Dynamic Development
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Main Page Dynamic Development

The Foundations of Developmental Biology

Gametogenesis

From Sperm and Egg to Embryo

Genetic Regulation of Development

Organizing the Multicellular Embryo

Generating Cell Diversity


Dynamic Development at a Glance


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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. 15.14; 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--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 B-Vg1 hybrid. The activin 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 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.


References

Browder, L.W., Erickson C.A. and Jeffery, W.R.. 1991. Developmental Biology. Third Edition. Saunders College Publishing. Philadelphia.

Dale, L., Matthews, G. and Colman, A. 1993. Secretion and mesoderm inducing activity of the TGF-beta-related domain of Xenopus Vg1. EMBO J. 12: 4471-4480.

Dale. L. and Slack, J.M.W. 1987. Regional specification within the mesoderm of early embryos of Xenopus laevis. Development 100: 279-295.

Dyson, S. and Gurdon, J.B. 1997. Activin signalling has a necessary function in Xenopus early development. Curr. Biol. 7: 81-84.

Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer. Sunderland, MA.

Kalthoff, K. 1996. Analysis of Biological Development. McGraw-Hill. New York.

Kessler, D.S. and Melton, D.A.. 1994. Vertebrate embryonic induction: mesodermal and neural patterning. Science 266: 596-604.

Kessler, D. S. and Melton, D. A. 1995. Induction of dorsal mesoderm by soluble, mature
Vg1 protein. Development 121: 2155-2164.

Roush, W. 1997. A developmental biology summit in the high country. Science 277: 639-640.

Shulte-Merker, S., Smith, J.C. and Dale, L. 1994. Effects of truncated activin and FGF receptors and of follistatin on the inducing activities of BVg1 and activin: does activin play a role in mesoderm induction? EMBO J. 13: 3533-3541.

Slack, J.M.W. 1991. The nature of the mesoderm-inducing signal in Xenopus: a transfilter induction study. Development 113: 661-669.

Slack, J.M.W. 1994. Inducing factors in Xenopus early embryos. Current Biology 4: 116-126.

Slack, J.M.W.et al.. 1984. Philos. Trans. R. Soc. Lond. [Biol] 307: 331-336.

Thomsen, G.H. and Melton. D.A.. 1993. Processed Vg1 protein is an axial mesoderm inducer in Xenopus. Cell 74: 433-441.

Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and Meyerowitz, E. 1998. Principles of Development. Current Biology. London.


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Leon Browder & Laurie Iten (Ed.) Dynamic Development
Last revised Wednesday, July 15, 1998