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Inducing factors in Xenopus early embryos
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Inducing factors in Xenopus early embryos J.M.W. Slack ICRF Developmental Biology Unit, Department of Zoology, Oxford University, South Parks Road, Oxford OX1 3PS, UK.
Recent results make it possible to postulate credible candidates for each of the known inducing signals that act to determine cell fate during Xenopus early development. Experiments on biological activity, expression patterns and inhibition of function suggest that Vg-1 and Wnt-11 may act as the primary mesoderm-inducing signals, FGF and activin may serve to relay their effects, and noggin may be a major component of the dorsalizing and neural-inducing signals from the organizer. The last few years have seen unparalleled progress in the unravelling of the early inductive interactions that specify the vertebrate body plan. Although this progress has regularly been reviewed [1,2], several very recent advances have filled in some more blank spaces in the picture. In this article, I shall try to set these new results in context and ask to what extent we now really understand what is going on.
The biology of inducing factors The following account very briefly reviews the biological basis of our present understanding of the early inductive interactions that take place following fertilization of the Xenopus egg. 'Induction' here describes the process by which one group of cells signals to a second group in such a way as to determine the developmental fate of the second; induction requires both a signalling capacity in the first set of cells and a competence to respond in the second. The inductive interactions during Xenopus early development are shown diagrammatically in Figure 1, which culminates in the situation at the end of gastrulation. By this time, two major embryonic tissue types have been formed and each
subdivided into several territories: the mesoderm, which gives rise to muscle, blood and mesenchyme, and the neural plate, which gives rise to the nervous system. For simplicity, we shall here exclude from consideration the anteroposterior patterning of the mesoderm and neural plate, which has been discussed in more detail elsewhere 13], and consider only induction of the mesoderm and its dorsoventral patterning. During its development in the mother, the Xenopus oocyte becomes polarized along its animal-vegetal dimension but remains radially symmetrical around this axis. The animal-vegetal polarization is clearly visible by a difference of pigmentation: the animal hemisphere is dark and the vegetal hemisphere light-coloured. The radial symmetry of the oocyte is broken at fertilization by the point of sperm entry. About half-way through the first cell cycle a cytoplasmic shift occurs, called the cortical rotation, which involves the movement of the plasma membrane plus a thin shell of underlying cytoplasm - the 'cortex' - relative to the internal cytoplasm [4]. The rotation occurs about the future left-right axis of the embryo, and is such that the dorsal side forms where vegetal cortex meets animal cytoplasm
Fig. 1. The sequence of inductive interactions in Xenopus embryos, deduced from 'biological' experiments. Yellow arrows show the general mesoderm-inducing signal ('make mesoderm'); the red arrow indicates the dorsovegetal organizer-inducing signal ('make organizer'); the purple arrow shows the third, dorsalization-inducing signal ('make dorsal mesoderm'); the white arrow represents neural induction. By the middle gastrula stage the neural plate and organizer are each subdivided into three regions (N1-N3 and 01-03, respectively).
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Inducing factors in Xenopus development and the ventral side forms on the side where animal cortex meets vegetal cytoplasm (Fig. lb). The direction of the rotation is determined by the point of sperm entry, which becomes the future ventral side. Cortical rotation is known to be essential for axis formation in the intact embryo, although not for the formation of ventral mesoderm - 'axis' here (and below) refers to the notochord, somites and neural plate, with their characteristic anterior-posterior pattern. If rotation is prevented by ultraviolet (UV) irradiation of the egg, the axis does not form, and the embryo is referred to as 'UVO' [4]. If UV irradiation is followed by an imposed rotation of the egg, the axis can be restored, with the new dorsoventral polarity corresponding to the imposed rotation [5]. Cortical rotation is thought to affect both the vegetal hemisphere, where it leads to the formation of a dorsovegetal signalling centre (DV centre - also known as the Nieuwkoop centre or organizer-inducing centre), and the animal hemisphere, where it leads to an enhanced competence to respond to signals from the DV centre on the dorsal side [6]. The fertilized egg cleaves to form a hollow ball of cells, the blastula, and during an extended period between about the 32-cell stage and the beginning of gastrulation, mesoderm is induced to form in the equatorial region of the embryo (Fig. c), as a result of signalling from the vegetal to the animal cells [7]. We know that at least some, and perhaps all, of the mesoderm is formed by induction, because of the comparison between the fate map and the specification map of the cleavage stages. In the fate map, which shows what happens in normal development, about half of the mesoderm arises from cells of the animal hemisphere, above the equatorial pigment boundary [8]. By contrast, in a specification map, which shows what tissue explants can do when cultured in isolation, no muscle or notochord is formed from explants taken from above the pigment boundary [9,101. This suggests that contact with vegetal cells is necessary for mesoderm formation, and indeed when animal and vegetal explants are combined, mesoderm formation is induced in the animal portion [11-13]. It is known that vegetal cells from the prospective dorsal quadrant (the DV centre) will induce the formation of axial mesoderm (notochord and segmented muscle), whereas the rest of the vegetal hemisphere induces mesoderm of a ventrolateral character (mesothelium, mesenchyme, blood cells) [13-15]. This led to the proposal that there are two mesoderm inducing signals: a general signal (yellow arrows in Fig. 1) operating all around the circumference and inducing ventral-type mesoderm, and a dorsovegetal signal, (red arrow in Fig. 1) restricted to the dorsal quadrant, inducing axial mesoderm [16]. The region that will form the axial mesoderm is the future dorsal blastopore lip, otherwise known as Spemann's organizer (the orange region O in Fig. 1). The general signal was presumed to operate around the whole
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circumference because it was known that embryos lacking an axis could be produced by UV irradiation of the fertilized egg, which inhibits cortical rotation and leads to the formation of a radially symmetrical embryo containing the normal amount of mesoderm, but all of it ventral in character [171. There is now good evidence that the dorsovegetal signal depends on the presence of a cytoplasmic determinant in the dorsovegetal quadrant of the egg. This is proposed to be localized to the vegetal hemisphere in the oocyte, perhaps as mRNA, and to be activated on the dorsal side only, as a result of cortical rotation, by translation or post-translational modification. It is known that UV irradiation of oocytes (as opposed to eggs) prevents subsequent axis formation in a way that is independent of the cortical rotation, as it cannot be rescued by an imposed rotation [18]. This effect could be due to destruction of the proposed localized maternal mRNA, and in fact two vegetally localized mRNAs with biological activity (vg-1 and wnt-11) have now been described (see below). There are also several studies showing that the dorsovegetal region of the early cleavage-stage embryo contains cytoplasm that can cause axis formation when it is injected into a UVO embryo or into the ventral side of a normal embryo [19,20]. A coordinated group of changes occur in the cells of the late blastula, involving breakdown of cleavage synchrony, onset of zygotic gene expression, an increase in cellular adhesiveness, and the commencement of an active expansion of the animal hemisphere. These changes are collectively called the 'mid-blastula' transition [21,22] (although they really occur in the late blastula), and a number of genes have recently been identified that are first expressed at this stage. They fall into three groups, the first being those that are expressed in a ring around the embryo, closely corresponding to the position of the mesoderm as defined earlier by specification mapping: brachyury [231 and snail [24]. The second group are expressed only in the organizer region: goosecoid [25], forkhead [261, lim [271, noggin [281, and, after a phase of universal expression, xnot [29]. Finally, there are those expressed only in the ventral-type territory, which at this stage is about three quarters of the embryo: wnt-8 [30,31] and xpo [32]. Doubtless there are many more markers to be discovered, but it is significant that, so far, the expression patterns correspond exactly to the earlier prediction from biological experiments that there are only two sorts of mesoderm: organizer-type and ventral-type. The diversification of the ventral mesoderm into several sub-domains that form different tissue types was ascribed to a third inductive process, dorsalization. The reason for supposing that this occurred in normal development was similar to the argument for mesoderm induction. Focusing attention on muscle formation, the fate map shows that about 60 % of muscle normally comes from the ventral half of the embryo [8]. But the specification map shows that explants from the ventral
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Current Biology 1994, Vol 4 No 2 half, and even intact ventral half-embryos, form little or no muscle [33]. Clearly, contact with the dorsal half is necessary for muscle formation in the ventral half. This was confirmed when it was shown that formation of both muscle and kidney (another mesodermal derivative) greatly increased in ventral mesoderm that was cultured in combination with dorsal (organizer) mesoderm 133,341. Dorsalization (indicated by the purple arrow in Fig. 1) occurs during gastrulation, and it is now known to be the process underlying the 'assimilative induction' of host mesoderm into the new axis in the original organizer graft of Spemann and Mangold [351. As the formation and patterning of the mesoderm appears to involve three more-or-less distinct inductive processes, our understanding of mesoderm formation as shown in diagrams such as Figure 1 is often referred to as the 'three signal model' (the three signals are shown by the coloured arrows in Fig. 1). Also occuring during gastrulation is neural induction, the most famous feature of the original organizer graft. For many decades, the textbook account of this has been that the neural plate becomes induced from ectoderm as it is underlain by the mesoderm. This undoubtedly happens, and the existence of an 'appositional' signal has been shown by several workers using molecular markers (for example, 1361). But recent work has shown that an important additional component of the process is a 'planar' signal emitted from the organizer, which travels in the plane of the outer cell layers and affects the animal-hemisphere ectoderm 1371. This signal alone is capable not only of inducing neural tissue but also of specifying anteroposterior pattern in the neural plate 138]. The appositional signal from the underlying archenteron roof (which develops from the organizer region) to the overlying ectoderm is now thought to be somewhat weaker, although it does synergize with the
planar signal and together they lead to the formation of a complete set of pattern elements [39]. In normal development, the region of the animal hemisphere that contributes to the organizer, and the region that forms the neural plate, are both in the dorsal midline and both are sensitized by the original cortical rotation to respond to their respective inducing stimuli [6]. But in abnormal situations produced by experimental grafting, it is possible to induce either an organizer or a neural plate to form from unsensitized ectoderm taken from elsewhere in the animal hemisphere or from UVO embryos (for example, see [15,311). This shows that it is ultimately the signals that control when and where structures will form, and that competence only affects the threshold at which a response can occur. In fact, we now know that receptors for some of the candidate factors are present all over the animal hemisphere (see below). To understand regional specification in the Xenopus embryo, it is thus of primary importance to understand the nature, distribution and mode of action of the inducing factors themselves. In essence, as shown in Figure 1, there are four signals defined by the biological experiments: three to induce and pattern the mesoderm (coloured arrows), and one to induce the neural plate (open arrow). This might suggest that we should need at least four substances to do the job - but there need not be a one-toone correspondence between signals and substances, and the final molecular answer is bound to be much more complicated than this simple model might suggest.
Identification of inducing factors There is general agreement in the field that the definitive proof that an inducing factor is responsible for a given process requires three types of evidence: the factor must be biologically active in an appropriate test
Inducing factors in Xenopus development
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system - the activity criterion; it must be expressed in the right place - the expression criterion; and specific inhibition of its action must prevent the process from occurring in vivo - the inhibition criterion. Biological activity has been assessed by various types of assay; a current list of factors with activity in one or more assays is provided in Table 1. The 'animal cap serial dilution assay' is a test for mesoderm induction and involves exposing animal pole explants from blastulae to serial dilutions of a test protein (Fig. 2) [40,41]. Mesoderm induction can be assessed visually, as the explants elongate and form vesicles. A more accurate indication of the character of the induction can be obtained by histology or by analysis of molecular markers. Factors active by this criterion are the activins and the fibroblast growth factors (FGFs) [42-451. The 'autoinduction assay' involves injection of the synthetic mRNA encoding the factor into the zygote, followed by extirpation of the animal cap at the blastula stage (Fig. 3d) [46]. If the RNA encodes a mesoderminducing factor, then the caps will induce themselves. This is often easier to do than an animal cap assay, as various factors are available as cDNA but not as purified protein. But the two assays do not always give .the same results: in autoinduction assays, the activins and Vg-1 are active [47-49], as are forms of FGF containing a secretory signal peptide - but forms of FGF without a signal peptide are much less active [50,511. The bone morphogenetic proteins (BMP2 and BMP4) also show good autoinduction activity, but they are barely active as proteins in the serial dilution assay [52,53]. Activins, Vg-1 and BMPs are all members of the transforming growth factor (TGFO) superfamily of factors. They share a common requirement for dimerization and proteolytic cleavage in order for an active molecule to be formed. As the BMPs seem much more active when expressed from RNA than they are when supplied to the cell surface as protein, it seems likely that their activity requires heterodimerization with other TGF[-like molecules, or some other intermolecular interactions that we currently do not understand. The 'axial rescue assay' involves injecting a synthetic mRNA into a UV-ventralized egg. If the encoded protein has either DV-centre or organizer activity, an axis will be formed. Active mRNAs will usually also produce a secondary axis if injected into the ventral side of a normal egg. This assay gives positive results for RNAs encoding activin, activin receptor, Vg-1, nodal, several of the Wnt genes, noggin, and the organizer-specific transcription factor goosecoid [25, 28,47-49,54,551. Excellent axial rescue is also produced by injection with lithium (Li+ ) ions [56]. This correlates with a dorsalizing effect of Li+ when administered to whole embryos at an early blastula stage [571, and is attributed to an inhibition of the inositol phosphate cycle, as embryos can be rescued from the effects of Li+ by co-injection of inositol [56]. Study of inositol phosphate metabolism has shown that the cycle is activated in whole embryos at the 32-64-cell stage, which is the
Fig. 2. The serial dilution assay using animal caps, where MIF is any mesoderm inducing factor. The expanded vesicles represent induced explants, and in this example the MIF has an activity of 8 units ml-'. With an inhibitor, which is present at the same concentration in all the wells, the titre is reduced to 1 unit ml- ', so the concentration of inhibitor present must be able to neutralize 7 units ml-1 of MIF. A similar assay can be used for neural induction using gastrula caps, or for dorsalization using gastrula mesoderm explants.
stage of maximum sensitivity to Li+, and that Li' does indeed reduce the formation of the intracellular second messenger inositol trisphosphate (IP3) [58]. The 'dorsalization assay', focusing on the dorsalizing signal operative at the gastrula stage, is similar in principle to the animal cap assay, but instead of a blastula stage cap the test tissue is a ventral marginal zone explant from a gastrula. As such explants always contain some uninduced ectoderm, it is important to take them from a sufficiently late stage that competence for mesoderm induction has been lost but competence for dorsalization remains. Activity in this assay is not shown by activins or FGFs or Li+ [34], but is shown by noggin [591]. The 'neuralization assay' is similar again, but here the test tissue is ectoderm from a gastrula. Again, there is a potential complication because axial mesoderm can itself induce neural tissue to form from competent ectoderm, so neural induction is often seen as a secondary consequence of axial mesoderm induction. In order to avoid this, the ectoderm should be taken after the stage at which mesodermal competence is lost. Although the 'folk lore' has it that half the substances on the laboratory bench are active in this test, in fact, using Xenopus ectoderm, only noggin is active [601. If the cells are first disaggregated, then FGF is also active [61], and if we count autoneuralization of ectoderm from RNA-injected eggs, then also active are two antagonists of activin a 'dominant-negative' mutated version of the activin receptor that prevents the action of the wild-type receptor and the activin-binding protein, follistatin ([621 and A. Hemmati-Brivanlou, personal communication). The final assay is the ventralization of the axis ('axial suppression') in normal embryos, provoked by injection
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Fig. 3. Assays based on overexpression of synthetic mRNA. of synthetic mRNA on the dorsal side of the egg. This occurs with BMP2 and BMP4 [48,49], and in a model system consisting of overexpression of the serotonin receptor followed by treatment with serotonin (A. Galione, personal communication). The serotonin receptor is known to activate the IP3 cycle, so this treatment represents the opposite of Li+ treatment, and further strengthens the case that axial development is normally promoted by repression of the IP3 cycle. With this assay, very surprising finding has been made: ventralization is also provoked at a later developmental stage, that is, after the mid-blastula transition, by the same procedures that have dorsalizing activity before the mid-blastula transition, namely treatment of late blastulae or early gastrulae with Li+, or overexpression of wnt-8 from an injected plasmid using a promoter activated at the mid-blastula transition [63,64]. The fact that there is such a close correlation between the effects of Li' and Wnt-8, both of which cause dorsalization before the mid-blastula transition and ventralization after it, enables us to predict that an important biochemical function of the Wnt proteins is to repress the IP 3 cycle. This is a significant prediction, as there are currently no ligands known in any system that have this activity. With such a number of active factors, there is obviously plenty of opportunity for interactions between them and the two interactions that are currently well documented both involve the FGFs. Each FGF alone behaves rather like the putative 'general mesoderminducing signal', causing induction of ventral-type mesoderm. Muscle is also formed, in an amount that increases with dose, but it is extremely rare to see notochord [65]. However, when FGF treatment is combined with Li+ or with wnt RNA injection, then axial induction is readily obtained [66,671; it is also obtained when basic FGF is overexpressed in the dorsal sector of the animal hemisphere, which has enhanced competence
as a result of the cortical rotation [50]. This type of result has led one group of workers to suggest that the DV signal consists of FGF plus Wnt, rather than of a single substance [21, although this now seems unlikely in view of the effects of dominant-negative FGF receptors (see below). The second known interaction is between FGF and activin. Activin alone produces remarkably sharp thresholds of response if animal cap cells are treated in a disaggregated state and then reaggregated [681, but treatment with activin plus FGF broadens these responses [60]. This group of workers therefore suggested that mesoderm induction could be achieved by orthogonal gradients of activin and FGF [691, but, as we shall see, this suggestion is not entirely compatible with the expression data. Expression studies Most of the factors mentioned above have been the subject of recent expression studies of their expression patterns. In some ways, this can be the most difficult type of evidence to assess. We do not necessarily know that RNA visualized by in situ hybridization is translated into protein in vivo. Nor do we know whether the protein is translated into a biologically available form. We do not usually even know what level of expression seen in a stained specimen corresponds to biological effectiveness. With these caveats we shall consider the evidence, which is summarized briefly in Table 2. The activins have provoked enormous interest because of their high biological potency, and intense efforts have been made to study their expression in the embryo. The results are now fairly clear cut: there is no maternal activin mRNA, but there is a small amount of activin protein in the early embryo [701, presumably
Inducing factors in Xenopus development
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having been taken up from the ovarian follicle cells, which are active sites of synthesis [71]. It is uncertain whether this embryonic activin could be resecreted from the blastomeres, and there is no evidence for it being vegetally localized. Zygotic expression of activins A and B commences at the mid-blastula transition and later becomes prominent in the head and anterior axis [71]. BMP2 and BMP4 are both expressed maternally without vegetal localization [72,73]. These expression studies do not provide any particular support for the suggestion that activins or BMPs act as primary mesoderm-inducing factors, but neither do they entirely rule out the possibility. Vg-1 is the product of a maternal mRNA that was identified by its restriction to the vegetal cortical region. On oocyte maturation, vg-1 RNA is released from the cortex and occupies a thicker region of cytoplasm, but still confined to the vegetal hemisphere [74]. This behaviour is also characteristic of other vegetally localized mRNAs that do not encode inducing factors, such as xcat-2 [75]. The change in location would be consistent with the possibility that vg-1 or another maternal mRNA is the target for the effect of UV in oocytes. As mentioned above, the effect of lW on oocytes cannot be rescued by an enforced cortical rotation, whereas the UV effect on eggs can be [18,5]. This could be explained by the fact that the RNA in oocytes is superficial, and therefore vulnerable to incident UV, but in the egg it is deeper and so less vulnerable. For many years, Vg-1 was thought to be biologically inactive, but it is now known that this is due to very inefficient processing and secretion. As mentioned above, TGFO-type factors need to dimerize and be proteolytically cleaved before they can become active. When a chimeric molecule is constructed from a BMP pro-region and a Vg-1 mature region, mature Vg-1 is produced and is active in the autoinduction and axial rescue assays [48,49]. Although this is a somewhat artificial situation, it does suggest that post-translational regulation of processing might be important in vivo. It is possible that processing commences all over the vegetal hemisphere after fertilization, in which case Vg-1 would be a good candidate for the general mesoderm-inducing signal. Alternatively, if we postulate some mechanism for processing that is dependent on the cortical rotation and occurs only in the dorsovegetal segment, then Vg-1 could be a candidate for the dorsovegetal signal. Three types of fibroblast growth factor have been found to be expressed in the early embryo: basic (b) FGF and embryonic (e) FGF are both expressed maternally but without vegetal localization [76,77] - zygotic expression of bFGF does not commence until the neurula stage [76]; eFGF and Int-2 (FGF-3) are both expressed from the mid-blastula transition onwards, in the mesoderm around the blastopore [77,78]. As with the activins, these data do not support the suggestion that FGFs function as primary mesoderm-inducing factors, but neither do they entirely rule it out.
Not all of the Wnt family of proteins have axial rescue activity; those that are known so far to do so are Wnt-1, Wnt-3A, Wnt-8, and, to a lesser extent, Wnt-11 [79]. Wnt-1 and Wnt-3A are not expressed in Xenopus until the neurula stage, when they appear in the anterior central nervous system [80]. Wnt-8 is first expressed at the mid-blastula transition in the ventrolateral part of the mesoderm [30,31], and, as mentioned above, ectopic expression in the prospective axis after the midblastula transition causes ventralization, the opposite of the early effect [64]. So Wnt-1, Wnt-3A and Wnt-8 are effectively ruled out as candidates for the dorsovegetal signal. Wnt-11 mRNA, on the other hand, has a similar localization to vg-1: it is found in the vegetal cortex of the oocyte and released into the deeper cytoplasm on maturation [79]. After the mid-blastula transition, it begins to be expressed in the mesoderm with a dorsal-ventral gradient of expression, but the maternal mRNA shows no localization to the dorsal side, so if this is to be a good candidate for the dorsovegetal signal, we would need to postulate a mechanism for its dorsal-specific translation, activation or secretion. Noggin mRNA is present maternally, although, like FGF and BMP mRNA, it is not vegetally localized, and so has no special claim to be the dorsovegetal factor. The zygotic expression pattern of noggin is more significant, as it begins at the mid-blastula transition and is then present in the organizer region and, later, the dorsal midline of the archenteron roof [28]. It is in exactly the temporo-spatial pattern predicted for the dorsalizing factor, and is also expressed appropriately for it to be responsible for at least some component of neural induction. As it has both of the corresponding biological activities, we must take very seriously the claim of noggin to encompass two essential properties of Spemann's organizer. In other words, noggin
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Current Biology 1994, Vol 4 No 2 may be both the dorsalizing and the neural induction factor. Space precludes any detailed consideration of expression patterns of the receptors for all these factors, but several have been cloned and their expression patterns studied. The FGF receptor type 1 [81-831, and various activin receptors [84-86], are present maternally and their RNAs are distributed uniformly (when compared to total RNA). As is the case for the inducing factors that are not vegetally localized, this means, in fact, that they are more abundant in the animal than in the vegetal hemisphere, because there is more total RNA in the animal hemisphere. Before the midgastrula transition, there is no regional restriction of expression. Functional inhibition of putative inducing factors Alas, genetic analysis of Xenopus is not possible, so we are not likely to be able to describe the phenotypes of mutations in any of the interesting genes, whether targeted or otherwise. This means that inhibition experiments have to be biochemically based, depending on inhibitors, neutralizing antibodies, antisense oligonucleotides or dominant-negative constructs. It is known that mesoderm induction, neural induction, dorsalization and maintenance of the organizer state are all inhibited by the drug suramin [87,88], which antagonizes the binding of several growth factors to their receptors. It has also been shown using a transfilter apparatus, in which animal and vegetal tissues are separated by a small liquid gap, that suramin or heparin can inhibit the mesoderm-inducing signal, whereas follistatin, a very avid inhibitor of activin, or a neutralizing antibody to bFGF, did not inhibit the signal 89]. Suramin and heparin are somewhat unspecific in their actions, but their effects do suggest that some sort of growth factor is responsible. On the other hand, the negative results with the more specific inhibitors suggest that neither activin nor bFGF is being secreted from the vegetal cells in this assay. More positive results have been obtained using dominant-negative receptors overexpressed from synthetic mRNAs. Studies have been published with a dominant negative FGF receptor [90,91] and a dominant-negative activin receptor [621. In each case, the construct encodes a receptor lacking most of the cytoplasmic domain and the dominant-negative action is ascribed to the formation of inactive dimers with the endogenous receptor. It is now known that the dominant-negative activin receptor inhibits the action of all of the TGFP superfamily members that are capable of inducing mesoderm, including activin itself and Vg-1 (D. Melton, personal communication). When overexpressed in whole embryos, it causes them to become 'empty sacs' containing little or no mesoderm [621. At the same time, there is an overproduction of neural tissue from the ectoderm. These results may be comparable to the effects of overexpressing the activin-binding protein
follistatin, which does not reduce mesoderm formation significantly although there are some pattern disturbances, perhaps reflecting the later, zygotic functions of activin. Once again there is a strong neuralizing effect on the ectoderm (A. Hemmati-Brivanlou, personal communication). These results seem compatible with those of the transfilter experiment, as they suggest that a TGF3-like molecule other than activin is necessary for mesoderm formation, whereas activin itself might function as a repressor of neural induction. The dominant-negative FGF receptor antagonizes the FGF receptor types that are found in the early embryo. The phenotype of overexpression is a reduction of mesoderm formation with associated defects in the posterior of the embryo [90,911. As there is a reduction in mesoderm formation, it seems hard to escape the conclusion that mesoderm induction requires FGF at some stage, even if FGF is not the factor produced by the vegetal cells to signal to the animal cells. The blockade of mesoderm formation is not total - in fact the head region is fairly normal - and to this extent the phenotype is less severe than that of the dominantnegative activin receptor. The normal head must count as evidence against the idea that the dorsovegetal signal consists of FGF plus Wnt, as a reduction of dorsovegetal signal should certainly affect the head: by comparison, the head is the first part of the body to be affected in the dose-response series of UV radiation to the egg [4].
Putting it all together It is always possible that novel molecules will be found that will render this analysis out of date. However, we do now know of enough components that approach satisfaction of the three criteria - activity, expression and inhibition - to provide a plausible model of the molecular basis for the sequence of inductions shown in Figure 1. So, Figure 4 shows the same diagram with the molecules named: the hypothetical cell states of Figure 1 (mesoderm, organizer, and so on) are presumed to be defined by combinations of transcription factors, whereas the inductive signals are tentatively identified as known secreted factors. The general mesoderm-inducing signal seems quite likely to be Vg-1: it has the appropriate activity, its mRNA is localized in the right place, and its activity is inhibited by the dominant-negative activin receptor, which is known to suppress mesoderm formation. It would, of course, be nice to have a more specific inhibition experiment, and we do still need some evidence that Vg-1 is actually processed in vivo and is secreted from vegetal cells. The organizer-inducing dorsovegetal signal seems most likely to be Wnt-11, or Wnt-11l plus Vg-l. The biological activity of Wnt-11 is appropriate although weak, and its localization is satisfactory, although we still need evidence of differential translation as a consequence of the cortical rotation. We also need an inhibition experiment to prove this proposed role of a Wnt protein. We do know that heparin
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antagonizes anterior-posterior axis formation [921, and that it also inhibits Wnt effects in tissue culture [931, but heparin is not a reagent of sufficiently high specificity for this to constitute proof. If we choose Vg-1 and Wnt-11 as front runners for the mesoderm-inducing signals, this might seem to leave us with no role for FGF or activin, which were presumed until recently to be responsible for the general and the dorsovegetal signals, respectively. If we accept that the dominant-negative FGF receptor really is specific for the FGF family, then it seems an inescapable conclusion that FGF is required for mesoderm formation
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in vivo. However, it need not act as the primary mesoderm-inducing signal, and in fact the evidence from expression data is against this suggestion. It may be that the primary signal causes release of FGF from cells, or exposure of the FGF receptor to a pre-existing extracellular ligand in the animal hemisphere. Perhaps FGF signalling is needed to amplify the primary signals or to relay them over several cell diameters. Of the FGFs, bFGF and eFGF are present maternally, and of these eFGF is the more attractive candidate for having such a role, in that it possesses a signal sequence that would cause it to be secreted. A similar argument for a relay role could be made for activin, although the evidence
Fig. 4. The sequence of inductive interactions in the Xenopus embryo. In this diagram, molecular markers of cell state are shown on the left and the inducing factors that bring about transitions between cell states are shown on the right. Justification for placement of the various factors is given in the text. Gene name abbreviations: bra, brachyury; Epi- 1, an epidermal marker; fkh, forkhead; gcd, goosecoid; lirm, Xlim 1; N-CAM, neural cell adhesion molecule; not, Xnot; twi, twist; sna, snail.
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Current Biology 1994, Vol 4 No 2 for its involvement in mesoderm induction is a little weaker, because the maternal levels seem so low and the dominant-negative effects may be attributable to Vg-1. The dorsalizing signal seems quite likely to be noggin, judging by activity and expression criteria. There is, as yet, no inhibition experiment to demonstrate the function of noggin. The organizer region is also responsible for neural induction, initially in the form of the planar signal and later, when it has become the archenteron roof, by the appositional route. Although dorsalization and neural induction have been considered to be separate processes [94], there is no particular reason to suppose that the same substances cannot be involved in both. Noggin does have neuralizing activity, and is expressed in the right place to be a neural inducer. There is however also considerable evidence that neural induction is provoked by removal of something, for example following cell disaggregation of blastula cells [95,961. Removal of activin seems to cause neuralization 162], and at the gastrula stage, if cells are already disaggregated, so does addition of FGF [61]. It is perfectly possible that each biologically defined 'signal' will correspond to a complex mixture of substances. But it is now possible to draw a simple diagram in which Vg-1 and Wnt-11 are the primary mesoderm-inducing signals, FGF and activin are secondary mesoderm-inducing signals, and noggin takes care of dorsalization and neural induction (Fig.4). This is where the game stands at present.
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Inducing factors in Xenopus early embryos J.M.W. Slack ICRF Developmental Biology Unit, Department of Zoology, Oxford University, South Parks Road, Oxford OX1 3PS, UK.
Recent results make it possible to postulate credible candidates for each of the known inducing signals that act to determine cell fate during Xenopus early development. Experiments on biological activity, expression patterns and inhibition of function suggest that Vg-1 and Wnt-11 may act as the primary mesoderm-inducing signals, FGF and activin may serve to relay their effects, and noggin may be a major component of the dorsalizing and neural-inducing signals from the organizer. The last few years have seen unparalleled progress in the unravelling of the early inductive interactions that specify the vertebrate body plan. Although this progress has regularly been reviewed [1,2], several very recent advances have filled in some more blank spaces in the picture. In this article, I shall try to set these new results in context and ask to what extent we now really understand what is going on.
The biology of inducing factors The following account very briefly reviews the biological basis of our present understanding of the early inductive interactions that take place following fertilization of the Xenopus egg. 'Induction' here describes the process by which one group of cells signals to a second group in such a way as to determine the developmental fate of the second; induction requires both a signalling capacity in the first set of cells and a competence to respond in the second. The inductive interactions during Xenopus early development are shown diagrammatically in Figure 1, which culminates in the situation at the end of gastrulation. By this time, two major embryonic tissue types have been formed and each
subdivided into several territories: the mesoderm, which gives rise to muscle, blood and mesenchyme, and the neural plate, which gives rise to the nervous system. For simplicity, we shall here exclude from consideration the anteroposterior patterning of the mesoderm and neural plate, which has been discussed in more detail elsewhere 13], and consider only induction of the mesoderm and its dorsoventral patterning. During its development in the mother, the Xenopus oocyte becomes polarized along its animal-vegetal dimension but remains radially symmetrical around this axis. The animal-vegetal polarization is clearly visible by a difference of pigmentation: the animal hemisphere is dark and the vegetal hemisphere light-coloured. The radial symmetry of the oocyte is broken at fertilization by the point of sperm entry. About half-way through the first cell cycle a cytoplasmic shift occurs, called the cortical rotation, which involves the movement of the plasma membrane plus a thin shell of underlying cytoplasm - the 'cortex' - relative to the internal cytoplasm [4]. The rotation occurs about the future left-right axis of the embryo, and is such that the dorsal side forms where vegetal cortex meets animal cytoplasm
Fig. 1. The sequence of inductive interactions in Xenopus embryos, deduced from 'biological' experiments. Yellow arrows show the general mesoderm-inducing signal ('make mesoderm'); the red arrow indicates the dorsovegetal organizer-inducing signal ('make organizer'); the purple arrow shows the third, dorsalization-inducing signal ('make dorsal mesoderm'); the white arrow represents neural induction. By the middle gastrula stage the neural plate and organizer are each subdivided into three regions (N1-N3 and 01-03, respectively).
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Inducing factors in Xenopus development and the ventral side forms on the side where animal cortex meets vegetal cytoplasm (Fig. lb). The direction of the rotation is determined by the point of sperm entry, which becomes the future ventral side. Cortical rotation is known to be essential for axis formation in the intact embryo, although not for the formation of ventral mesoderm - 'axis' here (and below) refers to the notochord, somites and neural plate, with their characteristic anterior-posterior pattern. If rotation is prevented by ultraviolet (UV) irradiation of the egg, the axis does not form, and the embryo is referred to as 'UVO' [4]. If UV irradiation is followed by an imposed rotation of the egg, the axis can be restored, with the new dorsoventral polarity corresponding to the imposed rotation [5]. Cortical rotation is thought to affect both the vegetal hemisphere, where it leads to the formation of a dorsovegetal signalling centre (DV centre - also known as the Nieuwkoop centre or organizer-inducing centre), and the animal hemisphere, where it leads to an enhanced competence to respond to signals from the DV centre on the dorsal side [6]. The fertilized egg cleaves to form a hollow ball of cells, the blastula, and during an extended period between about the 32-cell stage and the beginning of gastrulation, mesoderm is induced to form in the equatorial region of the embryo (Fig. c), as a result of signalling from the vegetal to the animal cells [7]. We know that at least some, and perhaps all, of the mesoderm is formed by induction, because of the comparison between the fate map and the specification map of the cleavage stages. In the fate map, which shows what happens in normal development, about half of the mesoderm arises from cells of the animal hemisphere, above the equatorial pigment boundary [8]. By contrast, in a specification map, which shows what tissue explants can do when cultured in isolation, no muscle or notochord is formed from explants taken from above the pigment boundary [9,101. This suggests that contact with vegetal cells is necessary for mesoderm formation, and indeed when animal and vegetal explants are combined, mesoderm formation is induced in the animal portion [11-13]. It is known that vegetal cells from the prospective dorsal quadrant (the DV centre) will induce the formation of axial mesoderm (notochord and segmented muscle), whereas the rest of the vegetal hemisphere induces mesoderm of a ventrolateral character (mesothelium, mesenchyme, blood cells) [13-15]. This led to the proposal that there are two mesoderm inducing signals: a general signal (yellow arrows in Fig. 1) operating all around the circumference and inducing ventral-type mesoderm, and a dorsovegetal signal, (red arrow in Fig. 1) restricted to the dorsal quadrant, inducing axial mesoderm [16]. The region that will form the axial mesoderm is the future dorsal blastopore lip, otherwise known as Spemann's organizer (the orange region O in Fig. 1). The general signal was presumed to operate around the whole
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circumference because it was known that embryos lacking an axis could be produced by UV irradiation of the fertilized egg, which inhibits cortical rotation and leads to the formation of a radially symmetrical embryo containing the normal amount of mesoderm, but all of it ventral in character [171. There is now good evidence that the dorsovegetal signal depends on the presence of a cytoplasmic determinant in the dorsovegetal quadrant of the egg. This is proposed to be localized to the vegetal hemisphere in the oocyte, perhaps as mRNA, and to be activated on the dorsal side only, as a result of cortical rotation, by translation or post-translational modification. It is known that UV irradiation of oocytes (as opposed to eggs) prevents subsequent axis formation in a way that is independent of the cortical rotation, as it cannot be rescued by an imposed rotation [18]. This effect could be due to destruction of the proposed localized maternal mRNA, and in fact two vegetally localized mRNAs with biological activity (vg-1 and wnt-11) have now been described (see below). There are also several studies showing that the dorsovegetal region of the early cleavage-stage embryo contains cytoplasm that can cause axis formation when it is injected into a UVO embryo or into the ventral side of a normal embryo [19,20]. A coordinated group of changes occur in the cells of the late blastula, involving breakdown of cleavage synchrony, onset of zygotic gene expression, an increase in cellular adhesiveness, and the commencement of an active expansion of the animal hemisphere. These changes are collectively called the 'mid-blastula' transition [21,22] (although they really occur in the late blastula), and a number of genes have recently been identified that are first expressed at this stage. They fall into three groups, the first being those that are expressed in a ring around the embryo, closely corresponding to the position of the mesoderm as defined earlier by specification mapping: brachyury [231 and snail [24]. The second group are expressed only in the organizer region: goosecoid [25], forkhead [261, lim [271, noggin [281, and, after a phase of universal expression, xnot [29]. Finally, there are those expressed only in the ventral-type territory, which at this stage is about three quarters of the embryo: wnt-8 [30,31] and xpo [32]. Doubtless there are many more markers to be discovered, but it is significant that, so far, the expression patterns correspond exactly to the earlier prediction from biological experiments that there are only two sorts of mesoderm: organizer-type and ventral-type. The diversification of the ventral mesoderm into several sub-domains that form different tissue types was ascribed to a third inductive process, dorsalization. The reason for supposing that this occurred in normal development was similar to the argument for mesoderm induction. Focusing attention on muscle formation, the fate map shows that about 60 % of muscle normally comes from the ventral half of the embryo [8]. But the specification map shows that explants from the ventral
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Current Biology 1994, Vol 4 No 2 half, and even intact ventral half-embryos, form little or no muscle [33]. Clearly, contact with the dorsal half is necessary for muscle formation in the ventral half. This was confirmed when it was shown that formation of both muscle and kidney (another mesodermal derivative) greatly increased in ventral mesoderm that was cultured in combination with dorsal (organizer) mesoderm 133,341. Dorsalization (indicated by the purple arrow in Fig. 1) occurs during gastrulation, and it is now known to be the process underlying the 'assimilative induction' of host mesoderm into the new axis in the original organizer graft of Spemann and Mangold [351. As the formation and patterning of the mesoderm appears to involve three more-or-less distinct inductive processes, our understanding of mesoderm formation as shown in diagrams such as Figure 1 is often referred to as the 'three signal model' (the three signals are shown by the coloured arrows in Fig. 1). Also occuring during gastrulation is neural induction, the most famous feature of the original organizer graft. For many decades, the textbook account of this has been that the neural plate becomes induced from ectoderm as it is underlain by the mesoderm. This undoubtedly happens, and the existence of an 'appositional' signal has been shown by several workers using molecular markers (for example, 1361). But recent work has shown that an important additional component of the process is a 'planar' signal emitted from the organizer, which travels in the plane of the outer cell layers and affects the animal-hemisphere ectoderm 1371. This signal alone is capable not only of inducing neural tissue but also of specifying anteroposterior pattern in the neural plate 138]. The appositional signal from the underlying archenteron roof (which develops from the organizer region) to the overlying ectoderm is now thought to be somewhat weaker, although it does synergize with the
planar signal and together they lead to the formation of a complete set of pattern elements [39]. In normal development, the region of the animal hemisphere that contributes to the organizer, and the region that forms the neural plate, are both in the dorsal midline and both are sensitized by the original cortical rotation to respond to their respective inducing stimuli [6]. But in abnormal situations produced by experimental grafting, it is possible to induce either an organizer or a neural plate to form from unsensitized ectoderm taken from elsewhere in the animal hemisphere or from UVO embryos (for example, see [15,311). This shows that it is ultimately the signals that control when and where structures will form, and that competence only affects the threshold at which a response can occur. In fact, we now know that receptors for some of the candidate factors are present all over the animal hemisphere (see below). To understand regional specification in the Xenopus embryo, it is thus of primary importance to understand the nature, distribution and mode of action of the inducing factors themselves. In essence, as shown in Figure 1, there are four signals defined by the biological experiments: three to induce and pattern the mesoderm (coloured arrows), and one to induce the neural plate (open arrow). This might suggest that we should need at least four substances to do the job - but there need not be a one-toone correspondence between signals and substances, and the final molecular answer is bound to be much more complicated than this simple model might suggest.
Identification of inducing factors There is general agreement in the field that the definitive proof that an inducing factor is responsible for a given process requires three types of evidence: the factor must be biologically active in an appropriate test
Inducing factors in Xenopus development
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system - the activity criterion; it must be expressed in the right place - the expression criterion; and specific inhibition of its action must prevent the process from occurring in vivo - the inhibition criterion. Biological activity has been assessed by various types of assay; a current list of factors with activity in one or more assays is provided in Table 1. The 'animal cap serial dilution assay' is a test for mesoderm induction and involves exposing animal pole explants from blastulae to serial dilutions of a test protein (Fig. 2) [40,41]. Mesoderm induction can be assessed visually, as the explants elongate and form vesicles. A more accurate indication of the character of the induction can be obtained by histology or by analysis of molecular markers. Factors active by this criterion are the activins and the fibroblast growth factors (FGFs) [42-451. The 'autoinduction assay' involves injection of the synthetic mRNA encoding the factor into the zygote, followed by extirpation of the animal cap at the blastula stage (Fig. 3d) [46]. If the RNA encodes a mesoderminducing factor, then the caps will induce themselves. This is often easier to do than an animal cap assay, as various factors are available as cDNA but not as purified protein. But the two assays do not always give .the same results: in autoinduction assays, the activins and Vg-1 are active [47-49], as are forms of FGF containing a secretory signal peptide - but forms of FGF without a signal peptide are much less active [50,511. The bone morphogenetic proteins (BMP2 and BMP4) also show good autoinduction activity, but they are barely active as proteins in the serial dilution assay [52,53]. Activins, Vg-1 and BMPs are all members of the transforming growth factor (TGFO) superfamily of factors. They share a common requirement for dimerization and proteolytic cleavage in order for an active molecule to be formed. As the BMPs seem much more active when expressed from RNA than they are when supplied to the cell surface as protein, it seems likely that their activity requires heterodimerization with other TGF[-like molecules, or some other intermolecular interactions that we currently do not understand. The 'axial rescue assay' involves injecting a synthetic mRNA into a UV-ventralized egg. If the encoded protein has either DV-centre or organizer activity, an axis will be formed. Active mRNAs will usually also produce a secondary axis if injected into the ventral side of a normal egg. This assay gives positive results for RNAs encoding activin, activin receptor, Vg-1, nodal, several of the Wnt genes, noggin, and the organizer-specific transcription factor goosecoid [25, 28,47-49,54,551. Excellent axial rescue is also produced by injection with lithium (Li+ ) ions [56]. This correlates with a dorsalizing effect of Li+ when administered to whole embryos at an early blastula stage [571, and is attributed to an inhibition of the inositol phosphate cycle, as embryos can be rescued from the effects of Li+ by co-injection of inositol [56]. Study of inositol phosphate metabolism has shown that the cycle is activated in whole embryos at the 32-64-cell stage, which is the
Fig. 2. The serial dilution assay using animal caps, where MIF is any mesoderm inducing factor. The expanded vesicles represent induced explants, and in this example the MIF has an activity of 8 units ml-'. With an inhibitor, which is present at the same concentration in all the wells, the titre is reduced to 1 unit ml- ', so the concentration of inhibitor present must be able to neutralize 7 units ml-1 of MIF. A similar assay can be used for neural induction using gastrula caps, or for dorsalization using gastrula mesoderm explants.
stage of maximum sensitivity to Li+, and that Li' does indeed reduce the formation of the intracellular second messenger inositol trisphosphate (IP3) [58]. The 'dorsalization assay', focusing on the dorsalizing signal operative at the gastrula stage, is similar in principle to the animal cap assay, but instead of a blastula stage cap the test tissue is a ventral marginal zone explant from a gastrula. As such explants always contain some uninduced ectoderm, it is important to take them from a sufficiently late stage that competence for mesoderm induction has been lost but competence for dorsalization remains. Activity in this assay is not shown by activins or FGFs or Li+ [34], but is shown by noggin [591]. The 'neuralization assay' is similar again, but here the test tissue is ectoderm from a gastrula. Again, there is a potential complication because axial mesoderm can itself induce neural tissue to form from competent ectoderm, so neural induction is often seen as a secondary consequence of axial mesoderm induction. In order to avoid this, the ectoderm should be taken after the stage at which mesodermal competence is lost. Although the 'folk lore' has it that half the substances on the laboratory bench are active in this test, in fact, using Xenopus ectoderm, only noggin is active [601. If the cells are first disaggregated, then FGF is also active [61], and if we count autoneuralization of ectoderm from RNA-injected eggs, then also active are two antagonists of activin a 'dominant-negative' mutated version of the activin receptor that prevents the action of the wild-type receptor and the activin-binding protein, follistatin ([621 and A. Hemmati-Brivanlou, personal communication). The final assay is the ventralization of the axis ('axial suppression') in normal embryos, provoked by injection
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Fig. 3. Assays based on overexpression of synthetic mRNA. of synthetic mRNA on the dorsal side of the egg. This occurs with BMP2 and BMP4 [48,49], and in a model system consisting of overexpression of the serotonin receptor followed by treatment with serotonin (A. Galione, personal communication). The serotonin receptor is known to activate the IP3 cycle, so this treatment represents the opposite of Li+ treatment, and further strengthens the case that axial development is normally promoted by repression of the IP3 cycle. With this assay, very surprising finding has been made: ventralization is also provoked at a later developmental stage, that is, after the mid-blastula transition, by the same procedures that have dorsalizing activity before the mid-blastula transition, namely treatment of late blastulae or early gastrulae with Li+, or overexpression of wnt-8 from an injected plasmid using a promoter activated at the mid-blastula transition [63,64]. The fact that there is such a close correlation between the effects of Li' and Wnt-8, both of which cause dorsalization before the mid-blastula transition and ventralization after it, enables us to predict that an important biochemical function of the Wnt proteins is to repress the IP 3 cycle. This is a significant prediction, as there are currently no ligands known in any system that have this activity. With such a number of active factors, there is obviously plenty of opportunity for interactions between them and the two interactions that are currently well documented both involve the FGFs. Each FGF alone behaves rather like the putative 'general mesoderminducing signal', causing induction of ventral-type mesoderm. Muscle is also formed, in an amount that increases with dose, but it is extremely rare to see notochord [65]. However, when FGF treatment is combined with Li+ or with wnt RNA injection, then axial induction is readily obtained [66,671; it is also obtained when basic FGF is overexpressed in the dorsal sector of the animal hemisphere, which has enhanced competence
as a result of the cortical rotation [50]. This type of result has led one group of workers to suggest that the DV signal consists of FGF plus Wnt, rather than of a single substance [21, although this now seems unlikely in view of the effects of dominant-negative FGF receptors (see below). The second known interaction is between FGF and activin. Activin alone produces remarkably sharp thresholds of response if animal cap cells are treated in a disaggregated state and then reaggregated [681, but treatment with activin plus FGF broadens these responses [60]. This group of workers therefore suggested that mesoderm induction could be achieved by orthogonal gradients of activin and FGF [691, but, as we shall see, this suggestion is not entirely compatible with the expression data. Expression studies Most of the factors mentioned above have been the subject of recent expression studies of their expression patterns. In some ways, this can be the most difficult type of evidence to assess. We do not necessarily know that RNA visualized by in situ hybridization is translated into protein in vivo. Nor do we know whether the protein is translated into a biologically available form. We do not usually even know what level of expression seen in a stained specimen corresponds to biological effectiveness. With these caveats we shall consider the evidence, which is summarized briefly in Table 2. The activins have provoked enormous interest because of their high biological potency, and intense efforts have been made to study their expression in the embryo. The results are now fairly clear cut: there is no maternal activin mRNA, but there is a small amount of activin protein in the early embryo [701, presumably
Inducing factors in Xenopus development
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having been taken up from the ovarian follicle cells, which are active sites of synthesis [71]. It is uncertain whether this embryonic activin could be resecreted from the blastomeres, and there is no evidence for it being vegetally localized. Zygotic expression of activins A and B commences at the mid-blastula transition and later becomes prominent in the head and anterior axis [71]. BMP2 and BMP4 are both expressed maternally without vegetal localization [72,73]. These expression studies do not provide any particular support for the suggestion that activins or BMPs act as primary mesoderm-inducing factors, but neither do they entirely rule out the possibility. Vg-1 is the product of a maternal mRNA that was identified by its restriction to the vegetal cortical region. On oocyte maturation, vg-1 RNA is released from the cortex and occupies a thicker region of cytoplasm, but still confined to the vegetal hemisphere [74]. This behaviour is also characteristic of other vegetally localized mRNAs that do not encode inducing factors, such as xcat-2 [75]. The change in location would be consistent with the possibility that vg-1 or another maternal mRNA is the target for the effect of UV in oocytes. As mentioned above, the effect of lW on oocytes cannot be rescued by an enforced cortical rotation, whereas the UV effect on eggs can be [18,5]. This could be explained by the fact that the RNA in oocytes is superficial, and therefore vulnerable to incident UV, but in the egg it is deeper and so less vulnerable. For many years, Vg-1 was thought to be biologically inactive, but it is now known that this is due to very inefficient processing and secretion. As mentioned above, TGFO-type factors need to dimerize and be proteolytically cleaved before they can become active. When a chimeric molecule is constructed from a BMP pro-region and a Vg-1 mature region, mature Vg-1 is produced and is active in the autoinduction and axial rescue assays [48,49]. Although this is a somewhat artificial situation, it does suggest that post-translational regulation of processing might be important in vivo. It is possible that processing commences all over the vegetal hemisphere after fertilization, in which case Vg-1 would be a good candidate for the general mesoderm-inducing signal. Alternatively, if we postulate some mechanism for processing that is dependent on the cortical rotation and occurs only in the dorsovegetal segment, then Vg-1 could be a candidate for the dorsovegetal signal. Three types of fibroblast growth factor have been found to be expressed in the early embryo: basic (b) FGF and embryonic (e) FGF are both expressed maternally but without vegetal localization [76,77] - zygotic expression of bFGF does not commence until the neurula stage [76]; eFGF and Int-2 (FGF-3) are both expressed from the mid-blastula transition onwards, in the mesoderm around the blastopore [77,78]. As with the activins, these data do not support the suggestion that FGFs function as primary mesoderm-inducing factors, but neither do they entirely rule it out.
Not all of the Wnt family of proteins have axial rescue activity; those that are known so far to do so are Wnt-1, Wnt-3A, Wnt-8, and, to a lesser extent, Wnt-11 [79]. Wnt-1 and Wnt-3A are not expressed in Xenopus until the neurula stage, when they appear in the anterior central nervous system [80]. Wnt-8 is first expressed at the mid-blastula transition in the ventrolateral part of the mesoderm [30,31], and, as mentioned above, ectopic expression in the prospective axis after the midblastula transition causes ventralization, the opposite of the early effect [64]. So Wnt-1, Wnt-3A and Wnt-8 are effectively ruled out as candidates for the dorsovegetal signal. Wnt-11 mRNA, on the other hand, has a similar localization to vg-1: it is found in the vegetal cortex of the oocyte and released into the deeper cytoplasm on maturation [79]. After the mid-blastula transition, it begins to be expressed in the mesoderm with a dorsal-ventral gradient of expression, but the maternal mRNA shows no localization to the dorsal side, so if this is to be a good candidate for the dorsovegetal signal, we would need to postulate a mechanism for its dorsal-specific translation, activation or secretion. Noggin mRNA is present maternally, although, like FGF and BMP mRNA, it is not vegetally localized, and so has no special claim to be the dorsovegetal factor. The zygotic expression pattern of noggin is more significant, as it begins at the mid-blastula transition and is then present in the organizer region and, later, the dorsal midline of the archenteron roof [28]. It is in exactly the temporo-spatial pattern predicted for the dorsalizing factor, and is also expressed appropriately for it to be responsible for at least some component of neural induction. As it has both of the corresponding biological activities, we must take very seriously the claim of noggin to encompass two essential properties of Spemann's organizer. In other words, noggin
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Current Biology 1994, Vol 4 No 2 may be both the dorsalizing and the neural induction factor. Space precludes any detailed consideration of expression patterns of the receptors for all these factors, but several have been cloned and their expression patterns studied. The FGF receptor type 1 [81-831, and various activin receptors [84-86], are present maternally and their RNAs are distributed uniformly (when compared to total RNA). As is the case for the inducing factors that are not vegetally localized, this means, in fact, that they are more abundant in the animal than in the vegetal hemisphere, because there is more total RNA in the animal hemisphere. Before the midgastrula transition, there is no regional restriction of expression. Functional inhibition of putative inducing factors Alas, genetic analysis of Xenopus is not possible, so we are not likely to be able to describe the phenotypes of mutations in any of the interesting genes, whether targeted or otherwise. This means that inhibition experiments have to be biochemically based, depending on inhibitors, neutralizing antibodies, antisense oligonucleotides or dominant-negative constructs. It is known that mesoderm induction, neural induction, dorsalization and maintenance of the organizer state are all inhibited by the drug suramin [87,88], which antagonizes the binding of several growth factors to their receptors. It has also been shown using a transfilter apparatus, in which animal and vegetal tissues are separated by a small liquid gap, that suramin or heparin can inhibit the mesoderm-inducing signal, whereas follistatin, a very avid inhibitor of activin, or a neutralizing antibody to bFGF, did not inhibit the signal 89]. Suramin and heparin are somewhat unspecific in their actions, but their effects do suggest that some sort of growth factor is responsible. On the other hand, the negative results with the more specific inhibitors suggest that neither activin nor bFGF is being secreted from the vegetal cells in this assay. More positive results have been obtained using dominant-negative receptors overexpressed from synthetic mRNAs. Studies have been published with a dominant negative FGF receptor [90,91] and a dominant-negative activin receptor [621. In each case, the construct encodes a receptor lacking most of the cytoplasmic domain and the dominant-negative action is ascribed to the formation of inactive dimers with the endogenous receptor. It is now known that the dominant-negative activin receptor inhibits the action of all of the TGFP superfamily members that are capable of inducing mesoderm, including activin itself and Vg-1 (D. Melton, personal communication). When overexpressed in whole embryos, it causes them to become 'empty sacs' containing little or no mesoderm [621. At the same time, there is an overproduction of neural tissue from the ectoderm. These results may be comparable to the effects of overexpressing the activin-binding protein
follistatin, which does not reduce mesoderm formation significantly although there are some pattern disturbances, perhaps reflecting the later, zygotic functions of activin. Once again there is a strong neuralizing effect on the ectoderm (A. Hemmati-Brivanlou, personal communication). These results seem compatible with those of the transfilter experiment, as they suggest that a TGF3-like molecule other than activin is necessary for mesoderm formation, whereas activin itself might function as a repressor of neural induction. The dominant-negative FGF receptor antagonizes the FGF receptor types that are found in the early embryo. The phenotype of overexpression is a reduction of mesoderm formation with associated defects in the posterior of the embryo [90,911. As there is a reduction in mesoderm formation, it seems hard to escape the conclusion that mesoderm induction requires FGF at some stage, even if FGF is not the factor produced by the vegetal cells to signal to the animal cells. The blockade of mesoderm formation is not total - in fact the head region is fairly normal - and to this extent the phenotype is less severe than that of the dominantnegative activin receptor. The normal head must count as evidence against the idea that the dorsovegetal signal consists of FGF plus Wnt, as a reduction of dorsovegetal signal should certainly affect the head: by comparison, the head is the first part of the body to be affected in the dose-response series of UV radiation to the egg [4].
Putting it all together It is always possible that novel molecules will be found that will render this analysis out of date. However, we do now know of enough components that approach satisfaction of the three criteria - activity, expression and inhibition - to provide a plausible model of the molecular basis for the sequence of inductions shown in Figure 1. So, Figure 4 shows the same diagram with the molecules named: the hypothetical cell states of Figure 1 (mesoderm, organizer, and so on) are presumed to be defined by combinations of transcription factors, whereas the inductive signals are tentatively identified as known secreted factors. The general mesoderm-inducing signal seems quite likely to be Vg-1: it has the appropriate activity, its mRNA is localized in the right place, and its activity is inhibited by the dominant-negative activin receptor, which is known to suppress mesoderm formation. It would, of course, be nice to have a more specific inhibition experiment, and we do still need some evidence that Vg-1 is actually processed in vivo and is secreted from vegetal cells. The organizer-inducing dorsovegetal signal seems most likely to be Wnt-11, or Wnt-11l plus Vg-l. The biological activity of Wnt-11 is appropriate although weak, and its localization is satisfactory, although we still need evidence of differential translation as a consequence of the cortical rotation. We also need an inhibition experiment to prove this proposed role of a Wnt protein. We do know that heparin
Inducing factors in Xenopus development
antagonizes anterior-posterior axis formation [921, and that it also inhibits Wnt effects in tissue culture [931, but heparin is not a reagent of sufficiently high specificity for this to constitute proof. If we choose Vg-1 and Wnt-11 as front runners for the mesoderm-inducing signals, this might seem to leave us with no role for FGF or activin, which were presumed until recently to be responsible for the general and the dorsovegetal signals, respectively. If we accept that the dominant-negative FGF receptor really is specific for the FGF family, then it seems an inescapable conclusion that FGF is required for mesoderm formation
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in vivo. However, it need not act as the primary mesoderm-inducing signal, and in fact the evidence from expression data is against this suggestion. It may be that the primary signal causes release of FGF from cells, or exposure of the FGF receptor to a pre-existing extracellular ligand in the animal hemisphere. Perhaps FGF signalling is needed to amplify the primary signals or to relay them over several cell diameters. Of the FGFs, bFGF and eFGF are present maternally, and of these eFGF is the more attractive candidate for having such a role, in that it possesses a signal sequence that would cause it to be secreted. A similar argument for a relay role could be made for activin, although the evidence
Fig. 4. The sequence of inductive interactions in the Xenopus embryo. In this diagram, molecular markers of cell state are shown on the left and the inducing factors that bring about transitions between cell states are shown on the right. Justification for placement of the various factors is given in the text. Gene name abbreviations: bra, brachyury; Epi- 1, an epidermal marker; fkh, forkhead; gcd, goosecoid; lirm, Xlim 1; N-CAM, neural cell adhesion molecule; not, Xnot; twi, twist; sna, snail.
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Current Biology 1994, Vol 4 No 2 for its involvement in mesoderm induction is a little weaker, because the maternal levels seem so low and the dominant-negative effects may be attributable to Vg-1. The dorsalizing signal seems quite likely to be noggin, judging by activity and expression criteria. There is, as yet, no inhibition experiment to demonstrate the function of noggin. The organizer region is also responsible for neural induction, initially in the form of the planar signal and later, when it has become the archenteron roof, by the appositional route. Although dorsalization and neural induction have been considered to be separate processes [94], there is no particular reason to suppose that the same substances cannot be involved in both. Noggin does have neuralizing activity, and is expressed in the right place to be a neural inducer. There is however also considerable evidence that neural induction is provoked by removal of something, for example following cell disaggregation of blastula cells [95,961. Removal of activin seems to cause neuralization 162], and at the gastrula stage, if cells are already disaggregated, so does addition of FGF [61]. It is perfectly possible that each biologically defined 'signal' will correspond to a complex mixture of substances. But it is now possible to draw a simple diagram in which Vg-1 and Wnt-11 are the primary mesoderm-inducing signals, FGF and activin are secondary mesoderm-inducing signals, and noggin takes care of dorsalization and neural induction (Fig.4). This is where the game stands at present.
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