Thomas et al., 1997). nents, in particular type I collagen (Gordon and Blobe, 2008), and
Homeostasis and Disease in renal fibrosis, by inducing EMT in renal epithelial cells, convert-
TGF-b superfamily signaling continues to function in fully devel- ing them to myofibroblasts, leading to tubular atrophy (Zeisberg
oped organisms where it is required for tissue homeostasis. A and Kalluri, 2004).
well-studied example is the maintenance of the vasculature, The importance of TGF-b’s involvement in the maintenance of
and relevant to this, mutations in a TGF-b receptor, Activin ECM is also evident in Marfan syndrome (MFS). This is a hereditary
receptor-like kinase 1 (ALK1), and a coreceptor, Endoglin, have congenital disorder resulting from connective tissue defects that
been linked to the disease Hereditary Hemorrhagic Telangiec- manifest in symptoms such as scoliosis (curved spine), arachno-
tasia (HHT) or Rendu-Oslr-Weber syndrome (ten Dijke and dactyly (long spidery fingers), dolichostenomelia (long slender
Arthur, 2007). Besides telangiectasis (formation of dilated blood limbs), pectus excavatum (sunken chest), or carinatum (protrusion
vessels at the surface of the skin and mucous membranes), of chest), and sometimes ectopia lentis (mispositioning of the crys-
patients often have arteriovenous malformations, nosebleeds, talline lens of the eyes) (Robinson et al., 2006). A large percentage
and gastrointestinal bleeding. Mutations in Endoglin are associ- of patients diagnosed with MFS harbor mutations in the gene
ated with HHT1 while mutations in ALK1 are linked to HHT2 with Fibrillin-1. Initially, this finding attributed the disorder to problems
the clinical diagnosis differing at the level of penetrance and with the structural roles of Fibrillins in the ECM (Robinson et al.,
localization of malformations. In most cases, the mutations are 2006). However, Fibrillin-1 is a member of the Fibrillin/Latent
thought to inactivate receptor function and disease occurs due TGF-b binding protein superfamily and members of this family
to haploinsufficiency (ten Dijke and Arthur, 2007). play an important role in regulating TGF-b ligands. Indeed, MFS
Interestingly, a subset of patients with HHT often also suffers type II is caused by mutations in TGFB2 and a related disease,
from juvenile polyposis (JP). JP is diagnosed when five or more Loeys-Dietz syndrome (LDS), which has overlapping symptoms
hamartomatous gastrointestinal polyps are found, usually in with MFS, has been shown to be associated with mutations in
the colon at a young age. Mutations in Smad4, a signal trans- either TGFB1 or TGFB2 (Gordon and Blobe, 2008).
ducer downstream of TGF-b superfamily signaling, and a BMP Due to the pleiotropic roles of TGF-b superfamily members, it
type I receptor, BMPR1A (ALK3), have been linked to the disease is not surprising that deregulation of TGF-b superfamily signals
(Levy and Hill, 2006). Interestingly, combined JP-HHT is linked to can lead to the progression of many cancers. It was the
mutations in Smad4 (Gallione et al., 2004). Intuitively, this makes discovery in 1996 that TGF-b exhibited a biphasic action during
sense due to the role of Smad4 downstream of BMPR1A/ALK3, skin carcinogenesis, inhibiting the formation of benign tumors,
ALK1, and Endoglin (see below). A study recently investigated but enhancing the progression to invasive spindle tumors (Cui
HHT patients that do not carry mutations in Endoglin or ALK1 et al., 1996), that crystalized the view that TGF-b plays both
and have not been diagnosed with combined JP and found tumor suppressive and tumor promoting roles in human cancer.
that 10% of these cases have mutations in Smad4 (Gallione Since that time these roles have been dissected in detail, but as
et al., 2006). Because JP is associated with a high risk of devel- this has been extensively reviewed recently (Massague, 2008), it
oping gastrointestinal cancers, HHT patients who have not been will not be discussed here.
diagnosed with JP may indeed be afflicted by the combined
syndrome and harbor this risk. The Molecular Mechanism of TGF-b Superfamily
Another disease associated with the maintenance of the Signaling
vasculature is primary pulmonary arterial hypertension (PAH). From the discussion above it is evident that TGF-b superfamily
This results from remodeling of pulmonary arteries leading to members play critical and diverse roles throughout embryonic
a constriction of the vessels that increases blood pressure and development in vertebrates and invertebrates, and that deregu-
decreases the efficiency of the heart in distributing oxygen-rich lated signaling contributes to many human diseases. These
blood to tissues around the body (ten Dijke and Arthur, 2007). ligands function by initiating new programs of gene expression
Prolonged stress to the heart due to PAH often leads to heart in responding cells and in this section we explain how this is
failure and premature death. Germline mutations in the BMP achieved. The best understood signaling pathway downstream
type II receptor, BMPR2, have been isolated in patients with of the receptors is relatively simple, but a considerable amount
PAH. The changes in BMP signaling associated with these muta- of combinatorial mixing and matching at the level of the ligands,
tions may contribute to an increased susceptibility of endothelial receptors, and Smads produces a large diversity in transcrip-
cells to apoptosis, leading to damage of vessels. The same tional outputs.

332 Developmental Cell 16, March 17, 2009 ª2009 Elsevier Inc.
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The Ligands comprising the two type I receptors Thickveins (Tkv) and Saxo-
The TGF-b superfamily ligands are secreted as precursors phone (Sax) and the type II receptor Punt appears to be costimu-
comprising a large prodomain and a C-terminal mature polypep- lated by a ligand heterodimer comprising Dpp and Scw to elicit
tide. Dimerization, which is stabilized by intermolecular disulphide highest levels of signaling (O’Connor et al., 2006).
bonds, requires the prodomains and thus occurs intracellularly. The TGF-b receptors are internalized constitutively in a cla-
The mature ligands are cleaved from the prodomain by furin-like thrin-dependent manner into EEA1-positive early endosomes
enzymes (Feng and Derynck, 2005). For the most part, the ligands and in a non-clathrin-dependent manner via caveolin-positive
homodimerize, but heterodimerization also occurs between lipid rafts (Di Guglielmo et al., 2003) (Figure 2B). Receptor
Nodal and BMP4 or BMP7 (Yeo and Whitman, 2001) or, in signaling and degradation are thought to occur in different
Drosophila, between Dpp and Scw (O’Connor et al., 2006). A cellular compartments. Receptor degradation occurs via the
major regulatory step in TGF-b superfamily signaling is the regu- lipid raft-caveolar internalization pathway. It is mediated via
lation of ligand accessibility by extracellular diffusible ligand- Smad7, which is a member of the Smad family (see below),
binding proteins. This is particularly important for the creation of which recruits the HECT domain E3 ubiquitin ligases Smurf1/2
gradients, such as the D/V BMP gradient as discussed in more to the activated type I receptors to degrade them (Feng and Der-
detail below. Examples of such ligand-sequestering molecules ynck, 2005). Receptor dephosphorylation also occurs, mediated
are the BMP antagonists Chordin/Short gastrulation (Sog), via the phosphatase PP1, which is targeted to the active type I
Noggin, Twisted gastrulation (Tsg), Crossveinless-2 (Cv-2), Scle- receptors with its regulatory subunit GADD34, again by Smad7
rostin, members of the DAN family, and Follistatin, an Activin (Shi et al., 2004). It is not clear in which cellular compartment
inhibitor that also interacts with BMPs and GDF8 (De Robertis this occurs, although it is tempting to speculate that it might be
and Kuroda, 2004). part of the receptor recycling pathway (Figure 2B). Active
The Receptors signaling is thought to occur in the endosomes (Di Guglielmo
TGF-b superfamily members require two different serine/threo- et al., 2003). Work on the Nodal/Activin pathway in Xenopus indi-
nine kinase receptors to signal, a type I and a type II. There are cates that receptor trafficking is controlled by the Ras GTPase
seven type I receptors (ALKs 1–7) and five type II receptors in family member Rap2 (Choi et al., 2008). In the absence of signal,
the human genome (for phylogenetic trees, see Schmierer and Rap2 directs the receptors into a recycling pathway via early
Hill, 2007). The ligand brings the receptors together in a heterote- sorting endosomes, which prevents their degradation and main-
trameric complex in which the type II receptors phosphorylate tains their levels at the plasma membrane. Upon Nodal/Activin
and activate the type I receptors (Figure 2A). For some ligand- stimulation, Rap2 delays receptor turnover to promote receptor
receptor interactions, coreceptors, such as Betaglycan, Endo- activity (Choi et al., 2008) (Figure 2B).
glin, and members of the EGF-CFC family, are also required The Smads
(Feng and Derynck, 2005). In the case of the TGF-b receptors, The most studied signaling pathway downstream of TGF-b
the ligand-receptor interaction is highly cooperative. The superfamily receptors is the Smad pathway. The Smads are
ligand-receptor complexes assemble through the recruitment a group of intracellular signaling molecules comprising the
of the low-affinity type I receptor by the ligand-bound high- receptor-regulated Smads (R-Smads) Smad1, 2, 3, 5, and 8,
affinity type II receptor, facilitated by direct type I-type II interac- the co-Smad Smad4, and the inhibitory Smads Smad6 and 7.
tions at the composite ligand-type II interface (Groppe et al., Upon ligand stimulation, the R-Smads are phosphorylated by
2008). In contrast, the BMP receptor complexes do not the type I receptors at two serines in an S-M/V-S motif at their
assemble cooperatively and the type I and type II extracellular extreme C termini (Massague et al., 2005). They then form both
domains do not interact directly. Instead they are linked via the homomeric and heteromeric complexes with Smad4 that accu-
ligand, and membrane localization may also promote ligand- mulate in the nucleus and directly regulate the transcription of
receptor assembly (Groppe et al., 2008). target genes (Figure 2A). The original view of TGF-b superfamily
It is becoming apparent that many different combinations of signaling pathways were that there were two branches: a BMP/
ligand-receptor interactions and type I and type II receptor pair- GDF branch signaling through ALKs 2, 3, and 6 and R-Smads
ings can occur, creating a huge potential diversity in the outputs 1, 5, and 8; and a TGF-b/Nodal/Activin branch signaling through
of the signaling pathways (Feng and Derynck, 2005). If the ligands ALKs 4, 5, and 7 and R-Smads 2 and 3 (Massague et al., 2005).
have different affinities for distinct receptor combinations, then it However, this is an oversimplification, since some GDFs, for
is easy to see how ligand dose-dependent responses may be example GDF8, 9, and 11, signal through ALKs 4, 5, and 7 (Schmi-
generated. For the most part, the type II and type I receptor dimers erer and Hill, 2007). Moreover, TGF-b activates both Smad2/3
in a ligand-receptor complex are assumed to be homodimers. and Smad1/5/8 in a variety of endothelial, epithelial, fibroblast,
However, in endothelial cells TGF-b activates both ALK1 and and tumor cells (Bharathy et al., 2008; Daly et al., 2008; Goumans
ALK5, and a complex has been proposed comprising these two et al., 2003; Liu et al., 2009). In epithelial cells this results in the
distinct type I receptors along with the TGF-b type II receptor TbRII formation of mixed R-Smad complexes, containing, for example,
(Goumans et al., 2003). Similarly, mixed receptor complexes activated Smad1 and Smad2 in addition to the canonical Smad2/
containing TbRII, ALK5, and either ALK2 or ALK3 have been 3-Smad4 complexes (Daly et al., 2008) (Figure 2A).
proposed to mediate a novel branch of TGF-b signaling in epithe- The mechanism whereby ligand stimulation leads to nuclear
lial cells (Daly et al., 2008). In this case, higher concentrations of accumulation of active Smad complexes is now well understood.
TGF-b are required to activate the putative heteromeric TbRII- The R-Smads and Smad4 constantly shuttle between the cyto-
ALK5-ALK2/3 receptor complex, compared with the canonical plasm and nucleus, both in unstimulated and ligand-stimulated
TbRII-ALK5 receptor complex. In Drosophila, a receptor complex cells, and nuclear accumulation of active Smad complexes is

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achieved by a profound decrease in Smad nuclear export rate factors (for reviews see Feng and Derynck, 2005; Ross and Hill,
and an increase in import rate compared with monomeric Smads 2008). Well-characterized examples of Smad2-interacting tran-
(Schmierer et al., 2008). The failure of Smad complexes to be scription factors are the FoxH1 family of winged-helix transcrip-
exported may result from their active retention in the nucleus. tion factors and members of the Mix family of homeobox proteins,
Consistent with this the transcriptional regulator TAZ has been Mixer, Milk, and Bix3, which recruit Smad2-Smad4 complexes to
demonstrated to bind nuclear Smad2/3-Smad4 complexes target gene promoters in response to Nodal signaling (Kunwar
and is recruited with them to TGF-b-responsive promoter et al., 2003; ten Dijke and Hill, 2004). Importantly, these two fami-
elements (Varelas et al., 2008). If TAZ is knocked down, Smad2 lies of transcription factors exhibit different expression patterns
and 3 do not accumulate in the nucleus upon TGF-b stimulation. and have different DNA-binding specificities (Hill, 2001), and
A prerequisite for Smad nuclear export during signaling is Smad thus they dictate cell-type-specific responses, mediating tran-
dephosphorylation, and several Smad phosphatases have been scription of different sets of genes in distinct cell types in
identified. Smad1 phosphatases include pyruvate dehydroge- response to Nodal signaling (Hoodless et al., 2001; Kunwar
nase phosphatase (PDP) and small C-terminal phosphatases et al., 2003; ten Dijke and Hill, 2004).
(SCP1, 2 and 3), while PPM1A can dephosphorylate both subsets All Smad-DNA binding interactions have been studied to date
of R-Smad (Chen et al., 2006; Knockaert et al., 2006; Lin et al., in the context of R-Smad-Smad4 complexes, but a considerable
2006). In none of these cases does knockdown of the phospha- number of TGF-b-induced genes do not require Smad4 for their
tase completely abolish Smad dephosphorylation when the regulation (Levy and Hill, 2005). It is possible that complexes con-
receptors are inactivated, leaving open the possibility that other taining only R-Smads, which form in the absence of Smad4, may
Smad C-terminal phosphatases remain to be discovered. be recruited to promoters of such genes. Indeed, Smad2/3 has
Activated Smad complexes bind to promoter sequences and been shown to interact with the nuclear IkB kinase a (IKKa) in
regulate transcription both positively and negatively (Figure 2C). the absence of Smad4 to regulate transcription of the Myc antag-
This has recently been extensively reviewed (Ross and Hill, onist Mad1 (Descargues et al., 2008). Furthermore, the transcrip-
2008), so we will just summarize the salient points here. Different tional regulator TRIM33/Tif1g/Ectodermin has been suggested to
activated Smad complexes have distinct DNA sequence speci- act as an alternative Smad4 to promote TGF-b-induced erythroid
ficities, explaining the diverse transcriptional responses of differentiation (He et al., 2006), although this is controversial as
different TGF-b superfamily members. The N-terminal MH1 TRIM33/Tif1g/Ectodermin, which is a RING-finger-containing
domains of Smad3 and 4 recognize the sequence 50 -GTCT-30 E3 ubiquitin ligase, was previously identified as a negative regu-
or its reverse complement, 50 -AGAC-30 , and complexes of lator of Smad4 (Dupont et al., 2005). The very recent discovery
Smad3 and Smad4 thus bind direct or inverted repeats of these that activated R-Smad complexes act in the absence of Smad4
Smad binding elements (SBEs) (Dennler et al., 1998). Smad1/5 to regulate the processing of a microRNA, miR-21, raises the
bind the GC-rich consensus 50 -GRCGNC-30 and when com- very interesting possibility that the Smads can additionally
plexed with Smad4, bind a combinatorial site comprising the regulate gene expression without directly affecting transcription
GC-rich element and an SBE spaced five base pairs apart (Pyro- (Davis et al., 2008).
wolakis et al., 2004). These complexes are stabilized by the tran- The Smads absolutely require chromatin to assemble the
scriptional regulator Schnurri, which contacts both Smads. basal transcription machinery and activate transcription, and
These Smad1/5-Smad4-Schnurri complexes are inhibitory in so predominantly act through chromatin remodeling. This is in
Drosophila, but activatory in mammalian tissue culture cells contrast to most transcription factors, which can directly recruit
(Yao et al., 2006). In fact in Drosophila, many Dpp-induced target the basal machinery to proximal promoters (Ross et al., 2006)
genes are not directly regulated by an activatory Smad1-Smad4 (Figure 2C). Smad-induced chromatin remodeling requires the
complex as they are for vertebrate BMP target genes, but are indi- histone acetylase p300, which specifically acetylates histone
rectly induced through repression of a transcriptional repressor H3 on lysines 9 and 18, and also the SWI/SNF component
Brinker (Brk). brk is repressed by Dpp signaling via a Schnurri- Brg1 (Ross et al., 2006; Xi et al., 2008). Undoubtedly other chro-
Smad1-Smad4 complex, and loss of Brk then derepresses the matin remodeling and histone modifying enzymes are also
Dpp target genes (Pyrowolakis et al., 2004). In the case of involved.
Smad2, although a spliced variant exists that binds DNA indistin- Non-Smad Signaling
guishably from Smad3, the major Smad2 isoform does not bind Although the Smads are the best understood signal transducers
DNA directly and thus most Smad2-Smad4 complexes are downstream of TGF-b superfamily receptors, other signaling
recruited to DNA through interactions with other transcription pathways can also be activated directly in response to TGF-b.

Figure 2. Summary of the Molecular Mechanism of TGF-b Superfamily Signaling

(A) Heterotetrameric receptor complexes are activated by ligand dimers (red and green triangles) and phosphorylate downstream effectors, the R-Smads
(Smad1/5/8/2/3). Phosphorylated R-Smads then form complexes with each other (right-hand side of figure) and/or the co-Smad, Smad4 (left-hand side of figure),
and accumulate in the nucleus to regulate target gene transcription. The function of the mixed R-Smad complexes in the nucleus is not yet clear (Daly et al., 2008).
Transcription factors (TF) cooperate with Smad complexes on DNA. Nonphosphorylated monomeric Smads shuttle in and out of the nucleus. Multimeric
complexes dissociate after R-Smad dephosphorylation in the nucleus and shuttle back to the cytoplasm. This is a receptor activity monitoring system.
(B) Receptors are internalized constitutively via clathrin-mediated endocytosis and caveolin-positive lipid rafts. Smad7/Smurf2-mediated degradation occurs in
lipid rafts while receptors are recycled to the plasma membrane through endosomes. Active signaling occurs in EEA1-positive early ensodomes where SARA
presents R-Smads to activated receptor complexes. PP1 dephosphorylates receptors in a Smad7-dependent manner. The Ras GTPase Rap2 promotes recy-
cling in the absence of signal and delays recycling in presence of signal.
(C) Smad-dependent transcription requires chromatin remodeling. Smads recruit the histone deacetylase p300 and Brg1-SWI/SNF complex. Nucleosome
remodeling and histone modification, for example histone H3 acetylation (AcH3), then allows the recruitment of the basal transcriptional machinery.

Developmental Cell 16, March 17, 2009 ª2009 Elsevier Inc. 335
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The type II receptor phosphorylates PAR6, a regulator of epithe-

lial polarity, leading to the dissolution of tight junctions, an initi-
ating step in EMT (Ozdamar et al., 2005). TGF-b can also activate
the ERK MAP kinase (MAPK) signaling pathway through the
ability of ALK5 to phosphorylate the scaffold protein ShcA, which
recruits Grb and Sos, thereby activating Ras and downstream
MAPKs (Lee et al., 2007). Moreover, two very recent papers
have described the mechanism whereby TGF-b induces JNK
and p38 MAPK signaling (Sorrentino et al., 2008; Yamashita
et al., 2008). In this case, TRAF6 interacts with the TGF-b recep-
tors and TGF-b induction leads to K63-linked ubiquitination of
TRAF6. This in turn leads to activation of the MAPKKK Tak1,
an upstream activator of JNK and p38. Finally, in mesenchymal
cells, but not epithelial cells, TGF-b activates the kinase PAK2
and this occurs via the activation of the small GTPases Rac1
and Cdc42 (Wilkes et al., 2003). The basis of this cell type spec-
ificity is now explained in a paper in this issue (Wilkes et al.,
2009). The authors show that in fact in both cell types TGF-b
can activate PAK2, but in epithelial cells activated PAK2 is
bound and inhibited by a complex of Erbin and the tumor
suppressor Merlin. This does not occur in mesenchymal cells
as they express very low levels of Erbin.

How Do TGF-b Superfamily Members Achieve

Their In Vivo Roles? Figure 3. BMP Signaling Gradients in Early Embryos
To achieve the in vivo roles in embryonic development summa- (A) mRNA and protein distributions in the Drosophila blastoderm and early
gastrula.
rized in the first section, TGF-b superfamily ligands must be
(B and C) BMP signal activity as detected by phospho-R-Smads in Drosophila
capable of functioning in a graded fashion as morphogens (for (P-Mad) (B) and zebrafish (P-Smad5) (C). In both cases, BMP signaling activity
example BMPs during D/V patterning in Drosophila and verte- is weak and diffuse over a broad area to begin with, but becomes increasingly
brates) and/or be able to signal in a highly dynamic manner localized, at the dorsal midline for Drosophila, and on the ventral side for zebra-
fish, with a gradient of activity diminishing toward the lateral regions.
both spatially and temporally (for example Nodal in L/R These images are taken from Rushlow et al. (2001) (B) and Tucker et al. (2008)
patterning and BMPs/GDFs during vertebrate limb develop- (C) with permission.
ment). Having outlined the intracellular signaling pathways in
the second section, we now highlight the known mechanisms lez-Gaitan, 2008). The Dpp gradient that specifies A/P patterning
that regulate the formation of ligand gradients and dynamic of the Drosophila wing obeys this paradigm. During larval devel-
signals, and those that regulate the downstream pathway to opment, Dpp is secreted in the wing imaginal disc from a central
interpret the signals and determine the specificity of the stripe of cells and forms a concentration gradient both anteriorly
responses. We discuss how BMPs are able to form gradients and posteriorly. This gradient has an approximately exponential
of ligand activity, which, over time, frequently sharpen to step- shape, governed by the rates of ligand diffusion, production, and
wise signals that ultimately define different tissue types (Suther- degradation, and the presence of an immobile fraction (Kicheva
land et al., 2003) (Figure 3). We examine how graded signals are and Gonzalez-Gaitan, 2008). The exact mechanism whereby the
sensed by receiving cells, leading to differential transcriptional ligand spreads is still a matter of debate. One proposed mecha-
responses according to ligand dose. In both of these areas, nism involves transcytosis (Kicheva et al., 2007), where Dpp is
mathematical modeling has proved to be a very powerful tool secreted from the producing cells and then endocytosed and
and this will be highlighted. We also discuss how the range of secreted by neighboring cells. An alternative mechanism is that
signaling is determined, newly discovered mechanisms whereby Dpp simply diffuses through the target tissue, with its diffusion
signal strength, duration, or both are modulated, and how other rate being restricted by heparan sulfate proteoglycans (Kicheva
signal transduction pathways modulate TGF-b superfamily and Gonzalez-Gaitan, 2008). In reality both mechanisms may be
signaling. All of these regulatory mechanisms provide qualitative relevant (Affolter and Basler, 2007). Interestingly, the staining
and quantitative differences in signaling that are capable of pattern of phosphorylated Mad (P-Mad), the downstream signal
increasing the diversity of functional outputs. transducer, in the wing disc does not correlate with Dpp ligand
Generating Gradients of TGF-b Superfamily Ligand concentration, suggesting that additional BMP ligands are
Activity involved. Indeed Gbb, which is broadly expressed in the wing,
Our current understanding of gradient formation derives mostly also signals in the wing disc, acting over long distances, while
from studies in Drosophila, and these studies will thus be a focal Dpp acts over a shorter range (Bangi and Wharton, 2006).
point for our discussion. The traditional view of morphogen In contrast to the wing disc, a gradient of Dpp activity forms in
gradients is that the ligand is secreted from a localized source, the early Drosophila embryo within a domain of uniform Dpp
diffuses across a field of cells, and forms a concentration mRNA expression in the dorsalmost 40% of the embryo. This
gradient in the target tissue (reviewed in Kicheva and Gonza- means that gradient formation cannot simply involve diffusion

336 Developmental Cell 16, March 17, 2009 ª2009 Elsevier Inc.
Developmental Cell

Review

of ligand away from a localized source, but instead involves redis- Very recently another molecule involved in D/V axis formation
tribution of ligand within the expression domain (Figure 3A). An in Xenopus embryos has been identified. ONT1, a member of the
important feature of the underlying mechanism is that Dpp acts Olfactomedin family of secreted proteins, has been shown to
together with another ligand, Scw, expressed uniformly in the maintain appropriate Chordin levels in dorsal regions by acting
blastoderm. Homomeric and heteromeric dimers of Dpp and as a scaffold between Chordin and BMP1/Tld-class proteases,
Scw form and bind with different affinities to the ligand antago- promoting Chordin cleavage and degradation (Inomata et al.,
nists Sog and Tsg (O’Connor et al., 2006). sog is expressed in 2008). This is required to prevent uncontrolled increases in Chor-
ventral lateral regions, while tsg is expressed in the dorsal half din levels that could shift the BMP gradient and lead to expan-
of the embryo (O’Connor et al., 2006) (Figure 3A). Dpp-Scw het- sion of the dorsal domain.
erodimers bind with high affinity to Sog and Tsg, forming How Gradients Are Sensed and Interpreted
a complex incapable of binding receptors, and are transported Ligand gradients must be quantitatively sensed and interpreted,
away from the source of Sog to the dorsal midline. There, they and several features of the TGF-b superfamily signaling pathway
are released from Sog and Tsg as a result of Sog cleavage by have been uncovered that help explain how this is accomplished.
a dorsally expressed metalloprotease, Tolloid (Tld), allowing the The Smad pathway is ideal for interpreting graded signals, as it
ligand dimer to activate receptor complexes containing the has no amplification steps (Schmierer and Hill, 2007). Moreover,
type I receptors Tkv and Sax (O’Connor et al., 2006). Tld-induced continuous shuttling of the Smads between the cytoplasm and
cleavage of Sog in more lateral regions results in rebinding of nucleus during signaling, mediated by cycles of receptor-induced
Dpp-Scw to other molecules of Sog and Tsg, due in part to higher Smad phosphorylation in the cytoplasm and constitutive Smad
levels of Sog in these regions. Homodimers of Dpp and Scw have dephosphorylation in the nucleus, allows the Smads to constantly
lower affinity for Sog and Tsg and thus are not transported as far monitor receptor activity (Schmierer and Hill, 2007). This concept
and are thought to elicit the weaker BMP signal in dorsal lateral has been explored by mathematical modeling, which has
regions of the embryo. Mathematical modeling suggests that confirmed that the levels of active Smad2-Smad4 complexes in
this mechanism can account for a BMP ligand concentration the nucleus directly reflect the activation level of the receptors
gradient. However, it predicts that over time the domain of at all times during signaling (Schmierer et al., 2008) (Figure 4). In
P-Mad would broaden and P-Mad levels would increase in inten- this way both the strength and duration of signaling is constantly
sity, whereas in reality, the dorsalmost region of high P-Mad monitored. The computational model was used to identify indi-
activity actually contracts, giving a step gradient (O’Connor vidual steps in the signaling cascade that, when modulated, can
et al., 2006) (Figure 3B). The most likely explanation is that there affect system output most significantly. The most important
is positive feedback in the system such that the initial Dpp-Scw step for positively influencing the peak concentration of nuclear
signal induces an as yet unidentified component that either active Smad2-Smad4 complexes is Smad2 phosphorylation,
reduces the interaction of receptors with an inhibitory factor or while Smad2 dephosphorylation, dissociation of active Smad
promotes ligand binding to receptors (Wang and Ferguson, complexes, and Smad4 nuclear export are the steps that have
2005). Very recently, type IV collagens have been shown to play the most negative influence (Schmierer et al., 2008). Consistent
a role in formation of the Dpp D/V gradient, promoting assembly with this, studies in Drosophila have shown that the nuclear export
of the Dpp/Scw-Sog-Tsg complex, and in the absence of Sog, rate of the Drosophila Smad4 Medea is promoted by SUMOyla-
facilitating Dpp/Scw-receptor interactions, thus amplifying the tion and, as a consequence, failure to SUMOylate Medea results
signal at the dorsal midline (Wang et al., 2008). in increased Dpp signaling range (Miles et al., 2008).
This mechanism for gradient formation might not be unique to Just as the levels of receptor activity are continuously moni-
the early Drosophila embryo, but may also function in early Xen- tored, so the ligand levels must be sensed accurately and
opus embryos, where the BMP ligands are also expressed in dynamically by the receptors. This is currently not well under-
a very broad domain, but nevertheless develop into a ventral- stood. It is tempting to speculate that constitutive receptor recy-
dorsal activity gradient (Little and Mullins, 2006). Xenopus ortho- cling through the endocytic pathway (Di Guglielmo et al., 2003;
logs of sog (known as Chordin in vertebrates), tsg, and tolloid Figure 2B) may act to monitor ligand levels in an analogous
have all been identified and biochemically the proteins have fashion to the Smads’ monitoring of receptor activity. A similar
been shown to act in the same way as in Drosophila (Oelgeschl- mechanism is known to be important for controlling the duration
ager et al., 2000; Piccolo et al., 1996, 1997; Sasai et al., 1995). of EGF signaling (Sigismund et al., 2008).
Mathematical modeling has been used to compare two possible Smad nucleocytoplasmic shuttling results in levels of acti-
mechanisms of BMP gradient formation in Xenopus embryos vated nuclear Smad complexes that reflect the extent of
(Ben-Zvi et al., 2008). One model assumes an inhibition-based receptor activation, but how is signal intensity ‘‘translated’’ into
mechanism whereby a gradient of BMP antagonists is created differential gene expression programs? An important determi-
over a uniform field of BMP ligands, and the other assumes nant is the affinity of Smad-binding sites for activated Smad
a shuttling-based mechanism where the ligands are transported complexes, and this has been directly demonstrated in the early
by inhibitors as described above for Dpp-Scw. The authors Drosophila embryo for the Dpp target gene race. Increasing the
found that while both mechanisms can generate a BMP activity affinity of the Smad-binding sites in its enhancer placed
gradient, the shuttling-based mechanism gives a sharper and upstream of a reporter gene broadened the reporter expression
more robust gradient. These models were tested experimentally pattern (Wharton et al., 2004). However, as discussed above,
and the evidence suggested that the shuttling-based mecha- many Dpp target genes in the Drosophila embryo are not acti-
nism could be involved in BMP gradient formation in Xenopus vated by Dpp per se, but are derepressed as a result of Dpp-
embryos. induced repression of the transcriptional repressor Brk. Multiple

Developmental Cell 16, March 17, 2009 ª2009 Elsevier Inc. 337
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Interestingly, cells along the A/P axis become competent to

respond to BMP signals in a temporal fashion such that as
gastrulation proceeds, progressively more caudal cells respond
to the BMP gradient and adopt their D/V identity. This temporal
regulation thus coordinates D/V with A/P patterning.
Determinants of Signaling Range
Several studies have revealed that distinct TGF-b family
members have very different signaling ranges in vivo (Chen and
Schier, 2001; Jones et al., 1996), and this seems to be determined
by the ligand prodomain, which is the least conserved part of the
protein. Prodomain swapping and mutation experiments have
demonstrated that these domains can affect the stability of these
proteins (Cui et al., 2001; Le Good et al., 2005). In most cases,
stability is affected during secretion of the ligands, and limiting
the concentration of the secreted product limits their range.
Indeed, the prodomain of the short-range zebrafish Nodal ligand
Cyc contains a lysosome-targeting region that destabilizes the
precursor and thus restricts Cyc activity (Tian et al., 2008). Addi-
tionally, BMP4 precursors are cleaved sequentially at two sites in
the prodomain. The first cleavage site activates the ligand, while
the second cleavage site affects stability and range (Cui et al.,
2001; Sopory et al., 2006). Posttranslational modifications of
domains, such as glycosylation, in addition to cleavage, also
contribute to ligand regulation (Le Good et al., 2005).
Signal range can also be influenced by the interaction of
ligands with secreted binding partners, and a good example of
this is Cv-2. Unlike Chordin, Cv-2 is not diffusible, but is tethered
Figure 4. Nucleocytoplasmic Shuttling of the Smads Acts as a Signal to the membrane by heparan sulfate proteoglycans and thus
Interpretation System
(A) Using the mathematical model of nucleocytoplasmic shuttling, receptor modulates BMP signals over a short range (Serpe et al., 2008).
levels were altered in a step-wise fashion as shown. The simulation demon- Studies focusing on the role of Cv-2 in the Drosophila wing indi-
strates that Smad nucleocytoplasmic shuttling couples the amount of nuclear cate that Cv-2 activates BMP signaling at low concentrations,
Smad2-Smad4 complexes to receptor activity, although with a time delay.
(B) Because of this delay, fluctuations in receptor activity are strongly damp-
but antagonizes it at higher concentrations (Serpe et al., 2008).
ened and do not cause corresponding fluctuations in the concentration of Moreover, Cv-2 binds BMP type I receptors as well as ligands.
nuclear Smad2-Smad4 complexes. As with other aspects of TGF-b superfamily signaling, mathe-
This image was taken from Schmierer et al. (2008) with permission.
matical modeling has been used to gain insights into the mech-
anism (Serpe et al., 2008). The model suggests that a transient
regulatory modules in the enhancer of brk bind repressive low-affinity tripartite complex forms and facilitates ligand-
Schnurri-Mad-Medea complexes, resulting in an inverse expres- receptor binding, but at high Cv-2 concentrations, this is
sion gradient relative to the Dpp activity gradient (Yao et al., compromised and it instead sequesters ligands to inhibit
2008). Some Dpp concentration-dependent transcription is signaling. A crystal structure of the Cv-2 N-terminal Von Wille-
then explained by the fact that Dpp-responsive genes have Brk brand factor type C domain 1 (VWC1) bound to BMP2 reveals
binding sites with different affinity in their enhancers. In addition, that Cv-2 sequesters ligand by blocking receptor binding sites
although loss of Brk-induced repression is sufficient for activa- (Zhang et al., 2008). Since Cv-2 is itself induced by BMP activity,
tion of some genes, others additionally require binding of activa- Cv-2 expression not only serves as a direct measure of BMP
tory Mad-Medea complexes (Affolter and Basler, 2007). For activity, but functions as part of a very effective feedback loop
Smad2-dependent responses in vertebrates, the affinity of acti- capable of maintaining signaling activity at an appropriate level
vated Smad2 for the DNA-bound Smad2-interacting transcrip- and preventing runaway signaling.
tion factors may play a role in interpreting the level of signal As in Drosophila, Cv-2 is expressed in regions of high BMP
intensity (Randall et al., 2004). signaling in both Xenopus and zebrafish embryos (Ambrosio
We have discussed mechanisms that allow cells to interpret et al., 2008; Rentzsch et al., 2006). The zebrafish Cv-2 also has
a signaling gradient. However, cells also need to be competent pro- and anti-BMP effects, and loss-of-function experiments
to respond to the signals. In early zebrafish embryos, this seems reveal that Cv-2 has mainly a signal-promoting function, since
to be regulated in a temporal fashion (Tucker et al., 2008). The Cv-2 morphants are moderately dorsalized (Rentzsch et al.,
developing embryo may be thought of as a four-dimensional 2006). However, in Xenopus, Cv-2 has a predominantly inhibitory
system, since besides acting in space, gradients may achieve role in BMP signaling, and may be part of a complex molecular
functional diversity by acting over time as well. Using heat shock circuit through its interactions with other secreted modulators
induction of Chordin to inhibit BMP signaling at different times of BMP signaling (Ambrosio et al., 2008). Cv-2 is capable of syn-
during zebrafish development, Tucker et al. (2008) tested how ergizing with the BMP-induced modulator Tsg to act as a local
time is involved in the response of tissues to the BMP gradient. feedback inhibitor on the ventral side of the embryo. It also

338 Developmental Cell 16, March 17, 2009 ª2009 Elsevier Inc.
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interacts with Chordin, and may exert a pro-BMP signaling influ- and tooth development (De Robertis and Kuroda, 2004). It now
ence by concentrating diffusable Chordin-BMP-Tsg in ventral turns out that the activity of the Smads themselves is directly
regions of the embryo (Ambrosio et al., 2008). influenced by RTK signaling. R-Smads and co-Smads have
Modulation of TGF-b Superfamily Signaling Activity a proline-rich linker that connects the MH1 and MH2 domains.
and Dynamics MAPKs activated downstream of RTKs phosphorylate several
Spatial and temporal control of Nodal signaling was recently serines and threonines in the Smad1 linker, which promotes
shown to be regulated by microRNAs in zebrafish and Xenopus binding of the E3 ubiquitin ligase Smurf1. This in turn enhances
(Choi et al., 2007; Martello et al., 2007). In zebrafish, miR-430 has degradation of Smad1, which reduces BMP signal transduction,
been shown to target both a Nodal ligand and Nodal antagonists, and curtails responses (Sapkota et al., 2007). The corollary of this
sqt and Lefty1/2, respectively, with its effects strongest on Lefty2 is that any signal that inhibits Smad1 linker phosphorylation
(Choi et al., 2007). miR-430 functions by balancing the expres- would prolong the duration of BMP signaling. Interestingly, the
sion of these positive and negative players to achieve optimal MAPK sites in the Smad1 linker act as priming sites for glycogen
signal levels required for organizer formation and germ layer synthase kinase 3 (GSK3), which is inhibited by Wnt signaling
specification (Choi et al., 2007). Nodal induces itself as well as (Fuentealba et al., 2007). GSK3 phosphorylation of linker serines
Lefty, and this auto-activatory-inhibitory loop limits signal range, and threonines close to the MAPK sites also destabilizes Smad1,
duration, and level, thus creating borders, patterns, or asymme- and thus it has been suggested that Wnt signaling synergizes
tries (Solnica-Krezel, 2003). The Nodal-Lefty mechanism fits with BMP signaling by stabilizing the Smads and increasing
what is known as the Turing reaction-diffusion model (reviewed the duration of BMP signaling (Fuentealba et al., 2007).
by Solnica-Krezel, 2003). This model explains how noise in an It is not only the Smads that can integrate signals from other
otherwise uniform diffusion system can become amplified by pathways; Smad-interacting transcription factors can also
the coexpression of a ligand and its antagonist. For this system perform this function. RTK signaling via Ras has long been
to work, the antagonist must be able to diffuse faster than the established as a requirement for Nodal/Activin-induced meso-
ligand, which thus creates short-range activation and long-range derm formation in Xenopus embryos, although it was unclear
inhibition. Changes in expression levels of either component, exactly how it modulated the Nodal signaling pathway. Recent
through, for example, the action of miR-430, would influence work has demonstrated that the target of RTK signaling in this
signal range, duration, and level. It will be interesting to see if case is the transcription factor p53 (Cordenonsi et al., 2007).
other microRNAs are involved in Nodal-Lefty regulation at later p53 is required for induction of a subset of Nodal target genes
stages, such as during L/R specification. and functions by interacting directly with Smad2 at the
In Xenopus, the Nodal signaling pathway has also been shown promoters of these genes. RTK signaling via Ras leads to phos-
to be modulated by the action of a microRNA, miR-15/16, the phorylation of p53 via the kinase CK13/d. This increases the
target of which is the type II Nodal receptor ACVR2A (Martello association of p53 with activated Smad2, and thus promotes
et al., 2007). While the expression of the miR-15/16 primary tran- the transcription of mesodermal target genes.
script is ubiquitous, processing of this transcript into mature A further informative example of signal integration at the level
functional miR-15 is inhibited by the Wnt/b-catenin pathway. of enhancer or promoter elements is the integration of Wnt and
Since b-catenin accumulates on the dorsal side of the embryo Dpp signals at the Leg Trigger (LT) enhancer of the Distalless
after fertilization, mature miR-15 is found in a gradient, with the (Dll) gene in Drosophila, which is involved in leg development
highest levels ventrally. This feature of miR-15 creates a reverse (Estella et al., 2008). The LT enhancer contains binding sites
expression gradient of ACVR2A, and hence restricts responsive- for TCF (which binds an activatory complex of b-catenin,
ness to Nodal ligands in a spatial manner and controls the size of Legless, and Pygopus in response to Wnt signaling), Mad, and
the organizer. Brk, and active transcription occurs only when both Wnt and
In both studies, whether balancing the levels of secreted Dpp signaling is high. This can be explained as follows. When
agonists and antagonists or spatially restricting responsive both signaling pathways are operating, the TCF and Mad sites
components in the pathway, microRNAs seem to provide bind their respective activatory complexes and the Brk site is
robustness to the signaling and can prevent transcriptional mis- unoccupied; thus, transcription is active. In cells exhibiting low
regulation of signaling components from causing catastrophic Dpp signaling, but high Wnt signaling, the binding of Brk to the
changes during early development. enhancer inhibits transcription. Conversely, in cells with high
Modulation of TGF-b Superfamily Signaling by Other Dpp signaling, but no Wnt signaling, the binding of activated
Signal Transduction Pathways Mad complexes is not sufficient for transcriptional activation.
Finally, it is important to consider that TGF-b superfamily In this way, the spatial domain of Dll transcription is defined by
signaling in any given cell does not occur in isolation, but is cells exhibiting simultaneously high Wnt and Dpp signaling.
subject to modulation by other signaling pathways. The duration
of TGF-b superfamily signaling can be regulated in this way, and Perspectives
in addition, transcriptional responses to TGF-b superfamily Our view of TGF-b superfamily signaling pathways has evolved
signals can be influenced due to the integration of multiple signals significantly since a decade ago, when the pathway was
at the promoters of target genes. perceived to be a three-step system of receptor activation by
It is well established that receptor tyrosine kinase (RTK) ligands followed by effector molecule activation through phos-
signaling can have an antagonistic effect on BMP signaling in phorylation, resulting in transcriptional activation or repression.
many developmental contexts, such as during neural induction, The tremendous effort by scientists around the world using
limb development, lung morphogenesis, cranial suture fusion, a combination of developmental, cellular, and molecular systems

Developmental Cell 16, March 17, 2009 ª2009 Elsevier Inc. 339
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Review

has contributed to our current understanding of the modulatory Brennan, J., Norris, D.P., and Robertson, E.J. (2002). Nodal activity in the node
governs left-right asymmetry. Genes Dev. 16, 2339–2344.
mechanisms that provide both robustness and diversity to this
signaling system. Chen, Y., and Schier, A.F. (2001). The zebrafish Nodal signal Squint functions
It is evident from our discussion that the model organisms and as a morphogen. Nature 411, 607–610.
systems used have different strengths, and thus have been Chen, H.B., Shen, J., Ip, Y.T., and Xu, L. (2006). Identification of phosphatases
exploited to address different questions. While playing to the for Smad in the BMP/DPP pathway. Genes Dev. 20, 648–653.
strengths of specific models is the most efficient way to gain
Chocron, S., Verhoeven, M.C., Rentzsch, F., Hammerschmidt, M., and
understanding, integration and consolidation of data between Bakkers, J. (2007). Zebrafish Bmp4 regulates left-right asymmetry at two
systems is now essential. For example, transport of BMPs within distinct developmental time points. Dev. Biol. 305, 577–588.
their expression domain is established as a mechanism for Choi, W.Y., Giraldez, A.J., and Schier, A.F. (2007). Target protectors reveal
gradient formation in the Drosophila blastoderm, and recent dampening and balancing of Nodal agonist and antagonist by miR-430.
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ACKNOWLEDGMENTS
De Robertis, E.M., and Kuroda, H. (2004). Dorsal-ventral patterning and neural
induction in Xenopus embryos. Annu. Rev. Cell Dev. Biol. 20, 285–308.
We thank all the members of the Hill lab, Megan Cully, Mike Howell, Erik Sahai,
Jim Smith, and Nic Tapon for very useful comments on the manuscript. The Dennler, S., Itoh, S., Vivien, D., ten Dijke, P., Huet, S., and Gauthier, J.M.
work in the Hill lab is funded by Cancer Research UK and the EU (MRTN- (1998). Direct binding of Smad3 and Smad4 to critical TGF b-inducible
CT-2004-005428). M.Y.W. acknowledges an ORS award. elements in the promoter of human plasminogen activator inhibitor-type
1 gene. EMBO J. 17, 3091–3100.
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