CONTENTS
Main Page Dynamic Development
The Foundations of Developmental
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Gametogenesis
From Sperm and Egg to Embryo
Genetic Regulation of Development
Organizing the Multicellular
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Generating Cell Diversity
Dynamic Development at a
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Developmental Biology Tutorial |
Embryonic Induction during Vertebrate Development: Mesoderm Induction
in Xenopus
The key to the middle kingdom
Vertebrate embryos rely extensively upon inductive interactions to diversify
the number of different kinds of cells in the embryo. Induction is the process
by which one group of cells produces a signal that determines the fate of
a second group of cells. This implies both the capacity to produce a signal
(ligand) by the inducing cells and the competence of the responding cells
to receive and interpret the signal via a signal transduction pathway.
Amphibians are the most extensively studied vertebrates for investigations
into embryonic induction. Most contemporary investigations have utilized
Xenopus. The two major inductive events during early Xenopus development
(Slack, 1994, Fig. 1) are:
- mesoderm induction
- neural induction
Mesoderm Induction: An Overview
Mesoderm induction occurs over an extended period of time in the equatorial
region of the embryo from about the 32-cell stage to the beginning of gastrulation.
The requirement of induction for production of mesoderm is evident by comparing
the embryonic fate map (which shows the fates of regions of the embryo during
normal development) to the specification map (which shows what tissue explants
can do in isolation) during cleavage stages.
The fate map shows that about half of the mesoderm arises from cells above
the equatorial pigment boundary, whereas no cells from above the pigment
boundary will form muscle or notochord in isolation. This implies that cells
above this boundary require contact with cells below the boundary (the vegetal
cells) to produce mesoderm, and that is what is observed when animal and
vegetal explants are combined using the procedure first developed by Nieuwkoop.
(See Browder et al., 1991, Figs. 12.1-12.4; Gilbert, 1997,
Fig. 5.14; Kalthoff, 1996, Figs. 6.2 and 9.19; Wolpert et al., 1998,
Figs. 3.16, 3.22 and 3.25)
The vegetal cells are not equal in their inductive capacities. The dorsal
vegetal cells (the Nieuwkoop Center) induce axial mesoderm of the dorsal
midline (notochord and segmented muscle), whereas the remaining vegetal
cells induce ventrolateral mesoderm (mesothelium, mesenchyme, blood cells).
(See Gilbert, Fig. 15.16; Kalthoff, Fig. 9.20; Wolpert, 1998, Fig.
3.27)
These observations have led to the proposal that there are two mesoderm-inducing
signals:
- a general vegetal signal that operates around the circumference and
induces ventral mesoderm; and
- a dorsal vegetal signal (emanating from the Niewkoop center) that induces
the axial mesoderm (i.e., Spemann's organizer).
The general vegetal signal apparently remains operational in ventralized
embryos produced by UV irradiation of fertilized eggs; these radially symmetrical
embryos have the normal amount of mesoderm, but all of it is ventral in
character. The dorsal vegetal signal is dependent upon dorsalization
factors.
The ventral mesoderm becomes further diversified into subdomains. This diversification
is apparently due to a third signal that dorsalizes the ventral mesoderm
closest to the organizer. Evidence in favor of this hypothesis again comes
from a comparison of the fate map and specification map: About 60% of muscle
is derived from ventral mesoderm (Slack, 1994). However, explanted ventral
mesoderm forms little or no muscle (Dale and Slack, 1987). Muscle is formed
by ventral mesoderm when it is juxtaposed with the organizer. This dorsalization
of the ventral mesoderm occurs during gastrulation.
(See Browder et al., Fig. 12.5, Gilbert, Fig. 15.17; Kalthoff,
Fig. 9.23; Wolpert et al., 1998, Fig. 3.26)
Can We Identify the Native Mesoderm Inducers?
A number of potential mesoderm inducers have been identified in the literature.
A partial list of inducers is shown in Table 1.
Table 1. Expression patterns of RNA encoding inducing factors
(After Slack, 1994)
| Factor |
Maternal |
Zygotic* |
| Activins A and B |
+** |
Neurula axis |
| Vg-1 |
Vegetal |
|
| BMP-2 and BMP-4 |
+ |
+ |
| FGF-2 |
+ |
Neurula axis |
| FGF-3 |
|
Blastopore, neural |
| FGF-4 |
+ |
Blastopore |
| Wnt1 and Wnt3A |
|
Neurula head |
| WntB |
|
Gastrula ventral |
| Wnt11 |
Vegetal |
Gastrula mesoderm |
| noggin |
+ |
Dorsal lip |
*Only the initial zygotic domain of expression is shown; many factors also
have later domains.
**Protein but not RNA detected.
+denotes expression detected by biochemical methods. |
Identification of inducers requires an assay to demonstrate their biological
activity. Induction is assessed by either morphological or molecular criteria.
A positive response is indicated by elongation of the explants and formation
of vesicles. A commonly-used molecular marker is muscle-specific alpha-cardiac
actin mRNA. A more recent assay is the detection of brachyury
(Xbra) mRNA.
One way to assay for inductive potential is by the "animal cap serial
dilution assay". This involves exposing isolated animal caps from blastulae
to serial dilutions of the suspected inducer (Slack, 1994, Fig. 2; ). Another
assay is the "autoinduction assay". By this procedure, RNA encoding
the suspected inducer is injected into the zygote, and the animal cap is
removed at the blastula stage. The animal cap will then induce itself.
(See Browder et al., Fig. 12.6)
Mesoderm induction is initiated before the zygotic genome is transcriptionally
active. Hence, any proteins that must be synthesized to initiate induction
must be encoded on oogenic mRNA, which is synthesized during oogenesis and
stored in the cytoplasm for later utilization. The expression patterns of
putative mesoderm inducers are shown in Table 1.
Considerable research has been conducted on the activins, which are very
potent mesoderm inducers. However, activin mRNA is not present in the zygote.
There is, however, a small amount of activin protein present. BMP-2, BMP-4,
noggin, and basic FGF transcripts are present in the zygote, although
they are not vegetally localized. Both Vg-1 and Wnt 11 messengers are present
and are localized to the vegetal hemisphere. Therefore, they have to be
given careful consideration as potential mesoderm inducers. The lack of
localization of the other transcripts should not rule them out as potential
inducers, however, because they could be subject to regulation that restricts
their translation to the vegetal hemisphere. Likewise the vegetally-restricted
transcripts Vg-1 and Wnt 11 could be candidates for the dorsovegetal signal
if their translation were restricted to the Nieuwkoop center.
Identification of the native inducers has been
frustrating. Although the identities of candidate inducers are known and
their genes have been cloned, direct tests of their roles using the genetic
approach that have been used so effectively with Drosophila and Caenorhabditis
have not been possible. Hence, indirect approaches using inhibitors have
been used extensively to provide hints as to their identities.
The drug suramin antagonizes growth-factor binding to their receptors, whereas
heparin binds to growth factors in the FGF family. Both of these agents
have been used to block the transmission of the mesoderm-inducing signal
in a transfilter apparatus (Slack, 1991, Fig. 1). On the other hand, follistatin,
an inhibitor of activin, and an antibody to bFGF were not inhibitory (Slack,
1991, Fig. 5). Thus, growth factors are apparently involved, but the roles
of activin and bFGF are questionable, at least based upon this assay.
Dominant-negative receptors overexpressed from synthetic messengers are
very powerful tools for studying intercellular signaling processes. These
messengers encode truncated receptors that form inactive dimers with the
endogenous receptors, thus inhibiting the signaling process. Expression
of a dominant-negative activin receptor in animal pole explants inhibits
the ability of both activin and processed Vg-1 to induce mesoderm. This
result suggests that both of these signaling molecues may be using the same
receptor. Thus, this result does not allow us to determine which of these
signaling molecules is essential for mesoderm induction.
However, overexpression of follistatin mRNA does not perturb mesoderm formation
(Kessler and Melton, 1994; Schulte-Merker et al., 1994). Follistatin
is an activin-binding protein. Its overexpression blocks activin function.
This results suggests that endogenous Vg-1, not activin, is involved in
mesoderm induction and is consistent with the observation by Slack (1991)
that follistatin does not inhibit the mesoderm-inducing signal from the
vegetal pole in trans-filter experiments.
Thus, the evidence for the involvement of Vg-1 in mesoderm induction is
strong. However, testing the putative role of Vg-1 in mesoderm induction
has been difficult, because Vg-1, like other TGF-ß-like proteins,
is synthesized as an inactive precursor that must dimerize through disulfide
linkages and must be processed by proteolysis to become active. Injection
of Vg-1 mRNA into embryos produces precursor, but no processed, biologically
active protein.
It has been possible to get embryos to process Vg-1 by injecting RNA that
contains the Vg-1 coding sequence linked to a sequence encoding a cleavage
site of bone morphogenetic protein (BMP). Injection of BMP2-Vg1 mRNA into
embryos resulted in induction of dorsal mesoderm in animal cap explants
(Thomsen and Melton, 1993). Another hybrid, BMP4-Vg1, was only weakly effective
(Dale et al., 1993).
More recently, Kessler and Melton (1995) have devised a means to make soluble,
biologically-active Vg1 to apply to animal pole explants. The Vg1 was made
from an activin ß B-Vg1 hybrid. The activin ß B element contained
both a signal sequence that mediates secretion and a cleavage site. This
facilitated both the processing and secretion of Vg1 protein in oocytes
injected with activin ß B-Vg1 mRNA (Fig. 1, Kessler and Melton, 1995).
Supernatant from injected oocytes was tested for its mesoderm-inducing activity
on explanted animal poles. The explants expressed mesoderm-specific markers,
and produced notochord, somitic muscle and neural tissue.
(See Gilbert, 1997, Fig. 15.19)
As we discussed previously, Vg-1 may be utilizing the activin receptor to
mediate its effects. To examine whether this may be the case, animal pole
explants of embryos that had been injected with a truncated activin receptor
(tAR) were treated with Vg1-containing supernatant. The truncated receptor
blocked mesoderm induction by mature Vg1. A truncated FGF receptor also
blocked induction by this factor. This suggests that the abilities of these
two receptors to block mesoderm induction in the embryo reflect their effects
on endogenous Vg1 signaling.
Does the activin receptor function as the endogenous Vg1 receptor? To examine
this possibility, a version of the Xenopus activin II receptor that
had an epitope of MYC attached to it (XARmyc) was expressed in Xenopus
oocytes. (This approach is called epitope tagging.) The binding of
radioactive Vg1 or activin to XARmyc was then assessed. The epitope tag
enabled the XARmyc with bound ligand to be immunoprecipitated using an antibody
to MYC. The specificity of binding was assessed by competition using excess
unlabeled ligand. As shown in Fig. 3 (Kessler and Melton, 1995), activin
binds avidly to the receptor, but Vg1 does not. Furthermore, unlabeled Vg1
fails to compete for activin binding.
These results suggest that
- the activin receptor is not a Vg1 receptor and
- the effects of the truncated activin receptor on Vg1 signaling are
probably mediated through an interaction between the truncated activin
receptor and an endogenous Vg1-specific receptor.
As we have previously discussed, follistatin is an inhibitor of activin.
Previous investigations of the effects of follistatin have utilized mammalian
follistatin. However, Kessler and Melton (1995) have examined the effects
of Xenopus follistatin on both normal development and response of
animal pole explants to activin and Vg1. Injection of follistatin mRNA into
embryos at the two-cell stage perturbed posterior development, but appeared
to enhance anterior dorsal development (Fig. 4). Normal expression of dorsal
mesodermal markers was detected. Thus, endogenous induction of dorsal mesoderm
is not inhibited, and a role for activin in dorsal mesoderm induction is
not supported by these results.
Follistatin was unable to block the effects of Vg1 on animal pole explants,
although it blocked the effects of activin (Fig. 5). Once again, these results
cast doubt on the role of activin and support a role for Vg1 in mesoderm
induction. These results may also indicate that follistatin is involved
in patterning of the mesoderm.
Now that we have thoroughly discredited activin, there is recent evidence
that may rehabilitate it. Dyson and Gurdon (1997) have demonstrated that
a new dominant-negative activin receptor construct does block activin
signaling but has no effect on Vg1 signaling. When 4-cell-stage embryos
were injected with messenger RNA encoding this dominant-negative activin
receptor, severe developmental defects were produced, including the absence
of a head. These defects could be rescued by co-injection of activin messenger
RNA. Dyson and Gurdon conclude that activin or an activin-like molecule
plays an essential role in mesoderm induction.
The jury is out regarding the respective roles of Vg1 and activin in mesoderm
induction. However, there is strong evidence that implicates both of them
in this process. At this time, we should consider the possibility that both
are involved.
Learning Objectives
- When does mesoderm induction begin? What does this timing tell you
about the source of the factors that are involved in early events in mesoderm
induction?
- Draw the fate map of a 32-cell Xenopus embryo.
- How does this fate map compare to the specification map?
- What is the animal cap assay that was developed by Nieuwkoop?
- Compare the inductive capacities of dorsal vegetal cells and ventral
vegetal cells.
- Describe the three signal model and discuss the evidence in favor of
this model.
- How many potential inducers can you name? Describe their expression
patterns.
- How have suramin, heparin and follistatin been used to identify the
native mesoderm inducers? Discuss the results obtained with these agents.
Discuss the effects of overexpression of follistatin mRNA.
- What is a dominant-negative receptor, and how has this technology been
used to identify native mesoderm inducers?
- How has soluble, biologically-active Vg1 been produced to test the
role of Vg1 in mesoderm induction?
- What is epitope tagging?
- Defend the following statement: Activin or an activin-like protein
plays an essential role in mesoderm induction.
Digging Deeper:
Recent Literature
Chang, C., Wilson, P.A., Mathews, L.S. and Hemmati-Brivanlou, A. A .
1997. Xenopus type I activin receptor mediates mesodermal but not
neural specification during embryogenesis. Development 124: 827-837.
Harger, P.L. and Gurdon, J.B. 1996. Mesoderm induction and morphogen gradients.
Sem. Cell and Develop. Biol. 7: 87-93.
Ryan, K., Garrett, N., Mitchell, A. and Gurdon, J.B. 1996. Eomesodermin,
a key early gene in Xenopus mesoderm differentiation. Cell 87: 989-1000.
Stennard, F., Carnac, G. and Gurdon, J.B. 1996. The Xenopus T-box
gene, Antipodean, encodes a vegetally localised maternal mRNA that
can trigger mesoderm formation. Development 122: 4179-4188.
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