Mesoderm induction and mesoderm-inducing factors in early amphibian development
1989; The Company of Biologists; Volume: 105; Issue: 4 Linguagem: Inglês
10.1242/dev.105.4.665
ISSN1477-9129
Autores Tópico(s)Congenital heart defects research
ResumoRecently, significant advances have been made in the analysis of mesoderm induction, one of the first inductive interactions in vertebrate development. In this article, I review these advances in the context of earlier work on the subject and go on to discuss what problems remain and how they might be approached. I shall concentrate on cell biological and embryological aspects of mesoderm induction rather than on the transcriptional control of genes which are activated in response to induction. This work has recently been reviewed elsewhere (Gurdon et al. 1989).Mesoderm induction probably occurs during the development of all vertebrates (Nieuwkoop et al. 1985; Smith, 1988) but the phenomenon has been most extensively studied in the Amphibia. This is for several reasons: amphibian embryos are large, making micro-dissection relatively easy; they are accessible to experimental manipulation at all developmental stages, from oogenesis to adulthood; development is rapid: the body plan is established and tissue-specific gene activation occurs within 24 h; amphibian embryos may be obtained in large numbers, which, together with their large size, makes it possible to extract enough material for biochemical analysis; and finally, the embryos do not grow - their early development consists of a series of cleavage divisions (see Fig. 1). This last point means, firstly, that it is possible to construct fate maps by injecting inert lineage tracers into selected blastomeres; because there is no growth the markers do not become diluted during development (see, for example, Jacobson & Hirose, 1978, 1981; Gimlich & Cooke, 1983; Gimlich & Gerhart, 1984; Heasman et al. 1984; Cooke & Webber, 1985; Dale & Slack, 1987a; Moody, 1987a,b; see also Fig. 2). Furthermore, since embryonic blastomeres survive on their yolk reserves, they will divide and even differentiate in a simple buffered salts solution. Thus it is possible to test defined molecules for their effects on differentiation without interference by poorly characterized serum components. In view of these advantages, this review deals exclusively with mesoderm induction in Amphibia.Mesoderm induction was discovered using the techniques of experimental embryology. At morula stages, the amphibian embryo seems to consist of only two cell types: prospective ectoderm in the animal hemisphere and prospective endoderm in the vegetal hemisphere (Fig. 3A; see Jones & Woodland, 1986). The evidence for this comes from experiments in which different regions of the embryo are dissected and cultured in isolation: even blastomeres of the equatorial 'marginal zone', which give rise to mesoderm in normal development, form epidermis if they are isolated before the 64-cell stage.If, however, they are dissected later than this they form mesoderm as well as epidermis (Nakamura & Matsuzawa, 1967; Nakamura et al. 1970b).One interpretation of this result is that mesoderm formation depends on an interaction between animal and vegetal blastomeres. This conclusion was confirmed by Ogi (1%7, 1%9) and by Nieuwkoop (1969), both of whom juxtaposed cells from the animal cap of the blastula-staged embryo with cells of the vegetal pole (Fig. 3B). When animal pole cells are cultured alone they form an atypical type of epidermis, while vegetal pole cells alone form poorly differentiated endoderm. Neither make mesoderm. Combinations of animal and vegetal pole cells, however, form a variety of mesodermal cell types.Neither Ogi nor Nieuwkoop had access to cell lineage markers and they differed in the interpretation of their experiment. Ogi, with tentative support from Nakamura et al. (1970a), was reminded of work on the seaurchin embryo indicating the existence of an animalvegetal double-gradient system (Horstadius, 1935). According to this view, apposition of animal and vegetal regions should result in regulation of the gradients, with both components giving rise to mesoderm. Nieuwkoop, however, regarded the mesoderm as being formed entirely from the ectodermal component, as a result of induction by prospective endoderm. He subsequently demonstrated that this view was correct by a quantitative analysis of the structures formed from animalvegetal combinations of blastomeres from Xenopus embryos (Sudarwati & Nieuwkoop, 1971) and by using [3H]thymidine to mark the animal pole component of combinations made from axolotl embryos (Nieuwkoop & Ubbels, 1972).Nieuwkoop made a second major contribution to the understanding of mesoderm induction by demonstrating that the type of mesoderm that forms in animal-vegetal combinations depends on the origin of the vegetal inducing cells (Boterenbrood & Nieuwkoop, 1973). Vegetal pole cells from the dorsal side of the axolotl blastula tended to induce dorsal cell types such as notochord and muscle while lateral and ventral vegetal blastomeres induced blood, a characteristic ventral cell type, along with mesenchyme and meso-thelium. Nieuwkoop concluded from this that the pattern of cell types in the mesoderm (Fig. 1) is determined, at least in part, by information derived from underlying vegetal blastomeres.The most widely used species in the study of amphibian development is Xenopus laevis, and the observations of Boterenbrood & Nieuwkoop (1973) on the axolotl have been confirmed for this species by Dale et al. (1985) and Dale & Slack (1987b). The results, however, appear to contradict the fate map of Xenopus. Ventral vegetal blastomeres induce little or no muscle from animal pole cells, yet most of the muscle of the embryo is formed from blastomeres of the 'ventral' half of the embryo (Keller, 1976; Cooke & Webber, 1985; Dale & Slack, 1987a; Moody, l987a,b; see Fig. 2).This incongruity may be resolved by the 'three signal' model of Slack and his colleagues (Smith & Slack, 1983; Slack et al. 1984; Smith et al. 1985; Dale & Slack, 1987b; see Fig. 4). The first two signals in this model are those of Boterenbrood & Nieuwkoop (1973), and are derived from the vegetal hemisphere of the embryo. One, on the dorsal side, induces predominantly notochord, perhaps with a small amount of muscle. The other, on the ventral side, induces blood, mesenchyme and meso-thelium. The third signal, however, originates from newly induced dorsal mesoderm. This signal acts within the prospective mesodermal germ layer to 'dorsalize' adjacent ventral mesoderm such that tissue that would have formed blood forms muscle instead. Only cells near the ventral midline of the embryo are out of range of the dorsalization signal and continue to form blood. Evidence for the existence of the dorsalization signal comes from experiments in which dorsal and ventral marginal zone regions of the early gastrula are juxta-posed. In isolation, dorsal marginal zone tissue forms notochord, with some muscle and neural tissue, while ventral marginal zone cells form blood, mesenchyme and mesothelium. In combinations, however, although the dorsal tissue continues to differentiate as noto-chord, the ventral marginal zone forms large amounts of muscle (Slack & Forman, 1980; Dale & Slack, 1987b).Dorsalization may be the major interaction at work in Spemann's 'organizer' graft (Spemann & Mangold, 1924), in which early gastrula dorsal marginal zone tissue (the 'organizer') is grafted into the ventral marginal zone region of a host embryo, causing the formation of a mirror-symmetrical double-dorsal larva. Perhaps the most dramatic aspect of this result is the formation of an additional neural tube, which arises from ventral ectoderm under the influence of a neural induction signal from involuting dorsal mesoderm (see Gimlich & Cooke, 1983; Jacobson, 1984). However, the earlier interaction is probably dorsalization, during which much of the original ventral mesoderm into which the graft was placed is induced to form muscle (Smith & Slack, 1983).Much of the recent interest in mesoderm induction stems from the discovery and purification of mesoderminducing factors (MIFs: see Smith, 1987; Slack et al. 1987; Kimelman & Kirschner, 1987; Rosa et al. 1988; Smith et al. 1988). It is ironic, therefore, that sources of mesoderm-inducing activity were discovered long before the phenomenon of mesoderm induction was properly defined. In 1953, Toivonen found that guineapig bone marrow could induce mesodermal cell types from isolated newt ectoderm and, a few years later, Saxen & Toivonen (1958) showed that HeLa cells have a similar effect. Indeed the discovery of these inducing factors may have influenced Nieuwkoop (1%9) in his interpretation of the results of animal-vegetal combinations.Since Nieuwkoop's work, other sources of mesoderm-inducing activity have been reported. One such is the carp swim-bladder (Kawakami, 1976; Kawakami et al. 1977), but the best-known is Tiedemann's 'vegetalizing factor', which has been purified from 9- to 11-day chick embryos (Born et al. 1972a). Experiments with this and other factors have provided some significant insights into mesoderm induction in amphibia, but there have been two problems with these inducers that have hindered their use as research tools. One is that they are usually assayed as an insoluble pellet, as the 'filling' of a sandwich in which test animal pole tissue is the bread. This makes dose-response experiments, for example, particularly difficult to interpret. The other problem is that these factors are 'heterogenous', derived from an inappropriate source (see Gurdon, 1987). It is difficult to understand the relevance of a factor from the carp swim-bladder to the events o_f early frog development.Recent work is overcoming these problems, but acceptance of the new mesoderm-inducing factors has been facilitated by the fact that they are related to known peptide growth factors. One class of MIFs is related to transforming growth factor type β (TGF-β ). This group includes XTC-MIF, which is derived from the Xenopus XTC cell line (Smith, 1987; Smith et al. 1988) and TGF-, β 2, obtained from pig platelets (Rosa et al. 1988). TGF-β 1 has no mesoderm-inducing activity (Slack et al. 1987; Kimelman & Kirschner, 1987), but it enhances the effect of the other group of MIFs, the heparin-binding growth factors (Kimelman & Kirschner, 1987). The main members of this second group are acidic and basic fibroblast growth factor (aFGF and bFGF), both of which are equally active in inducing mesoderm from isolated Xenopus animal pole tissue (Slack et al. 1987; Kimelman & Kirschner, 1987; Slack et al. 1988).The usual test for mesoderm-inducing activity involves culturing isolated animal pole tissue in dilute solutions of the factors. In the absence of MIFs, the test tissue differentiates as epidermis, but in their presence mesodermal cell types arise (see Smith, 1987). Both groups of MIF are active at picomolar concentrations, but the assay reveals a significant difference between them. The TGF-β class of MIF induces a variety of mesodermal cell types including notochord, muscle, kidney, mesenchyme and mesothelium (Smith et al. 1988). FGF is capable of inducing all these cell types except notochord, the most dorsal mesodermal tissue (Godsave et al. 1988). This has led to the suggestion that the 'dorsal' signal in Slack's model (Fig. 4) is TGF-β - like and that the 'ventral' signal is FGF-like (Dale & Slack, 1987b). To confirm suggestions such as this, it is first necessary to demonstrate that TGF-β - and FGF-like molecules are present in the Xenopus embryo in the predicted amounts, in the predicted regions, and at the appropriate stages.Definitive TGF-β '2 and XTC-MIF transcripts or protein have not yet been identified in Xenopus embryos, but a maternal mRNA that is restricted to the vegetal hemi-sphere of the egg (see Rebagliati et al. 1985) has been shown to code for a factor related to TGF-β (Weeks & Melton, 1987). This mRNA, designated Vgl, constitutes 0·05-0&1% of the poly(A)+ RNA pool (Rebagliati et al. 1985). An open reading frame of 1080 bases encodes a protein of 41·8 × 103 Mr of which the carboxy-terminal 120 amino acids show 38% homology to TGF-β 1 and in which the positions of the cysteine residues are conserved. Interestingly, Vgl shows greatest similarity to the deduced sequence of the decapentaplegic (dpp) gene product of Drosophila, another member of the TGF-β family (Padgett et al. 1987). There is a 48% match in the carboxy-terminal 114 amino acids, and the two share a potential glycosylation site.Judging from changes in the spatial distribution of Vgl mRNA, the protein encoded by this message is an excellent candidate for an endogenous mesoderm-inducing factor. During early oogenesis, the message, as revealed by in situ hybridization, is distributed uniformly within the oocyte (Melton, 1987). As oogenesis proceeds the RNA moves towards the vegetal pole of the oocyte and eventually is localized as a thin cortical shell. Following fertilization the message is released from its cortical position and it spreads towards the animal pole. This spread is restricted by the cleavage divisions of the embryo such that at the early blastula stage the only cells to contain substantial amounts of Vgl transcripts are the vegetal blastomeres, those responsible for inducing adjacent equatorial cells to form mesoderm (Weeks & Melton, 1987).Although the circumstantial evidence is compelling, there is no direct evidence at present that Vgl mRNA does code for a mesoderm-inducing factor. It is possible, for example, that it is an inactive homologue of an endogenous inducing factor in the way that TGF-β 1 is an inactive homologue of the inducing factor TGF-, β 2. One way to investigate this might be to make Vgl protein by amplified expression in Chinese Hamster ovary cells, as described for TGF-β 1 by Gentry et al. (1987). Alternatively, Vgl RNA might be micro-injected into the animal hemisphere of early Xenopus embryos. This should cause the formation of ectopic mesoderrn in a manner similar to microinjected XTC cell poly (A)+ RNA (Cooke et al. 1987; Woodland & Jones, 1987).Even if Vgl protein is not a mesoderm-inducing factor, study of the message will yield important results concerning the mechanisms by which mRNAs are localized within early embryos. For example, Pondel & King (1988) have recently shown that Vgl RNA is concentrated 35- to SO-fold in detergent-insoluble extracts of Xenopus oocytes, whereas histone H3 mRNA is equally distributed between detergent-insoluble and -soluble fractions. The detergent-insoluble extracts are enriched for cytokeratins and vimentin, suggesting that Vgl RNA may be localized through an interaction with the cytoskeleton. In support of this, Vgl RNA is found to be released to the detergent-soluble fraction after ovulation; this is when cortical cytokeratin filaments break down (Klymkowsky et al. 1987) and Vgl RNA spreads towards the animal pole (Weeks & Melton, 1987). Using a different approach, Yisraeli & Melton (1988) have shown that Vgl message transcribed in vitro is translocated to the vegetal hemisphere after being injected into immature Xenopus oocytes. Localization does not require translation of Ygl. This technique should make it possible to map the 'vegetal translocation' sequence on the Ygl message, which may, for example, recognize intermediate filaments. It will then be interesting to screen Xenopus oocyte cDNA libraries for clones with similar sequences. Analysis of such clones may reveal additional localized mRNAs which could code for mesoderm-inducing factors.Work on FGF indicates that both the mRNA and the protein are present in the early embryo. In their original paper, Kimelman & Kirschner (1987) described a cDNA from a Xenopus oocyte library that contained sequences closely related to human and bovine basic FGF. This cDNA hybridized to a 1&5 kb RNA in Xenopus oocytes, eggs and early embryos, but the level of this transcript varied through early development. The message was abundant in oocyte preparations (which included follicle cells), but had decreased by 95% at the fertilized egg stage. It increased again at the midblastula transition, when embryonic transcription commences (Newport & Kirschner, 1982), and remained fairly steady at least until the late neurula stage.The RNA detected by Kimelman & Kirschner's original cDNA is large enough to encode a protein the size of bFGF but, although it contains a short open reading frame encoding a peptide domain homologous to the third exon of mammalian bFGFs, sequences homologous to the first and second exons are absent. Recently, however, use of a different probe has revealed two additional RNA species (Kimelman et al. 1988). One of these, of approximately 2&1 kb, is present continuously from the oocyte to midgastrula stages. The other is a 4&2 kb species which is present in the oocyte and is transcribed again at neurula stages. A 4&3 kb cDNA corresponding to this larger species was isolated and sequence analysis indicated that the primary translation product is a 155-residue protein with an overall homology to human bFGF of 89% . When this protein was synthesized in a TI expression system and purified by heparin-Sepharose chromatography it was shown to be as effective as bovine bFGF in inducing muscle from isolated Xenopus animal pole regions.To demonstrate that bFGF protein is present in the Xenopus embryo, both Kimelman et al. (1988) and Slack & Isaacs (1989) have passed extracts of eggs and blastulae through heparin-Sepharose columns before eluting the bound material with high concentrations of NaCl. Slack & Isaacs (1989) were able to show that the eluted material had mesoderm-inducing activity which could be blocked by antibodies to bFGF but not to aFGF or TGF-β . Furthermore, active fractions from an HPLC heparin affinity column contained material identifiable as bFGF by use of specific antibodies after Western blotting. Kimelman et al. (1988) did not assay their heparin-Sepharose eluates for mesoderm-inducing activity, but they also showed by immunological criteria that FGF-like-protein was present. Both groups estimated the total amount of FGF present in the Xenopus embryo by inspection of Western blots reacted with anti-FGF antibodies, but they differed in their conclusions. Kimelman et al. (1988) calculated that a single embryo contains 100 Pp FGF, a concentration of approximately 70ng ml- . Slack & Isaacs (1989), however, estimate a total concentration of about 7 ng m1- 1 bFGF. Both concentrations are certainly high enough for FGF to be a natural mesoderm inducer, but further work is required to discover which estimate is nearer the mark. Calculations of amounts of protein from the intensities of bands on gels are probably subject to errors of at least a factor of three, so the apparent difference may not be significant. It is noteworthy, however, that the antibody used by Slack & Isaacs (1989) may not recognize Xenopus FGF as efficiently as human; on the other hand, their figure is corroborated by the levels of biological activity that can be recovered from the embryo.The spatial distribution of FGF in the Xenopus embryo has not yet been studied. The obvious prediction is that, like Vgl, the factor is concentrated in the vegetal hemisphere of the embryo. There is, however, one difficulty with understanding how localized FGF might act as an inducing factor. TGF-, β -like molecules, including Ygl (Weeks & Melton, 1987), carry a signal sequence which directs their secretion from the cell, thus allowing them to act on their neighbours (reviewed by Massague, 1987). Bovine and Xenopus FGF carry no such sequence (Abraham et al. 1986; Kimelman et al. 1988) and cannot be secreted from cells through the usual pathway. For example, NIH 3T3 cells transfected with a plasmid containing cDNA that encodes bovine bFGF synthesize large amounts of bFGF, but this remains associated with the cells in an inactive form, and the cells themselves appear morphologically normal. However, when a heterologous secretory signal sequence is fused to the bFGF cDNA, the cells become morphologically transformed and tumorigenic, although no bFGF can be detected in the culture medium (Rogeli et al. 1988). A similar experiment has been carried out with acidic FGF and Swiss 3T3 cells (Jaye et al. 1988). Again, no FGF could be detected in medium conditioned by the transfected cells but, even in the absence of a signal peptide, several traits characteristic of the transformed phenotype were expressed. The authors suggest, as has also been suggested for cells expressing PDGF encoded by v-sis, that the growth factor may stimulate its receptor in an internal compartment (Leal et al. 1985; Keating & Williams, 1988).It is not possible to draw conclusions about the way FGF acts in Xenopus embryos from these experiments on tissue-culture cells, although there are several intriguing possibilities. For example, FGF may act during normal development in an autocrine fashion. In this case, prospective mesodermal cells would both synthesize and respond to the factor, perhaps on receipt of a TGF-, β -like factor. The observation that exogenous FGF acts as an inducing factor on isolated animal pole regions might then be coincidental, not implying anything about the nature of the vegetal inducer.It is encouraging that FGF and TGF-, β -like molecules are present in the Xenopus embryo, but much work remains to be done before either is proved to be a natural mesoderm inducer. In the case of TGF-, β, the first task is to identify and characterize a member of the family which is both present in the embryo and which has mesoderm-inducing activity. There are three candidates at present: the Vgl protein, the Xenopus equivalent of TGF-, β 2, and XTC-MIF. The mRNA for the first of these is present in the embryo at the right time and place but it is not yet known whether the protein has inducing activity or, indeed, whether the RNA is translated. The other two are known to be mesoderm-inducing factors, but their presence in the embryo must be established, followed by an analysis of their spatial distribution. FGF is known to be present in the embryo and to be active as a mesoderm inducer, but its spatial distribution has not yet been studied.Definitive proof that bFGF or TGF-, β -like factors are the natural mesoderm inducers requires the elimination of these factors from the embryo. Recently, techniques have been developed that should make this possible. Embryological experiments indicate that the vegetal hemisphere acquires the ability to induce mesoderm from animal pole regions at least as early as the 64-cell stage (Jones & Woodland, 1987). This is about 3 h earlier than the midblastula transition, when embryonic transcription begins (Newport & Kirschner, 1982), so the mRNA for inducing factors must be synthesized during oogenesis. Shuttleworth & Colman (1988) have demonstrated that such maternal messages can specifically be eliminated from the oocyte simply by microinjecting appropriate antisense oligonucleotides. The oligonucleotides form a DNA-RNA duplex with the target message and this acts as a substrate for an endogenous RNase H activity. The technique works most effectively when oligonucleotides are injected into oocytes rather than fertilized eggs or early embryos, because an RNA duplex unwinding activity appears soon after fertilization (Rebagliati & Melton, 1987; Bass & Weintraub, 1987). However, it is possible to overcome this problem by ablating mRNA in oocytes as outlined above and then maturing the oocytes invivo before fertilizing them in vitro, as described by Holwill et al. (1987). The result of such experiments, if mRNAs for endogenous mesoderm-inducing factors are ablated early enough, should be the development of mesodermless embryos.It may not be possible to eliminate mRNAs for inducing factors before synthesis of substantial amounts of the factors themselves. If this is the case it may be possible to interfere with induction in vivo by microinjecting specific antibodies to MIFs into the vegetal hemisphere of early embryos.It is important to discover the identity and analyse the spatial distribution of endogenous mesoderm-inducing factors in the amphibian embryo, but to explain how the right mesodermal cell type forms in the right place it is also necessary to study the responses of animal pole cells to these factors. Most recent studies of mesoderm induction in Xenopus have used muscle, the most abundant mesodermal cell type (Cooke, 1983), as a marker of mesoderm formation (for example, Gurdon et al. 1985; Sargent et al. 1986; Kimelman & Kirschner, 1987; Rosa et al. 1988; Gurdon, 1988, 1989). However, as mentioned above, many other mesodermal cell types are formed in response to induction, both in animal-vegetal combinations (Dale et al. 1985) and in response to purified mesoderm-inducing factors (Smith et al. 1988; Godsave et al. 1988; J. Cooke & J. C. Smith, unpublished observations). The only mesodermal cell type that is not formed in response to MIFs is blood (Smith, 1987; K. Symes & J. C. Smith, unpublished observations), and it seems likely that this is because erythrocyte differentiation requires a late-acting permissive signal from hepatic endoderm, not because MIFs do not specify ventral mesoderm (Deparis & Jaylet, 1984).The embryological experiments already described indicate that different mesodermal cell types are formed in response to different regions of the vegetal hemisphere of the embryo. Dorsal mesodermal cell types are formed in response to dorsal vegetal pole blastomeres and intermediate or ventral cell types in response to ventral vegetal pole blastomeres. This series of cell types is also observed in response to decreasing concentrations of mesoderm-inducing factors. Thus high concentrations (10-25 ng ml •1) of XTC-MIF induce notochord, and progressively lower concentrations induce muscle, followed by mesenchyme and mesothelium (Smith et al. 1988). Like Godsave et al. (1988) I consider mesenchyme, and particularly mesothelium, to represent ventral differentiation, even though large amounts of mesenchyme are found, for example, in the head of Xenopus. This is because mesenchyme and mesothelium form more frequently in combinations of ventral vegetal blastomeres with animal pole regions than in combinations involving dorsal vegetal blastomeres (Dale & Slack, 1987b). A similar dorsal-to-ventral response curve is seen with bFGF, with the significant difference that even the highest concentrations of this factor only rarely induce notochord (Godsave et al. 1988).One conclusion from these results, already mentioned above, is that a TGF-β -like factor may act as the 'dorsal' component of Slack's three-signal model (Fig. 4), with bFGF, being unable to induce notochord, representing the ventral component. The data also suggest that the vegetal hemisphere of the embryo might contain graded distributions of one or both factors, with the highest concentrations at the dorsal side. However, there is no evidence for a graded distribution of the mRNA for the only potential MIF for which such information is available: in situ hybridization studies with Vgl probes have not revealed an asymmetrical distribution in the dorsoventral axis (D. A. Melton, personal communication). Such studies need to be repeated with the other candidates for endogenous mesoderm-inducing factors, but embryological evidence suggests that the gradient hypothesis in its simplest form, in which mRNAs for inducing factors are arranged in an asymmetric fashion, is unlikely to be true. The dorsoventral axis of Xenopus is established at fertilization, with dorsal structures usually forming opposite the site of sperm entry (Black & Gerhart, 1985). This determinative event is linked to a series of cytoplasmic shifts in which the entire subcortical cytoplasm rotates about 30 ° with respect to the cell membrane, with that in the vegetal hemisphere moving away from the dorsal side (Vincent et al. 1986). According to a strict localization model these shifts might move determinants to the dorsal side of the embryo. However, this interpretation is not consistent with experiments in which fertilized Xenopus eggs are tilted or centrifuged, so as to displace the contents of the cytoplasm. Embryos subjected to this treatment become either double-dorsal or 'head-heavy' (Black & Gerhart, 1986; Cooke, 1986, 1987) and this cannot be explained by the redistribution of existing determinants for two reasons. First, in the formation of twinned embryos, it is not clear why centrifugation would only move half of the axis-forming molecules. Secondly, in both experiments, the results imply that centrifugation has caused greater amounts of XTC-MIF-like factors to be created. This would not be predicted by a simple gradient model. It may be that regions specified by centrifugation as dorsal translate mRNA for inducing factors more efficiently. Alternatively, there may be graded post-translational modifications of proteins which affect their activity, or the receptors for inducing factors may become differentially localized. These possibilities require investigation.Another difficult problem is that of how different concentrations of MIFs induce different types of cell differentiation. This question is not restricted to mesoderm induction in the amphibian embryo. A similar problem exists in systems as different as the chick limb, where a graded signal from the posterior margin of the limb bud (perhaps retinoic acid: Tickle et al. 1982; Thaller & Eichele, 1987) specifies different digits along the anteroposterior axis (Tickle et al. 1975; Smith et al. 1978), and in the insect egg, where position along the anteroposterior axis is specified by the concentration of the bicoid gene product (Driever & Nlissleinolhard, 1988).A measure of our ignorance on this topic, that of the interpretation of positional information (Wolpert, 1969), is that it is not even clear whether such threshold phenomena operate at the single cell level or at
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