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Induction and early amphibian development
Induction and early amphibian development J.C. Smith Laboratory of Embryogenesis, National Institute for Medical Research, London, UK Current Opinion in Cell Biology 1989, 1:1061-1070
Introduction It has been clear for almost a century that cell-cell interactions play an important role in embryonic development. This conclusion originated largely through experiments by Spemann and his colleagues, most notably with their discovery of the organizer, and Spemann's own description of this work has recently been re-published (Spemann, Embr)~onic Development and Induction. Garland Publishing, 1988). In addition, Hamburger has written a fascinating eyewitness account covering the same period (Hamburger, The Heritage of Experimental EmbD~91ogy. Oxford University Press, 1988). For a field this venerable, it may seem surprising that it is only in the last 2 or 3 years that significant progress has been made in coming to understand the molecular basis of cell-cell interactions during development. As Kay and I have discussed, this is quite simply because the problem is very ditficult and the appropriate techniques were not available [Kay and Smith, Development 1989, (suppl):l-2]. Now that the techniques are available, dramatic progress is being made. This review summarizes what has been achieved during the last 18 months using amphibian embryos. Amphibian embryos offer great advantages to the experimental embryologist in being big, readily available in large numbers, and accessible to manipulation at all developmental stages. In addition, small pieces of early embrYos will survive and differentiate in simple balanced salt solutions, because they can survive on their yolk reserves. Thus putative 'inducing factors' can be tested without risk of interference by poorly defined serum components. The early development of the frog Xenopus laevis is described in Fig. 1. So far very little work has been done to analyse inductive interactions in other vertebrates, such as chickens and mice; the next year, however, should see lessons learnt from the amphibia being applied to the amniotes.
A sequence of interactions
It would be remarkable ff the great complexity of the vertebrate body could arise from a single inductive interac-
tion. In fact, a great deal of experimental embryological work indicates that development consists of a sequence of inductive interactions (see Slack, From Egg to Embr)~. Cambridge University Press, 1983). Experimental embryology is not an exact science, and data are often open to more than one interpretation. Nevertheless, a consensus view, providing a framework on which to discuss recent work, might begin with the 'three-signal model' for mesoderm formation and be followed by neural induction [Smith et eL, J Emboxgl Exp Morpbol 1985, 89 (suppl):317-331]. This scheme is outlined in Fig. 2; it is not possible to describe here all the work on which it is based, but this is reviewed elsewhere [1] (see also Smith,
In Developmental Biology: a Comprehensive Synthesis, Vol 5, edited by Browder. Plenum, 1988, pp 79-125). The first two signals of the three-signal model for mesoderm formation occur during blastula stages, when the fertilized egg has divided to form 64-1024 cells. These signals are produced by cells of the vegetal hemisphere of the embryo, and cause the formation of mesoderm, and perhaps some endoderm, from overlying equatorial and animal hemisphere cells. Two signals are believed to be involved because dorsal and vegetal blastomeres induce quite different types of mesoderm from test animal cap tissue. Dorsal vegetal blastomeres induce predominantly dorsal mesodermal ceil types, such as notochord and muscle, whereas ventral vegetal blastomeres induce predominantly ventral cell types like mesenchyme and blood (reviewed in [1]; for a different view see [2]). As I discuss below, with the recent advent of molecular markers for anterior and posterior mesoderm it may be more appropriate to describe the signals as 'antero-dorsar and 'postero-ventral'. The third signal of the model is produced by newly induced dorsal mesoderm cells, and is responsible for subdividing a large region of general ventral mesoderm into, for example, somite muscle, pronephros, lateral plate mesoderm and blood. This interaction can be demonstrated by juxtaposing extreme ventral mesoderrn with extreme dorsal; the dorsal tissue remains dorsal and continues to differentiate as notochord, while the ventral tissue is 'dorsalized' and forms muscle (Dale and Slack, Development 1987, 100:279-295). 'Dorsalization' is the first interaction to oc-
Abbreviations
bFGr--~basic fibroblast growth factor; c/UVlP--cyclic adenosine monophosphate; cDNA--complementary DNA; r--GF~fibroblast growth factor; NCAM--neural cell adhesion molecule; NF--neurofilament; mRNA--messenger RNA; NIFs--neural inducing factors; PI--phosphatidylinositol; PKC--protein kinase (7; TGF~transforming growth factor;, TPA--12-o-tetradecanoylphorbol-13-acetate;XTC-MIr-~xTC-mesoderm-inducingfactor. (~) Current Science Lid ISSN 0955-0674
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Cell differentiation tissue (Smith, Development 1987, 99:3-14), and by the discovery that members of the fibroblast growth factor (FGF) family had similar properties (Slack et al., Nature 1987, 326:197-200; Kimelman and Kirschner, Cell 1987, 51:869-877). Subsequent work has suggested that the XTC-mesoderm-inducing factor (XTC-MIF) might be related to transforming growth factor (TGF)-[3. Firstly, antibodies against TGF-[32 (but not against TGF-[31) inhibRed ,,, 80% of the activity of XTC-conditioned medium [3]. Secondly, TGF-~2, but not TGF-~I, was found to be active as a mesoderm inducer [3]. And finally, purification of XTC-MtF revealed some properties in common with TGF-[3 [4]. Complete characterization of XTC-MIF has not yet been achieved, but circumstantial evidence suggests that it is not simply Xenopus TGF-[32 (see [4]).
cur in Spemann's organizer graft (Smith and Slack, JEnv bo~ol Exp Morpho11983, 78:299-317). Dorsalization probably occurs just before and during gastrulation. At the same time, different regions of the mesoderm are believed to acquire information about their antero-posterior positions. The mechanism by which this occurs is unknown. Neural induction is also believed to occur during gastmlation, with dorsal mesodermal cells instructing overlying ectoderm to form nervous system, transmitting information about antero-posterior position at the same time. This version of the inductive events occurring during early amphibian development accounts for much of the experimental embryology carried out until the end of 1987. Recent, more molecular, work has supported some of the interpretations but other data will necessitate changes in the models.
Mesoderm induction
Although TGF-~I alone has no mesoderm-inducing activity (Slack et al., 1987; Kimelman and Kirschner, 1987) [3], it has been reported to act synergistically with members of the FGF family (Kimelman and Kirschner, 1987). For reasons that are not yet clear, this result has not been confirmed [3]; the results may depend on the source of TGF-]3].
Mesoderm-inducing factors The recent interest in mesoderm induction in amphibian embryos was inspired in part by the observation that conditioned medium from a Xenopus cell line called XTC could induce mesoderm from isolated animal pole
It is, of course, important to show that mesoderm-inducing factors derived from tissue culture cells or, in the case of FGF, from cows' brains, are present in the Xenoptts embryo at the appropriate stages and in the predicted regions. In the case of FGF, Kimelman and Kirschner
~ .~2~ --. • ~:'A
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(e)
(f) ~ //(~)
Neural tube .Notochord
~,~
//,.-3Y.k.-~\\ ((/
~
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ndoderm~ ~
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Somite Epidermis
Lateral plate Blood island
Fig. 1. The early development of Xenopus laevis. The Xenopus egg (a) has a diameter of about 1.4 mm. The animal hemisphere, which lies upwards by gravity, is heavily pigmented, and the vegetal hemisphere is pale. Sperm entry occurs in the animal hemisphere, and the side on which the sperm enters becomes the ventral-posterior half of the embryo. Ninety minutes after fertilization a rapid series of cleavage divisions begins. As a result, the embryo forms a hollow ball of cells, the blastula (b; 5 h after fertilization). During gastrulation (c; 11 h after fertilization: viewed from the vegetal hemisphere of the embryo) the three germ layers of the embryo take up their definitive positions, a process completed by the neurula stages, by which time tissue-specific gene activation has started (d; 18 h after fertilization). After formation of the neural tube the embryo elongates (e; 27 h after fertilization). The body plan of the embryo is now complete. (0 Shows the location of the major cell types. (a-e) Adapted from Nieuwkoop and Faber, Normal Table of Xenopus laevis, 2nd edn. North Holland, 1987.
Induction and early amphibian development Smith (1987) were able to isolate an oocyte complementary DNA (cDNA) which contained sequences homologous to exon I1] o f human and bovine basic FGF (bFGF). This clone hybridized to a 1 kb RNA that was present in abundance in the oocyte and in midblastula to neurula stages, but the RNA lacked sequences homologous to the first and second exons of human bFGF. Recently, however, Kimelman and Kirschner and their colleagues [5] have isolated a 4.3 kb cDNA from a neumla library which contains the entire Xenoptts bFGF polypeptide. This transcript, which, like bovine and human bFGF, lacks a classic secretory signal sequence, is present in the oocyte and is then undetectable until early neurula stages. Interestingly, Volk et aL [6] have shown that the
,
Oogenesis
Fertilization
abundant 1 kb transcript described by Kimelman and Kirschner in fact encodes an antisense transcript to part of exon IlI o f bFGF RNA and that an open reading frame in the sense orientation encodes a 24 kD protein which is conserved in humans. The function of this protein is unknown. Kimelman et al. [5], as well as Slack and Isaacs [7], have also shown that bFGF protein is present in the Xenopus egg and early embryo. The m'o groups differ somewhat in their estimates of how much bFGF is present, but both figures are consistent with the factor being a natural inducer of mesoderm. No information is yet available about the spatial distribution of bFGF mRNA and protein.
@
Mesodermal induction Dorsalization
Gastrulation
Neural induction
Fig. 2. The sequence of inductive interactions in early amphibian development. During oogenesis differences arise between the animal CA) and vegetal (V) halves of the egg. These include, for example, the translocation of Vgl messenger RNA to the vegetal pole (see text). Fertilization initiates a 30° rotation of subcortical cytoplasm, in a direction defined by the position of sperm entry. This direction of rotation in turn defines which region of the vegetal hemisphere acquires dorsovegetal (DV) signalling ability and which ventrovegetal (VV). During mesoderm induction the dorsovegetal region induces the organizer (O) from the animal half of the embryo and the ventral region induces a general ventral mesoderm (M) which is subdivided into several regions during dorsalization, under the influence of the organizer. During gastrulation the mesoderm acquires antero-posterior positional values (represented as 01 to 04 for the organizer) and these are transmitted to the overlying ectoderm as part of the neural induction process. This produces neuroepithelium with homologous craniocaudal positional values (N1 to N4) while the uninduced animal pole material becomes epidermis (E).Adapted from Smith ~ci Prog 1985, 69:511-532).
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Cell differentiation No member of the TGF-fl family which is known to have mesoderm-inducing activity has been demonstrated to be present in the early Xenopzcs embryo. One strong candidate for an endogenous inducing factor is, however, the protein product of the localized messenger RNA (mRNA) Vgl (Rebagliati et al., Cell 1985, 48:599--605; Melton, Nature 1987, 328:80-82). Vgl RNA is restricted to the vegetal region of the oocyte and early embryo, and codes for a factor related to TGF-IB (Weeks and Melton, Cell 1987, 51:861--867). The mRNA is translated during oogenesis and early development and is glycosytated [8,9]. Although a less abundant glycosylated form (42 kD) has a roughly uniform distribution within the embryo a more abundant, larger (44 kD), form is restricted to the vegetal hemisphere [9]. By analogy with other members of the TGF-]3 family, this large form would be expected to be processed to yield a smaller (17kD) protein which is secreted as the active factor. Such a protein could not be detected in normal embryos [8,9], although if large amounts of full-length Vgl mRNA were injected into the embryo a little of the 17 kD form became visible [9]. Vgl mRNA is initially distributed uniformly within the developing oocyte (Melton, 1987) and the question of how it becomes localized is an important problem in cell biology. Pondel and King [10] have shown that Vgl mRNA is concentrated in detergent-insoluble extracts of Xenopus oocytes whereas histone H3 mRNA is equally distributed between soluble and insoluble extracts. Thus localization may involve interaction between mRNA and elements of the cytoskeleton. Yisraeli and Melton [11] have recently shown that Vgl mRNA transcribed in vitro is translocated to the vegetal hemisphere after being injected into immature Xenopus oocytes. By deleting parts of the message it should be possible to map the 'vegetal translocation' sequence, which may, perhaps, recognize components of the cytoskeleton. These results are summarized in Table 1, which makes clear how much work remains to be done in characterizing the normal in vivo mesoderm-inducing factors in the amphibian embryo. For members of the FGF family it is necessary to discover if the factors are localized, whether they escape from the cell, and if so how, in the absence of a secretory signal sequence, they do so. For members of the TGF-I3 family, one must discover whether the Vgl protein has mesoderm-inducing activity and whether any of the other members of the family, known to have activity, are present in the embryo. Finally, in order to confirm the role of any of these factors in early development it is necessary to eliminate them from the embryo and observe the effects of their absence. This may be difficult to achieve using antisense RNA techniques because synthesis of bFGF and Vgl protein occurs through a long period of oogenesis. Thus sufficient amounts of inducing factors may have been synthesized well before experimental intervention is possible. Biological effects of mesoderm-inducing factors Descriptions of the effects of XTC-MIF and bFGF suggest that the former resembles the dorsal mesoderm-induc-
ing signal of the three-signal model (Fig. 2) and FGF the ventral. The main evidence cited is simply that at high concentrations XTC-MIF induces notochord and neural tissue with reasonable frequency, whereas FGF does so only" very rarely [4,12] (Green et al., Development, in press). However, the observation that lower concentrations of XTC-MIF (0.2-1.0 ng/ml) have effects on the differentiation of animal pole cells very similar to those of bFGF [4] (Green et al., in press) might suggest that the difference between the two factors is quantitative rather than qualitative. Further evidence that the two factors do induce qualitatively different types of mesoderm comes from studying the timing of the gastmlation movements induced° by the two factors. Symes and Smith (Develop ment 1987, 101:339-349) demonstrated that XTC-MIF induces isolated animal pole tissue to undergo gastrutationlike movements, and that the time of onset of these movements coincided with the time at which the dorsal lip of the blastopore appeared in sibling embryos, irrespective of the stage at which they had been exposed to the factor. FGF also induces gastrulation-like movements but these are less dramatic than those induced by XTC-MIF and it is harder to determine their time of onset. However, when inducing factors are injected into the blastocoels of host embryos, thus converting the whole animal hemisphere to mesoderm, it is much easier to time the onset of ectopic gastrulation behaviour, and it is clear that gastrulation induced by bFGF occurs about 1.5 h later than that induced by XTC-MIF, and at a time corresponding to the formation of the ventral lip of the blastopore [13]. Importantly, this time does not depend on the concentration of either factor, indicating that the difference is qualitative rather than merely quantitative. A final piece of evidence regarding the identity of the dorsal and ventral mesoderm-inducing signals is considered below, under 'dorsalization'. Early responses to mesoderm induction The different tissues formed in response to mesoderm induction are usually assessed by histological analysis some days after treatment with inducing factors (see, for example, [4]). Alternatively, if one is interested in the expression of specific genes such as muscle, one can carry out RNase protection assays to detect specific responses within 5-7h (Gurdon et al., Cell 1985, 41:913-922). Using earlier muscle-specific marker genes such as M_)~D [14] it is possible to measure responses even sooner than this. But is the expression of genes specific to a certain mesodermal cell type a direct response to inducing factors or is this a somewhat later event resulting from intervening cell interactions? Attempts to answer this question have used XTC-MIF, FGF and the natural inducer from vegetal pole cells, and the answer is not yet clear. Symes et aL [15] exposed dispersed Xenopus blastomeres to XTC-MIF before reaggregating them at the early-to-mid gastrula stage, by which time they had lost competence to respond to the factor (Green et aL, in press). Although the dispersed cells were able to respond to XTC-MIF in the sense that they suppressed epidermal differentiation they did not form muscle, suggesting that some inter-
Induction and early amphibian development Smith
Table 1. Mesoderm-inducing factors in Xenopus. Present in early embryo? Factor
Family
Active?
RNA
Protein
Localized?
Vgl °
TGF-~ TGF-I] TGF-]] TGF-~ FGF FGF FGF FGF FGF
Unknown Yes With FGF Yes Yes Yes Yes Yes Yes
Yes Unknown Unknown Unknown Unknown Yes Unknown Unknown Unknown
Yes Unknown Unknown Unknown No Yes Unknown Unknown Unknown
Yes Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
XTC-MIFt TGF-~I:I: TGF-I]2§ aFGF~ bFGF¶ ECDGF** INT-2tt kFGFtt
"Rebagliati et aL (Ceil 1985, 48:599-605); Weeks and Melton (Cell 1987, 51:861-867); Melton (Nature 1987, 328:80-82) [8,9,11]; 1"Smith (Development 1987, 99:3-14) [3,4]; :[:Kimelman and Kirschner (Cell 1987, 51: 869-877); §13]; ,[Slack et aL (Nature 19/37, 326:197-200) [7,22]; ¶Slack et al. {1987); Kimelman and Kirschner (1987) [5,7]; *'Slack et al. (1987}; HPaterno et al. (Development 1989, 106:79-83).
cellular communication might be required during induction to activate mesoderm-specific genes. In a conceptually similar experiment Gurdon [16] exposed animal pole blastomeres to vegetal tissue under conditions in which the animal cells would not divide or contact each other. Again, muscle differentiation did not occur. In control experiments, where animal pole cells were allowed to contact each other, muscle was formed. A different experimental design and a different inducing factor gave different results for Godsave and Slack [17]. Here, single animal pole cells were exposed to FGF and then cultured on a substrate coated with laminin and fibronectin in a medium containing 2 mg/ml y-globulin. The single cells divided to give clones of eight to 100 cells and in many cases mesoderm differentiation occurred. The authors suggest that provided the correct factors are supplied, single cells are capable of responding to mesoderm induction by forming mesodermal cell types. It is also possible, however, that even in clones as small as eight cells sufficient cell-cell contact occurs to provide the necessary secondary interactions. A more direct approach to studying the early response to induction was undertaken by Rosa [18]. This most important work has discovered a homeobox-containing gene M ~ 1, which is activated in response to XTC-MIF. It is not detectably activated in response to FGF and this provides another piece of evidence that these two factors have qualitatively different effects. Three results indicate that activation of M/x 1 is an immediate early response to induction. First, Mix.1 mRNA is detectable within 30min of treatment of animal pole regions with XTC-MIF; second, the gene is activated in the absence of protein synthesis, and, finally, the gene is activated in dispersed cells. Perhaps the most remarkable aspect of the work is that during normal development Mix. 1 transcripts are expressed throughout the vegetal hemisphere of the blastula and early gastrula, in a region which will become
prospective endoderm and only part of the future mesoderm. This suggests that the initial response to XTC-MIF and perhaps other mesoderm-inducing factors is not to form mesodermal cell types but to become 'vegetal', and that formation of, for example, large amounts of muscle in explants exposed to XTC-MIF depends upon further interactions between induced cells. These interactions may themselves be initiated by the product of the Mix. 1 gene.
Transduction of the mesoderm induction signal Since mesoderm-inducing factors resemble mammalian growth factors, it seems probable that their modes of action will be similar. Thus Gillespie et al. [19] have demonstrated that the early Xenopus embryo has highaffinity binding sites for t25I-acidic FGF and that their temporal pattern of expression matches the period over which animal pole cells are competent to respond to the factor. Although FGF receptor density is highest in the equatorial region of the embryo, from which the mesoderm is formed, the. receptor is present at significant levels in all parts of the blastula. Initially, this observation appeared slightly surprising, because vegetal pole cells do not form mesoderm in response to mesoderm-inducing factors (Smith, unpublished observations). However, in view of the results of Rosa [18], cited above, it seems possible that by the time of assay vegetal pole cells will already have responded to an endogenous inducing factor by becoming 'vegetal', and that additional mesoderminducing signals merely reinforce this developmental decision. Although little is known about the signal tmnsduction pathways for mesoderm-inducing factors, some indirect evidence suggests that inositol lipid metabolism is involved. This evidence originated with the observation of Kao et aL (Nature 1986, 322:371-373) that brief treat-
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1066 Cell differentiation ment of 32-ceU stage Xenoptts embryos with LiCI has a striking anteriorizing effect, such that the whole tadpole becomes 'head-heavy' (see also [20]). The phenotype of embryos such as this could be interpreted as being due to an increase in the extent of dorsal mesoderm induction, because Kao and Elinson [21] later showed that the entire equatorial region of lithium.treated embryos had acquired organizer activity. This could occur either through increased production of factor or by increased responsiveness of animal hemisphere cells. Experiments by Slack et al. [22] and Cooke et al. [23], with FGF and XTC-MIF, respectively, favour the latter possibility, because lithium increases the sensitivity of responding cells to both these factors although it has no mesoderminducing activity itself. The best-understood action of lithium on cell signalling events is its effect on inositol lipid metabolism (see Drummond, Trends Pharmacol Sci 1987, 8:129-133), where it blocks the recycling of inositol phosphates to m)~inositol and therefore indirectly depletes levels of phosphatidylinositol (PI) bisphosphate. This is significant because on receipt of an extracellular stimulus PI bisphosphate is hydrolysed to diacytglycerol and inositol trisophosphate, which, respectively, stimulate protein kinase C activity and release of intraceUular calcium. Data indicating that the head-heavy embryos resulting from lithium treatment are indeed caused through lack of m)~ inositol come from Busa and Gimlich [24], who were able to 'rescue' lithium-treated embryos by injecting m.~ inositol into Xenopus blastomeres at the 32-cell stage. Treatment of embryos with phorbol myristate acetate, a functional analogue of diacylglycerol, also abolished the lithium effect [24]. At first sight these biochemical data would seem to be at odds with the suggestion that lithium enhances the response to mesoderm-inducing factors, because lack of m.)ginositol, and thus of PI bisphosphate, would be expected to depress the response to an external signal rather than enhance it. In addition, since XTC-MIF and bFGF appear to have different effects on responding cells, it is slightly unexpected that they should share a second messenger pathway, as implied by the resuits of Slack et aL [22] and Cooke et aZ [23]. Clearly, more work needs to be done here: in particular, it will be necessary to measure levels of PI cycle intermediates in different regions of both normal and lithium-treated embryos. It is worth noting, for example, that in GH3 cells lithium causes an agonist-induced elevation of diacylglycerol and thus of protein kinase C (Drummond and Raeburn, BiochemJ 1984, 224:129-136). A different approach to studying second messenger pathways in mesoderm induction was taken by Whitman and Melton [25], who showed that animal caps dissected from embryos which had received injections of polyoma virus middle T mRNA would form mesoderm in the absence of exogenous signals. The ability of middle T to transform fibroblasts is believed to be associated with its interactions with two cellular tyrosine kinases, pp60 c-src and pp62C--~% and, interestingly, with its interaction with a PI(3) ldnase. The role of PI(3) phosphate in cellular
signalling is not yet known, although it could act as a regulator of PI remover (Whitman et aL, Nature 1988, 332:644-446). Furore work should address which, if any, of these signalling pathways is activated during mesoderm induction in vivo.
Dorsalization Dorsalization is the least studied and least understood inductive interaction in early amphibian development. One hopeful approach to the problem, however, comes from experiments in which animal pole explants exposed to mesoderm-inducing factors are tested for Spemann organizer activity: that is, for the ability to 'dorsalize' adjacent ventral mesoderm. In one series of experiments (Cooke et aL, Development 1987, 101:893-908) [26], animal pole tissue induced by XTC-MIF was grafted to the ventral region of host embryos and shown to be capable of inducing almost complete secondary axes; bFGF-induced tissue tested in the same way lacked organizer activity. This result is important firstly because it offers a way to search for a dorsalizing signal through differential screening of cDNA libraries, and, secondly, because it provides further evidence that XTC-MIF induces dorsal mesoderm and bFGF ventral. However, slightly different results, albeit using a different assay, were obtained by Ruiz i AItalba and Melton [27]. They tested for organizer activity by implanting induced animal pole tissue into the blastocoels of host embryos. Consistent with Cooke's results [26], blastula ectoderm treated with XTC-MIF induced anterior structures and sometimes complete secondary axes. However, rather than lacking organizer activity, ectoderm treated with bFGF tended to induce posterior structures. The reason for the difference between the two laboratories is unclear, although it probably relates to the design of the assays. However, both sets of results clearly demonstrate that mesoderm induced by XTC-MIF differs from that induced by bFGF, and the data from Ruiz i Altalba and Melton [27] provide an interesting introduction to the question of antero-posterior patterning in Xenopu~
Antero-posterior pattern formation Antero-posterior pattern formation in the amphibian embryo first occurs in the mesoderm, and, until recently, analysis of this process has been hampered through lack of markers; although Sharpe et aL (Cell 1987, 50:749--758) have shown that expression of the homeobox-containing gene XIHBox 6is restricted to the posterior region of the neural plate, this is a late and rather indirect indicator of mesodermal position. Now, important work by Ruiz i Altalba and Melton [27-29] has shown that a Xenopus gene containing a homeobox of the even-skipped class, called Xbox3, provides a suitable antero-posterior mesodermal marker and may even be a determinant.
Induction and early amphibian development Smith 1067 )doox3 is expressed during m'o phases of Xenopus development, the first during gastmla and neumla stages and the second at tailbud and tadpole stages. Most work has concentrated on the first period of expression, when Xhox3 transcripts are found in a graded distribution along the antero-posterior axis of the mesoderm, with the highest concentration at the posterior end [28]. Two lines of evidence indicate that Xbox3 is involved in antero-posterior pattern. Firstly [28], levels of Xbox3 are lowered in embryos treated with LiCl,which 'anteriorizes' them, and elevated in embryos treated with ultraviolet light at the one-cell stage, which 'posteriorizes' them (see Scharf and Gerhart, Dev Biol 1980, 79:191-198; Cooke and Smith, Development 1987, 99:197-210). Secondly [29], microinjection of Xtoox3, but not control, RNA into prospective anterior regions of developing embryos produces embryos which fail to form anterior structures. The most recent work by Ruiz i Altalba and Melton [27] has shown that Xtoox-3 is activated, as might be expected for a mesoderm-specific gene, by mesoderm-inducing factors. Interestingly, however, it is activated to much higher levels by bFGF than by XTC-MIF. This indicates that XTC-MIF induces mesoderm that is more anterior in nature than does bFGF. This conclusion adds to, rather than contradicts, earlier data indicating that the two factors induce, respectively, dorsal and ventral tissue. Thus XTC-bIIF should be regarded as inducing antero-dorsal mesoderm and bFGF postero-ventral, a conclusion consistent with the normal fates of the 'dorsal' and 'ventral' halves of the early gastmla (Dale and Slack, Development 1987, 99:527-551). One intriguing observation made by Ruiz i Altalba and Melton [27] is that high concentrations of XTC-MIF induce less Xbox3 than lower concentrations. This implies, firstly, that higher concentrations of XTC-MIF induce more anterior structures. This is consistent with the observation that higher concentrations of XTC-MIF induce more dorsal tissue [4] (Green et at., in press). Of equal interest, however, is the bell-shaped response curve of )¢7oox3 to XTC-MIF that is implied by these results. Since XToox3 is activated very rapidly in response to XTCMIF and bFGF it may be that this shape of curve is intrinsic to the early response mechanism. Alternatively, the initial activation of Xbox3, which occurs at the mid-blastula stage, may not be specific to a particular antero-posterior position, but may be modulated during gastmlation, as antero-posterior position is established. This behaviour would be analogous to that of pair-role genes in Drosophila such as hairy, fushi-tarazu and even-sk~ed (Akam, Development 1987, 101:1-22; Ingham, Nature 1988, 355:25-34). Clearly, much remains to be done in coming to understanding how antero-posterior position is assigned during development. However, markers such as Xtoox3 should be of great use in approaching the problem.
Neural induction The final inductive interaction described in Fig. 2 is neural induction, during which a signal from dorsal mesoderm acts on overlying ectoderm to induce the nervous system. Recent evidence is confirming the original views of Spemann and of Nietxwkoop et al. (J Exp Zool 1952, 120:1-108) that neural induction involves at least two steps, although the precise nature and outcome of these steps have not been determined. The first recent indication that two steps might be involved came from work by Sharpe et at (1987), who found that axial mesoderm induced the expression of a posterior nervous system marker, XlHbox6,, more readily in dorsal ectoderm than in ventral ectoderm. An analogous conclusion was reached by London et at. [30] with respect to an epidermis-specific antigen, EpH, whose expression in normal development is complementary to that of neural markers. London etal. [30] found that even at the eight-cell stage, but particularly after the onset of gastrulation, isolated ventral animal pole blastomeres go on to express Epil very strongly, but dorsal blastomeres do so only weakly. One interpretation of these observations is that signals acting early in development predispose dorsal ectoderm to neural development and divert it from epidermal differentiation. One such signal was recently demonstrated by Savage and Phillips [31]: the signal is derived from the dorsal lip of the blastopore, travels through the plane of the ectoderm, and suppresses expression of Epil. Dixon and Kintner [32] have investigated the signals required for neural induction by using the neural markers neural cell adhesion molecule (NCAM; Kintner and Melton, Development 1987, 99:311-325) and NF-3 (Charnas et at, Soc Neurosci Abstract 1987, 450:15). Like Savage and Phillips [31] they show that one component of the neural induction signal is derived from tissue near the dorsal lip of the blastopore and that this signal travels in the plane of the ectoderm. However, they also show that the signal acts synergistically with a later stimulus provided by involuting dorsal mesoderm, and that together the two signals are sufficient to induce NCAM and NF-3 almost to the levels found in normal embryos. Interestingly, the second stimulus has very little effect in the absence of the first, and this correlates well with the result of Otte et at [33] described below. The idea that two signals are required for neural induction may explain the otherwise perplexing results of Sato and Sargent [34]. These authors dispersed the blastomeres of whole Xenoptts embryos between the early blastula and early gastrula stages. This procedure prevents mesoderm induction from occurring, and muscle and other mesodermal tissues do not differentiate (Sargent et at, Dev Biol 1986, 114:238-246) [15]. However, despite the absence of mesodermal tissues a neural marker, NCAM, was expressed to a high level. This re-
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Cell differentiation suit might be explained if the dispersion regime removed only the second signal of Dixon and Kintner [32]; the first signal, perhaps derived from dorsal vegetal tissue without the need for induction, might still be produced and be sufficient to induce expression of NCAbl.
Transduction of neural-inducing stimuli The search for neural-inducing factors (NIFs) has not met with the same success as the search for mesoderm-inducing factors. One reason for this may be, of course, that neural induction requires two signals and screening single factors would therefore be ineffective. One may speculate, however, that the first of Dixon and Kintner's [32] signals is related to a dorsal mesoderm-inducing factor, and indeed XTC-MIF is known to induce abundant neural tissue in addition to mesoderm [4] (Green et al., in press). It is possible that XTC-MIF provides one neuralinducing signal directly and a second, through the induction of dorsal mesoderm, indirectly. While the search for NIFs continues, progress has been made in coming to understand the second messenger pathways that the NIFs must use. Otte et al. [35] have measured protein kinase C (PKC) activity in Xenopus presumptive neural plate at the early gastmla stage and at the end of gastmlation, after induction has occurred. They found that membrane-bound PKC activity increased significantly in the induced tissue while the cytoplasmic activity decreased (see also [36]). This suggested that translocation of PKC activity might be involved in neural induction. To confirm this the authors treated early gastmla ectoderm with 12-o-tetradecanoytphorbol13-acetate (TPA), a potent activator of PKC. TPA indeed causes a translocation of PKC activity from the cytosol to the membrane fraction and, as has also been shown in urodele embryos (Davids et at, Rouxs Arch D e v Biol 1987, 196:137-140), caused neural induction to occur. More recent work by Otte et al. [33] has demonstrated that neural induction by TPA is enhanced by subsequent treatment with cyclic adenosine monophosphate (cAMP) analogues, although by themselves cAMP analogues have very little effect. These results are immediately reminiscent of those of Dixon and Kinmer [32], and suggest that the first signal proposed by these authors on embryological grounds acts through PKC and the second through the cAMP pathway.
verted to anterior-specified neural tissue. This might occur through one or both of the two signals proposed by Dixon and Kinmer [32] and Otte et al. [33]. Then, during 'neural transformation', anterior neural tissue would progressively be specified as being more posterior. Recent work by Sive et al. [37] supports this model. The cement gland is the most anterior specialization of the ectoderm in X e n o p u s and, like neural tissue (which is located just posterior to the cement gland), arises through induction (see also Jamrich and Sato, Development 1989, 105:779-786). Interestingly, the first tissue in the embryo to be specified as cement g l a n d - - that is, to form cement gland if it is isolated from the embryo - - is actually fated to become neural plate in normal development. This implies that the initial anterior specification as cement gland is progressively superseded by more posterior fates. One candidate for a posteriorizing factor is retinoic acid, a molecule also believed to act as a morphogen in, for example, the developing chick limb (Thaller and Eichele, N a t u r e 1987, 327:625-628). Durston et al. [38] have shown that retinoic acid converts anterior neural tissue to posterior, and that the Xenoptts embryo contains the appropriate levels of this retinoid.
Conclusions The period reviewed in this article has seen dramatic advances in our understanding of inductive interactions in amphibian development. The questions that remain will clearly be difficult to solve, but for the first time one can pose the questions at the molecular level in a meaningful way. The next year should see further significant advances.
Acknowledgements I am grateful to Jeremy Green, Coston Guex and James Howard for their helpful comments.
Annotated references and recommended reading • ••
Regionalization of the nervous system Although markers for different positions along the antero-posterior axis of the Xenopus nervous system are becoming available (such as XlHbox6. Sharpe et al., 1987), little is known about how antero-posterior regionalization occurs. Work by Nietrwkoop et al. (1952) had suggested that regionalization is a distinct step in neural induction. First, competent ectoderm would be con-
Of interest Of outstanding interest
1. e
SMrrH JC: M e s o d e r m induction and m e s o d e r m - i n d u c i n g factot's in early amphibian development. Development 1989, 105:665-677. A recent r ~ i e w describing work on mesoderm induction in more detail than is possible here. 2. •
PIERCEKE, BROTHERSAJ: Dorsal and ventral cells of cleavagestage Xenopus e m b r y o s s h o w t h e same ability to ind u c e n o t o c h o r d a n d somite formation. Dev Biol 1988, 126:228-232. Describes the surprising result that ventral ~getal blastomeres induce notochord and muscle ~ith the same frequency as dorsal vegetal ceils.
Induction and early a m p h i b i a n d e v e l o p m e n t Smith 3-
ROSA F, ROBERTS AB, DANIELPOURD, DART LL, SPORN /~B, DAWIDIB: Mesoderm induction in amphibians: the role of TGF-[32-1ike factors. Science 1988, 239:783-785. TGF-132, but not TGF-131,is effec~'e as a mesoderm inducer in Xenopux Conditioned medium from the XTC cell lJne has TGFI~ activity, and its inducing actMty can be blocked by antibodies to TGF.132 but not to TGF-13t. The paper shows that a member of the TGF-13 family can induce mesoderm in the absence of other factors.
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4. ••
SMrI'HJC, YAQOOB M, SYMF.SK: Purification, partial characterization and biological effects of the XTC mesoderm-inducing factor. Development 1988, 103:591-600. Purified XTC mesoderm-inducing factor has properties in common with TGF-13. High concentrations of factor induce dorsal mesoderm, including notochord, and lower concentrations induce ventral mesoderm. 5. ••
KL~mLMAN D, ABRAHAMJA, HA.M'ARANTAT, PAL1SITM, KIP.SCHNER MW: The presence of fibroblast growth factor in the frog egg: its role as a natural mesoderm inducer. Science 1988, 242:1053-1056. A cDNA done encoding the entire Xenopus bFGF protein gas isolated. When expressed in E colt; Xenopus bFGF was ac~'e as a mesoderm inducer. Xenopus embl3"os were shog~ to contain FGF in sufficient amounts to act as a mesoderm inducer. 6. •
VOLK R, KOSTERM, POTLNGA, HARTMAN~I~ KNOCHELW: An antisense transcript from the Xenopus laevis bFGF gene coding for an evolutionary conserved 24 kD protein. EMBO J 1989, 8:2983-2988. A l k b cDNA pr~'iously identified by Kimelman and Kirschner (Cell 1987, 51:869-877) and thought to represent an unspliced fragment of bFGF pre-mRNA actually encodes an unkno~aa 24 kD protein. The mRNA has partial bFGF sequences in the 3' untranslated region, but in the anti.sense orientation.
7.
SLACKJMW, ISAACSI-IV: Presence of basic fibroblast growth factor in the early Xenopus embryo. Development 1989, 105:147-153. Mesoderm-inducing actMty extracted from Xenopus ovary, eggs and embryos was identified as bFGF. There is sut~cient bFGF for the molecule to act as a natural mesoderm inducer. Like [5] this article demonstrates that a puta~'e inducing factor is present in the Xenopus embt3~o. oo
8. •
DALE L, ~t~TI'ItEWS G, TABE L, CO,tAN A: Developmental expression of the protein product of Vgl, a localized maternal mRNA in the frog Xenopus laevtg FAIBOJ 1989, 8:1057-1065. Vgl mRNA is translated in the Xenopus oocyte and embryo to form 45 and 43.5 kD proteins, which ate glycosylated forms of a 40 kD protein. Synthesis is restricted to the vegetal hemisphere but both proteins dif. fuse into the animal hemisphere. See also [9]. 9. •
TANNAHILLD, M.ELTON D& Localized synthesis of the Vgl protein during early Xenopus development. Development 1989, 106:775-785. Vgl mRNA is translated in Xenopus oocytes, eggs and early embt)~3s. There are 2 glycosytated forms, 1 of 44 kD together with a less abundant 42 kD form. Diffusion of the larger form into the animal hemisphere seems to be restricted. Processing of the Vgl protein into a 17 kD form is only detected if Vgl is over-expressed. PONDEL/~D, KING ~L!2 Localized maternal mRNA related to transforming growth factor 13 mRNA is concentrated in a cytokeratin-enriched fraction from Xenopus oocytes. Proc Natl Acad Sci USA 1988, 85:7612-7616. Vgl ~ unlike histone It3 mRNA, assodates with elements of the cytoskeleton. This association may be linked vdth the translocation of the message to the vegetal pole. 10. •
11. •0
YISRAELI JK, ~LTON DA: The maternal mRNA Vgl is correctly localized foUowing injection into Xenopus oocytes. Nature 1988, 336:592-595. Vgl mRNA is translocated to the vegetal pole after being microinjected into Xenopus ooeytes. Translocation does not require protein synthesis. The ~ork shows that naked RNAcan be recognized by the translocation
machinery of the oo~te, making it possible to map the tmuslocation recognition sequences. 12. •
GODSAVESF, ISAACS HV, SLACKJMW: Mesoderm.inducing factors: a small class of molecules. Development 1988, 102:555-556. Mesoderm-indudng factors from a variety of sources were sho~-n to have similar properties. 13.
COOKE J, S.~mH JC: Gastrulation and larval pattern in Xenopus after blastocoelic injection of a Xenopu~derived inducing factor: experiments testing models for the normal organization of mesoderm. Dev Biol 1989, 131:383--400. The effects of injecting me.soderm-inducing factors into the blastocoels of Xenopus embr)x3s ate described. Gastmhtion-like movements in. duced by XTC-MIF occur about 1.5 h earlier than those induced by bFGF.
•
HOPWOODNO, PLUCKA, GURDONJB: MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. FAIBOJ 1989, 11:3409-3417. M)~D represents an early marker of muscle differentiation in Xenopux 14. •
15.
SYMESK, YAQOOB M, SMITH JC: Mesoderm induction in Xenopus laev& responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells. Development 1988, 104:609-618. This paper and [16] suggest that contact is required bet~x'~en animal pole cells ff they ate to respond to induction by forming mesc~erm. This paper also shows that suppression of epidermal differentiation can occur in single ceils in the absence of cell dMsion.
•
16. Gt/m~N JB: Cell movements and a community effect in • tissue morphogenesis. Nature 1988, 336:772-774. See [15]. The requirement for cell contact is referred to as the 'community effect'. 17. GODSAVESF, STACKJMW: Clonal analysis of mesoderm in• duction in Xenopus laevt~ Dev Biol 1989, 134:486--490. Single animal pole cells exposed to bFGF go on to differentiate as mesodermal cell types. ROSAFM: Mio¢l, a homeobox mRNA inducible by mesodcrm inducers, is expressed mostly in the presumptive endoderreal cells of Xenopus embryos. Cell 1989, 57:965-974. M/x:/, a homeobox-containing gene, is activated in animal pole cells in response to XTC-MIF. M/x:1 transcripts in normal embryos are expressed mostly in presumptive endoderm. The first 'immediate early' genetic response to induction to be discovered. 18. ••
19. •
Gna.ESt'rELL, P^~RNO GD, SLACKJMW: Analysis of cornpetence: receptors for fibroblast growth factor in early Xenopus embryos. Development 1989, 106:203-208. Provides spatial and temporal analyses of FGF receptor distribution in the early Xenopus embryo. 20. •
COOKEJ, SMrrH EJ: The restrictive effect of early exposure to lithium upon body pattern in Xenopus development, studied by quantitative anatomy and immunofluorescence Development 1988, 102:85-99. A detailed anatomical and histological description of dorso-antetior enhanced Xenopus embryos resulting from LiCI treatmenL 21. •
KAO KR, ELLNSONRP: The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos. Dev Biol 1988, 127:64-77. All regions of the mesoderm of LiCl-treated dorso-anterior enhanced Xenopus embryos ha~e organizer activity. 22. •
SLACKJMW, ISAACSI-W, DAm.~GTON BG: Inductive effects of libroblast growth factor and lithium ion on Xenopus blastula ectoderm. Development 1988, I03:581-590. LiCI has no mesoderm-inducing actMty alone, but acts s)~ergistically with fibroblast growth factor, such that muscle formation is enhanced. See also [23]. 23. •
COOK~J, SY~mSK, S.~m'HEJ: Potentiation by the lithium ion of morphogenetic responses to a Xenopus inducing factor. Development 1989, 105:549-558.
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Cell differentiation LiCI acts synergistically with XTC-MIF. See also [22]. 24. •
BUSAWB, GIML/Ctl RL: Lithium-induced teratogenesis in flog embryos prevented by a polyphosphoinositide q-cle intermediate or a diacylglycerol analog. Dev Biol 1989, 132:315-324. Formation of dorso-amerior enhanced Xenopus embryos after injection of HC! is pre'vented by co-injection of m)~inositol or by subsequent treatment with phorbol myristate acetate.
WHITMANM, MELTON DA: Induction of mesoderm by a viral oncogene in early Xenopus embryos. Science 1989, 244:803--806. Microinjection of middle T mRNA into early embryos causes isolated ectodermal tissue to form mesoderm. In frog embwos, middle T associates with cellular activities similar to those observed in pob'omatransformed mouse cells. The results offer a powerful route to stud)~ng second messenger pathv~)~ in mesoderm induction. 25. eo
26. ••
COOKEJ: Mesoderm.inducing factors and Spemann's organizer phenomenon in amphibian development. Development 1989, 107:229--241. Animal pole cells exposed to XTC-MIF,but not to bFGF, are effective as Spernann organizers. The completeness of the secondary axis induced by XTC-MIF.treated tissue is set by graft size and factor concentration. Provides further information about the specification of the antero-posterior axis. 27. ••
Rum I ALTA1BA A, MELTONDA: Interaction between peptide growth factors and homeobox genes in the estabIishment of antero-posterior polarity in frog embryos. Nature 1989, 341:33-38. XT.r~x3 is induced to high levels by bFGF and lower levels by XTCMIF. Animal pole explants treated vdth XTC-MIF induces anterior duplications when implanted into the blastocoels of host embryos and FGF-treated explants induce posterior duplications. The first molecular anab~is of antero-posterior pattern formation in Xenopu,~ RUm I ALTALBA A, MELTONDA: Bimodal and graded expression of the Xenopus homeobox gene X h o x 3 during embryonic development. Development 1989, 106:173-183. XT.xgx3is expressed during 2 periods of Xenopus development: during gastrula stages and during tailbud stages. During gastrula stages Xlmx3 is expressed in a gradient, ~ith low l~els anteriorly and high levels posteriorly. 28. •
31. •
SAVAGER, PtnLHPS CR: Signals from the dorsal blastopore lip region during gastrulation bias the ectoderm toward a nonepidermal pathway of differentiation in Xenopus laevtx Dev Biol 1989, 133:157-I68. The blastopore lip region inhibits expression of Epil in dorsal ectoderm. The signal travels through the plane of the ectoderm sheet (see [32]). 32. •
DIXONJE, K2Xq'~'ERCPc Cellular contacts required for neural induction in Xenopus embryos: evidence for two signals. Development 1989, 106:749-757. 2 signals ~ere found to be responsible for neural induction. The first, and most effective, travels through the plane of the ectoderm from the blastopore lip region. The second is derived from in~oluting dorsM mesoderm, and acts s3aaergisticallyxxith the first. 33.
OTTEAP, VAN RUN P, HE1DEVELDM, VAN DR/El. P,, DUF:S'I'ON AJ: Neural induction is mediated by cross.talk between the protein kinase C and q'clic AMP pathways. Cell 1989, 58:641-648. Adenyl c3"claseacuity and cAMP levels increase after neural induction. Although cA_MPanalogues have no neural inducing activity they act ~aaergistically ~Sth TPA, an activator of PKC. IAke [32], the results imply that neural induction consists of ~'o steps, one actix-atedby PKC and one acting through the c_AMPpathx~'ay. e e
8ATO SM, S.M~.GENT TO: Development of neural inducing capacity in dissociated Xenopus embryos. Dev Biol 1989, 134:263-266. When Xenopus embryos are dispersedbem-een early clea~uge and early gastmla stages mesoderm induction is prevented, but this does not interfere with neural differentiation. 34. •
35. •
OTI'EAP, KOSTERCH, SNOEKGT, DUK~ON AJ: Protein kinase C mediates neural induction in Xenopus laevts, Nature 1988, 334:618-620. PKC actMty is translocated from cytosol to membrane fractions during neural induction. TPA, an activator of PKC, causes neural induction.
36. •
DAVIDSM: Protein kinases in amphibian ectoderm induced for neural differentiation. Rou.xs Arch Dev Biol 1988, 197:339-344. PKC actMty increases in Xenopus gastmla ectoderm treated ~ith a crude neuraLinducing factor. 37.
SlXX HI, HATrORI K, WEL'Cr~,UB 1t: Progressive determination during formation of the anteroposterior axis in Xenopus laevix Cell 1989, 58:171-180. The first cells in the embryo to be specified to form cement gland eventually form neural plate instead. The results suggest that the anteroposterior axis of Xenopus is determined progressb,'ely,with anterior specification being superseded by posterior. o e
Rum I ALTALBAA, MELTONDA: Involvement of the Xenopus homeobox gene X h o x 3 in pattern formation along the antero-posterior axis. Cell 1989, 57:317-326. Microinjeetion of symhetic AT.~gx3mRNA, but not control RNA, into prospec~-e anterior regions of Xenopus embryos causes anterior defects. The results imply that Xhox3 pla~ a role in specifying antero-posterior posidon in Xenopus 29. e e
38.
DURSTONAJ, TP.IMEIZMANSJP*I, HAGE %V'J,HENDRIKSHFJ, DE VRIESNJ, ttEIDEVELDM, Nml.~l~OOP PD: Retinoic acid causes an anteroposterior transformation in the developing central nervous system. Nature 1989, 340:140-144. Retinoic acid gxs applied to developing Xenopus embtTos, causing transformation of anterior neural structures to posterior. Measurements of retinoic acid levels in the embryo are consistent with the suggestion that the molecule acts as a morphogen. oo
LO,,a~ONC, AKEV, S P,, PHIU.U'S C: Expressions of Epll, an epidermis-specific marker in Xenopus laevis embryos, is specified prior to gastruladon. Dev Biol 1988, 129:380-389. Blastomeres derived from ventral halves of early Xenopus embryos express the epidermal marker Epil strongly; blastomeres from the dorsal half do so only weakly. 30. •
Introduction It has been clear for almost a century that cell-cell interactions play an important role in embryonic development. This conclusion originated largely through experiments by Spemann and his colleagues, most notably with their discovery of the organizer, and Spemann's own description of this work has recently been re-published (Spemann, Embr)~onic Development and Induction. Garland Publishing, 1988). In addition, Hamburger has written a fascinating eyewitness account covering the same period (Hamburger, The Heritage of Experimental EmbD~91ogy. Oxford University Press, 1988). For a field this venerable, it may seem surprising that it is only in the last 2 or 3 years that significant progress has been made in coming to understand the molecular basis of cell-cell interactions during development. As Kay and I have discussed, this is quite simply because the problem is very ditficult and the appropriate techniques were not available [Kay and Smith, Development 1989, (suppl):l-2]. Now that the techniques are available, dramatic progress is being made. This review summarizes what has been achieved during the last 18 months using amphibian embryos. Amphibian embryos offer great advantages to the experimental embryologist in being big, readily available in large numbers, and accessible to manipulation at all developmental stages. In addition, small pieces of early embrYos will survive and differentiate in simple balanced salt solutions, because they can survive on their yolk reserves. Thus putative 'inducing factors' can be tested without risk of interference by poorly defined serum components. The early development of the frog Xenopus laevis is described in Fig. 1. So far very little work has been done to analyse inductive interactions in other vertebrates, such as chickens and mice; the next year, however, should see lessons learnt from the amphibia being applied to the amniotes.
A sequence of interactions
It would be remarkable ff the great complexity of the vertebrate body could arise from a single inductive interac-
tion. In fact, a great deal of experimental embryological work indicates that development consists of a sequence of inductive interactions (see Slack, From Egg to Embr)~. Cambridge University Press, 1983). Experimental embryology is not an exact science, and data are often open to more than one interpretation. Nevertheless, a consensus view, providing a framework on which to discuss recent work, might begin with the 'three-signal model' for mesoderm formation and be followed by neural induction [Smith et eL, J Emboxgl Exp Morpbol 1985, 89 (suppl):317-331]. This scheme is outlined in Fig. 2; it is not possible to describe here all the work on which it is based, but this is reviewed elsewhere [1] (see also Smith,
In Developmental Biology: a Comprehensive Synthesis, Vol 5, edited by Browder. Plenum, 1988, pp 79-125). The first two signals of the three-signal model for mesoderm formation occur during blastula stages, when the fertilized egg has divided to form 64-1024 cells. These signals are produced by cells of the vegetal hemisphere of the embryo, and cause the formation of mesoderm, and perhaps some endoderm, from overlying equatorial and animal hemisphere cells. Two signals are believed to be involved because dorsal and vegetal blastomeres induce quite different types of mesoderm from test animal cap tissue. Dorsal vegetal blastomeres induce predominantly dorsal mesodermal ceil types, such as notochord and muscle, whereas ventral vegetal blastomeres induce predominantly ventral cell types like mesenchyme and blood (reviewed in [1]; for a different view see [2]). As I discuss below, with the recent advent of molecular markers for anterior and posterior mesoderm it may be more appropriate to describe the signals as 'antero-dorsar and 'postero-ventral'. The third signal of the model is produced by newly induced dorsal mesoderm cells, and is responsible for subdividing a large region of general ventral mesoderm into, for example, somite muscle, pronephros, lateral plate mesoderm and blood. This interaction can be demonstrated by juxtaposing extreme ventral mesoderrn with extreme dorsal; the dorsal tissue remains dorsal and continues to differentiate as notochord, while the ventral tissue is 'dorsalized' and forms muscle (Dale and Slack, Development 1987, 100:279-295). 'Dorsalization' is the first interaction to oc-
Abbreviations
bFGr--~basic fibroblast growth factor; c/UVlP--cyclic adenosine monophosphate; cDNA--complementary DNA; r--GF~fibroblast growth factor; NCAM--neural cell adhesion molecule; NF--neurofilament; mRNA--messenger RNA; NIFs--neural inducing factors; PI--phosphatidylinositol; PKC--protein kinase (7; TGF~transforming growth factor;, TPA--12-o-tetradecanoylphorbol-13-acetate;XTC-MIr-~xTC-mesoderm-inducingfactor. (~) Current Science Lid ISSN 0955-0674
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Cell differentiation tissue (Smith, Development 1987, 99:3-14), and by the discovery that members of the fibroblast growth factor (FGF) family had similar properties (Slack et al., Nature 1987, 326:197-200; Kimelman and Kirschner, Cell 1987, 51:869-877). Subsequent work has suggested that the XTC-mesoderm-inducing factor (XTC-MIF) might be related to transforming growth factor (TGF)-[3. Firstly, antibodies against TGF-[32 (but not against TGF-[31) inhibRed ,,, 80% of the activity of XTC-conditioned medium [3]. Secondly, TGF-~2, but not TGF-~I, was found to be active as a mesoderm inducer [3]. And finally, purification of XTC-MtF revealed some properties in common with TGF-[3 [4]. Complete characterization of XTC-MIF has not yet been achieved, but circumstantial evidence suggests that it is not simply Xenopus TGF-[32 (see [4]).
cur in Spemann's organizer graft (Smith and Slack, JEnv bo~ol Exp Morpho11983, 78:299-317). Dorsalization probably occurs just before and during gastrulation. At the same time, different regions of the mesoderm are believed to acquire information about their antero-posterior positions. The mechanism by which this occurs is unknown. Neural induction is also believed to occur during gastmlation, with dorsal mesodermal cells instructing overlying ectoderm to form nervous system, transmitting information about antero-posterior position at the same time. This version of the inductive events occurring during early amphibian development accounts for much of the experimental embryology carried out until the end of 1987. Recent, more molecular, work has supported some of the interpretations but other data will necessitate changes in the models.
Mesoderm induction
Although TGF-~I alone has no mesoderm-inducing activity (Slack et al., 1987; Kimelman and Kirschner, 1987) [3], it has been reported to act synergistically with members of the FGF family (Kimelman and Kirschner, 1987). For reasons that are not yet clear, this result has not been confirmed [3]; the results may depend on the source of TGF-]3].
Mesoderm-inducing factors The recent interest in mesoderm induction in amphibian embryos was inspired in part by the observation that conditioned medium from a Xenopus cell line called XTC could induce mesoderm from isolated animal pole
It is, of course, important to show that mesoderm-inducing factors derived from tissue culture cells or, in the case of FGF, from cows' brains, are present in the Xenoptts embryo at the appropriate stages and in the predicted regions. In the case of FGF, Kimelman and Kirschner
~ .~2~ --. • ~:'A
..
(e)
(f) ~ //(~)
Neural tube .Notochord
~,~
//,.-3Y.k.-~\\ ((/
~
'~I'~
ndoderm~ ~
/
Somite Epidermis
Lateral plate Blood island
Fig. 1. The early development of Xenopus laevis. The Xenopus egg (a) has a diameter of about 1.4 mm. The animal hemisphere, which lies upwards by gravity, is heavily pigmented, and the vegetal hemisphere is pale. Sperm entry occurs in the animal hemisphere, and the side on which the sperm enters becomes the ventral-posterior half of the embryo. Ninety minutes after fertilization a rapid series of cleavage divisions begins. As a result, the embryo forms a hollow ball of cells, the blastula (b; 5 h after fertilization). During gastrulation (c; 11 h after fertilization: viewed from the vegetal hemisphere of the embryo) the three germ layers of the embryo take up their definitive positions, a process completed by the neurula stages, by which time tissue-specific gene activation has started (d; 18 h after fertilization). After formation of the neural tube the embryo elongates (e; 27 h after fertilization). The body plan of the embryo is now complete. (0 Shows the location of the major cell types. (a-e) Adapted from Nieuwkoop and Faber, Normal Table of Xenopus laevis, 2nd edn. North Holland, 1987.
Induction and early amphibian development Smith (1987) were able to isolate an oocyte complementary DNA (cDNA) which contained sequences homologous to exon I1] o f human and bovine basic FGF (bFGF). This clone hybridized to a 1 kb RNA that was present in abundance in the oocyte and in midblastula to neurula stages, but the RNA lacked sequences homologous to the first and second exons of human bFGF. Recently, however, Kimelman and Kirschner and their colleagues [5] have isolated a 4.3 kb cDNA from a neumla library which contains the entire Xenoptts bFGF polypeptide. This transcript, which, like bovine and human bFGF, lacks a classic secretory signal sequence, is present in the oocyte and is then undetectable until early neurula stages. Interestingly, Volk et aL [6] have shown that the
,
Oogenesis
Fertilization
abundant 1 kb transcript described by Kimelman and Kirschner in fact encodes an antisense transcript to part of exon IlI o f bFGF RNA and that an open reading frame in the sense orientation encodes a 24 kD protein which is conserved in humans. The function of this protein is unknown. Kimelman et al. [5], as well as Slack and Isaacs [7], have also shown that bFGF protein is present in the Xenopus egg and early embryo. The m'o groups differ somewhat in their estimates of how much bFGF is present, but both figures are consistent with the factor being a natural inducer of mesoderm. No information is yet available about the spatial distribution of bFGF mRNA and protein.
@
Mesodermal induction Dorsalization
Gastrulation
Neural induction
Fig. 2. The sequence of inductive interactions in early amphibian development. During oogenesis differences arise between the animal CA) and vegetal (V) halves of the egg. These include, for example, the translocation of Vgl messenger RNA to the vegetal pole (see text). Fertilization initiates a 30° rotation of subcortical cytoplasm, in a direction defined by the position of sperm entry. This direction of rotation in turn defines which region of the vegetal hemisphere acquires dorsovegetal (DV) signalling ability and which ventrovegetal (VV). During mesoderm induction the dorsovegetal region induces the organizer (O) from the animal half of the embryo and the ventral region induces a general ventral mesoderm (M) which is subdivided into several regions during dorsalization, under the influence of the organizer. During gastrulation the mesoderm acquires antero-posterior positional values (represented as 01 to 04 for the organizer) and these are transmitted to the overlying ectoderm as part of the neural induction process. This produces neuroepithelium with homologous craniocaudal positional values (N1 to N4) while the uninduced animal pole material becomes epidermis (E).Adapted from Smith ~ci Prog 1985, 69:511-532).
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Cell differentiation No member of the TGF-fl family which is known to have mesoderm-inducing activity has been demonstrated to be present in the early Xenopzcs embryo. One strong candidate for an endogenous inducing factor is, however, the protein product of the localized messenger RNA (mRNA) Vgl (Rebagliati et al., Cell 1985, 48:599--605; Melton, Nature 1987, 328:80-82). Vgl RNA is restricted to the vegetal region of the oocyte and early embryo, and codes for a factor related to TGF-IB (Weeks and Melton, Cell 1987, 51:861--867). The mRNA is translated during oogenesis and early development and is glycosytated [8,9]. Although a less abundant glycosylated form (42 kD) has a roughly uniform distribution within the embryo a more abundant, larger (44 kD), form is restricted to the vegetal hemisphere [9]. By analogy with other members of the TGF-]3 family, this large form would be expected to be processed to yield a smaller (17kD) protein which is secreted as the active factor. Such a protein could not be detected in normal embryos [8,9], although if large amounts of full-length Vgl mRNA were injected into the embryo a little of the 17 kD form became visible [9]. Vgl mRNA is initially distributed uniformly within the developing oocyte (Melton, 1987) and the question of how it becomes localized is an important problem in cell biology. Pondel and King [10] have shown that Vgl mRNA is concentrated in detergent-insoluble extracts of Xenopus oocytes whereas histone H3 mRNA is equally distributed between soluble and insoluble extracts. Thus localization may involve interaction between mRNA and elements of the cytoskeleton. Yisraeli and Melton [11] have recently shown that Vgl mRNA transcribed in vitro is translocated to the vegetal hemisphere after being injected into immature Xenopus oocytes. By deleting parts of the message it should be possible to map the 'vegetal translocation' sequence, which may, perhaps, recognize components of the cytoskeleton. These results are summarized in Table 1, which makes clear how much work remains to be done in characterizing the normal in vivo mesoderm-inducing factors in the amphibian embryo. For members of the FGF family it is necessary to discover if the factors are localized, whether they escape from the cell, and if so how, in the absence of a secretory signal sequence, they do so. For members of the TGF-I3 family, one must discover whether the Vgl protein has mesoderm-inducing activity and whether any of the other members of the family, known to have activity, are present in the embryo. Finally, in order to confirm the role of any of these factors in early development it is necessary to eliminate them from the embryo and observe the effects of their absence. This may be difficult to achieve using antisense RNA techniques because synthesis of bFGF and Vgl protein occurs through a long period of oogenesis. Thus sufficient amounts of inducing factors may have been synthesized well before experimental intervention is possible. Biological effects of mesoderm-inducing factors Descriptions of the effects of XTC-MIF and bFGF suggest that the former resembles the dorsal mesoderm-induc-
ing signal of the three-signal model (Fig. 2) and FGF the ventral. The main evidence cited is simply that at high concentrations XTC-MIF induces notochord and neural tissue with reasonable frequency, whereas FGF does so only" very rarely [4,12] (Green et al., Development, in press). However, the observation that lower concentrations of XTC-MIF (0.2-1.0 ng/ml) have effects on the differentiation of animal pole cells very similar to those of bFGF [4] (Green et al., in press) might suggest that the difference between the two factors is quantitative rather than qualitative. Further evidence that the two factors do induce qualitatively different types of mesoderm comes from studying the timing of the gastmlation movements induced° by the two factors. Symes and Smith (Develop ment 1987, 101:339-349) demonstrated that XTC-MIF induces isolated animal pole tissue to undergo gastrutationlike movements, and that the time of onset of these movements coincided with the time at which the dorsal lip of the blastopore appeared in sibling embryos, irrespective of the stage at which they had been exposed to the factor. FGF also induces gastrulation-like movements but these are less dramatic than those induced by XTC-MIF and it is harder to determine their time of onset. However, when inducing factors are injected into the blastocoels of host embryos, thus converting the whole animal hemisphere to mesoderm, it is much easier to time the onset of ectopic gastrulation behaviour, and it is clear that gastrulation induced by bFGF occurs about 1.5 h later than that induced by XTC-MIF, and at a time corresponding to the formation of the ventral lip of the blastopore [13]. Importantly, this time does not depend on the concentration of either factor, indicating that the difference is qualitative rather than merely quantitative. A final piece of evidence regarding the identity of the dorsal and ventral mesoderm-inducing signals is considered below, under 'dorsalization'. Early responses to mesoderm induction The different tissues formed in response to mesoderm induction are usually assessed by histological analysis some days after treatment with inducing factors (see, for example, [4]). Alternatively, if one is interested in the expression of specific genes such as muscle, one can carry out RNase protection assays to detect specific responses within 5-7h (Gurdon et al., Cell 1985, 41:913-922). Using earlier muscle-specific marker genes such as M_)~D [14] it is possible to measure responses even sooner than this. But is the expression of genes specific to a certain mesodermal cell type a direct response to inducing factors or is this a somewhat later event resulting from intervening cell interactions? Attempts to answer this question have used XTC-MIF, FGF and the natural inducer from vegetal pole cells, and the answer is not yet clear. Symes et aL [15] exposed dispersed Xenopus blastomeres to XTC-MIF before reaggregating them at the early-to-mid gastrula stage, by which time they had lost competence to respond to the factor (Green et aL, in press). Although the dispersed cells were able to respond to XTC-MIF in the sense that they suppressed epidermal differentiation they did not form muscle, suggesting that some inter-
Induction and early amphibian development Smith
Table 1. Mesoderm-inducing factors in Xenopus. Present in early embryo? Factor
Family
Active?
RNA
Protein
Localized?
Vgl °
TGF-~ TGF-I] TGF-]] TGF-~ FGF FGF FGF FGF FGF
Unknown Yes With FGF Yes Yes Yes Yes Yes Yes
Yes Unknown Unknown Unknown Unknown Yes Unknown Unknown Unknown
Yes Unknown Unknown Unknown No Yes Unknown Unknown Unknown
Yes Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
XTC-MIFt TGF-~I:I: TGF-I]2§ aFGF~ bFGF¶ ECDGF** INT-2tt kFGFtt
"Rebagliati et aL (Ceil 1985, 48:599-605); Weeks and Melton (Cell 1987, 51:861-867); Melton (Nature 1987, 328:80-82) [8,9,11]; 1"Smith (Development 1987, 99:3-14) [3,4]; :[:Kimelman and Kirschner (Cell 1987, 51: 869-877); §13]; ,[Slack et aL (Nature 19/37, 326:197-200) [7,22]; ¶Slack et al. {1987); Kimelman and Kirschner (1987) [5,7]; *'Slack et al. (1987}; HPaterno et al. (Development 1989, 106:79-83).
cellular communication might be required during induction to activate mesoderm-specific genes. In a conceptually similar experiment Gurdon [16] exposed animal pole blastomeres to vegetal tissue under conditions in which the animal cells would not divide or contact each other. Again, muscle differentiation did not occur. In control experiments, where animal pole cells were allowed to contact each other, muscle was formed. A different experimental design and a different inducing factor gave different results for Godsave and Slack [17]. Here, single animal pole cells were exposed to FGF and then cultured on a substrate coated with laminin and fibronectin in a medium containing 2 mg/ml y-globulin. The single cells divided to give clones of eight to 100 cells and in many cases mesoderm differentiation occurred. The authors suggest that provided the correct factors are supplied, single cells are capable of responding to mesoderm induction by forming mesodermal cell types. It is also possible, however, that even in clones as small as eight cells sufficient cell-cell contact occurs to provide the necessary secondary interactions. A more direct approach to studying the early response to induction was undertaken by Rosa [18]. This most important work has discovered a homeobox-containing gene M ~ 1, which is activated in response to XTC-MIF. It is not detectably activated in response to FGF and this provides another piece of evidence that these two factors have qualitatively different effects. Three results indicate that activation of M/x 1 is an immediate early response to induction. First, Mix.1 mRNA is detectable within 30min of treatment of animal pole regions with XTC-MIF; second, the gene is activated in the absence of protein synthesis, and, finally, the gene is activated in dispersed cells. Perhaps the most remarkable aspect of the work is that during normal development Mix. 1 transcripts are expressed throughout the vegetal hemisphere of the blastula and early gastrula, in a region which will become
prospective endoderm and only part of the future mesoderm. This suggests that the initial response to XTC-MIF and perhaps other mesoderm-inducing factors is not to form mesodermal cell types but to become 'vegetal', and that formation of, for example, large amounts of muscle in explants exposed to XTC-MIF depends upon further interactions between induced cells. These interactions may themselves be initiated by the product of the Mix. 1 gene.
Transduction of the mesoderm induction signal Since mesoderm-inducing factors resemble mammalian growth factors, it seems probable that their modes of action will be similar. Thus Gillespie et al. [19] have demonstrated that the early Xenopus embryo has highaffinity binding sites for t25I-acidic FGF and that their temporal pattern of expression matches the period over which animal pole cells are competent to respond to the factor. Although FGF receptor density is highest in the equatorial region of the embryo, from which the mesoderm is formed, the. receptor is present at significant levels in all parts of the blastula. Initially, this observation appeared slightly surprising, because vegetal pole cells do not form mesoderm in response to mesoderm-inducing factors (Smith, unpublished observations). However, in view of the results of Rosa [18], cited above, it seems possible that by the time of assay vegetal pole cells will already have responded to an endogenous inducing factor by becoming 'vegetal', and that additional mesoderminducing signals merely reinforce this developmental decision. Although little is known about the signal tmnsduction pathways for mesoderm-inducing factors, some indirect evidence suggests that inositol lipid metabolism is involved. This evidence originated with the observation of Kao et aL (Nature 1986, 322:371-373) that brief treat-
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1066 Cell differentiation ment of 32-ceU stage Xenoptts embryos with LiCI has a striking anteriorizing effect, such that the whole tadpole becomes 'head-heavy' (see also [20]). The phenotype of embryos such as this could be interpreted as being due to an increase in the extent of dorsal mesoderm induction, because Kao and Elinson [21] later showed that the entire equatorial region of lithium.treated embryos had acquired organizer activity. This could occur either through increased production of factor or by increased responsiveness of animal hemisphere cells. Experiments by Slack et al. [22] and Cooke et al. [23], with FGF and XTC-MIF, respectively, favour the latter possibility, because lithium increases the sensitivity of responding cells to both these factors although it has no mesoderminducing activity itself. The best-understood action of lithium on cell signalling events is its effect on inositol lipid metabolism (see Drummond, Trends Pharmacol Sci 1987, 8:129-133), where it blocks the recycling of inositol phosphates to m)~inositol and therefore indirectly depletes levels of phosphatidylinositol (PI) bisphosphate. This is significant because on receipt of an extracellular stimulus PI bisphosphate is hydrolysed to diacytglycerol and inositol trisophosphate, which, respectively, stimulate protein kinase C activity and release of intraceUular calcium. Data indicating that the head-heavy embryos resulting from lithium treatment are indeed caused through lack of m)~ inositol come from Busa and Gimlich [24], who were able to 'rescue' lithium-treated embryos by injecting m.~ inositol into Xenopus blastomeres at the 32-cell stage. Treatment of embryos with phorbol myristate acetate, a functional analogue of diacylglycerol, also abolished the lithium effect [24]. At first sight these biochemical data would seem to be at odds with the suggestion that lithium enhances the response to mesoderm-inducing factors, because lack of m.)ginositol, and thus of PI bisphosphate, would be expected to depress the response to an external signal rather than enhance it. In addition, since XTC-MIF and bFGF appear to have different effects on responding cells, it is slightly unexpected that they should share a second messenger pathway, as implied by the resuits of Slack et aL [22] and Cooke et aZ [23]. Clearly, more work needs to be done here: in particular, it will be necessary to measure levels of PI cycle intermediates in different regions of both normal and lithium-treated embryos. It is worth noting, for example, that in GH3 cells lithium causes an agonist-induced elevation of diacylglycerol and thus of protein kinase C (Drummond and Raeburn, BiochemJ 1984, 224:129-136). A different approach to studying second messenger pathways in mesoderm induction was taken by Whitman and Melton [25], who showed that animal caps dissected from embryos which had received injections of polyoma virus middle T mRNA would form mesoderm in the absence of exogenous signals. The ability of middle T to transform fibroblasts is believed to be associated with its interactions with two cellular tyrosine kinases, pp60 c-src and pp62C--~% and, interestingly, with its interaction with a PI(3) ldnase. The role of PI(3) phosphate in cellular
signalling is not yet known, although it could act as a regulator of PI remover (Whitman et aL, Nature 1988, 332:644-446). Furore work should address which, if any, of these signalling pathways is activated during mesoderm induction in vivo.
Dorsalization Dorsalization is the least studied and least understood inductive interaction in early amphibian development. One hopeful approach to the problem, however, comes from experiments in which animal pole explants exposed to mesoderm-inducing factors are tested for Spemann organizer activity: that is, for the ability to 'dorsalize' adjacent ventral mesoderm. In one series of experiments (Cooke et aL, Development 1987, 101:893-908) [26], animal pole tissue induced by XTC-MIF was grafted to the ventral region of host embryos and shown to be capable of inducing almost complete secondary axes; bFGF-induced tissue tested in the same way lacked organizer activity. This result is important firstly because it offers a way to search for a dorsalizing signal through differential screening of cDNA libraries, and, secondly, because it provides further evidence that XTC-MIF induces dorsal mesoderm and bFGF ventral. However, slightly different results, albeit using a different assay, were obtained by Ruiz i AItalba and Melton [27]. They tested for organizer activity by implanting induced animal pole tissue into the blastocoels of host embryos. Consistent with Cooke's results [26], blastula ectoderm treated with XTC-MIF induced anterior structures and sometimes complete secondary axes. However, rather than lacking organizer activity, ectoderm treated with bFGF tended to induce posterior structures. The reason for the difference between the two laboratories is unclear, although it probably relates to the design of the assays. However, both sets of results clearly demonstrate that mesoderm induced by XTC-MIF differs from that induced by bFGF, and the data from Ruiz i Altalba and Melton [27] provide an interesting introduction to the question of antero-posterior patterning in Xenopu~
Antero-posterior pattern formation Antero-posterior pattern formation in the amphibian embryo first occurs in the mesoderm, and, until recently, analysis of this process has been hampered through lack of markers; although Sharpe et aL (Cell 1987, 50:749--758) have shown that expression of the homeobox-containing gene XIHBox 6is restricted to the posterior region of the neural plate, this is a late and rather indirect indicator of mesodermal position. Now, important work by Ruiz i Altalba and Melton [27-29] has shown that a Xenopus gene containing a homeobox of the even-skipped class, called Xbox3, provides a suitable antero-posterior mesodermal marker and may even be a determinant.
Induction and early amphibian development Smith 1067 )doox3 is expressed during m'o phases of Xenopus development, the first during gastmla and neumla stages and the second at tailbud and tadpole stages. Most work has concentrated on the first period of expression, when Xhox3 transcripts are found in a graded distribution along the antero-posterior axis of the mesoderm, with the highest concentration at the posterior end [28]. Two lines of evidence indicate that Xbox3 is involved in antero-posterior pattern. Firstly [28], levels of Xbox3 are lowered in embryos treated with LiCl,which 'anteriorizes' them, and elevated in embryos treated with ultraviolet light at the one-cell stage, which 'posteriorizes' them (see Scharf and Gerhart, Dev Biol 1980, 79:191-198; Cooke and Smith, Development 1987, 99:197-210). Secondly [29], microinjection of Xtoox3, but not control, RNA into prospective anterior regions of developing embryos produces embryos which fail to form anterior structures. The most recent work by Ruiz i Altalba and Melton [27] has shown that Xtoox-3 is activated, as might be expected for a mesoderm-specific gene, by mesoderm-inducing factors. Interestingly, however, it is activated to much higher levels by bFGF than by XTC-MIF. This indicates that XTC-MIF induces mesoderm that is more anterior in nature than does bFGF. This conclusion adds to, rather than contradicts, earlier data indicating that the two factors induce, respectively, dorsal and ventral tissue. Thus XTC-bIIF should be regarded as inducing antero-dorsal mesoderm and bFGF postero-ventral, a conclusion consistent with the normal fates of the 'dorsal' and 'ventral' halves of the early gastmla (Dale and Slack, Development 1987, 99:527-551). One intriguing observation made by Ruiz i Altalba and Melton [27] is that high concentrations of XTC-MIF induce less Xbox3 than lower concentrations. This implies, firstly, that higher concentrations of XTC-MIF induce more anterior structures. This is consistent with the observation that higher concentrations of XTC-MIF induce more dorsal tissue [4] (Green et at., in press). Of equal interest, however, is the bell-shaped response curve of )¢7oox3 to XTC-MIF that is implied by these results. Since XToox3 is activated very rapidly in response to XTCMIF and bFGF it may be that this shape of curve is intrinsic to the early response mechanism. Alternatively, the initial activation of Xbox3, which occurs at the mid-blastula stage, may not be specific to a particular antero-posterior position, but may be modulated during gastmlation, as antero-posterior position is established. This behaviour would be analogous to that of pair-role genes in Drosophila such as hairy, fushi-tarazu and even-sk~ed (Akam, Development 1987, 101:1-22; Ingham, Nature 1988, 355:25-34). Clearly, much remains to be done in coming to understanding how antero-posterior position is assigned during development. However, markers such as Xtoox3 should be of great use in approaching the problem.
Neural induction The final inductive interaction described in Fig. 2 is neural induction, during which a signal from dorsal mesoderm acts on overlying ectoderm to induce the nervous system. Recent evidence is confirming the original views of Spemann and of Nietxwkoop et al. (J Exp Zool 1952, 120:1-108) that neural induction involves at least two steps, although the precise nature and outcome of these steps have not been determined. The first recent indication that two steps might be involved came from work by Sharpe et at (1987), who found that axial mesoderm induced the expression of a posterior nervous system marker, XlHbox6,, more readily in dorsal ectoderm than in ventral ectoderm. An analogous conclusion was reached by London et at. [30] with respect to an epidermis-specific antigen, EpH, whose expression in normal development is complementary to that of neural markers. London etal. [30] found that even at the eight-cell stage, but particularly after the onset of gastrulation, isolated ventral animal pole blastomeres go on to express Epil very strongly, but dorsal blastomeres do so only weakly. One interpretation of these observations is that signals acting early in development predispose dorsal ectoderm to neural development and divert it from epidermal differentiation. One such signal was recently demonstrated by Savage and Phillips [31]: the signal is derived from the dorsal lip of the blastopore, travels through the plane of the ectoderm, and suppresses expression of Epil. Dixon and Kintner [32] have investigated the signals required for neural induction by using the neural markers neural cell adhesion molecule (NCAM; Kintner and Melton, Development 1987, 99:311-325) and NF-3 (Charnas et at, Soc Neurosci Abstract 1987, 450:15). Like Savage and Phillips [31] they show that one component of the neural induction signal is derived from tissue near the dorsal lip of the blastopore and that this signal travels in the plane of the ectoderm. However, they also show that the signal acts synergistically with a later stimulus provided by involuting dorsal mesoderm, and that together the two signals are sufficient to induce NCAM and NF-3 almost to the levels found in normal embryos. Interestingly, the second stimulus has very little effect in the absence of the first, and this correlates well with the result of Otte et at [33] described below. The idea that two signals are required for neural induction may explain the otherwise perplexing results of Sato and Sargent [34]. These authors dispersed the blastomeres of whole Xenoptts embryos between the early blastula and early gastrula stages. This procedure prevents mesoderm induction from occurring, and muscle and other mesodermal tissues do not differentiate (Sargent et at, Dev Biol 1986, 114:238-246) [15]. However, despite the absence of mesodermal tissues a neural marker, NCAM, was expressed to a high level. This re-
1068
Cell differentiation suit might be explained if the dispersion regime removed only the second signal of Dixon and Kintner [32]; the first signal, perhaps derived from dorsal vegetal tissue without the need for induction, might still be produced and be sufficient to induce expression of NCAbl.
Transduction of neural-inducing stimuli The search for neural-inducing factors (NIFs) has not met with the same success as the search for mesoderm-inducing factors. One reason for this may be, of course, that neural induction requires two signals and screening single factors would therefore be ineffective. One may speculate, however, that the first of Dixon and Kintner's [32] signals is related to a dorsal mesoderm-inducing factor, and indeed XTC-MIF is known to induce abundant neural tissue in addition to mesoderm [4] (Green et al., in press). It is possible that XTC-MIF provides one neuralinducing signal directly and a second, through the induction of dorsal mesoderm, indirectly. While the search for NIFs continues, progress has been made in coming to understand the second messenger pathways that the NIFs must use. Otte et al. [35] have measured protein kinase C (PKC) activity in Xenopus presumptive neural plate at the early gastmla stage and at the end of gastmlation, after induction has occurred. They found that membrane-bound PKC activity increased significantly in the induced tissue while the cytoplasmic activity decreased (see also [36]). This suggested that translocation of PKC activity might be involved in neural induction. To confirm this the authors treated early gastmla ectoderm with 12-o-tetradecanoytphorbol13-acetate (TPA), a potent activator of PKC. TPA indeed causes a translocation of PKC activity from the cytosol to the membrane fraction and, as has also been shown in urodele embryos (Davids et at, Rouxs Arch D e v Biol 1987, 196:137-140), caused neural induction to occur. More recent work by Otte et al. [33] has demonstrated that neural induction by TPA is enhanced by subsequent treatment with cyclic adenosine monophosphate (cAMP) analogues, although by themselves cAMP analogues have very little effect. These results are immediately reminiscent of those of Dixon and Kinmer [32], and suggest that the first signal proposed by these authors on embryological grounds acts through PKC and the second through the cAMP pathway.
verted to anterior-specified neural tissue. This might occur through one or both of the two signals proposed by Dixon and Kinmer [32] and Otte et al. [33]. Then, during 'neural transformation', anterior neural tissue would progressively be specified as being more posterior. Recent work by Sive et al. [37] supports this model. The cement gland is the most anterior specialization of the ectoderm in X e n o p u s and, like neural tissue (which is located just posterior to the cement gland), arises through induction (see also Jamrich and Sato, Development 1989, 105:779-786). Interestingly, the first tissue in the embryo to be specified as cement g l a n d - - that is, to form cement gland if it is isolated from the embryo - - is actually fated to become neural plate in normal development. This implies that the initial anterior specification as cement gland is progressively superseded by more posterior fates. One candidate for a posteriorizing factor is retinoic acid, a molecule also believed to act as a morphogen in, for example, the developing chick limb (Thaller and Eichele, N a t u r e 1987, 327:625-628). Durston et al. [38] have shown that retinoic acid converts anterior neural tissue to posterior, and that the Xenoptts embryo contains the appropriate levels of this retinoid.
Conclusions The period reviewed in this article has seen dramatic advances in our understanding of inductive interactions in amphibian development. The questions that remain will clearly be difficult to solve, but for the first time one can pose the questions at the molecular level in a meaningful way. The next year should see further significant advances.
Acknowledgements I am grateful to Jeremy Green, Coston Guex and James Howard for their helpful comments.
Annotated references and recommended reading • ••
Regionalization of the nervous system Although markers for different positions along the antero-posterior axis of the Xenopus nervous system are becoming available (such as XlHbox6. Sharpe et al., 1987), little is known about how antero-posterior regionalization occurs. Work by Nietrwkoop et al. (1952) had suggested that regionalization is a distinct step in neural induction. First, competent ectoderm would be con-
Of interest Of outstanding interest
1. e
SMrrH JC: M e s o d e r m induction and m e s o d e r m - i n d u c i n g factot's in early amphibian development. Development 1989, 105:665-677. A recent r ~ i e w describing work on mesoderm induction in more detail than is possible here. 2. •
PIERCEKE, BROTHERSAJ: Dorsal and ventral cells of cleavagestage Xenopus e m b r y o s s h o w t h e same ability to ind u c e n o t o c h o r d a n d somite formation. Dev Biol 1988, 126:228-232. Describes the surprising result that ventral ~getal blastomeres induce notochord and muscle ~ith the same frequency as dorsal vegetal ceils.
Induction and early a m p h i b i a n d e v e l o p m e n t Smith 3-
ROSA F, ROBERTS AB, DANIELPOURD, DART LL, SPORN /~B, DAWIDIB: Mesoderm induction in amphibians: the role of TGF-[32-1ike factors. Science 1988, 239:783-785. TGF-132, but not TGF-131,is effec~'e as a mesoderm inducer in Xenopux Conditioned medium from the XTC cell lJne has TGFI~ activity, and its inducing actMty can be blocked by antibodies to TGF.132 but not to TGF-13t. The paper shows that a member of the TGF-13 family can induce mesoderm in the absence of other factors.
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4. ••
SMrI'HJC, YAQOOB M, SYMF.SK: Purification, partial characterization and biological effects of the XTC mesoderm-inducing factor. Development 1988, 103:591-600. Purified XTC mesoderm-inducing factor has properties in common with TGF-13. High concentrations of factor induce dorsal mesoderm, including notochord, and lower concentrations induce ventral mesoderm. 5. ••
KL~mLMAN D, ABRAHAMJA, HA.M'ARANTAT, PAL1SITM, KIP.SCHNER MW: The presence of fibroblast growth factor in the frog egg: its role as a natural mesoderm inducer. Science 1988, 242:1053-1056. A cDNA done encoding the entire Xenopus bFGF protein gas isolated. When expressed in E colt; Xenopus bFGF was ac~'e as a mesoderm inducer. Xenopus embl3"os were shog~ to contain FGF in sufficient amounts to act as a mesoderm inducer. 6. •
VOLK R, KOSTERM, POTLNGA, HARTMAN~I~ KNOCHELW: An antisense transcript from the Xenopus laevis bFGF gene coding for an evolutionary conserved 24 kD protein. EMBO J 1989, 8:2983-2988. A l k b cDNA pr~'iously identified by Kimelman and Kirschner (Cell 1987, 51:869-877) and thought to represent an unspliced fragment of bFGF pre-mRNA actually encodes an unkno~aa 24 kD protein. The mRNA has partial bFGF sequences in the 3' untranslated region, but in the anti.sense orientation.
7.
SLACKJMW, ISAACSI-IV: Presence of basic fibroblast growth factor in the early Xenopus embryo. Development 1989, 105:147-153. Mesoderm-inducing actMty extracted from Xenopus ovary, eggs and embryos was identified as bFGF. There is sut~cient bFGF for the molecule to act as a natural mesoderm inducer. Like [5] this article demonstrates that a puta~'e inducing factor is present in the Xenopus embt3~o. oo
8. •
DALE L, ~t~TI'ItEWS G, TABE L, CO,tAN A: Developmental expression of the protein product of Vgl, a localized maternal mRNA in the frog Xenopus laevtg FAIBOJ 1989, 8:1057-1065. Vgl mRNA is translated in the Xenopus oocyte and embryo to form 45 and 43.5 kD proteins, which ate glycosylated forms of a 40 kD protein. Synthesis is restricted to the vegetal hemisphere but both proteins dif. fuse into the animal hemisphere. See also [9]. 9. •
TANNAHILLD, M.ELTON D& Localized synthesis of the Vgl protein during early Xenopus development. Development 1989, 106:775-785. Vgl mRNA is translated in Xenopus oocytes, eggs and early embt)~3s. There are 2 glycosytated forms, 1 of 44 kD together with a less abundant 42 kD form. Diffusion of the larger form into the animal hemisphere seems to be restricted. Processing of the Vgl protein into a 17 kD form is only detected if Vgl is over-expressed. PONDEL/~D, KING ~L!2 Localized maternal mRNA related to transforming growth factor 13 mRNA is concentrated in a cytokeratin-enriched fraction from Xenopus oocytes. Proc Natl Acad Sci USA 1988, 85:7612-7616. Vgl ~ unlike histone It3 mRNA, assodates with elements of the cytoskeleton. This association may be linked vdth the translocation of the message to the vegetal pole. 10. •
11. •0
YISRAELI JK, ~LTON DA: The maternal mRNA Vgl is correctly localized foUowing injection into Xenopus oocytes. Nature 1988, 336:592-595. Vgl mRNA is translocated to the vegetal pole after being microinjected into Xenopus ooeytes. Translocation does not require protein synthesis. The ~ork shows that naked RNAcan be recognized by the translocation
machinery of the oo~te, making it possible to map the tmuslocation recognition sequences. 12. •
GODSAVESF, ISAACS HV, SLACKJMW: Mesoderm.inducing factors: a small class of molecules. Development 1988, 102:555-556. Mesoderm-indudng factors from a variety of sources were sho~-n to have similar properties. 13.
COOKE J, S.~mH JC: Gastrulation and larval pattern in Xenopus after blastocoelic injection of a Xenopu~derived inducing factor: experiments testing models for the normal organization of mesoderm. Dev Biol 1989, 131:383--400. The effects of injecting me.soderm-inducing factors into the blastocoels of Xenopus embr)x3s ate described. Gastmhtion-like movements in. duced by XTC-MIF occur about 1.5 h earlier than those induced by bFGF.
•
HOPWOODNO, PLUCKA, GURDONJB: MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. FAIBOJ 1989, 11:3409-3417. M)~D represents an early marker of muscle differentiation in Xenopux 14. •
15.
SYMESK, YAQOOB M, SMITH JC: Mesoderm induction in Xenopus laev& responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells. Development 1988, 104:609-618. This paper and [16] suggest that contact is required bet~x'~en animal pole cells ff they ate to respond to induction by forming mesc~erm. This paper also shows that suppression of epidermal differentiation can occur in single ceils in the absence of cell dMsion.
•
16. Gt/m~N JB: Cell movements and a community effect in • tissue morphogenesis. Nature 1988, 336:772-774. See [15]. The requirement for cell contact is referred to as the 'community effect'. 17. GODSAVESF, STACKJMW: Clonal analysis of mesoderm in• duction in Xenopus laevt~ Dev Biol 1989, 134:486--490. Single animal pole cells exposed to bFGF go on to differentiate as mesodermal cell types. ROSAFM: Mio¢l, a homeobox mRNA inducible by mesodcrm inducers, is expressed mostly in the presumptive endoderreal cells of Xenopus embryos. Cell 1989, 57:965-974. M/x:/, a homeobox-containing gene, is activated in animal pole cells in response to XTC-MIF. M/x:1 transcripts in normal embryos are expressed mostly in presumptive endoderm. The first 'immediate early' genetic response to induction to be discovered. 18. ••
19. •
Gna.ESt'rELL, P^~RNO GD, SLACKJMW: Analysis of cornpetence: receptors for fibroblast growth factor in early Xenopus embryos. Development 1989, 106:203-208. Provides spatial and temporal analyses of FGF receptor distribution in the early Xenopus embryo. 20. •
COOKEJ, SMrrH EJ: The restrictive effect of early exposure to lithium upon body pattern in Xenopus development, studied by quantitative anatomy and immunofluorescence Development 1988, 102:85-99. A detailed anatomical and histological description of dorso-antetior enhanced Xenopus embryos resulting from LiCI treatmenL 21. •
KAO KR, ELLNSONRP: The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos. Dev Biol 1988, 127:64-77. All regions of the mesoderm of LiCl-treated dorso-anterior enhanced Xenopus embryos ha~e organizer activity. 22. •
SLACKJMW, ISAACSI-W, DAm.~GTON BG: Inductive effects of libroblast growth factor and lithium ion on Xenopus blastula ectoderm. Development 1988, I03:581-590. LiCI has no mesoderm-inducing actMty alone, but acts s)~ergistically with fibroblast growth factor, such that muscle formation is enhanced. See also [23]. 23. •
COOK~J, SY~mSK, S.~m'HEJ: Potentiation by the lithium ion of morphogenetic responses to a Xenopus inducing factor. Development 1989, 105:549-558.
1069
1070
Cell differentiation LiCI acts synergistically with XTC-MIF. See also [22]. 24. •
BUSAWB, GIML/Ctl RL: Lithium-induced teratogenesis in flog embryos prevented by a polyphosphoinositide q-cle intermediate or a diacylglycerol analog. Dev Biol 1989, 132:315-324. Formation of dorso-amerior enhanced Xenopus embryos after injection of HC! is pre'vented by co-injection of m)~inositol or by subsequent treatment with phorbol myristate acetate.
WHITMANM, MELTON DA: Induction of mesoderm by a viral oncogene in early Xenopus embryos. Science 1989, 244:803--806. Microinjection of middle T mRNA into early embryos causes isolated ectodermal tissue to form mesoderm. In frog embwos, middle T associates with cellular activities similar to those observed in pob'omatransformed mouse cells. The results offer a powerful route to stud)~ng second messenger pathv~)~ in mesoderm induction. 25. eo
26. ••
COOKEJ: Mesoderm.inducing factors and Spemann's organizer phenomenon in amphibian development. Development 1989, 107:229--241. Animal pole cells exposed to XTC-MIF,but not to bFGF, are effective as Spernann organizers. The completeness of the secondary axis induced by XTC-MIF.treated tissue is set by graft size and factor concentration. Provides further information about the specification of the antero-posterior axis. 27. ••
Rum I ALTA1BA A, MELTONDA: Interaction between peptide growth factors and homeobox genes in the estabIishment of antero-posterior polarity in frog embryos. Nature 1989, 341:33-38. XT.r~x3 is induced to high levels by bFGF and lower levels by XTCMIF. Animal pole explants treated vdth XTC-MIF induces anterior duplications when implanted into the blastocoels of host embryos and FGF-treated explants induce posterior duplications. The first molecular anab~is of antero-posterior pattern formation in Xenopu,~ RUm I ALTALBA A, MELTONDA: Bimodal and graded expression of the Xenopus homeobox gene X h o x 3 during embryonic development. Development 1989, 106:173-183. XT.xgx3is expressed during 2 periods of Xenopus development: during gastrula stages and during tailbud stages. During gastrula stages Xlmx3 is expressed in a gradient, ~ith low l~els anteriorly and high levels posteriorly. 28. •
31. •
SAVAGER, PtnLHPS CR: Signals from the dorsal blastopore lip region during gastrulation bias the ectoderm toward a nonepidermal pathway of differentiation in Xenopus laevtx Dev Biol 1989, 133:157-I68. The blastopore lip region inhibits expression of Epil in dorsal ectoderm. The signal travels through the plane of the ectoderm sheet (see [32]). 32. •
DIXONJE, K2Xq'~'ERCPc Cellular contacts required for neural induction in Xenopus embryos: evidence for two signals. Development 1989, 106:749-757. 2 signals ~ere found to be responsible for neural induction. The first, and most effective, travels through the plane of the ectoderm from the blastopore lip region. The second is derived from in~oluting dorsM mesoderm, and acts s3aaergisticallyxxith the first. 33.
OTTEAP, VAN RUN P, HE1DEVELDM, VAN DR/El. P,, DUF:S'I'ON AJ: Neural induction is mediated by cross.talk between the protein kinase C and q'clic AMP pathways. Cell 1989, 58:641-648. Adenyl c3"claseacuity and cAMP levels increase after neural induction. Although cA_MPanalogues have no neural inducing activity they act ~aaergistically ~Sth TPA, an activator of PKC. IAke [32], the results imply that neural induction consists of ~'o steps, one actix-atedby PKC and one acting through the c_AMPpathx~'ay. e e
8ATO SM, S.M~.GENT TO: Development of neural inducing capacity in dissociated Xenopus embryos. Dev Biol 1989, 134:263-266. When Xenopus embryos are dispersedbem-een early clea~uge and early gastmla stages mesoderm induction is prevented, but this does not interfere with neural differentiation. 34. •
35. •
OTI'EAP, KOSTERCH, SNOEKGT, DUK~ON AJ: Protein kinase C mediates neural induction in Xenopus laevts, Nature 1988, 334:618-620. PKC actMty is translocated from cytosol to membrane fractions during neural induction. TPA, an activator of PKC, causes neural induction.
36. •
DAVIDSM: Protein kinases in amphibian ectoderm induced for neural differentiation. Rou.xs Arch Dev Biol 1988, 197:339-344. PKC actMty increases in Xenopus gastmla ectoderm treated ~ith a crude neuraLinducing factor. 37.
SlXX HI, HATrORI K, WEL'Cr~,UB 1t: Progressive determination during formation of the anteroposterior axis in Xenopus laevix Cell 1989, 58:171-180. The first cells in the embryo to be specified to form cement gland eventually form neural plate instead. The results suggest that the anteroposterior axis of Xenopus is determined progressb,'ely,with anterior specification being superseded by posterior. o e
Rum I ALTALBAA, MELTONDA: Involvement of the Xenopus homeobox gene X h o x 3 in pattern formation along the antero-posterior axis. Cell 1989, 57:317-326. Microinjeetion of symhetic AT.~gx3mRNA, but not control RNA, into prospec~-e anterior regions of Xenopus embryos causes anterior defects. The results imply that Xhox3 pla~ a role in specifying antero-posterior posidon in Xenopus 29. e e
38.
DURSTONAJ, TP.IMEIZMANSJP*I, HAGE %V'J,HENDRIKSHFJ, DE VRIESNJ, ttEIDEVELDM, Nml.~l~OOP PD: Retinoic acid causes an anteroposterior transformation in the developing central nervous system. Nature 1989, 340:140-144. Retinoic acid gxs applied to developing Xenopus embtTos, causing transformation of anterior neural structures to posterior. Measurements of retinoic acid levels in the embryo are consistent with the suggestion that the molecule acts as a morphogen. oo
LO,,a~ONC, AKEV, S P,, PHIU.U'S C: Expressions of Epll, an epidermis-specific marker in Xenopus laevis embryos, is specified prior to gastruladon. Dev Biol 1988, 129:380-389. Blastomeres derived from ventral halves of early Xenopus embryos express the epidermal marker Epil strongly; blastomeres from the dorsal half do so only weakly. 30. •