Download regionally specific induction by the spemann

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REGIONALLY SPECIFIC INDUCTION
BY THE SPEMANN–MANGOLD
ORGANIZER
Christof Niehrs
Eighty years ago, Spemann and Mangold discovered the extraordinary inductive potency of the
dorsal blastopore lip in amphibian embryos. Many inducers released by this organizer have now
been identified and they typically encode antagonists of bone morphogenetic protein, Nodal or
Wnt growth factors. The different expression domains of these growth factors and their
antagonists create signalling gradients, which pattern the early embryo in a combinatorial fashion
and explain the regional specificities of head, trunk and tail organizers. New findings indicate that
both quantitative and qualitative mechanisms account for regionally specific organizer function.
DORSAL BLASTOPORE LIP
The region in amphibian
embryos where gastrulation
begins. Also known as the
Spemann–Mangold organizer.
ORGANISIN
An elusive compound that was
predicted by Dalq and Pasteels in
the1930s to be emitted by the
Spemann–Mangold organizer
and its derivatives. Its regional
distribution followed sulfhydrylrich proteins.
WNTS
A growth-factor family that acts
through seven transmembrane
receptors.
Division of Molecular
Embryology, Deutsches
Krebsforschungszentrum,
Im Neuenheimer Feld 280,
69120 Heidelberg, Germany.
e-mail:
[email protected]
doi:10.1038/nrg1347
NATURE REVIEWS | GENETICS
The Spemann–Mangold organizer consists of a small
group of cells in vertebrate embryos, the morphogenetic
and inductive properties of which are of paramount
importance for the early establishment of the vertebrate
body plan. Grafted to the ventral side of a host embryo,
the DORSAL BLASTOPORE LIP, which corresponds to the organizer in amphibians, induces a twinned embryo1. The
organizer differentiates into various midline tissues (see
below), and can be subdivided into head, trunk and tail
organizers on the basis of their different inducing abilities. The various models that were historically put forward to explain regionally specific induction by these
different organizers can be divided into two classes.
Quantitative models proposed gradients of single
inducers, such as the elusive ‘ORGANISIN’, which emanate
from homogenous organizer cells. By contrast, qualitative models postulated that the organizer and its derivatives contain distinct cell subpopulations, which emit
different inducers2. Recent studies that address the role
of transforming growth factor β (TGF-β) and WNT signalling have shed light on these old questions. On the
basis of these studies, I argue in this review that both
qualitative and quantitative models apply: the organizer
contains distinct cell populations, which emit different
inducers and set up growth-factor signalling gradients
that account for regionally specific induction in the
vertebrate embryo.
The literature on the Spemann–Mangold organizer is
vast, including a new monograph3, and recent reviews
that cover the regulation of organizer formation1, mechanisms that govern its GASTRULATION movements4, neural
induction5 and patterning6. Therefore, I restrict my discussion to the molecular nature of the inducing factors
released by the organizer that account for regionally specific inductions and how they act in a combinatorial fashion to pattern the anterior–posterior and dorsal–ventral
axes of the vertebrate embryo. I focus on three growthfactor classes: BONE MORPHOGENETIC PROTEINS (BMPs), Wnt
and NODAL, which have pre-eminent roles in this context.
Other important signals, such as fibroblast growth factor
(FGF) and retinoic acid, are covered elsewhere7,8. I
include data from all principal vertebrate model systems,
each of which has contributed to our understanding of
embryonic axis formation.
Form, function and conservation
As little as a few dozen cells of an amphibian embryo
that harbours the dorsal blastopore lip is sufficient to
induce the development of a respectable miniature tadpole that contains all axial structures and a central nervous system (CNS). The organizer cells recruit and
organize neighbouring cells into a harmoniously patterned SECONDARY EMBRYONIC AXIS. The function of the
organizer is to establish the three vertebrate body axes
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a
— anterior–posterior (AP), dorsal–ventral (DV) and
left–right (LR) — in all germ layers (ECTODERM, MESODERM
AND ENDODERM). The most prominent feature of the AP
axis is the pattern of the CNS, forebrain, midbrain, hindbrain and spinal cord. The DV mesodermal axis is patterned to form axial (notochord), paraxial (somite),
intermediate and lateral plate mesoderm. The first evidence for organizer subdivision came from Spemann
himself, who found that transplantation of the early GASTRULA lip (PRESUMPTIVE PRECHORDAL MESENDODERM, PME)
into the BLASTOCOEL of host embryos resulted in the formation of secondary heads. By contrast, late gastrula lips
(PRESUMPTIVE CHORDAMESODERM) induced secondary trunks.
Tissues that correspond to the Spemann–Mangold
organizer have been identified in the chick and mouse as
HENSEN’S NODE and in fish as the shield. As in the frog, distinct head, trunk and tail organizers have been recognized
in other vertebrates9–18 (FIG. 1).
cm
PME
an
GASTRULATION
dc
A morphogenetic process that
leads to the formation of the
germ layers and the body plan.
BONE MORPHOGENETIC
PROTEINS
ae
b
(BMPs). A subfamily of the
transforming growth factor
β-superfamily.
cm
an
PME
sh
NODALS
A subfamily of the transforming
growth factor β-superfamily.
SECONDARY EMBRYONIC AXIS
c
A twin embryo that is induced
by transplantation of the
Spemann–Mangold organizer
or by manipulation of organizer
effectors.
ep
d
The embryonic stage when the
central nervous system forms
the neural tube.
AVE
In amphibians and zebrafish, the
top-most pole of the embryo.
ANTERIOR ENDODERM
An embryonic tissue that is
derived from the
Spemann–Mangold
organizer/Hensen’s node, which
will form the foregut and
pharynx.
EPIBLAST
The outer layer of the
blastoderm in the chicken; the
epiblast gives rise to the
definitive embryonic tissues.
HYPOBLAST
The inner layer of the
blastoderm in the chicken,
which covers the yolk; the
hypoblast gives rise to
extraembryonic tissues.
PRIMITIVE STREAK
An elongated structure that is
formed by an accumulation of
cells, through which cells
gastrulate.
ECTODERM, MESODERM AND
ENDODERM
The three germ layers that give
rise to all somatic tissues in
animals.
GASTRULA
The embryonic stage when the
germ layers aquire the final
position relative to each other
through a complex process of
morphogenetic movements.
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| JUNE 2004 | VOLUME 5
ps
an
hb
NEURULA
ANIMAL POLE
hn
ae
an
ps
n
PME cm
Regionally specific induction
PME
an
ps
n
cm
Figure 1 | Comparative diagram of Spemann–Mangold
organizer development in (a) Xenopus laevis, (b)
zebrafish, (c) chick and (d) mouse gastrulae. Left side,
early gastrulae; right side, late gastrulae/early NEURULAE. Sagittal
views are shown. The early gastrulae in a and b are shown with
the ANIMAL POLE to the top, dorsal to the right. In all other panels,
anterior points to the left, dorsal to the top. a | In X. laevis, the
organizer is located in the upper dorsal blastopore lip. Its different
cell populations are the leading edge cells, which give rise to
ANTERIOR ENDODERM (ae; yellow). Prechordal mesendoderm
(PME; brown) is derived from the deep cells (dc; brown) of the
Spemann–Mangold organizer and underlies the anterior neural
plate (an; purple) in the late gastrula. The last cells to involute
are chordamesodermal cells (cm; green). b | In zebrafish, the
organizer is located in the shield (sh), which contains the
indicated cell populations. c | The chick embryo is a bilayered
structure that is composed of the EPIBLAST (ep; blue) and the
extraembryonic HYPOBLAST (hb; flesh coloured). At the onset
of gastrulation, a full-length PRIMITIVE STREAK (ps) with
Hensen’s node (hn; the chick organizer; orange) at its tip has
formed. Both contain precursors of PME and chordamesoderm.
During gastrulation, cells ingress through the node, form the
PME and chordamesoderm and displace the hypoblast
anteriorly. d | In the mouse, the equivalent of the
Spemann–Mangold organizer is located in the primitive streak
and Hensen’s node. A supporting signalling centre resides in
the anterior visceral endoderm (AVE; yellow), which juxtaposes
the prospective anterior neural plate. The primitive streak with
the node (n; the mouse organizer; orange) forms at the
posterior end of the embryo. Similar to the chick, both streak
and node contain precursors of PME and chordamesoderm.
The streak elongates during gastrulation while cells emigrate
through the node and form the axial mesendoderm that
displaces the AVE. At the end of gastrulation, the PME
underlies the anterior neural plate and is followed posteriorly by
chordamesoderm. Modified with permission from REF. 20 ©
(2001) Elsevier Science Ltd.
The Spemann–Mangold organizer is the region where
gastrulation movements originate. The first organizer
cells to migrate end up anteriorly whereas the last ones
will localize to the posterior end of the embryo.
Therefore, the organizer is not a homogenous tissue
but a dynamic structure; while cells migrate during gastrulation, they acquire different fates, inducing properties and gene-expression profiles16,19. Prospective PME
cells are among the first to gastrulate and they are fated
for foregut and head mesenchyme. Transplantation
experiments in all vertebrate model systems that have
been tested indicate that these cells have the most
potent head-inducing activity20. The homeobox gene
gsc is a marker for PME. The chordamesodermal cells
are the next to involute, they give rise to notochord,
have trunk- and tail-inducing activity and express the
marker Xnot/flh.
In contrast to these contiguous tissues, the mouse
anterior visceral endoderm (AVE) and chicken anterior
hypoblast are never part of the node, although they are
essential for anterior neural induction. Mouse AVE and
chick anterior hypoblast are considered to be equivalent
and they give rise to extraembryonic structures. The
anteriorly migrating prospective PME displaces the AVE
during gastrulation (FIG. 1). Both tissues express common markers and secreted growth-factor antagonists
(for example, Cerb-l and Dkk1), which might regulate
the adjacent neuroectoderm. Removal of the AVE or
PME in early gastrulae inhibits the expression of forebrain markers. Chimeric mice, in which developmental
regulatory genes are specifically deleted in the AVE,
characteristically show anterior CNS deficiencies21.
However, in transplantation experiments, the inducing
ability of the AVE/anterior hypoblast/anterior endoderm is poor in all vertebrates22–24. An exception is the
rabbit AVE, which can induce forebrain markers, albeit
in heterologous transplantations to the chick epiblast14.
It was therefore proposed that rather than being an
important neural-inducing tissue, the AVE and its
equivalent in other vertebrates might prime the neuroectoderm for neural induction25, protect the forebrain
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Wnt↑
or
BMP↑
or
Nodal↑
Wnt↓ and BMP↓
or
Nodal↓ and BMP↓
BMP↓
BMP↑ and Wnt↑
or
Nodal↑ and Wnt↑
or
Nodal↑ and BMP↑
Figure 2 | Combinatorial action of Wnts, bone morphogenetic proteins and Nodals
during axis formation. The effect of overexpression or reduction of the three signals alone or in
combination is indicated. Overexpression of Wnt or of low doses of bone morphogenetic protein
(BMP) or Nodals inhibits head development. Higher Nodal or BMP doses interfere with trunk
formation as well. Reduction of BMP signalling alone induces secondary trunks, whereas
combined reduction of BMPs and either Wnt or Nodal induces extra heads. The combined
action of growth-factor overexpression leading to ectopic tails (bottom) has only been
demonstrated in zebrafish so far.
PRESUMPTIVE PRECHORDAL
MESENDODERM
An embryonic tissue that is
derived from the
Spemann–Mangold
organizer/Hensen’s node, which
will form the pharynx and head
mesenchyme.
BLASTOCOEL
A fluid-filled cavity that develops
in the interior of the blastula in
amphibian embryos.
PRESUMPTIVE
CHORDAMESODERM
An embryonic tissue that is
derived from the
Spemann–Mangold
organizer/Hensen’s node, which
will form the notochord.
HENSEN’S NODE
A condensation of cells in the
primitive streak in, for example,
chick and mouse embryos, that
contains organizer activity.
NATURE REVIEWS | GENETICS
from posteriorizing factors23, prevent ectopic organizer
formation26 or promote anterior positional identity27.
As the organizer was for decades considered to be the
source of a powerful instructive agent, it initially came as
a surprise, but is now widely accepted, that its main molecular function is to secrete antagonists to growth factors
of three main classes: BMPs, Wnts and Nodals, which
are inhibitory to all or part of the organizer. The growthfactor antagonists typically bind to the growth factors
directly and inhibit their receptor interaction or signal
transduction, thereby protecting the organizer from
these inhibitory growth factors. Exceptions are the Nodal
antagonist Lefty, which interacts with the Nodal receptor,
and Dickkopf1 (Dkk1), which interacts with the Wnt
receptor LRP5/6. The antagonism of growth-factor signalling does not occur in an all-or-nothing fashion but in
a graded as well as a combinatorial fashion (see below).
This quantitative combinatorial pattern of growth-factor
antagonism is the key to understanding the organizer’s
regionally specific induction.
Head organizer
In Xenopus laevis, an extra head is induced when either
BMPs and Wnts or BMPs and Nodals are simultaneously inhibited (FIG. 2). Conversely, overexpression of
Wnts, BMPs or Nodals leads to head defects. This led to
the suggestion that head induction requires triple inhibition of all three signalling pathways28. The headinducing PME expresses secreted BMP antagonists such
as noggin and follistatin, Wnt antagonists including
Dkk1, Frzb-1 and Crescent, as well as the Nodal antagonists antivin and Lefty. Indeed, the first head inducer to
be identified, Cerberus, is a multifunctional antagonist
that binds and inhibits BMPs, Wnts and Nodals and is
necessary and sufficient for head induction28,29. An
important test for the triple-inhibition model is to
demonstrate for each growth-factor class that its inhibition is required in vivo for head structures to form. This
test has been met most prominently for Wnt signalling
and in three species — X. laevis, zebrafish and mouse
(FIG. 3). Furthermore, insulin-like growth-factor signalling is necessary and sufficient for anterior neural
induction in X. laevis, and its signalling proceeds
through the intracellular blockage of both Wnt and
BMP signalling30.
Inhibition of BMP signalling is also necessary for
the formation of anterior structures as chordin–/–noggin–/– and Dkk1+/–noggin+/– compound mutant mice
show head defects31,32. However, convincing evidence of
a requirement for Nodal antagonists in head induction
is lacking. Neither Lefty/Cerberus-like mouse double
mutants26 nor antivin-morphant zebrafish embryos33
show head defects. In both cases, the anti-Nodals
instead function as negative-feedback inhibitors that
control mesoderm formation. Furthermore, although a
hallmark of the Spemann–Mangold head organizer is to
induce and to pattern the neural tissue regionally along
its AP axis, Nodals and Activin do not directly regionalize or induce neuroectoderm other than the floor plate.
Instead, Nodals are powerful inducers of mesoderm and
endoderm, and thereby indirectly affect neural induction and patterning34. Their early role in mesoderm
induction does not exclude a later effect of Nodals during head induction through mesoderm or endoderm;
double knockouts of other Nodal antagonists might
shed light on whether or not this is the case.
So, there is now good evidence in all vertebrates that
effectors of the head organizer inhibit BMPs and Wnts
both in neuroectoderm and mesoderm and that the
primary role of Nodals is to regulate mesoderm and
endoderm (TABLE 1).
Trunk organizer
Our understanding of the secreted effectors of the organizer began with the discovery of the trunk inducer,
when it was realized that secondary trunks can be
elicited in X. laevis by overexpression of BMP inhibitors1
(FIG. 2). Since then, it has become clear that a common
feature of both head and trunk organizers is BMP inhibition. The trunk-inducing prospective chordamesoderm expresses various BMP antagonists, such as
chordin, noggin and follistatin1. BMP antagonism is not
only sufficient, but is also required for trunk formation,
as shown in zebrafish double mutants for the transcription factors bozozok and chordin: these embryos only
form tails35. Wnt antagonists are typically not expressed
or are much more weakly expressed in the trunk than in
the head organizer. Indeed, zygotic Wnt signalling is
required for the expression of the trunk mesodermal
markers brachyury (Xbra)36 and MyoD37. By contrast,
notochord formation requires Wnt inhibition.
Secondary axes that are induced by anti-BMPs alone
typically lack a notochord, whereas co-inhibition of
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BMPs and Wnts induces a notochord38,39. As mentioned
above, Nodals have a pivotal role in mesoderm induction and patterning, including the induction of secreted
organizer effectors. The evidence that the trunk organizer requires Wnt and Nodal signalling but inhibition
of BMPs is summarized in TABLE 1.
a
wt
hdl
b
Dkk1–/–
c
Membrane
Cytoplasm
Dkk1
Krm
wnt8
Lrp6
Fz
axin
Dsh
β-catenin
tcf3
Anterior neural genes
(for example, Six3, Hesx1)
TAILBUD STAGE
The embryonic stage when
neurulation is completed and
tail formation begins, visible by
an emerging tail primordium.
BLASTULA
An early-stage embryo that is
composed of a hollow ball of
cells.
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| JUNE 2004 | VOLUME 5
β-catenin signalling
Figure 3 | Genetics of the role of Wnt/β
in the head organizer. Zebrafish and mouse mutants, as
well as antisense/antibody-treated Xenopus laevis embryos,
helped to reveal the requirement for active inhibition of Wnt/
β-catenin signalling in the head organizer. a | In zebrafish
headless (hdl), the transcriptional repressor tcf3 is mutated94
(see scheme in c). The mutant embryos (lower embryo) show
loss of forebrain and upper jaw. b | In Dkk1 knockout mice, the
anterior part of the head fails to develop111. c | Schematized
Wnt/β-catenin signalling pathway. Components in red are the
products of genes for which loss-of-function studies have
provided direct evidence for a role in the head organizer:
Dkk1 (REFS 92,111), Krm (REF. 101), wnt8 (REFS 80,81), Lrp6
(REF. 115), axin (REFS 95,96), β-catenin (REF. 119), tcf3 (REF. 94),
Six3 (REF. 112). For clarity, some components of the pathway
have been omitted. Part a reproduced with permission from
REF. 94 © (2000) Macmillan Magazines Ltd. Part b reproduced
with permission from REF. 111 © (2001) Cell Press. Part c
modified with permission from REF. 101 © (2002) The
Company of Biologists Ltd.
Tail organizer
The tail organizer was long neglected and frequently
grouped together with the trunk organizer as the
‘trunk/tail’ organizer. One reason for this is that experimental manipulations often lead to the induction of
trunks and tails with similar frequency, an observation
that was first made in Spemann’s transplantation experiments. It was therefore assumed that the organizer
induces a field that might become either the trunk or
the tail. Both the trunk and the tail contain the same
axial organs (spinal cord, notochord and somites) and
the tail develops from the tailbud relatively late in
embryogenesis, so a separate tail inducer at the gastrula stage was not considered. Rather, tail development was thought to be a continuation of gastrulation
and trunk induction that was regulated by a late-acting
trunk organizer of weaker potency. Molecular support
for this mechanism was that Activin, a relative of Nodal,
induces tails at a lower dose than it induces trunks40. On
the other hand, there are qualitative differences between
trunk and tail induction. For example, activation of
the FGF pathway characteristically induces tail-like
structures but not trunks in X. laevis and chick41–43.
Moreover, tail-organizer activity resides in tailbuds both
in X. laevis 44 and chick45. Slack and colleagues have
extensively investigated tail formation in X. laevis and
their conclusion was that the tailbud arises at the neurula
stage as the result of interactions between the neural
plate and a posterior mesodermal territory17. They also
showed that Wnt, Notch46 and BMP47 signalling are all
required for tail formation.
As the consensus was that the earliest time when a
distinct tail organizer can be distinguished is around
the TAILBUD STAGE, it came as a surprise that in zebrafish,
the ventral margin of the late BLASTULA stage can induce
ectopic tails when transplanted to the animal pole of
host embryos18. However, not only the timing but also
the location of the tail organizer discovered in this study
were unexpected: the ventral margin is a tissue that does
not become part of the ‘shield’, which is considered to be
the fish equivalent of the Spemann–Mangold organizer.
Furthermore, inactivation of the fish organizer does not
affect ‘ectopic’ tail formation, which implies that the two
organizers are indeed independent. However, the
induced tails are always incomplete as they lack a notochord and a floor plate. Therefore, it seems that the tail
organizer in zebrafish develops from an interaction of
the dorsal margin, which harbours the trunk organizer,
and the ventral margin, which specifies the tail-like characteristic of the outgrowth as well as the somitic component. Indeed, ventral and dorsal marginal zone cells meet
at the end of epiboly, at which point they can interact.
However, one important caveat in these experiments is
that they did not show that tail formation requires the
ventral margin because the margin regenerates readily
after ablation.
The Thisse laboratory also showed that Wnt, BMP
and Nodal signalling are involved in this tail-organizer
activity. All three signals were known to be required for
tail development in zebrafish, X. laevis and mouse
(TABLE 1). The interesting finding was that misexpressing
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Table 1 | Wnt, BMP and Nodal signalling and regionally specific induction by the Spemann–Mangold organizer
Fish
Frog
Chick
Mouse
Wnt/β-catenin signalling*
Head inhibiting
Head defect in tcf3 axin,
LoF94–96; anteriorized in
Wnt8, LoF80,81
Head defect in Dkk1, Krm
CNS posteriorized by
LoF92,101; big heads in Frzb GoF, Wnts79
β-catenin LoF; head induction
by Dkk1 and BMP inhibition38
Head defect in Dkk1 and Six3
LoF111,112
Trunk promoting
Trunk defect in wnt8 LoF80,81
Muscle defect in Wnt LoF37;
direct induction of posterior
CNS markers in Wnt GoF78
No primitive streak in Wnt3 LoF113;
trunk defect in Wnt3a LoF82
Tail promoting
Tail defect in wnt8 LoF18,80;
tail induction in Wnt GoF18
Tail defect in Wnt LoF37;
tail induction in Wnt GoF46
AP gradient
Dose-dependent brain
patterning93,97
Nuclear β-catenin gradient
and dose-dependent CNS
patterning78
Head inhibiting
Head defect in chordin/boz
double mutants or bmp
over-expression68,35,75
Head defect in Chordin LoF,
BMP GoF102,103; head induction
by Dkk1 and BMP inhibition92
Trunk inhibiting
Trunk defect in chordin LoF35; noggin, chordin, or dominant– Chordin induces and BMP4
axial mesoderm induction in negative BMP receptor
inhibits primitive streak108
bmp LoF68,70
induce trunk104–106
Tail promoting
Tail defect in tld LoF98; tail
induction in bmp GoF18,35
Tail defect in Bmp LoF47
DV gradient
Mesoderm and CNS
patterning74,75,99
Phospho-Smad1 gradient56;
mesoderm and CNS
patterning65,67
Head inhibiting
Only anterior CNS left in
nodal LoF embryos57
Head defect in nodal GoF;
head induction by Cerberusshort and Wnt- or BMP
inhibitors28
Mesoderm inducing
Trunk defect in oep and
nodal LoF64,100
Mesoderm induction by
activin GoF107; trunk defect
by nodal LoF52
Tail promoting
Tail defect in oep LoF100; tail
induction in nodal GoF18
Tail induction in activin GoF40
DV/AP gradient
AP, DV and AV mesoderm
patterning57,58, 59
DV mesoderm patterning50,52
Direct induction of spinal
cord in Wnt GoF79
Tail defect in Wnt3a, Wnt5a,
Tcf1, Lrp6 LoF114,115,82,116
Dose-dependent CNS
patterning79
Dose-dependent tail defect in
allelic Wnt3a-mutant series82
BMP signalling
Headless chordin/noggin and
noggin/Dkk1 double LoF31,32
Tail defect in Bmp4 LoF76;
primitive-streak defect in LoF of
putative BMP inhibitor
amnionless117
Nodal signalling
Activin/nodal signalling
Axial defect in nodal LoF118,61
sufficient and necessary for
primitive-streak induction109,110
Phospho-Smad1
gradients55,53,56
AP patterning of the
primitive streak61
*Only zygotic Wnt signalling at gastrula stage is considered. AP, anterior–posterior; AV, animal–vegetal; DV, dorsal–ventral; GoF, gain of function; LoF, loss of function.
Growth-factor signalling gradients
on this AP axis. Much of this AP and DV axial patterning occurs during gastrulation and is regulated by the
Spemann–Mangold organizer. How can a three-partite
head–trunk–tail organizer account for this complex pattern? One answer is that BMPs, Wnts and Nodals act in
a concentration-dependent fashion within these regions
to orchestrate axial patterning.
So, differential inhibition by the organizer of BMP, Wnt
and Nodal signals explains the regionally specific axial
inductions that occur during vertebrate development.
Therefore, a qualitative mechanism is clearly operating:
different growth-factor antagonists act in different organizers. However, head, trunk and tail are not uniform
structures but form an AP continuum. For example, the
head CNS consists of forebrain, midbrain and anterior
hindbrain; the trunk CNS contains both hindbrain and
spinal cord; the tailbud is a particularly complex organ,
and in X. laevis, molecular markers divide the chordoneural hinge into three AP domains48. The DV pattern in the mesoderm and ectoderm is superimposed
Nodal signalling gradient. There is a consensus that
Nodals are important for mesoderm and endoderm
induction in all vertebrates and that a gradient of Nodal
signalling governs early axis formation34,49. However, the
exact axis along which this gradient exerts its patterning
effect is controversial; in X. laevis, it is believed to be primarily the DV axis, in zebrafish, either the AP or the
animal–vegetal (AV) axis and in the mouse, it is thought
to be the AP axis of the primitive streak (see TABLE 1 for a
summary).
In X. laevis, the Nodal relative Activin has long been
known to induce a DV range of mesodermal tissues (for
these growth factors in combination, but not alone,
leads to the generation of extra tails, which are, again,
incomplete18 (FIG. 2). Therefore, the combination of all
three growth factors is involved in the newly discovered
tail-organizer activity, at least in zebrafish.
NATURE REVIEWS | GENETICS
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example, blood, muscle, notochord) in ectodermal animal caps at increasing doses and has served as a model for
50,51
MORPHOGENS
. Nodals function through the same signalling pathway as Activin and show comparable effects.
X. laevis embryos express a multitude of Nodal relatives
(Xnr1, -2, -4, -5, -6; derriere), which can heterodimerize
and cooperatively induce mesoderm and endoderm.
When Nodal signalling is inhibited, for example, by
injecting the anti-Nodal reagent Cerberus-short (cerb-s),
Antivin or dominant–negative Nodals, endoderm and
mesoderm formation are inhibited34,49. When different
Cerb-s mRNA doses are injected, expression of the mesodermal marker Xbra is lost in a progressive DV fashion52.
This correlates with a wave of Nodal signalling that
sweeps from the dorsal to the ventral side from early
to mid-gastrulation, as detected with anti-phosphoSmad2 antibodies. Together, these studies indicate that a
temporal mechanism might generate the DV pattern
(FIG. 4; REF. 53). The results in X. laevis therefore indicate a
DV gradient of Nodal signalling in the mesoderm.
However, there is also evidence for an AV Nodal gradient in X. laevis. At the early gastrula stage, PME precursors are located more vegetally, notochord precursors
more animally and induction of PME markers requires
higher Activin doses than induction of a notochord
marker. Furthermore, high Activin doses induce endoderm but lower doses induce mesoderm50. Conversely,
incomplete antisense inhibition of VegT, a T-box transcription factor that functions upstream of nodals,
results in embryos that lack endoderm, whereas complete inhibition blocks both endoderm and mesoderm
formation in X. laevis 54. These findings support the
view that an AV gradient of Nodal signalling operates to
pattern the germ layers. Consistent with this model, no
phosphorylated Smad2 is detected by antibody staining
in the animal region, whereas there are intermediate levels in the mesoderm and high levels in the endoderm of
X. laevis gastrulae55,56 (FIG. 4).
In zebrafish, similar results were obtained as in
X. laevis. The overexpression of low doses of the Nodal
antagonist Antivin depletes the endoderm and the prechordal plate, whereas increasing doses deplete the axial,
paraxial and ventral mesoderm, indicating a role in DV
mesodermal patterning57. Furthermore, increasing
Antivin doses progressively anteriorizes the CNS of
embryos. The highest doses remove all neural fates
except the forebrain and eyes. By contrast, overexpression of Nodal posteriorizes embryos. So, it seems that
Nodal signalling regulates AP patterning of the CNS57.
Two other zebrafish studies obtained similar results, but
placed their emphasis on mesoderm rather than CNS
patterning. They concluded that a Nodal signalling gradient regulates patterning along the AV axis that runs
between the animal pole and the embryonic margin
(FIG. 1). High levels of Nodal signalling induce the prechordal marker gsc, whereas lower levels induce the
notochord marker not58. Similarly, the reduction in
Nodal signalling by expressing the Nodal antagonist
lefty converts PME to notochord progenitors, and doing
likewise with an allelic series of nodal mutants shifts
mesodermal progenitors at the ventral and lateral margin towards the VEGETAL POLE59. These results indicate that
Nodal signalling in zebrafish patterns cell fates along the
AV as well as the DV axis.
Analyses of mice that are mutant for nodal or components of the Nodal pathway confirm that in the mouse, as
in other vertebrates, Nodal signalling is required for
definitive mesoderm and endoderm formation34. The
phenotypes of nodal null and hypomorphic mutants are
consistent with a requirement for high nodal signalling in
PME and the foregut, whereas lower levels are required
for notochord and hindgut differentiation60. Similarly,
conditional activation of the Nodal transmitter Smad2 in
the epiblast disrupts PME, whereas compound mutants
between the conditional Smad2 and Smad3 knockout
mice also lack the notochord. It was concluded that a
Nodal signalling gradient regulates AP patterning61.
There are three reasons for the confusion over which
embryonic axis is regulated by Nodal. First, there are different naming traditions for axes in different vertebrates.
For example, unlike in the frog and fish, there is no AV
axis in the mouse gastrula: the proximal–distal axis of
the mouse egg cylinder, a term that is not used in the
other vertebrates, might be the closest equivalent to the
AV axis. Furthermore, DV patterning of the mesoderm
receives a lot of attention in X. laevis and zebrafish,
A
Ectoderm
Lm
Mesoderm
MORPHOGEN
A substance that is active in
pattern formation, the spatial
concentration or activity of
which varies, and to which cells
respond differently at different
threshold concentrations.
VEGETAL POLE
In amphibians and zebrafish, the
bottom-most, yolk-rich pole of
the embryo.
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| JUNE 2004 | VOLUME 5
Endoderm
V
V
Stage 9.5
Stage 10
Mu
No
D
Stage 10.5
Figure 4 | Formation of Nodal gradients in Xenopus laevis. Staining with anti-phospho-Smad2 antibodies reveals regions of active
Nodal signalling. (Left) A vegetal–animal (V, A) gradient is detected in early gastrula55,56, which might be responsible for patterning the
germ layers into endoderm and mesoderm. (Right) A dorsal–ventral (D, V) wave of phospho-Smad2 is detected during early
gastrulation, which indicates that a temporal mechanism might generate a DV Nodal gradient53 (stages 9.5, 10 and 10.5 of early
embryonic development are depicted in the figure). This Nodal gradient sets a bone morphogenetic protein (BMP) signalling gradient in
motion, through induction of BMP antagonists. The BMP signalling gradient patterns the mesoderm in notochord (No), muscle (Mu) and
lateral plate (Lm) mesoderm (far right panel). Modified with permission from REF. 53 © (2001) The Company of Biologists Ltd.
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NATURE REVIEWS | GENETICS
but not in the mouse and chick. In the mouse, the DV
axis is not even considered as a separate issue21, partly
because at the beginning of gastrulation, the DV and
AP axes are still collapsed on a single dorsal–anterior/
ventral–posterior axis in all vertebrates. Even in the
X. laevis community, the issue of DV- versus AP-axis definition is controversial62. The second source of confusion
over which axis is regulated by Nodal signalling arises
from the fact that some authors study mesoderm patterning, whereas others study CNS (AP) patterning by Nodal:
the latter is an indirect and secondary consequence of the
former. In particular, the effect of Nodal on AP patterning
is opposite for the mesoderm and the CNS. Formation of
the most anterior part of the mesoderm (PME) and CNS
(forebrain) require the highest and lowest Nodal levels,
respectively, although they lie adjacent after gastrulation.
This apparent paradox arises because before gastrulation, the two tissues are at opposite ends of the AV axis
(FIG. 1). The third source of confusion is that the axes in
mesodermal patterning are not clearly separated; for
example, PME-notochord patterning has been considered under DV patterning in the frog50, AV patterning in
fish59 and AP patterning in the mouse61. This confusion
highlights the need for a common axis nomenclature for
the early vertebrate embryo.
The dual DV and AV Nodal gradient in the mesoderm
and endoderm that is observed in the frog and fish (FIG. 4)
is crucial to set up two secondary signalling gradients of
BMPs and Wnts. Low Nodal signalling induces growth
factors that inhibit the organizer — for example, BMP4
in zebrafish18 and Wnt8 in X. laevis and zebrafish18,52
— whereas increasing doses progressively induce the
diffusible antagonists Chordin, Dkk1 and Cerberus63.
Their ability to induce Wnts, BMPs and Nodals, as
well as the respective antagonists at different doses, complicates the analysis of the role of Nodals and can lead to
confusing results (FIG. 5). For example, zebrafish mutants
with impaired Nodal signalling develop a well-patterned
CNS in the absence of most organizer mesoderm, which
is sometimes taken as an argument against a CNSpatterning role for the organizer64. However, residual
Nodal signalling is still present in these mutants. Certain
mesodermal markers continue to be expressed, and, furthermore, the phenotype of embryos that are injected
with high doses of Antivin mRNA is more severe than
that of any nodal mutant. Importantly, such highAntivin-injected embryos fail to develop CNS pattern
and only differentiate a single eye57.
blood formation65. In the ectoderm, no BMP is required
for neural fates, low BMP for the neural margin fate (for
example, future neural crest) and high BMP for epidermal fates66,67. Staining for phosphorylated Smad1 allows
this DV signalling gradient to be visualized56.
In zebrafish, mutant analysis has provided independent evidence for a BMP gradient in DV patterning of
the mesoderm and ectoderm. Mutations in bmp2b,
(REFS 68,69), bmp7, (REFS 70,71) and smad5 (REF. 72) result in
strong dorsalization, whereas chordin mutants are ventralized73. A gradient of bmp2/4/7 transcripts results
from the interaction of autoregulatory BMPs with dorsal antagonists, such as Chordin, and patterns both the
ectoderm and mesoderm69,74,75.
In the mouse and chick, the evidence for a BMP gradient that operates in DV patterning is limited. Mouse
Bmp4 mutants show gastrulation defects, and most
embryos fail to form a mesoderm. The few embryos that
survive beyond gastrulation show truncated posterior
structures, including extra-embryonic mesoderm derivatives76. These results were interpreted as Bmp4 patterning
the proximal–distal axis of the epiblast that is converted
during gastrulation into the AP axis of the embryo.
BMP signalling gradient. A ventralizing BMP signalling
gradient is well characterized in X. laevis and zebrafish. BMPs that are expressed widely in the embryo
(bmp2, -4, -7) and BMP antagonists that are expressed in
the Spemann–Mangold organizer and all its derivatives
along the AP axis generate this signalling gradient. The
secreted BMP antagonists attenuate BMP signalling such
that the organizer and its axial derivatives are the sink of
the gradient, and pattern is generated in all germ layers at
a distance. As shown in X. laevis, in the mesoderm, no
BMP is required for notochord, low BMP is required for
muscle and higher BMP is required for lateral plate and
Wnt signalling gradient. Before gastrulation, early Wnt
signalling is required in conjunction with early Nodal
signals to induce the organizer in lower vertebrates and
the primitive streak in amniotes1. As shown in X. laevis
and zebrafish, during gastrulation, Wnts are powerful
posteriorizing factors that antagonize and interact with
the Spemann–Mangold organizer to generate the AP
pattern. Similar to BMP signalling, the interaction of
widely expressed Wnts with Wnt antagonists generates a
Wnt signalling gradient. However, in contrast to BMP
antagonists, the expression of Wnt antagonists is typically
restricted to the anterior endoderm and PME.
Spemann
organizer
BMP
Wnt
Nodal
GF
antagonists
Nodal
Figure 5 | Cross-regulation of growth-factor signalling at
the early gastrula stage. A Xenopus laevis embryo in lateral
view is depicted. Low and high Nodal signalling induces
growth factors and growth-factor (GF) antagonists in the
Spemann–Mangold organizer, respectively. Interaction of
growth factors with their antagonists leads to secondary
signalling gradients (see FIG. 6).
VOLUME 5 | JUNE 2004 | 4 3 1
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Conclusions
Wnt
BMP
Figure 6 | Double-gradient model of embryonic axis
formation. The model shows how perpendicular activity
gradients of Wnts and bone morphogenetic proteins (BMPs)
regulate head-to-tail and dorsal–ventral patterning. The colour
scales of the arrows indicate the signalling gradients; arrows
indicate the spreading of the signals. Patterning begins at
gastrula stages, but for clarity, it is depicted in an early
amphibian neurula. The formation of head, trunk and tail
requires increasing Wnt activity. Note that Nodal signals
are not included here, because their effects on the
anterior–posterior patterning of ectoderm are indirect.
Modified with permission from REF. 78 © (2001) The Company
of Biologists Ltd.
AP patterning by Wnts is best characterized in neuroectoderm in which, as first shown in X. laevis, Wnts
directly posteriorize cell fate77. In X. laevis neuralized animal caps, different doses of Wnt3a induce different AP
markers, and in whole embryos, overexpression of antagonists progressively inhibits posterior markers78. An
endogenous AP gradient of Wnt/β-catenin signalling is
detected in the presumptive neural plate of the X. laevis
gastrula78. In the chick, Wnts also act directly and in a
graded manner on anterior neural cells to induce their
progressive differentiation into caudal forebrain, midbrain and hindbrain cells79. Conversely, increasing doses
of Wnt8 morpholino oligonucleotides in zebrafish progressively delete posterior neural fates80,81. In the mouse,
allelic combinations of mutants for Wnt3a and Vestigial
tail (a hypomorphic mutation of Wnt3a) show dosedependent posterior truncations, which indicates that
a Wnt signalling gradient might specify AP fates 82.
This effect on the entire trunk–tail highlights that
Wnt-regulated AP patterning is not restricted to neuroectoderm but, similar to BMP signalling, affects all
germ layers in a coordinate fashion. For example, notochord and heart formation also require Wnt inhibition39,83, and both tissues originate from precursor cells
that are part of, or close to, the organizer. Similarly,
expression of the anterior endodermal markers Hex and
Blimp1 is inhibited in X. laevis embryos that are injected
with inhibitory anti-Dkk1 antibodies84. So, data from all
vertebrates studied so far support the view that a posteriorizing Wnt activity gradient is operative in vertebrate
AP patterning during gastrulation (TABLE 1).
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| JUNE 2004 | VOLUME 5
Growth-factor antagonists that are secreted by the
Spemann–Mangold organizer are at the heart of a
three-dimensional coordinate system of positional
information that functions during axial patterning in
the vertebrate gastrula. The Nodal signalling gradient(s)
is crucial to set this process in motion by inducing both
Wnt and BMP growth factors as well as their antagonists at different doses (FIG. 5). A modernized doublegradient model can therefore be proposed78, in which
orthogonal BMP and Wnt gradients pattern the DV and
AP axis (FIG. 6). In the classical models that were put forward for amphibian embryos, the AP-graded factor was
proposed to be both mesoderm-inducing as well as
posteriorizing2. The two processes were indistinguishable because mesoderm induction is accompanied by
induction of posteriorizing factors. Today, mesoderm
induction and posteriorization can be uncoupled,
with Nodals inducing mesoderm, and Wnts, FGF and
retinoic acid acting as posteriorizing agents. Gastrulation elaborates the DV–AV Nodal gradient into two
orthogonal gradients of BMP and Wnt. BMP antagonists
are expressed in all organizer derivatives, particularly in
the chordamesoderm, which undergoes convergent
extension movements and therefore spans the entire AP
axis. By contrast, Wnt antagonists are expressed predominantly in the anterior mesendoderm, which leads
gastrulating cells anteriorly and ends up in a rostral
position. A similar double-gradient model was proposed for induction and patterning of the neural crest,
with BMP and Wnt signalling regulating DV and AP
patterning, respectively85. Orthogonal morphogen gradients also operate during Drosophila melanogaster
embryogenesis, in which wingless and the BMP homologue decapentaplegic specify DV and AP compartment
boundaries in the wing, respectively86.
An important feature of this patterning system is
that Nodal, BMP and Wnt signals cross-regulate each
other. Nodals induce other Nodals, Wnts and BMPs,
as well their antagonists, as discussed above. Similarly, Xwnt8 expression requires BMP signalling87,88.
Furthermore, head and trunk organizers mutually regulate each other both positively and negatively and
thereby stabilize their domains. With regard to positive
regulation, in mice that are double-mutant for chordin
and noggin, the expression of head-organizer markers in
AVE is affected, even though both genes are not expressed
there but in the primitive streak and node31. On the other
hand, overexpression of the head-promoting genes cerberus and Blimp1 blocks trunk-organizer formation28,89.
Conversely, X. laevis Admp, which encodes a BMP that is
expressed in the trunk organizer, is required to repress
ectopic head-organizer gene expression90. It is probable
that these positive and negative interactions are important for coordinating regulation of the embryonic axes
and might account for the observed regeneration of the
organizer19,91.
Cross-regulation of Wnt and BMPs is also one
explanation for why BMP signalling not only regulates
DV patterning, but is also essential for head and tail
formation — that is, AP patterning. However, another
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REVIEWS
answer is that at the head and tail ends, the DV axis is
not orthogonal to the AP axis but opposite to it.
Therefore, at the body ends, dorsalization becomes
equivalent to anteriorization. An example is the telencephalon, which different authors have defined as either
an anterior subdivision or a dorsal compartment of the
rostral forebrain, and Wilson and colleagues concluded
that it is both dorsal and anterior74. So, at the body ends,
the patterning effects of ventralizing BMPs and posteriorizing Wnts become qualitatively indistinguishable.
Consistent with this, Dkk1 overexpression can rescue
head formation in X. laevis embryos that are posteriorized by Bmp4 overexpression92 and bmp4 can substitute
wnt8 in conjunction with nodal during tail induction in
zebrafish18.
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Competing interests statement
The author declares that he has no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez: http://www.ncbi.nih.gov/Entrez
bmp2 | bmp4 | bmp7 | bozozok | brachyury | chordin | Crescent |
derriere | Dkk1 | flh | follistatin | Frzb-1 | gsc | Hex | noggin | Six3 |
Smad2 | Smad3 | smad5 | tcf3 | Wnt3a | Xnot
FURTHER INFORMATION
Axeldb: http://www.dkfz-heidelberg.de/molecular_embryology/
axeldb.htm
Dynamic development:
http://www.ucalgary.ca/UofC/eduweb/virtualembryo/dev_biol.html
The zebrafish information network: http://zfin.org/cgi-bin/
webdriver?MIval=aa-ZDB_home.apg
Xenbase: http://www.xenbase.org/
Access to this links box is available online.
www.nature.com/reviews/genetics