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Mesoderm induction:
from caps to chips
David Kimelman
Abstract | Vertebrate mesoderm induction is one of the classical problems in developmental
biology. Various developmental biology approaches, particularly in Xenopus and zebrafish,
have identified many of the key factors that are involved in this process and have provided
major insights into how these factors interact as part of a signalling and transcription-factor
network. These data are beginning to be refined by high-throughput approaches such as
microarray assays. Future challenges include understanding how the prospective
mesodermal cells integrate the various signals they receive and how they resolve this
information to regulate their morphogenetic behaviours and cell-fate decisions.
Blastula
A stage during which the
embryo undergoes cleavage to
become multicellular. The late
blastula stage precedes
gastrulation.
Gastrula
A stage during which the
embryo undergoes major
morphogenetic changes, which
positions the endoderm on
the inside, the mesoderm in the
middle and the ectoderm on
the outside.
Organizer
A signalling centre in a
vertebrate embryo comprising
a group of cells that secrete
signalling factors or inhibitors
of signalling factors, which
changes the fate of the
surrounding cells.
Department of Biochemistry,
Box 357350, University of
Washington, Seattle,
Washington 98195-7350,
USA
e-mail: kimelman@
u.washington.edu
doi:10.1038/nrg1837
The vertebrate mesoderm produces a wide range of tissues including the muscles, heart, vasculature, blood,
kidney, gonads, dermis and cartilage, and it also has a
major role in the morphogenetic movements of gastrulation. The study of mesoderm formation originated
with Pieter Nieuwkoop’s classical experiments in the
amphibian embryo, nearly 40 years ago. Nieuwkoop
showed that explanted tissue from the bottom of the
blastula-stage embryo (the vegetal cap) could convert
prospective ectodermal cells taken from the top of the
embryo (the animal cap) into mesodermal tissues that
normally reside at the equator, demonstrating that the
mesoderm is formed by a mechanism called induction1.
Induction refers to a process in which extracellular signals bring about a change from one cell fate to another
in a particular group of cells. In the classical view, induction occurs when one group of cells signal to a different
set of cells that respond by changing fate. In practice, it
can be more complex than this. In zebrafish, for example, the prospective mesodermal cells are involved in
sending and receiving the inducing signals.
Before Nieuwkoop’s seminal experiments, the first
inductive event in the amphibian embryo was thought
to involve gastrula-stage signals that originate in the
organizer and pattern the mesoderm and ectoderm
(reviewed in REF. 2). Although subsequent experiments
have led to the subdivision of mesoderm induction into
initiation, maintenance and patterning events, here I
use mesoderm induction in a broad sense to encompass
all these steps because all three take place concurrently
in the late blastula/early gastrula embryo. Nieuwkoop’s
original assay provided a valuable tool for studying
embryonic signalling that is still in constant use today.
360 | MAY 2006 | VOLUME 7
Because animal caps can be easily isolated and grown in
a simple buffer solution, and because conversion of animal cap explants to mesoderm can be readily monitored
at the macroscopic level, this assay provided a valuable
means for identifying the first mesoderm-inducing factors (reviewed in REF. 3). With the development of more
sophisticated methods for measuring gene expression
and manipulating the amount of endogenous signalling
in Xenopus, the utility of the animal cap-explant assay for
understanding the basic biology of inductive processes
has continued to expand.
Although the field of mesoderm induction began
with studies in amphibians, today it is impossible to
discuss this topic without including the genetic studies in zebrafish that have contributed important new
discoveries to this area. Comparison of the two species,
which is also the primary focus of this review, provides
interesting insights into conserved aspects of mesoderm
induction, as well as changes in the induction mechanisms to accommodate different embryological features.
Studies in Xenopus and zebrafish also provide a valuable
foundation for understanding mesoderm induction in
other species, such as birds and mammals. We can now
begin to develop a clear understanding of the molecular interactions that underlie this essential process in
embryonic development.
Here I first introduce the principal signalling factors
that are involved in mesoderm induction, with emphasis
on members of the Nodal family, which function to initiate the formation of the mesoderm. I discuss how these
factors function at different times and in various combinations to regulate different regions within the embryo.
I conclude by examining some new approaches that are
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Animal
Xenopus
Ventral
Dorsal
Zebrafish
Ectoderm
Mesoderm
Endoderm
YSL
Vegetal
express a marker for a different domain than is predicted
by the fate map7. This result probably reflects the fact
that embryonic cells are exposed to multiple signals
(including signalling factors and their inhibitors) as
they migrate in the embryo; therefore, their eventual
fate might represent the sum of these external influences,
rather than their position at a particular point in time.
Atrium
Tail
somites
Trunk
somites
Notochord
Pronephros
Blood
Blood
Head
Tail somites
Trunk somites Notochord
Pronephros
Head
Erythroid
Ventricle
Myeloid
Figure 1 | Fate maps of Xenopus and zebrafish embryos at the late blastula/early
gastrula stage. Prospective mesodermal territories are shown in red. Note that in
zebrafish the bottom layer of the mesoderm sits on top of the extra-embryonic yolk
syncytial layer (YSL), and that mesodermal cells are intermixed with endodermal cells
(green). The fate maps are based on REFS 5,125,126.
being used to gain further insights into the mechanism
of mesoderm formation and regulation.
Notochord
A rod-shaped structure that
runs along the dorsal axis of
the embryo, separating the
muscle blocks. It is one of
the defining features of the
phylum Chordata, to which
vertebrates belong.
Fate map
A map that shows which
tissues are likely to develop
from different regions of the
embryo.
Forming the mesoderm
In both zebrafish and Xenopus embryos, mesoderm
induction creates a zone of mesodermal cells at the
equator of the embryo (often called the marginal zone).
Whereas the mesoderm and endoderm are regionally
distinct in Xenopus, in zebrafish the endoderm and
mesoderm precursors are mixed (FIG. 1). In addition,
whereas all the cells in the early Xenopus gastrula contribute to the final embryo, in zebrafish the embryo sits
on top of a yolk cell that does not undergo cleavage. This
difference in architecture between the two species has
major consequences for the mechanism of mesoderm
induction, as discussed below.
In addition to establishing the mesodermal zone,
inductive processes pattern the embryo along what is
commonly called ‘the dorsal–ventral axis’ as well as the
animal–vegetal axis (FIG. 1). Because the ‘dorsal’ side produces both anterior (head) and dorsal (notochord) fates,
whereas the ‘ventral’ side produces both posterior (tail)
and ventral (for example, pronephros) fates, the equatorial axis in the pre-gastrula embryo might be better
termed the ‘dorsoanterior–ventroposterior axis’ (for an
excellent discussion of the complexities in labelling the
pre-gastrula axis see REF. 4). Regardless of the nomenclature, complex morphogenetic movements during
gastrulation bring mesodermal cells into their correct
position within the post-gastrula embryo.
Although fate maps are often drawn with the late
blastula/early gastrula mesoderm demarcated into
regions of defined fate, careful lineage labelling of
zebrafish embryos at these stages has demonstrated that
mesodermal derivatives outside the organizer region are
highly intermixed5,6. Similarly, a recent study in Xenopus
has shown that more cells in the early gastrula embryo
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Signalling factors in mesoderm induction
It is likely that all of the families of signalling factors that
are important for mesoderm induction have been identified, although all of the crucial individual factors might
not yet have been determined. A surprising feature is the
complexity of signalling factors that are used by
the embryo for inducing mesoderm (TABLE 1). From the perspective of the embryo, there might be evolutionary advantages to having multiple factors with overlapping functions.
From the perspective of the experimentalist, however,
this degree of redundancy can make loss-of-function
studies much more challenging.
As a first approximation one can say that: the Nodal
family is involved in initiating mesoderm formation,
FGFs (Fibroblast growth factors) and Wnts are involved
in maintaining the mesodermal state, and BMPs (Bone
morphogenetic proteins) are involved in patterning the
mesoderm. This, however, is an oversimplification of what
these factors actually do. For example, in various experimental models, FGFs, BMPs and Wnts have been shown
to be sufficient for initiating mesoderm formation8–13,
and Nodal family members have been shown to be
involved in patterning the mesoderm14,15. These results
indicate that extracellular signals do not necessarily
have rigidly separated functions in mesoderm induction, and instead indicate that these signals might work
in a partially overlapping way to form and pattern
the mesoderm. Because the Nodal family functions as the
main initiating stimulus for mesoderm induction16, it
is the main focus of the discussion below (for a more
thorough discussion of the roles of Wnts, BMPs and
FGFs see REFS 17–19). A description of the intracellular
signalling pathways that are used by each of these factors
can be found in BOX 1.
Models for mesoderm induction in fish and frogs
Recent studies have revealed how the mesoderm-inducing
signal described by Nieuwkoop1 is localized to the vegetal
hemisphere in Xenopus embryos. During oogenesis, transcripts that encode the T-box transcription factor VegT
are localized to the vegetal pole through a complex process
that involves specific RNA-binding factors and cytoskeletal elements (reviewed in REF. 20). At fertilization, VegT
transcripts are released from the vegetal pole and slowly
diffuse upwards. Because the third cleavage plane passes
through the equator of the embryo before the transcripts
leave the vegetal hemisphere, the VegT transcripts are
trapped there, and therefore the subsequently translated
VegT protein is restricted to the vegetal half. At the start of
zygotic transcription at the 4,000-cell stage, which is called
the mid-blastula transition, VegT activates the transcription of the Xenopus nodal related (Xnr) genes, which then
initiate mesoderm formation21–25 (FIG. 2).
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In zebrafish, the Nodal genes squint (sqt; also known
as ndr1) and cyclops (cyc; also known as ndr2) (BOX 1)
initiate mesoderm formation, but their transcription is
activated by an unknown signal originating in the extraembryonic yolk syncytial layer (YSL; reviewed in REF. 26)
(FIG. 2). The zebrafish VegT orthologue has been identified, and although it has an important role in forming the
trunk musculature as discussed below, it is not maternal
and it does not activate nodal gene expression27. It is still
unclear how a YSL-specific signal is actually produced,
although it seems unlikely to use the same mechanism
as is used by Xenopus VegT. The YSL does not even form as
a distinct entity until approximately the 1,000-cell stage,
and the YSL nuclei and cytoplasm have the same origin
as the overlying embryonic cells (called the blastomeres)
that form the mesendoderm. Therefore, if a transcript
were restricted to the vegetal pole during oogenesis and
released at fertilization, it would probably be inherited by
both the YSL and the blastomeres. It is possible that there
is a mechanism that anchors a transcript (or protein) to
the yolk and keeps it inactive until the YSL is formed; at
that point the factor could be released into the YSL and
activated to initiate mesoderm formation.
Nodal signalling: are gradients important?
As described below, the embryo potentially has gradients
of Nodal activity along the dorsal–ventral and animal–
vegetal axes. The existence, significance and role of these
Nodal gradients in mesoderm induction continue to be
hot topics of debate. In Xenopus, several lines of evidence
indicate that Nodal signalling is stronger on the dorsal
than on the ventral side of the pre-gastrula embryo. In
Xenopus embryos, a ‘dorsalizing’ activity moves from the
vegetal pole towards one side of the embryo soon after
fertilization (reviewed in REF. 28), and evidence from
experiments in which the early vegetal cytoplasm was
removed from the embryo indicates that there is a similar
mechanism in zebrafish29,30. The net result of this movement is the stabilization of β-catenin, an intracellular Wnt
pathway component (BOX 1), on the future dorsal side
of the embryo (FIG. 3). This asymmetrical stabilization of
β-catenin is essential for the formation of all dorsal and
anterior structures (REFS 31,32; reviewed in REFS 28,33).
Among the many targets of β-catenin are members of the
Nodal gene family called the Xenopus nodal-related factors; for this reason, the Xnr genes are expressed earlier
and/or at a higher level on the dorsal side of the embryo
(FIG. 3). For example, Xnr5 and Xnr6 are transcribed on
the dorsal side of the embryo in response to β-catenin
as early as the 256-cell stage, well before most embryonic genes begin to be transcribed at the mid-blastula
transition34. These two Xnr genes, as well as Xnr1, Xnr2
and Xnr4, which are first transcribed at the mid-blastula
transition, are expressed in a dorsal–ventral gradient in
the late blastula stages35,36. Consequently, Nodal signalling is at least transiently enriched dorsally, as indicated
by increased phosphorylation (and so activation) of the
Nodal intracellular factor Smad2 (BOX 1) on the dorsal
Table 1 | Candidate zygotic signalling molecules in mesoderm induction*
Signal family
Xenopus
Mouse
Chick
Zebrafish (mutant/morpholino phenotype)
Nodal
Xnr1
Nodal
Nodal
Sqtठ(cyclopia/dorsal mesoderm defects)
Xnr2
Gdf3
Vg1
Cycठ(cyclopia)
Xnr4
Vg1 (N.D.)
Xnr5
Xnr6
Activin
Derriére
Vg1
FGF
Fgf3
Fgf3
Fgf8?
Fgf3 (N.D.)
Fgf4
Fgf9
Fgf4
Fgf8‡ (cerebellum and mild posterior defects)
Fgf8
Fgf24‡ (pectoral fin defect||)
Fgf8
BMP
Bmp2
Bmp7
Bmp2
Bmp2b‡ (severe dorsalization)
Bmp4
Bmp2
Bmp4
Bmp4 (N.D.)
Bmp7
Bmp4
Bmp7
Bmp7‡ (severe dorsalization)
Admp
Admp (dorsalized)
Wnt8
Wnt8c
Wnt8 (posterior mesoderm and neural defects)
Admp
Wnt ¶
Wnt8
Wnt3
Wnt3a (no effect#)
Wnt3a
*There is no horizontal relationship between the genes listed in this table. ‡Zebrafish with mutations in these genes have been
identified. §sqt;cyc double mutants have no head or trunk mesoderm, and no endoderm. ||fgf24MO in fgf8 mutants causes loss of the
most posterior mesoderm. ¶Members of the canonical (β-catenin stabilizing) family of Wnts are listed. #Loss of wnt3a enhances loss
of wnt8. Admp, Anti-dorsalizing morphogenetic protein; BMP, Bone morphogenetic protein; Cyc, Cyclops; FGF, Fibroblast growth
factor; Gdf3, Growth/differentiation factor; N.D., not determined; Sqt, Squint; Xnr, Xenopus nodal related.
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Box 1 | Intercellular signalling pathways
Many of the essential details of the intracellular pathways that are used by the four signalling factors discussed in this
review have been identified and are shown here (see figure). Below I discuss key aspects of each of the main pathways;
for more extensive discussions of these pathways see REFS 119,120,121.
Nodal and BMP
Both the Nodal and BMP (bone morphogenetic protein) pathways are activated when a ligand binds a specific
heterotetramer that is composed of two type I and two type II receptors, as is the case with all members of the
Transforming growth factor-β (TGFB) family. In zebrafish, mutants have been identified in two Nodal ligands: squint (sqt)
and cyclops (cyc), and in two BMP ligands: swirl (bmp2b) and snailhouse (bmp7). The type II receptor phosphorylates a
cytoplasmic domain on the type I receptor, activating the type I receptor to phosphorylate a Smad factor. Whereas
Smad2 and Smad3 are specific mediators of Nodal (including Activin and Vg1) pathways, Smad1, Smad5 (Somitabun in
zebrafish) and Smad8 are mediators of the BMP pathway. When these Smads are phosphorylated they bind Smad4 and
translocate to the nucleus where they bind to specific DNA-binding factors. The Smad factors also have a weak DNAbinding ability, and it is the combination of the DNA-binding specificity of the molecular partner together with the
DNA-binding specificity of the Smads that allows the transcriptional activation of specific targets. For example, in the
zebrafish Nodal pathway, the binding of Smads to the trancription factor Bonnie (Bon) is an essential step in endoderm
formation, whereas the binding of Smads to Schmalspur (Sur) is important for mesoderm formation. The Nodal pathway
is unique in that it requires an extracellular membrane-bound EGF–CFC cofactor called One-eyed pinhead (Oep) in
zebrafish, Xcr1/Frl1, Xcr2 and Xcr3 in frogs, Cryptic in chicks and Cripto and Cryptic in mice122,123. Although these
factors are required for Nodal and Vg1 signalling124, it remains unclear why Nodal and Vg1 specifically require these
cofactors to signal whereas related ligands such as Activin do not.
Canonical Wnt
The Wnt pathway is divided into at least two main branches, including the canonical (β-catenin-dependent) pathway
and the non-canonical pathway(s), which is independent of β-catenin. In the canonical Wnt pathway, the Wnt ligand
binds to a Frizzled/LRP heterodimer, which mediates the intracellular response. Exactly how these receptors activate
the downstream pathway is still unclear, but it involves G-protein signalling, LRP phosphorylation on its cytosolic
C terminus, and the activity of Dishevelled (Dsh). The net result of these factors is the disruption of a large protein
machine called the β-catenin destruction complex — which phosphorylates β-catenin on its N terminus — causing it to
be ubiquitylated and then rapidly degraded by the proteasome. The destruction complex is composed of many proteins,
including the central scaffolding protein Axin (Masterblind in zebrafish), the Adenomatous polyposis coli (APC) protein,
and the kinase Glycogen synthase kinase 3 (Gsk3). Several studies have suggested that Wnt signalling disrupts the
complex by causing one or more proteins to leave the complex, preventing the phosphorylation and ubiquitylation of
β-catenin. When β-catenin is not degraded, it accumulates and translocates to the nucleus, where it binds members
of the Tcf/LEF1 family of DNA binding factors (including Headless (Hdl) in zebrafish), and recruits transcriptional
activators to the promoter.
FGF
Binding of the ligand to the Fibroblast growth factor (FGF) receptor (Fgfr) results in receptor dimerization and transphosphorylation of the receptor’s cytosolic domain. The phosphorylated receptor recruits proteins that activate the G-protein
Ras, which then activates the kinase Raf. Raf phosphorylates and activates Mek, which subsequently phosphorylates and
activates MAP kinase (Mapk). Mapk enters the nucleus where it phosphorylates and activates target transcription factors.
Wnt
BMP
FGF
Fgfr
Frizzled
Type II
Type I
Type I
EGF–CFC
Type II
FGF
Wnt
Fgfr
BMP
Nodal
LRP
Nodal
Ras
Dsh
G
P
Smad1,5,8 Smad4
APC
Smad2,3 Smad4
P
Gsk3
P
Raf
P
Axin
Mek
Mapk
β-Catenin
P
P
Smad2,3 Smad4
Mapk
P
Smad1,5,8 Smad4
β-Catenin
P
Tcf/LEF
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Xenopus
Zebrafish
sqt and cyc
YSL
Xnrs
VegT
Figure 2 | Models for activation of Nodal signalling in Xenopus and zebrafish.
In Xenopus, the vegetally localized transcription factor VegT (blue) activates the
transcription of the Xenopus nodal-related genes (Xnrs) in the vegetal hemisphere, which
then initiate mesoderm formation. In zebrafish, signals from the yolk syncytial layer (YSL;
blue) activate the transcription of the Nodal genes squint (sqt) and cyclops (cyc), which
then initiate mesoderm formation.
Morpholinos
Antisense oligonucleotides that
are stable and are commonly
used in zebrafish and Xenopus
to inhibit either the translation
or splicing of mRNAs.
Spemann’s organizer
A signalling centre in
amphibians that is created on
the dorsal side of the late
blastula embryo. The
equivalent centre in fish is
called the shield.
side of the late blastula embryo37,38. However, by the early
gastrula stages, phosphorylated Smad2 levels are equal
across the dorsal–ventral axis37,38. Therefore, there is a
period of time during which Nodal signalling is asymmetrical across the dorsal–ventral axis, and this could be
important for establishing asymmetrical gene expression
along this axis.
In zebrafish, the Nodal gene sqt has been proposed
to be directly activated by β-catenin, resulting in dorsal
expression of sqt for a brief time before its expression
throughout the mesendoderm39. Whether or not this
causes a transient dorsal–ventral asymmetry in phosphorylated Smad2 levels has not yet been determined.
Another potential mechanism for regulating asymmetrical zebrafish nodal expression has recently been
reported. Maternal sqt transcripts seem to be transported
to the dorsal side of the zebrafish embryo as early as the
4-cell stage, presumably allowing the Sqt protein to accumulate dorsally before the onset of zygotic transcription40. Morpholino-mediated knockdown experiments
indicate that a loss of maternal and zygotic Sqt causes
a more severe phenotype than loss of the zygotic Sqt
alone. However, embryos that genetically lack maternal
and zygotic Sqt were reported to have no more severe
defects than embryos that lack only zygotic Sqt 41, and
embryos that lack only maternal Sqt show no early patterning defects (S. Dougan, personal communication).
Therefore, the importance of localized maternal sqt
might depend on the genetic background of the embryo
and/or epigenetic effects.
Several elegant experiments in Xenopus using Activin
as a Nodal pathway stimulant have shown that variations
in the level of Nodal activity can regulate dorsal–ventral
patterning, with low levels inducing ventral fates and
high levels inducing dorsal fates42–44. These results are
consistent with the idea that Nodal activity is higher dorsally than ventrally, at least in the pre-gastrula embryo.
Nevertheless, it has been difficult to ascertain whether
the Nodal pathway actually regulates dorsal–ventral
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patterning. Studies in frogs using different doses of a synthetic Nodal-specific inhibitor (the truncated Cerberus
protein) seemed to indicate that ventral mesoderm was
most readily eliminated by small reductions in Nodal
signalling, whereas dorsal mesoderm was only eliminated with higher doses of Nodal antagonist 35. These
are the only data so far that support the importance of
a dorsal–ventral Nodal gradient in Xenopus. Similarly,
no evidence has yet been presented that a reduction of
Nodal signalling on the dorsal side results in a respecification of dorsal to ventral fates. Surprisingly, reduction
of Nodal signalling using various sqt and cyc mutants
in zebrafish produced the opposite result; the dorsal
region was the most sensitive to a loss of Nodal signalling 45. In this case, reduced Nodal signalling resulted
in a reduction in dorsal mesoderm formation but not a
respecification to ventral mesodermal fates. Therefore,
there is no compelling evidence at this point that a
temporal/spatial gradient of Nodal signalling regulates
differences in dorsal–ventral mesodermal fates in either
fish or frogs.
Studies in zebrafish have indicated that Nodal gradients are involved in patterning the mesoderm along the
animal–vegetal axis. Differences in Nodal levels are proposed to separate head mesoderm from the notochord
within the organizer14,45, and to separate ventricular and
atrial myocardial precursors46. This concept is generally
consistent with Xenopus studies showing that partial
Nodal or Activin inhibition causes head defects without
affecting the notochord47–50, and with the existence of
a vegetal to animal gradient of phosphorylated Smad2,
indicating that a Nodal gradient might also pattern
the embryo along the animal–vegetal axis in frogs37,38.
Further studies will need to determine to what extent
differences in Nodal-signalling levels specify different
mesodermal tissue fates in vivo.
The enigmatic Vg1
The Xenopus Transforming growth factor-β (TGFB) family member Vg1 was originally identified as an excellent
candidate to be the vegetally derived Nieuwkoop signal
(REF. 51; reviewed in REF. 52). Vg1 transcripts are localized
to the vegetal pole in a similar way to VegT. Moreover, the
mature region of the Vg1 pro-protein attached to a different pro-domain can function as a potent mesoderminducing agent. However, the originally identified Vg1
pro-protein has no activity and endogenous mature
Vg1 is not detectable in Xenopus embryos. Although
there is evidence that Vg1 has an important role in
chick mesoderm induction53,54 and a Vg1-related protein Growth/differentiation factor 3 (Gdf3) is essential
for mesoderm formation in the mouse55, until recently
Vg1 was shown to be crucial only for establishing
left–right asymmetry in Xenopus 56–58. However, Birsoy
et al. showed that embryos with depleted Vg1 lack head
and notochord structures, which is most likely due to
a reduction in the expression of Spemann’s organizer
genes, particularly the BMP and Wnt inhibitors15.
Moreover, the authors resolved the issue of the inactive
Vg1 pro-protein by demonstrating the existence of a
second Vg1 allele, which is active as an inducing agent 15.
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Xenopus
Zebrafish
Dorsalizing
activity
β-Catenin
Nodal
activity
Postfertilization
Maternal
squint
Blastula
?
Pre-gastrula
Figure 3 | Establishing the dorsal–ventral axis. In Xenopus (left panels) a dorsalizing
activity moves from the vegetal pole to one side of the embryo after fertilization, and a
similar mechanism seems to occur in zebrafish (right panels). During the blastula stages,
this dorsalizing activity stabilizes β-catenin on what will be the future dorsal side of the
embryo. When zygotic transcription of the Xenopus Nodal genes begins, the β-catenin
creates an asymmetry in Nodal expression, which results in elevated phosphorylated
Smad2 levels on the dorsal side of the pre-gastrula embryo. In zebrafish, maternal squint
transcripts are localized to the future dorsal side by the 4-cell stage, and zygotic squint is
initially transcribed dorsally. It is not yet clear whether this creates a significant
asymmetry in phosphorylated Smad2 levels.
Although this work has re-established Vg1 as an important member of the Nodal family of mesoderm inducers, it raises the question as to why so many members of
this family are used in Xenopus (TABLE 1). In zebrafish,
the vg1 transcript is maternal but not localized59, and
so far there is no evidence that it is involved in either
mesoderm induction or left–right patterning. Therefore,
if zebrafish Vg1 is essential for Nodal signalling as in
frogs, chicks and mice, it either functions as a ubiquitous
factor or the processing of the pro-protein to the mature
ligand is spatially regulated.
Beyond Nodals: maintenance and patterning
Evidence from both fish and frogs demonstrates that the
head (anterior) is regulated differently from the trunk
and tail (posterior). For example, inhibition of FGF
signalling in both systems eliminates the trunk and tail
without causing severe head defects, indicating that
these two regions of the body use different regulatory
circuits60,61 (FIG. 4). Mesoderm induction in the head
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activates various transcription factors and secreted
inhibitors of the BMP, Wnt and Nodal pathways that are
essential for patterning the embryo33. Meanwhile, trunk
mesoderm induction activates signalling factors of the
FGF and Wnt families (BOX 1; TABLE 1), along with several
transcription factors. Among the transcription factors
are at least three members of the T-box family, Xbra (in
frogs) or No tail (Ntl; in fish), VegT (in frogs) or Spadetail
(Spt; in fish), and Tbx6 (in fish and frogs), which function combinatorially to regulate mesoderm formation62.
Whereas zebrafish that lack spt (also known as tbx16)
or ntl function have major defects within the trunk or
tail, respectively, fish that lack both T-box genes fail
to form the trunk and tail mesoderm, demonstrating
that these factors function early and redundantly in
the initial stages of mesoderm induction63. Among the
targets of the T-box genes are FGFs, which function in
an autoregulatory loop to maintain T-box gene expression64–66 (FIG. 4). Zygotic Wnt signalling not only limits
the size of the Spemann’s organizer 67, it is also necessary
for the maintained expression of the T-box genes68–70
(FIG. 4). Why both FGFs and Wnts are needed to maintain
the mesoderm is still not clear.
In addition to Nodals, Wnts and FGFs, several members of the BMP family and their inhibitors are involved
in mesoderm patterning (REFS 71,72; reviewed in REF. 73)
(BOX 1; TABLE 1). Surprisingly, both the dorsal and ventral sides of the embryo simultaneously express BMPs
and BMP inhibitors, indicating that the regulation of
mesodermal patterning by BMPs is complex. Zebrafish
embryos that are mutant for bmp2b or frog embryos with
depleted Bmp4 and Bmp7 fail to form tails26,74–76, which
is consistent with the proposal that BMPs are essential
regulators of tail development 77,78 (FIG. 4).
Many lines of evidence indicate that the embryonic
mesoderm of frogs and fish is subdivided into three
principal domains: the head, trunk and tail. As discussed above, the trunk and tail require FGF signalling
and functional T-box genes, whereas the head does not.
The trunk and tail are also under separate regulatory
control, although the regulation of tail formation seems
to be different between fish and frogs. In Xenopus, the
initiation of tail outgrowth involves the BMP and Notch
signalling pathways, coupled with distinct changes
in gene expression as the tail forms 79–82. Although
BMP signalling is also required for tail formation in
zebrafish75,76,78, there is no evidence for Notch signalling in this process, nor are there any reported changes
in gene expression when the tail begins to develop.
However, zebrafish tail formation clearly involves a
pathway that is distinct from head or trunk formation,
because zebrafish embryos with deficient Nodal signalling form essentially normal tails, even though the head
and trunk mesoderm are absent 83.
Depletion of the dorsally expressed BMP ligand
Admp (Anti-dorsalizing morphogenetic protein) in
Xenopus and zebrafish also results in embryos with
defects in posterior development 84–86. Moreover, an
important new analysis of Xenopus embryos that lack
dorsal (Admp) and ventral (Bmp2, Bmp4 and Bmp7)
BMPs revealed that interactions between the BMPs and
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Maternal
β-catenin
Medium
Nodal
High
Nodal
Low Nodal (frog)
Low Nodal/YSL signal (fish)
FGF
Wnt
T-box genes
FGF
Wnt
T-box genes
BMP
Head
Trunk
Tail
Figure 4 | A model for patterning the embryonic body in Xenopus and zebrafish.
The fish and frog body (shown here as a generic embryo) is divided into three principal
domains: the head, trunk and tail, and some of the key signalling events are shown. In
zebrafish, the yolk syncytial layer (YSL) signal seems to be redundant, with Nodal
signalling inducing the formation of the tail. Note that the role of Wnt signalling within
the trunk is uncertain. BMP, Bone morphogenetic protein; FGF, Fibroblast growth factor.
their inhibitors on both the dorsal and ventral sides
of the embryo are essential for establishing normal
dorsal–ventral patterning 86,87.
It is still unclear how the posterior dorsal (tail
mesoderm), posterior ventral (the posterior blood
islands) and ventral (pronephros) fates (FIG. 1) are
specified. Although BMP signalling is necessary for
the formation of the ventral mesodermal fates, there
is no compelling evidence that a gradient of BMP signalling distinguishes between the different tissues that
form on the ventral side. This remains an important
area of investigation.
Temporal aspects of mesoderm induction
Mesoderm induction occurs over several hours during
which embryonic cells are exposed to shifting concentrations of inducing signals and their inhibitors. Even if a
cell begins to head towards a particular fate decision, it
might change fate if it receives new external influences.
But once a cell becomes irreversibly committed to a
specific fate it is said to be determined.
An important area of research involves understanding
when cells are able to respond to the different mesoderminducing signals and when they become committed to a
particular fate. A detailed analysis of cell commitment in
Xenopus showed that cells in the early gastrula embryo
are labile, expressing genes that mark multiple germ layers and regions88. By the end of gastrulation, however,
cells seem to be more committed to a specific fate, as
judged by gene expression. How and when cells become
committed is still mostly unknown. One mechanism
that limits the ability of Nodal-type signals to affect the
mesoderm after the mid-gastrula stage in Xenopus
involves phosphorylation of residues in the middle of the
Smad2 protein, which keeps Smad2 out of the nucleus and
therefore prevents it from activating gene expression89.
Many studies in Xenopus and zebrafish have examined how signalling pathways influence embryonic gene
expression by using overexpression of specific signalling molecules, overexpression of dominant-negative
366 | MAY 2006 | VOLUME 7
receptors, morpholino oligonucleotides and zebrafish
embryos that are mutant for one component of a particular signalling pathway. Although these results have
been informative, they do not provide information
about the important temporal requirement for the
signalling pathway as all of these approaches alter
the signalling pathway throughout the entire process
of mesoderm induction, and therefore there is not
much known in this area in general. Fortunately, new
approaches are being developed that will allow the
time requirement for different signalling factors to be
addressed. Relatively specific drugs that inhibit the
FGF (SU5402 (REF. 90)) and Nodal (SB505124 (REF. 91))
pathways are commercially available. For example, the
FGF inhibitor has been used to examine a wide variety of processes, including the regulation of the T-box
genes by FGF in the zebrafish mesoderm, Xenopus
neural induction, Xenopus forebrain development, and
zebrafish tooth development 64,92–94.
As an alternative approach, transgenic zebrafish
that express inhibitors of the canonical Wnt and BMP
pathways under heat-shock control have been developed95,96. For example, this was used to show that the
major role for BMP signalling in patterning the mesoderm occurs during the late blastula/early gastrula
period, but that BMP signalling continues to operate
during the mid–late gastrula stages to specify ventral
tail-fin formation95. Similarly, studies with secreted
natural inhibitors of FGF signalling in frogs indicated
that the FGF-signalling pathway functions during the
gastrula stages primarily to regulate mesodermal gene
expression, whereas FGF signalling largely regulates
morphogenesis after the gastrula stages97. These types
of temporal analysis provide a framework for examining changes in mesodermal gene expression in order
to understand how and why the mesoderm changes its
ability to respond to inducing signals.
Combinatorial signalling
Mesodermal cells respond to a cacophony of signals,
but somehow they need to integrate this information
to make a specific fate decision. How the different signalling pathways combine to regulate gene expression
in the mesoderm is still poorly understood. Different
signalling pathways might to some extent regulate
each other’s intracellular networks but, most likely,
much of the interaction occurs through changes in
gene expression. Although there is some evidence for
genes that are directly regulated by multiple signalling pathways using promoter analysis (for example,
Wnt and Nodal signalling regulating the Xenopus twin
promoter 98 and BMP and Wnt signalling regulating
the zebrafish tbx6 promoter11), for the most part it is
not known how many mesodermal genes are regulated
by just one pathway and how many are regulated by
more than one pathway. Promoter analysis is feasible
in zebrafish and Xenopus using injected plasmid DNA,
although it is best done using technology that allows
transgenic Xenopus embryos to be produced in the
F0 generation99; nevertheless, these methods are still
laborious. Moreover, with the exception of binding
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a
Primitive
streak
Vg1 and
Wnt8c
Vg1 and
Wnt8c
Nodal
Nodal
Cerberus
Cerberus
Hypoblast
b
6.5 DPC
5.5 DPC
AVE
Nodal inhibitors
Wnt inhibitors
Wnt3
AVE
Brachyury
Nodal
Proximal
Anterior
Wnt3
Posterior
Distal
Figure 5 | Mesoderm induction in the chick and mouse. a | In the chick embryo, Vg1 and Wnt8c cooperate to activate
Nodal expression. Nodal activity is blocked initially by the secreted Nodal-binding protein Cerberus, which is expressed in
the underlying hypoblast (green circles). The movement of the endoblast (white circles) displaces the Cerberus-expressing
hypoblast, allowing Nodal to function, which causes mesoderm and endoderm cells to ingress through the primitive
streak. Modified with permission from REF. 103 © (2004) Company of Biologists Ltd. b | In 5.5 day postcoitum (DPC) mouse
embryos, Wnt3 is initially expressed in the posterior extra-embryonic tissue (the visceral endoderm). The anterior visceral
endoderm (AVE) initially forms in the distal region of the embryo. By 6.5 DPC, the AVE, which secretes Nodal and Wnt
inhibitors, has moved to the anterior side. Wnt3 is now expressed in the posterior epiblast, where the primitive streak
forms, and in the posterior visceral endoderm. Wnt3 activates the expression of genes such as Nodal and Brachyury in the
posterior epiblast. Nodal is also expressed throughout the epiblast at 5.5 DPC (not shown). Modified with permission from
REF. 105 © (2005) Academic Press.
Primitive streak
The site of major
morphogenetic movements
during gastrulation in reptiles,
birds and mammals. The
mesoderm, as well as the
endoderm, moves through this
structure as it ingresses.
sites for the Tcf factor (a transcriptional activator in
the Wnt pathway; BOX 1), it is still not possible to easily
recognize elements that respond to the FGF, Nodal or
BMP pathways because each of these pathways regulate
several DNA-binding proteins and it is typically not clear
which binding protein interacts with a specific promoter.
For these reasons, few mesodermal promoters have been
studied in any detail, leaving much of the molecular
network of mesoderm induction incomplete.
A recent paper has brought to light a new interesting example of combinatorial signalling in the
mesoderm, which would not have been obviously
revealed by promoter studies. As discussed above, the
T-box transcription factor Xbra is a crucial regulator
of posterior mesoderm formation. The specificity of
Xbra-mediated regulation of gene expression is altered
by its binding to Smad1, an intracellular mediator of
the BMP pathway 100 (BOX 1). Because BMP signalling
is high ventrally and low dorsally, targets of Xbramediated activation are likely to be different on the dorsal
and ventral sides of the embryo. It will be interesting
NATURE REVIEWS | GENETICS
to see how general this type of mechanism is for regulating the activity of transcription factors in different
regions of the embryo.
Mesoderm induction in chicks and mice
The basic process of mesoderm induction is generally
conserved among all vertebrates, although the basic
embryonic architecture is different between species (for a
more extensive discussion comparing mesoderm induction in chick and mouse to fish and frogs see REF. 3). A
direct comparison of chicks and mice to fish and frogs
reveals interesting commonalities and differences. For
example, in chicks, a combination of the TGFB factor
Vg1 and canonical Wnt (Wnt8c) signalling has been
proposed to initiate the formation of the initial axial
structure, the primitive streak, albeit with the caveat that
there are no loss-of-function data yet that test the exact
role of Vg1 signalling in chicks54 (FIG. 5a). Intriguingly,
one of the earliest targets of Vg1 and Wnt8c is the chick
Nodal gene101, which parallels the observations in fish
and frogs that Nodal expression is a key step in the
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Epiblast
The portion of the mouse
embryo that will become the
definitive embryo (as opposed
to extra-embryonic tissues).
Amniotes
Include reptiles, birds and
mammals, which all have a
protective membrane (the
amnion) surrounding the
embryo that prevents it from
desiccating.
mesoderm-induction process (see above). However, an
important difference is that the chick extra-embryonic
structure called the hypoblast, which initially lies under
the future primitive streak, secretes the Nodal inhibitor
Cerberus, which blocks Nodal function102. Only when
the hypoblast is physically displaced away from the posterior region of the embryo, where the primitive streak
forms, is Nodal able to function in forming the primitive
streak, together with Fgf8 and Chordin103 (FIG. 5a). This
mechanism of hypoblast displacement is proposed to
be essential for ensuring that the primitive streak forms
only in the posterior region of the embryo102,103. It is also
possible that the gradual movement of Nodal inhibitors
away from the primitive streak establishes a gradient of
Nodal activity within the streak itself 102.
In mice, understanding mesoderm induction is complicated by the fact that the primitive streak forms on
one side of the embryo (proximal to the site of implantation and in the region of the future posterior end
of the embryo), whereas a separate crucial signalling
centre, the anterior visceral endoderm (AVE), initially
forms distal to the site of implantation and then moves
to the opposite side from the primitive streak (the future
anterior end of the embryo; FIG. 5b). A complex series
of signalling interactions that involve Nodal, BMP and
Wnt signalling sets up these two centres (reviewed
in REF. 104).
Gdf3, a Vg1-like gene, is expressed in the very early
mouse embryo, paralleling the very early expression of
Vg1 in frogs, fish and chick. As with zebrafish vg1, but
unlike Vg1 in frogs and chicks, Gdf3 expression is uniform within the cells that will give rise to the embryo55.
Surprisingly, Gdf3-null mutant mice are often viable,
with approximately one-third showing a range of morphological defects, which can be explained by alterations
in Nodal signalling 55. In general terms this result fits with
loss-of-function experiments in frogs, indicating that
Xenopus Vg1 works together with the Nodal factors15,
and it differs from studies in chicks, indicating that Vg1
is the key initiator of Nodal expression and primitive
streak formation53,54,103. These differences might reflect
the ways in which the use of these separate TGFB factors
has evolved to establish the mesoderm in embryos that
have different topologies.
A recent study challenges the view that the mouse
primitive streak is initially established in a radial pattern and then refined to a localized site in the proximal
posterior region of the epiblast, potentially through
inhibitory activities of the AVE105. Instead, the authors
find that Wnt3, which is essential for the formation
of the primitive streak and mesoderm and for Nodal
expression106, is initially expressed in tissues next to the
proximal posterior region of the epiblast before the AVE
has moved into the future anterior region, and not in a
radial pattern throughout the proximal region as was
previously thought. Wnt3 then regulates downstream
genes such as Nodal and the T-box gene Brachyury (and
potentially Wnt3 itself) in the adjacent proximal posterior epiblast (FIG. 5b). How Wnt3 expression is initially
restricted to the proximal posterior region remains
unanswered.
368 | MAY 2006 | VOLUME 7
In an interesting parallel to the function of the
chick hypoblast, the AVE secretes inhibitors of Nodal
signalling, Cerberus-like and Lefty1, that are essential
for restricting the primitive streak to the posterior end
of the embryo by limiting the region in which Nodal
signalling can function107,108 (FIG. 5b). Moreover, these
Nodal inhibitors are essential for the normal patterning
of the streak because mutants that lack both Nodal inhibitors have an expansion of the anterior mesoderm and a
concomitant loss of posterior mesodermal tissues108. The
AVE also expresses inhibitors of Wnt signalling 109, which
also limits the domain of Wnt3 signalling. Therefore,
an essential role of the AVE is to secrete inhibitors that
restrict the primitive streak to one side of the embryo.
Because different experimental approaches are often
used in different species, a direct comparison of mesoderm induction between vertebrates is complicated.
Nonetheless, it is possible to make some basic generalizations. First, the same families of signalling factors
are involved in all vertebrates, as are the T-box genes,
although there are some significant differences in which
molecular players are involved (TABLE 1). Second, localized
maternal determinants have no role or a reduced role in
axis specification and mesoderm induction in amniotes,
and the polarity of the amniote embryo is not fixed until
the beginning of gastrulation. Third, extra-embryonic
structures have an important role in the early intercellular
signalling processes in amniotes, whereas the fish extraembryonic structure, the YSL, initiates only the Nodal
pathway, and frogs do not have extra-embryonic structures. Finally, the effect of inhibiting signalling pathways
in the mouse embryo produces much more severe effects
than in fish and frogs. For example, inhibition of BMP
signalling 110,111 or elimination of Wnt3 (REF. 106) in mouse
embryos causes a failure of mesoderm formation, whereas
inhibition of these pathways in fish and frogs results only
in truncations of the body. Schier and Talbot have suggested that the differences in the roles of the signalling
factors in these different species are due to a much greater
role for cross-regulation between the signalling pathways
in mouse embryos than in fish and frogs, so that if one
pathway is inhibited, the others are affected as well26.
Future directions
Many studies on mesoderm induction have involved
identifying a new gene either through an unbiased
screen or homology searches that are based on what
is known from other species, and analysing its regulation by the signalling pathways that are discussed in
this review. An alternative approach involves altering
one of the signalling pathways by molecular or genetic
methods and then analysing the expression and function of a handful of marker genes, typically by RT-PCR.
Although the zebrafish and Xenopus genomes are still
not completely sequenced, it has recently become
possible to use genomic methods, such as microarray screens and genome-based expression screens, to
analyse mesoderm induction. For example, Xenopus
embryos that lack the T-box transcription factor
VegT were examined using microarrays, identifying
99 known and novel genes that are activated by VegT, of
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+ 100 ng ml–1 Activin
Heart and
cartilage
+ 10–50 ng ml–1 Activin
Notochord
+ 10–50 ng ml–1 Activin + RA
Pronephros
–1
+ 5–10 ng ml Activin
Muscle
0.1–1 ng ml–1 Activin + Il3
Lymphocyte
–1
0.1–1 ng ml Activin + Scf
Erythrocyte
–1
0.1–1 ng ml Activin + Ang2
Vascular
tissue
Figure 6 | Production of different mesodermal cell types from animal cap explants. Animal caps are excised from
Xenopus embryos at the mid-blastula stage and cultured in a saline solution. In the absence of inducers, the explants will
form primitive epidermis. Addition of different factors will enhance the formation of specific mesodermal cell types.
RA, Retinoic acid; Il3, Interleukin 3; Scf, Stem cell factor; Ang2, Angiopoietin 2. Modified with permission from REF. 114 ©
(2003) Elsevier Science.
which 13 were shown to be direct targets of VegT within the
mesendoderm112. Among these direct VegT targets,
the transcription factors Snail, Hesr1 (hairy/enhancerof-split related) and Esr4 (enhancer-of-split related 4)
were shown to be essential for proper embryonic
morphology. A similar approach using the drug SU5402
to block FGF signalling in Xenopus identified 43 FGFregulated genes, of which 26 were novel113. One of the
43 genes (Xmig6) was shown to be required for muscle differentiation, whereas a novel G-protein coupled receptor
(encoded by Xgpcr4) seems to be involved in the morphogenesis of gastrulation. These approaches demonstrate
the value of microarrays for finding novel genes that are
regulated by the signalling and transcription factors that
are involved in mesoderm induction, and for establishing
regulatory relationships between known genes.
Although initial experiments are likely to identify
targets when a pathway is activated or blocked throughout the entire mesoderm-induction period (as with the
elimination of VegT in the above example), the development of transgenic fish and frog embryos that contain
inducible activators and inhibitors, as well as the use
of specific pharmacological reagents, will facilitate the
genome-wide examination of changes in gene expression when a signalling pathway is activated or blocked
at a specific time in development. Because the functions
of the different signalling factors are constantly changing over developmental time as discussed above, the
use of microarray analysis combined with the ability
to temporally regulate each of the signalling pathways
will show how the transcriptional network changes to
allow each of the signals to regulate different processes
as embryogenesis proceeds.
Microarray analysis is not without its drawbacks. One
of the problems is that when whole embryos are analysed,
many non-mesodermal cells contribute to the signal. For
NATURE REVIEWS | GENETICS
genes that are expressed broadly throughout the mesoderm this might not be a problem, but many mesodermal
fates are represented by only a small subset of cells from
the entire embryo. One probable solution is to return
to Nieuwkoop’s original assay. When animal caps are
removed from embryos and treated with different factors such as Activin, which activates the Nodal signalling
pathway, different cell fates are induced depending on the
concentration of the factors used (reviewed in REF. 114)
(FIG. 6). Although the cell populations in these explants
typically are not made up of single cell types, they are
enormously enriched in a particular tissue compared
with the whole embryo. Moreover, because the explants
can be maintained in culture for up to 2 months115, not
only can the early events in mesoderm induction be
examined, but longer-term changes in gene expression
can also be analysed. Although animal cap explants have
occasionally been used in zebrafish, they are technically
more challenging and it is not yet clear whether the same
approach will be useful. In zebrafish the use of transgenic
lines that express a fluorescent protein from a cell-type
specific promoter combined with fluorescent cell sorting
might be a more feasible alternative.
Because mesoderm induction happens early in
embryogenesis, it has been particularly easy to manipulate this process by injecting pools of synthetic RNAs
into Xenopus embryos to identify genes that regulate
mesoderm formation. Previous attempts used pooled
libraries of cDNA that is attached to a promoter for the
SP6 RNA polymerase (reviewed in REF. 116). Although
some interesting genes have been identified in this way,
there were several problems with this approach. Even in
the most highly normalized library many cDNAs, especially abundant ones, were overrepresented. In addition,
particularly for longer mRNAs, many cDNAs are not full
length. Because of these problems, it was necessary to
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REVIEWS
inject relatively large pools of synthetic RNAs to sample
enough different cDNAs. Because there is a limit to the
amount of RNA that can be injected into an embryo,
these methods select for genes that produce an effect at
very low doses, which might explain why many of the
genes found this way encode signalling factors.
With increasing information from large-scale
sequencing projects, it is now possible to produce a
library that contains full-length clones of many genes to
test them in small pools by overexpression in the early
embryo116,117. One large-scale approach of this type
identified 64 Xenopus genes that affected the mesoderm,
from genes that caused defects in the morphogenesis
of gastrulation to those that produced an extra axis117.
Although some of these genes are well-known factors
such as VegT, others are either novel or have not been
studied in the context of mesoderm induction before.
Given the amount of data that are generated using
both classical and genomics-based approaches it is
increasingly important to develop databases to connect and cross reference the data. One valuable effort
in Xenopus called the Xenopus Mesendoderm Network
shows the known molecular interactions as a generegulatory circuit, which can be constantly modified as
new data become available118. Because data are of variable reliability and sometimes conflict, curating such a
database is a major challenge, but it still represents the
best approach for understanding the underlying circuitry in the mesoderm. Other databases with searchable
gene-expression patterns not only allow all genes with
a mesodermal expression pattern to be found, but also
provide connections to the relevant literature (Axeldb
for Xenopus and ZFIN for zebrafish).
Conclusions
The field of mesoderm induction has advanced a great
deal in the almost 40 years since its origin, identifying the
key signalling factors and establishing the groundwork
for understanding their roles in forming the mesoderm.
Genomic approaches will probably identify additional
key players downstream of these signals, including those
that are involved not only in cell-fate decisions but also
in the equally essential process of mesodermal morphogenesis, which transforms the spherical embryo into the
final embryonic body plan.
Because the mesoderm gives rise to many cell fates
and because it involves a wide variety of morphogenetic
movements, it continues to provide a wealth of important
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Acknowledgements
I wish to thank A. Schier, E. Amaya, U. Pyati, and D. Szeto for
critical comments on this manuscript; C. Stern, H. Isaacs,
G. Lieschke, R. Behringer and A. Schier for providing valuable
information; and S. Dougan and J. Heasman for communicating unpublished results. D.K.’s work on mesoderm induction
is supported by the US National Science Foundation and
National Institutes of Health.
Competing interests statement
The author declares no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
bmp2b | Bmp4 | cyc | Ntl | Spt | sqt | Xnr5 | Xnr6
FURTHER INFORMATION
Axeldb: http://www.dkfz-heidelberg.de/molecular_
embryology/axeldb.htm
Xenopus Mesendoderm Network: http://www.nottingham.
ac.uk/biology/Genetics/staff/rogerpatient/networks/
mesendoderm/mesendoderm.htm
ZFIN: http://zfin.org
Access to this links box is available online.
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