BMP Antagonists and Neural Induction

Bart JL Eggen, The Rockefeller University, New York, New York, USA Ali Hemmati-Brivanlou, The Rockefeller University, New York, New York, USA
In vertebrates, during the gastrula stage of embryonic development, the dorsal part of the ectoderm (the animal pole) is fated to form the neural plate, whereas the ectoderm located at the ventral side will primarily form epidermis. Embryonic factors known as BMP antagonists play a critical role in the processes of neural and epidermal induction.

Secondary article
Article Contents
. Introduction . Inhibition of Activin Signalling . BMPs as Neural Inhibitors and Epidermal Inducers . Neural Inducers that Bind and Inhibit BMPs . Evolutionary Conservation . Summary . Acknowledgements

Introduction
During vertebrate gastrulation, the three germ layers (ectoderm, mesoderm and endoderm) are formed. The dorsal ectoderm gives rise to the entire nervous system, while the ventral side will generate epidermis. Thus at the onset of gastrulation the neuroectoderm is anked by two boundaries: the boundary between neural and epidermal within the ectoderm and a boundary between ectoderm and mesoderm in the equatorial region (Figure 1). The formation of neural tissue is mediated by signals secreted by a group of cells located in the dorsal blastopore lip of a gastrula embryo. In the 1920s, Spemann and Mangold showed in a series of ground-breaking experiments that the dorsal blastopore lip of a gastrula stage newt embryo, when transplanted to the ventral side of a host embryo, can induce a second axis. In the ectopic axis the entire nervous system (with the exception of the oor plate) is derived from the host tissue and not from the graft. Based on these results, Spemann and Mangold postulated that the dorsal blastopore lip is responsible for the induction of the nervous system in the adjacent dorsal ectoderm, and termed this region the organizer (reviewed in Harland and Gerhart, 1997). The equivalent of the organizer has been identied in other species; these include the Hensen node in chick, the node in mouse and the embryonic shield in zebrash. When the presumptive ectoderm of a Xenopus laevis blastula (the animal cap region) is explanted and cultured, it gives rise solely to epidermis. However, when recombined with a dorsal lip, these explants dierentiate into neural tissue, showing that the organizer is sucient for neural induction. Surprisingly, however, when subjected to prolonged dissociation, ectodermal cells (which had never been exposed to organizer tissue) form neural tissue. Here the ectoderm adopts a neural fate in the absence of the source of the neuralizing signal: the organizer (Harland and Gerhart, 1997). This apparent contradiction has been solved over the course of the last few years. A remarkable set of discoveries has led to the identication of several key players in the process of neural induction and a mechanism of action unexpected from
Ventral ectoderm Dorsal ectoderm

Lateral Lateral mesoderm Organizer mesoderm

Endoderm

(a)

(b)

Neural inducers: noggin, chordin, follistatin and Xnr3 Epidermal inducers: BMP2, BMP4 and BMP7 BMP receptor Embryonic ectodermal cell
BMPs

Nucleus
Smad1 Smad4

Smad7 P Smad1 Smad4 P Smad1 P P

Induction of epidermal genes Inactive complex

Smad1 Smad6

(c)
Figure 1 Model for amphibian ectodermal patterning during gastrulation. (a) Dorsal view of a X. laevis gastrula embryo. During gastrulation, the organizer (red) secretes factors (including noggin, chordin, follistatin and Xnr3) that bind directly to, and inhibit epidermal inducers (including BMP2, BMP4 and BMP7). The border of the dorsal and ventral ectoderm is enlarged in (b). (b) In the dorsal ectoderm, factors secreted by the organizer inhibit epidermal differentiation by binding to epidermal inducers. In the ventral ectoderm, these BMP antagonists are absent and epidermal inducers can bind to their receptors without interference, and epidermal differentiation takes place. Signalling in ventral ectodermal cells downstream of these receptors is illustrated in (c). (c) BMP signalling in a ventral ectodermal cell. BMP signals are transduced via type I and II serine/threonine receptors. BMP-activated type I receptors activate the pathway-specific effector Smad1, which forms a complex with regulatory Smad4; this complex translocates to the nucleus where the expression of epidermal genes is induced and neural gene expression is inhibited, ultimately resulting in epidermal induction in the ventral ectoderm. In the Smad signal transduction cascade, several inhibitory Smads have been identified; Smad7 (and perhaps Smad8) inhibits signalling of TGFb growth factors at the receptor level; Smad6 competes with Smad1 for Smad 4, resulting in an inactive Smad1/Smad6 heteromer.

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BMP Antagonists and Neural Induction

these earlier embryological experiments. In this entry we describe the experiments leading to the identication and functional characterization of the factors involved in ectodermal patterning. Since most of these studies have been performed in the amphibian X. laevis, this entry focuses on experiments performed in this species. Finally, we place these observations in an evolutionary context by comparing them to ndings with proteins that have potentially similar activities in other species.

Inhibition of Activin Signalling

Formation of neural tissue by the embryonic ectoderm can be the result of either direct or indirect induction. For example, the addition of growth factors like activin or Vg1 to animal cap explants induces cells to form dorsal mesoderm, similar to the cell types present in the organizer. Subsequently these mesodermal cells act as an organizer by inducing the surrounding cells to form neural tissue. In this case, the induction of neural tissue is dened as indirect, as it is dependent on the formation of an intermediate cell type (dorsal mesoderm), which in turn induces neural tissue. By contrast, a direct neural inducer can induce animal caps to form neural tissue without the requirement for other cell types to form (Baker and Harland, 1997). The rst example of a direct neural inducing agent was a dominant negative type II activin receptor, which lacks the intracellular domain and inhibits signalling from activin and related transforming growth factor b (TGFb) family members. Inhibition of signalling by this receptor indeed blocks the formation of mesoderm in the intact embryo (Hemmati-Brivanlou and Melton, 1997). The surprising observation, however, was that inhibition of signalling through the activin receptor, both in embryos and in animal cap explants, resulted in expression of neural cell adhesion molecule (NCAM), a marker of neural tissue (Hemmati-Brivanlou and Melton, 1997). This result implied that interference with TGFb signalling not only inhibits mesoderm formation but also causes direct extensive induction of neural tissue. The results obtained with the dominant negative receptor allowed an analogy with cell dissociation experiments. The common denominator between cell dissociation experiments and interference with TGFb signalling was the elimination of a putative secreted neural inhibitor. In cell dissociation experiments, the inhibitor would be diluted such that it would no longer aect the dissociated embryonic cells, while the same was achieved in intact ectodermal explants in which a subset of cellular signalling was inhibited by the truncated activin receptor. This line of reasoning led to the proposal of the default model of neural induction. This model postulated that cells of the embryonic ectoderm would dierentiate as neural tissue in the complete absence of signalling from their neighbours, and thus neural fate
2

represents the default fate of the ectoderm. This also implies that epidermis, the other possible fate that ectodermal cells can adopt, is induced. This is in contrast with conclusions drawn by classical embryologists, which suggest the epidermis to be the default, and neural the induced, state. The dominant negative activin receptor pointed to the TGFb superfamily of ligands as potential inhibitory candidates. Further experiments broadened the default model to all germ layers of the blastula, as cells of the bottom part of the sphere (vegetal pole), which normally give rise to endodermal derivatives only, would also become nerve cells in the presence of the truncated activin receptor. This suggested that the default fate of all early embryonic cells is neural and implies that all frog blastomeres will adopt a neural fate unless specied otherwise by instructive signals (i.e. a signal that activates a signal transduction pathway). In order to generate neural tissue, this secreted inhibitor needs to be inhibited. Perhaps the most important prediction of the model was the existence of epidermal inducers in vivo in the ectoderm and antagonists of these inhibitors secreted by the organizer (Hemmati-Brivanlou and Melton, 1997).

BMPs as Neural Inhibitors and Epidermal Inducers

To isolate factors with neural inhibitory and epidermalinducing activity, Wilson and Hemmati-Brivanlou (1995) used the dissociated animal cap assay. Dissociated animal cap cells autoneuralize and provide a powerful tool to test putative factors for their ability to inhibit neural induction and induce epidermis. This is achieved by adding back puried factors to dissociated cells and challenging them to adopt other fates than neural. The potential neural inhibitor/epidermal-inducing factor was expected to be a member of the TGFb family of growth factors, as the dominant negative activin receptor interfered with TGFb signalling. The rst logical choice of a member of the TGFb family to test for epidermalization of dissociated animal cap cells was activin, as a dominant negative activin receptor neuralizes embryos and animal caps. By adding puried recombinant activin protein to dissociated animal cap cells, the formation of neural tissue is blocked. However, as previously demonstrated, instead of adopting an epidermal fate the cells adopt a mesodermal fate. This result showed that activin is a neural inhibitor which is more likely to be involved in the formation of the boundary between the dorsal ectoderm and the mesoderm. Based on the fact that it was inhibited by the truncated activin receptor, the TGFb family member bone morphogenetic protein 4 (BMP4) was another potential epidermal inducer. This argument was supported by the fact that BMP4 RNA is present in the entire animal pole at early gastrula stages. Furthermore, it had been shown that

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BMP Antagonists and Neural Induction

BMP4 ventralizes embryonic mesoderm. This function could potentially parallel the activity of BMP4 in the ectoderm, as epidermis is derived from ventral ectoderm and neural tissue from dorsal ectoderm. When added to dissociated cells, BMP4 both suppressed neuralization and induced epidermis at concentrations of 15 pmol L 2 1. This led to the conclusion that neural suppression and epidermal induction can be mediated by a single secreted factor and fullled the most stringent requirement of the default model (Wilson and Hemmati-Brivanlou, 1995). Is BMP4 the only TGFb endowed with epidermalinducing activity? BMP4 is a member of the large family of TGFb growth factors. At blastula stages, transcripts of two related molecules, BMP2 and BMP7 are detected throughout the animal pole. In gastrula stage embryos, BMP2 and BMP7 are expressed throughout the ectoderm and marginal zones. Recently it has been shown that BMP2 and BMP7, like BMP4, can induce epidermis in dissociated animal caps, although the activity of BMP7 is weak. The dominant negative activin receptor, as well as the truncated BMP receptor, which also neuralizes animal caps, can block the activity of all three BMPs in both intact and dissociated ectodermal explants, reinforcing the involvement of BMPs in these processes (Suzuki et al., 1997).

Neural Inducers that Bind and Inhibit BMPs

The discovery that BMPs directly block neural induction and induce epidermis solved the rst part of the puzzle: a neural inhibitor/epidermal inducer was identied. However, the question as to how neural inducers from the organizer act with respect to BMPs remained to be addressed. The neural inhibitory action of BMPs presumably requires the presence of anti-BMPs to block BMP function in the dorsal ectoderm and to allow for neuralization to occur. This issue was solved when it was shown that neural inducers secreted by the organizer have the ability to block the action of BMPs by specically binding and inactivating them (Sasai and De Robertis, 1997). Independent strategies led to the identication of several proteins with neural inducing activity, namely noggin, follistatin, chordin and Xenopus nodal related 3 (Xnr3). The rst endogenous, direct neural inducer identied was noggin. Noggin was identied in a screen for factors that are able to rescue ultraviolet (UV) ventralized embryos (Baker and Harland, 1997). Follistatin was identied as a protein able to bind and inhibit activin with high anity. Since a dominant negative activin receptor is able to neuralize intact embryos, follistatin was an obvious candidate to test in the context of neural induction. Indeed, follistatin is expressed in the Spemann organizer and displays direct neuralizing activity (Hemmati-Brivanlou

and Melton, 1997). It was recently shown that, in addition to activin, follistatin also inhibits the function of BMP7. Chordin was identied in a screen for organizer-specic factors (Sasai and De Robertis, 1997) and also displays direct neural-inducing ability. Biochemical analyses showed that noggin and chordin antagonize BMP4 by direct high-anity binding. An interesting observation is that binding of noggin and chordin to BMP4 can be competed by BMP2 and to a lesser extent by BMP7, suggesting that these neural inducers also bind other BMPs. This result also agrees with the ability of both BMP2 and (to a lesser extent) BMP7 to epidermalize dissociated caps. Binding of noggin and chordin prevents binding of BMP4 to its receptor and inhibits BMP signalling in the dorsal side of the embryo, leading to neural dierentiation of the dorsal ectoderm. A complicating factor is that BMPs form biologically active heterodimers when tested in an osteogenic dierentiation assay, with the BMP4/7 heterodimer being the most active. Chordin has been shown to bind this heterodimer with the same high anity with which it binds to BMP4 (Baker and Harland, 1997; Sasai and De Robertis, 1997). The molecular mechanism by which follistatin and Xnr3 neuralize remains to be determined. It has been shown that follistatin binds activin with high anity; activin, however, induces mesoderm and is not an epidermal inducer. Another study has demonstrated that follistatin can interfere with BMP7 activity; since BMP7 heterodimerizes with BMP4, an attractive possibility is that follistatin additionally inhibits BMP4 through binding to BMP7. Though follistatin has been shown to bind to BMP4 under certain conditions, it has not been shown that follistatin can inhibit BMP4 activity. Additional experiments will also be required to address the exact mechanism of function of Xnr3 (Baker and Harland, 1997). The observations that multiple BMPs with similar functions are expressed in the ectoderm, and that several neural inducers are expressed in the organizer, implies some degree of redundancy. However, the expression patterns of these factors (both BMPs and neural inducers) is overlapping but not identical. In addition, BMPs form heterodimers that may dier both in their activity as well as in their binding specicities to dierent neural inducers. The default model predicts that inhibition of the neural inhibitor at any level of the signal transduction pathway should lead to the same outcome: the expression of the neural fate. The four neural inducers mentioned above are factors that inhibit the pathway from outside the cell (in a non-cell-autonomous fashion). At the membrane level, we have already mentioned that interference with the reception of the signal by using dominant negative activin or BMP receptors also neuralizes. Recently, a family of novel genesrequiredforintracellularsignaltransductionofTGFb growth factors was identied and collectively named Smads (Figure 1). So far, the family of Smad proteins in vertebrates contains nine members which can be divided
3

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BMP Antagonists and Neural Induction

, 1998): (1) into three groups (reviewed in Massague pathway-specic eector Smads which are activated by ligand-dependent dimerization of TGFb receptors that mediate intracellular signalling of specic TGFb ligands these include Smads: 1, 3, 5; (2) regulatory smads, for which only one candidate, Smad4, has been identied that functions as an obligatory partner for all other Smads; (3) inhibitory Smads, Smad6, 7 and 8, which inhibit the function of the eector Smads. Smad2 and 3 transduce signals on behalf of the activin/TGFb pathway, while Smads 1, 5 and maybe 9 transduce signals on behalf of the BMPs. Thus, overexpression of Smad1, 5 and 9 in dissociated ectodermal explants leads to the inhibition of neural fate and induction of epidermis, mimicking BMP eects. Smad6 interferes with BMP signalling by specically binding to Smad1, preventing complex formation between Smad1 and Smad4. Smad7 (and perhaps 8) acts through nonselective binding to receptors, preventing activation of eector Smads by these receptors and thereby blocking both activin and BMP4 signalling. Overexpression of Smad6, 7 or 8 interferes with BMP signalling and, as predicted by the default model, leads to the induction of , 1998). neural markers in animal caps (Massague Finally, at the end of the pathway the eector Smads, associated with Smad4, translocate to the nucleus and aect transcription. The Smad2Smad4 complex has been shown to associate with the transcription factor FAST1 in response to activin. For the BMP pathway, two immediate early response genes of the homeobox family, msx1 and Xvent1, have been identied. Consistent with their aliation to the BMP pathway, msx1 has been shown to inhibit neural formation and induce epidermis in dissociated , 1998). ectodermal explants (Massague

Evolutionary Conservation
Homologues of several of the molecular players described above have been cloned in many other species, ranging from Drosophila melanogaster to humans. In D. melanogaster, the polarity of the dorsalventral axis is reversed with respect to vertebrates. The nervous tissue develops at the ventral side of the embryo and is derived from ventrolateral blastoderm cells. Two genes identied based on their requirement for correct dorsalventral pattern formation are decapentaplegic (dpp) and short gastrulation (sog). The dpp gene is required for patterning dorsal cell fates: in embryos that lack DPP, a cell fate typical of ventrally derived cells is acquired by dorsal and dorsolateral cells. The opposite occurs when the sog gene is mutated: ventrolateral cells in sog 2 embryos adopt a dorsal fate with a compromised nervous system. DPP is the y homologue of BMP2/4, and SOG is the homologue of chordin. These genes are in fact functionally interchangeable between Xenopus and Drosophila. Although the y
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homologues of noggin and follistatin have not yet been characterized, noggin can inhibit DPP function in Drosophila embryos. Thus, despite a possible reversal of the dorsalventral axis between arthropods and chordates, the molecular mechanisms involved in ectodermal patterning seem to be conserved (Ferguson and Anderson, 1996). In the urochordate ascidian Halocynthia roretzi, a homologue for BMP2/4, termed HrBMPb, has been identied (Miya et al., 1997). The injection of RNA encoding HrBMPb in cells that normally give rise to neural tissue changes their fate into epidermis, resulting in a loss of anterior neural structures in the larva. These results suggest a conserved function for the BMP homologue HrBMPb as a neural inhibitor and epidermal inducer in ascidian development. In zebrash, a number of mutants with abnormal dorsalventral patterning have been recently identied, including dino and swirl. Dino displays a ventralized phenotype. The dino mutation turned out to be the zebrash chordin gene and the mutant has since been renamed chordino. In situ hybridization showed that chordino is expressed in the zebrash organizer, the embryonic shield. The swirl mutation displays a dorsalized phenotype and is caused by a mutation in the zebrash BMP2 gene. Furthermore, the chordino and swirl mutations interact genetically as is observed for dpp and sog in D. melanogaster. Thus in zebrash, correct dorsalventral patterning also requires interactions between BMPs and BMP antagonists (Schier, 1997). The situation in amniotes is still unclear. In the chick embryo, pellets of COS cells overexpressing chordin do not lead to induction of ectopic neural tissue. The chordin expressed by these cells can induce an ectopic axis (primitive streak), thus demonstrating that chordin derived from the mammalian cells is still active (Streit et al., 1998). As is the case with X. laevis, misexpression of BMP4 inhibits the formation of the dorsal axis. These observations suggest that regulation of BMP signalling by chordin plays a role in formation of the primitive streak and that chordin is not sucient to induce neural tissue. This surprising observation could mean that the mechanism of neural induction in chick is dierent from that in X. laevis. Another potential explanation is that the concentration of chordin available to bind BMPs, although high enough to induce an ectopic primitive streak, is not sucient to induce neural tissue. More experiments will be required to further clarify this issue. In mammals, both cell culture and in vivo studies have been used to address the role of BMPs and their inhibitors in ectodermal patterning. In P19 embryonic carcinoma cells, overexpression of a truncated activin receptor results in neural dierentiation (Wilson and Hemmati-Brivanlou, 1997). BMP4 inhibits the neural inducing ability of retinoic acid when copresented to these cells, and addition of BMP4 induces epidermal markers. In mouse embryos, both BMP4 and the type I BMP receptor appear to be critical around gastrulation (reviewed in

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BMP Antagonists and Neural Induction

Hogan, 1996). In mice with a BMP4 null mutation, the majority of the homozygous embryos die around gastrulation. Embryos with a null mutation of the type I BMP receptor also die before gastrulation. Although these data indicate that BMP signalling plays a critical role during early development in mammals, it does not help solve the function of BMP signalling during this process. Knockout mice for noggin and follistatin do not show an elimination of the nervous system, suggesting that either these factors are not involved in neural induction in the mouse or, more likely, that redundancy between multiple BMP antagonists prevents the generation of a severe phenotype by single gene knockouts.

Acknowledgements
Work from the laboratory of AHB is supported by NIH grant 32105-01.

References
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Summary
Recent work has largely solved the molecular mechanism of neural induction, at least in amphibians, as originally described by Spemann and Mangold in 1924. The current, widely supported model is that neural induction of the dorsal ectoderm is a result of the inhibition of soluble epidermal inducers. The action of these epidermal inducers is blocked by factors secreted by the Spemann organizer. The mechanism underlying this inhibition involves direct binding of these organizer molecules to the epidermal inducers, thereby inactivating them (Figure 1). The inhibition of epidermal induction reveals the default state of the ectoderm, which is neural. Good candidates for epidermal inducers include homo- and heterodimers of BMP2, BMP4 and BMP7. Neural inducers with the correct expression pattern and function include noggin, chordin, follistatin and Xnr3. It is important to emphasize that the neuralization described here is only the very rst step in the long and complicated process that ultimately results in the development of a fully organized and functional nervous system.

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