early specification for body position in mes-endodermal regions of an amphibian embryo

Cell Differentiation, 17 (1985) 1-12 1 Elsevier Scientific Publishers Ireland, Ltd.

CDF 00314

Review

Early specification for body position in mes-endodermal regions of an amphibian embryo

J o n a t h a n C o o k e

National Institute for Medical Research, Mill Hill, London, N W7 1AA, U.K.

(Received 5 March 1985)

Specification for development of the body pattern in the amphibian embryo has usually been thought of as a prolonged process, initiated from an ooplasmic Iocalisation of some kind in what will become the dorsal-anterior midline. The evidence has been interpreted as suggesting that this initial Iocalisation is centred in what will become anterior endoderm, but gives rise by an inductive process in early blastula stages to an overlying organising centre which eventually controls the genesis of mesodermal pattern. Neurectoder- real development (especially, the position and pattern of the central nervous system) is seen as controlled considerably later, by inductive signals from submigrating mesoderm at gastrulation. Current work tends to confirm that this sequence of inductive influences can occur at least in experimental situations. It also suggests, however, that in the normal development of the rather small egg of Xenopus , genesis of positional cues that specify the body pattern contributions within the more vegetal material (mes-endoderm) is a rather rapid, widespread and direct consequence of events occurring in the interval between fertilisation and cleavage. Possible molecular bases of early nuclear responses to position within egg material, and the more problematic nature of the positional system itself, are discussed.

fertilisation; cleavage; primary induction; organiser; egg fragments; developmental rate; nuclear proteins; homoeo box sequence; positional cues

Introduction

Spemann's concept of the organiser region, as a localisation controlling the development of the amphibian embryo, rested principally on two sets of observations made in his laboratory and in subsequent related work (Spemann, 1902, 1938; see also Fankhauser, 1948; Brice 1958). These concerned interactions around the equatorial egg regions that appeared to be necessary to specify the overall plan of development, and then later interactions whereby the pattern in neurectoderm was co-ordinated with that in underlying mesoderm at gastrulation.

Vertical partitioning of the very large, yolky urodele egg into two approximately equal-sized fragments was accomplished by a variety of tech- niques at stages from before cleavage up to the onset of gastrulation, followed by the isolated development of one or both fragments. The results led to the definition of a relatively restricted sector which could control the formation of a complete and harmonious, though small, edition of the larval body plan in those fragments which received a share of it. Fragments which could be assumed to lack material from this sector, although cleaving and remaining healthy in a cellular sense, gastru-

lated late or not at all and failed to produce axial

0045-6039/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland, Ltd.

mesodermal patterns or nervous systems. Their development thus appeared to approximate to radial symmetry. Intermediate forms were sometimes seen, however, with axial but anteriorly incomplete bodies, and assessment of the presumptive plane of bilateral symmetry in eggs relative to the plane of experimental partitioning was poor or absent. The definition of the extent of the special region, controlling the activation and polarity of pattern formation, was thus arbitrary ( - 4 /5 of the 'dorsal' half, for instance), but the experiments were at least held to indicate that normal specification of pattern was the result of prolonged and progressive spatial interactions during blastula and gastrula stages. This is because the behaviour of both classes of fragments, in 'regulating' to whole- ness or in developing as if they lacked all information for pattern, appeared to deviate greatly from the contribution they would have made to the whole development in terms of the presumptive fate map, and such behaviour was recorded from fragments isolated at times up to the onset of gastrulation.

Later, in the classic experiments carried out at early gastrula stages, the dorsal lip of the blasto- pore was found to exert two controlling functions when used as a graft to a distant part of a host marginal zone (Spemann and Mangold, 1924); it re-arranged the pattern of development in sur- rounding mesoderm to produce a partial extra body plan centred on itself, and in co-ordination with this it induced a second edition of the nervous system rudiment, appropriately positioned in relation to the additional mesodermal pattern although almost entirely constructed of cells from the host neurectodermal layer. Spemann himself seems never to have wished clearly to distinguish between interactions controlling patterns of development within major cell layers (germ layers) and those co-ordinating pattern between such layers, referring to these all as 'inductions'. But the dorsal lip region, itself firmly committed to develop the antero-dorsal mesodermal and anterior endodermal regions, was now established as occupying at least a very early and controlling position in the hierarchy of such interactions. Spemann regarded it as in some way 'descended' from the controlling region already defined at the pre-cleavage stage in

the earlier experiments. But was this a descent by direct inheritance of

an ooplasmic localisation, i.e. cellular descent, or was it the result of an early inductive effect upon the marginal zone from another level in the egg, occurring at early cellularised stages? In extensive subsequent experiments, Nieuwkoop and his col- leagues have shown in both urodele and anuran amphibians that interactions along the vegetal- animal egg axis (parallel with gravity and normal to that considered so far), result in progressive specification of an equatorial region to produce the mesodermal cell population at gastrulation (Sudarwati and Nieuwkoop, 1971; Nieuwkoop, 1973). The dorsal blastoporal lip, so far defined as possessing the fullest 'organiser' capacity, arises at the mesodermal/endodermaljunction. The vegetal component has been shown to dominate and control the meridian on which the dorsal lip and thus the midline of mesodermal pattern develops in surgical recombinations of animal and vegetal regions from blastulae. If these experiments are in- formative about normal development therefore, the dorsal lip represents simply that mesodermal region which is induced by the ultimately controlling localisation inherited in cleavage. This primary reference point for pattern lies in future endoderm and is determined to form the anterior boundary of its pattern.

Fig. 1 outlines the sequence of interactions proposed to occur within and between the zones of the amphibian egg material which will produce the germ layers, in giving rise to the pattern of the body. The rough time courses shown for the specification (not the final determination) of pattern within each layer, in relation to stages of cleavage and morphogenesis, are those that have hitherto been assumed. The diagram also shows in outline a current view of the mesodermal fate map - the way in which contributions to the normal plan of structures are disposed in the equatorial region of the egg before the physical re-organisation of gastrulation (mesoderm invagination and migration). This map, for the anuran Xenopus, resulting from reconstructions of the contributions from strictly lineage-marked early blastomeres to the larval pattern, differs appreciably from what was assumed hitherto. It is shown as relevant to

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Fig. 1. Transfer of information for the body plan during Xenopus development. Schematic view of embryo's structure from future body's left side. Heavy arrow head and asterisk, off drawing, show respectively the vegetal centre of dorsalisation and the meridian of sperm entry (in the idealised case). Two sets of short solid arrows indicate, respectively, the induction by endoderm of mesoderm within the deep layers of the marginal zone during blastula stages, and the induction of nervous system in neurectoderm by submigrating axial mesoderm during gastrulation, end = endoderm; 1 pl = lateral plate mesoderm; som = s o m i t e mesoderm (notochord and prechordal territories not indicated); ns = nervous system region; a = anterior; p = posterior. Long arrow indicates mesodermal involution, migration and stretching in gastrulation.

the interpretation of the more recent experiments discussed later. The following interactions have all been recently confirmed as able to occur in experimentally manipulated development: mesodermal induction from an endodermal site of primary information for pattern (Gimlich and Gerhart, 1984), the capacity of the mesodermal dorsal lip to adjust the fate of adjacent mesoderm in relation to itself (Smith and Slack, 1983), and the control of neurectodermal fate in founding the nervous system by late-acting induction from mesoderm (Gimlich and Cooke, 1983). By experimentally manipulated development we mean that in which materials coming from non-adjacent positions within their eggs of origin are placed adjacent by microsurgery, or where grafted material is used to 'rescue' responding material in a host embryo deprived of its endogeneous capacity to develop pattern normally. Thus a question remains, for each of the above-demonstrated spatial interactions, as to whether they are the main mechanism

in the normal course of pattern establishment, or are simply the extreme operation under abnormal circumstances of a 'backup' mechanism which plays a minor, regulatory or refining role to ensure the accuracy of normal development. The impli- cation of the latter supposition is that in the normally developing egg the information which can be given by induction is there already in the 'responding' region, via other mechanisms which involve the direct inheritance of some spatial organisation established early in the material of the dividing egg.

The evidence that inductive patterning of neurectodermal development by submigrating mesoderm is also the mechanism of the normal development has been very strong ever since the discovery of this effect after grafting of the dorsal lip. Ex- periments have led to various allied models of how a transfer of correctly patterned spatial information is ensured during the process of gastrulation (Nieuwkoop et al., 1952; Toivonen, 1978; but see Suzuki et al., 1984, for a somewhat different view). The source of spatial information for pattern in the neurectodermal regions of the embryo is not the central concern of this article however, and this question, including recent work using strictly lineage-linked tracers to follow in situ clones, has been reviewed elsewhere (Cooke, 1985a). Here it is sufficient to say that although the evidence does not preclude some spatial bias or heterogeneity of tendencies in neurectoderm, primarily inherited in the process of cleavage, it is unlikely that these are important in normal development.

The normal course of pattern establishment earlier on, in those egg zones giving rise to endoderm and mesoderm, remains problematic. As to normal origin of information for mesoderm, recombination and isolation experiments in the laboratory of Nakamura (1978) for instance, have indicated that the emergence of competence to develop mesoderm occurs at rather earlier stages than Nieuwkoop's view of its induction would lead us to expect, and that the field specified to be mesodermal material already shows spatially het- erogeneous tendencies for differentiation at its inception. But the location and layout for the presumptive mesodermal pattern in the pre-gastrula has usually been misunderstood, and there has

been no reliable marker for identification of dif- ferentiated mesoderm other than notochord and somite muscle in small explants. The situation is also complicated by a pronounced timing, or rate gradient programmed into the material along the body plan (see later section) with respect to the onset of the cell behaviours of gastrulation and then differentiation.

Similarly the importance, for normal development, of spatial interactions over a long time span within the mesodermal territory is unclear. Al- though the dominance of the dorsal lip in being able to 'upgrade' the mode of differentiation in mesoderm artificially placed next to it has been rigorously demonstrated, we do not know how big a part such interaction plays in normal development or even in producing the partially twinned body plan that follows implantation of an ectopic dorsal lip. The observed pattern duplication might largely be explicable in terms of spatial redeploy- ments of mesodermal cell groups already specified as to their contributions in the normal single plan, and responding to the implanted organiser in terms of locomotory-adhesive behaviour rather than by changing their courses of differentiation. Extensive studies combining the Spemann type of grafting operation with the creation of large marked mesodermal clones in hosts, whose normal contribution to their pattern is known, are required to investigate this issue and are only now in progress (A. Smallcombe and J. Cooke, unpublished data). Results to hand suggest that in Xenopus at least, both spatial redeployment and change of specification in host mesoderm can occur, but their relative importances remain unclear.

Our data about mes-endodermal development until recently, therefore, have left us uncertain as to how rapid and widespread is the deployment of those spatial cues (in the broad sense, positional information, Wolpert, 1971) which specify the normal plan of contributions to the body and lead to the proper determinations at each location. The most that could safely be said is that the inner two germ layers, together, possess the requisite spatial information much earlier than neurectoderm, at stages from the onset of cleavage into blastula. Slack (1983) has emphasised a useful conceptual distinction that has long been implicit in embryo-

logical literature, by use of the term specification, in contradistinction to subsequent determination, of cellular material. Specification represents the stable possession of information which, in the absence of further naturally occurring or experimentally imposed stimuli, will lead to the development of particular contributions to a presumptive pattern. Determinative events are those whereby a state of specification is irrevocably acted upon (at the cellular level) so that the options available to the cell's epigenetic apparatus are foreclosed. Note that states of specification can be transient in normal development. Thus we are quite confident that material normally forming the nervous system passes through a phase when it is specified as epidermis, and fairly confident that at least most of the normal mesodermal material also passes, more briefly, through such a phase. Biologists most interested in molecular mechanisms of differentiation itself will tend to be most interested in later determination events in embryogenesis, since they are concerned with cellular 'decision-taking' or switch-like processes rather than with the possibly variable histories of cellular information leading up to these. Those most interested in the mechanisms whereby the correct spatial pattern for the organism is arranged will, by contrast, be most interested in the normal time course of specifications during early development, since this most directly reveals the performance and dynamics of the unknown patterning mechanism.

The experimental procedure which most nearly reveals specification states at any one time in development is the creation of large isolates, followed by the rapid restoration of cellular continu- ity in the resulting part-embryos. The 'normal fate map' for the material of the egg at the time of isolations should be understood in as quantitative a way as possible, so that the patterns made by the isolates can be compared with the contributions they would otherwise have made to the normal whole body pattern. The recent results of such an experimental series (Cooke and Webber, 1985a) will be described in the next section. They reveal that, in the relatively small and rapidly developing egg of Xenopus at least, the information that leads to pattern specification across the mes-endodermal zones is often present at very early cleavage stages,

in a widespread and stable configuration ap- proaching that in which it is finally interpreted to give normal body balance. This contrasts with the classical view, though other recent workers have realised that this discrepancy exists after observa- tion of the gross morphology of isolate develop- ments (Kageura and Yamana, 1983). The implica- tions for the mechanism of pattern establishment in these eggs will be briefly discussed, but for a fuller discussion of this and of possible causes of earlier observations (e.g. Cooke, 1981) that indicated slower, more regulative spatial organisation, see Cooke and Webber (1985a).

Body patterns in 4-cell stage isolates

In assaying specification from early stages in amphibian mes-endoderm we are helped by the fact that the egg regions concerned are disposed in two contiguous zones whose relative movements in gastrulation etc. cause only rather local slippage between them, even though two concentric tissue layers are finally produced. This shows up in the disposition of large marked clones at the larval stage, derived from the in situ injection of single early blastomeres whose material contributes to both mesodermal and endodermal layers. The patches of descendent cells within mesoderm and endoderm are largely contiguous and adjacent. This contrasts with such clones that contribute to both mesodermal and neurectoderm layers, where the movements of gastrulation cause great geomet- rical re-alignment of regions before the tissue con- tacts that mediate induction (cp. Fig. 1, the reverse directionality of the body axis in mes-endodermal vs. neurectodermal fate maps). The state of the primary information, used in specifying the axial pattern of the body in mes-endoderm and disposed around the vegetal regions and marginal zone, can therefore be studied by making isolates in the vertical plane that is normal to that for bilateral symmetry. The little understood animal- vegetal interactions that co-ordinate mesodermal and endodermal contributions to this pattern might occur within the material of each isolate at times before or after the procedure for preparing it. In fact, it will be argued that the results indicate that

any mesodermal induction from endodermal regions must take the form of a pattern of differen- tial stimuli arising around the marginal zone and giving the induced zone its regionality at the out- set, rather than being a single stimulus over one meridian (the dorsal midline) that serves only to locate a mesodermal 'organiser'.

Fig. 2a shows the disposition of the contributions to the normal plan of mesoderm, in the egg

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Fig. 2. The fate map, the second cleavage plane and the partial body plans formed by cell pairs isolated across that plane. (a) Pre-gastrular mesodermal fate map from future body's left side, with the typical partitioning made by the vertical second cleavage plane. The disposition of materials for segments is indicated in the somite area, and the pronephric territory lying at its lower boundary with the lateral plate region is shown stippled. Notochord territory is indicated by small open circles, with prechordal head region lying below its vegetal end. (b) Mesodermal body patterns seen after isolated development of the respective cell pairs. Numbers of somite cell-populations segmented are shown in relation to the number of somite territories indicated across the map of (a), though the real number is expanded in the posterior end of the series. Pattern parts are indicated as in (a), but within outlines of the body forms. Headparts anterior to ear vesicle, as well as heart and notochord, are always and only found in the dorso-anterior isolate pattern. The posterior isolate pattern is sometimes less axial than shown.

marginal zone, by reference to an outline of that plan as it exists by axial larval stages. This has been established by utilising the fact that the plane for future bilateral symmetry in anuran development is set up in relation to the egg meridian defined by the sperm entry point (SEP) and the animal/vegetal axis. The SEP is visible, and its statistical relation to the future body's symmetry can be reinforced by appropriate tipping of pre- cleavage eggs in gravity (Gerhart et al., 1981). Eggs that begin development with particular patterns of cleavage planes and thus subdivisions of their material can therefore be selected and particular early blastomeres filled with inert heritable tracers to found large clones in normally developing animals. Mes-endodermal pattern contributions of such clones from each position, founded at stages up to 32-cell or later, are rather regular and predictable, with considerable clonal coherence. The outline map of Figs. 1 and 2 is that deduced from such 4-32-cell clonal contributions to notochord, somite segment, pronephric and lateral plate patterns, and is of course limited by the inherent variability of the cleavage planes in relation to the egg's cytoplasmic structure and pattern-forming events. The only artificial parti- tion shown, with a solid line on the map of Fig. 2a, is our best estimate of that separating contributions from either side of the second cleavage plane. This is vertical, but at right angles to the first which co-incides with that for right-left symmetry in the eggs selected. The SEP or posterior midline of pattern and the dorso-anterior midline or site of the long-recognised organising centre lie on opposite meridians. The pattern of ultimate specifications for mesoderm lying inbetween is such that a rather complex but definite subdivision of the presumptive body plan is made by this frontal cleavage plane that leads to the 4-cell stage.

When the reciprocal pairs of cells separated by this second cleavage are prevented from re-adher- ing via their new cell membranes, in circumstances that encourage rapid re-formation of moru la / blastula isolates without visible blastocoelic surface, their development continues at a tempo very close to normal. In a large proportion ( > 50%) of such reciprocal pairs of isolates, both members create part-bodies whose mesoderms are close rep-

licas, qualitatively and quantitatively, of the respective contributions that they would have made to the whole body without the early separation (Fig. 2b). The blastomere pairs centred on the dorsoLanterior specified meridian (usually re- ferred to as a dorsal half egg but not accurately thus described), always make such a part-pattern corresponding to fate. Their partners centred on the sperm entry meridian (more meaningfully called posterior than ventral egg halves) frequently do this, but a minority give radially symmetrical, apparently patternless forms, and the remainder give a series of posterior axial part-patterns corresponding to larger or smaller sectors of the normal fate map centred on the sperm entry position. This behaviour of pattern specification in the reciprocal isolates is represented in Fig. 3a and b in abstract form. The patterns are assayable directly in mesoderm because of its readily scorable histo- differentiations and architecture in sections, but the endoderms of isolates give every appearance of corresponding, in terms of body position values, with their mesoderm patterns and with the reciprocal 4-cell contributions to the normal endoderm.

The cell cycle schedule, thus the total number of cells in the embryo structure at any time, is not affected by partitioning of cleavage products (or any other operation) in amphibian pre-larval development. We can be sure that there has been no regulation towards small, harmonious pattern in either of the reciprocal types of part-body, but frequently a retention of information leading to almost exactly the presumptive contribution even in the posterior isolate. This statement is possible because of an intimate knowledge of the sizes and proportions of the cell populations that have founded the mesodermal pattern elements in intact development, at a particular early axial stage (st. 28-30, see Cooke and Webber, 1985a, b). Normal fate mapping and quantitation of mesodermal pattern in isolates and co-fertilised sibling controls have all been carried out at this particular familiar stage. Thus we can state for example that notochords of dorso-anterior isolates are of normal full size, and that the quantitative assignment of cell numbers to particular members of the somite segment series is reciprocal and partial in the two

types of isolate pattern, in the way that one might expect from an understanding of the normal map. The pro-nephric formations in the most axial but notochordless posterior part-patterns are larger than those in dorso-anterior part patterns, as would statistically be expected from the division of the normal map by the second cleavage plane.

Such results might tempt us to imagine a series of plasms, specific to each mes-endodermal pattern element, somehow appropriately localised within the egg by the events that follow fertilisation. But a wealth of surgical recombination and explantation data show that the potencies of cells from these regions are not thus restricted until much later stages (see, e.g. Forman and Slack, 1980, and reviews by Holtfreter and Hamburger, 1955, and Nieuwkoop, 1973). The experiments just described are revealing merely the early presence and stability in the profile of some spatial variable across the egg which acts normally as specifying

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Fig. 3. Behaviour of the positional system in the Xenopus egg after early transection, in comparison with behaviour expected from a reaction-diffusion mediated signal gradient. (a) Arbi- trarily drawn profile to represent the normal spatial distribu- tion of a graded variable state, distributed across the egg and leading to correctly proportioned and positioned contributions to the body plan within mes-endodermal material. (b) The response of the positional system shown in (a) to transection at the 4-cell stage. This indicates that the system frequently exists in a rather stable condition at this stage, close in configuration to that which is ultimately ' interpreted' as the normal pattern. Various degrees of information loss, or 'relaxation' in the gradation, may occur in the posterior (left) isolate, however. (c) In the expected behaviour of a source-diffusion or reaction-diffusion controlled morphogen gradient, early transection of the material leads to divergent results depending upon precise kinetics; either the regulative restoration of profile or its loss by flooding or deactivation. The actual system studied in these experiments shows no regulation, but is quite resistant to information loss.

information (see Fig. 3a and b), to be interpreted only gradually and progressively into the determined pattern. The originally reported results from isolations by constrictions (see Introduction), have led pattern specification in the marginal region of the amphibian egg to be regarded as a classic case of control by a physiological or morphogen gradient. In contemporary terms, this would be either a positional information field established by prolonged, dynamic source-diffusion or reaction-diffusion mechanisms, triggered by an isolated localisation at the 'organiser' region (Wolpert, 1971; Gierer and Meinhardt, 1972; Gierer, 1981), or else a series of specific inductions beginning from that region (Spemann, 1938; Nieuwkoop, 1973). This has been the interpretation of the reported tendency of isolated egg parts either to give rise regulatively to a miniature complete pattern, or to behave as if they had never acquired, or had entirely lost, such information about body position as would normally have been available to them. Such behaviour is again represented, in Fig. 3c, in terms of the dynamics of a system giving rise to a graded signal whose profile will normally be translated into pattern. This shows the behaviour, in response to early partitioning, of the general reaction-diffusion or source-diffusion class of model system that has been proposed as controlling morphogen signal gradients (see above refs.), with the added assumption that some necessary 'trigger' or polarising localisation is either included in, or excluded from, particular members of isolate pairs. The Xenopus results, by contrast, indicate that by the 4-cell stage (essentially, in pre-cellularised material) there often exists information whose profile tends neither to regulate nor to 'relax' when the extremes of the system are severed from one another. The crucial result telling us this is the close co-incidence of the normal fate-map division by the second cleavage plane with the reciprocal part-patterns often developed in pairs of isolates.

If we believe that only the endodermal egg region is in fact the primary site of information for body pattern, then it follows that mesodermal pattern formation occurs via a widespread set of inductive influences, under fairly local control from subjacent yolky endoderm all around the egg,

rather than by operation of a dynamic gradient field under 'diffusion' from the dorsal lip region. If the latter were the mechanism, then isolates deprived of the dorsal lip-producing region at the 4-cell stage, although perhaps giving rise to a mesodermal cell population, could have no access to information with which to produce partial mesodermal patterns in accordance with fate.

We cannot at present say whether the rather different behaviour (at once less stable and more regulative) on record foi" the urodele embryo is a genuine difference, associated with the strategy of a larger, slower-developing egg, or is at least partly to be explained by a lack of quantitative and internal analysis of the tissues actually produced in urodele isolates and consequent misinterpreta- tion.

If in this particular version of vertebrate development the diffusion or reaction-diffusion-mediated gradient field is not a good model for the positional apparatus, what then is the latter's cellular nature? We have so far described only isolation procedures that avoid the experimental juxtaposition of material from different original positions in the fertilised egg. Other procedures, however, offer a body of evidence that the spatial information at early stages is of the form of a smooth gradation of state, whose levels are ultimately 'read' as body position, but where certain interactions between onset of cleavage and gastrulation can adjust levels when extreme values have been abnormally healed together. There is not space in this article to describe these other observations and their logical interpretation in detail, but they are the subject of a further experimental paper (Cooke and Webber, 1985b). Two key observations are depicted diagrammatically in Fig. 4. Biologists thinking about this system must try to imagine or discover how some continuously variable cell-structural feature, not dependent upon cel- lularisation for the maintenance of gradations, might be arranged around the egg by the coherent sliding movements of deep-lying plasm in relation to the surface components. Such a physical re- organisation is normally instituted in relation to gravity and the SEP, halfway through the first cleavage interval, and rather rapidly translated into the stable variable that constitutes the 'mem-

ory for body position'. This primary variable is interpreted within the more vegetal part of the embryo to found the contributions to the body plan, and therefore it corresponds with the idea of 'position value' in positional information models following Wolpert's (1971) formulation. But the cellular machinery used initially to set up the information must here be rather different from those proposed for other systems (or, previously, for this one, e.g. Cooke, 1972), because of the rapidity with which it is set up, and its stability and lack of dependence upon continued contact with the normal 'boundary ' regions (see Figs. 3b and 4c).

The nature of the events in relation to gravity and sperm entry that are the immediate precursor of this initial specifiation for pattern, is imper- fectly understood. They are discussed by Gerhart et al. (1981), Scharf and Gerhart (1980, 1983),

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Fig. 4. Behaviour of the positional system in the Xenopus egg following two different early perturbations. (a) Arbitrarily drawn profile for a positional variable as in Fig. 3a, with the sector normally surpassing the threshold intensity for notochord specification drawn in (small circles). Proportion of the normal single notochord is quite accurately controlled. (b) When a doubled version of the early post-fertilisation events occurs, spontaneously or due to experimental manipulation of gravity orientation, two separate notochord territories can be induced which each approach normal extent. The total proportion of notochord tissue in such twinned embryos is always considerably more than normal, but usually significantly less than twice normal, while the total mesodermal cell population is normal or slightly less than normal. (c) When the early plasmatic movements in eggs are attenuated by progressive doses of UV-irradiation to the vegetal hemisphere, a series of partial but dorso-anteriorly truncated body patterns results. These imply the stability of incomplete profiles of the positional variable, as shown. Small arrow represents threshold value in the positional system for notochord specification or induction.

Neff et al. (1984) and Elinson (1984). But in these publications the older assumption is made that what is to be explained is only the setting up of one restricted region, the future organiser centre, that breaks the egg's radial symmetry and then controls the build-up of pattern by one or other of the slow, progressive but regulative mechanisms that have been modelled in the literature. What now needs to be explained is the rapid institution and storage of a graded trace of information over much of the egg.

On this view of the early events, the organiser region does retain its special status even though preliminary specifications exist elsewhere. This is shown by its properties in grafting operations (whatever their precise mechanism, see Introduc- tion). Individual eggs that have failed to achieve the degree of activation that corresponds with this region and its pattern contribution, due to at- tenuation or disturbance of the pre-cleavage events, cannot subsequently upgrade their spectrum of position values to specify complete bodies (Scharf and Gerhart, 1983; Cooke, 1985b). Only the grafting in of an adequately activated region from a normal egg, followed by intercellular transfer of information that replaces the effects of the normal early events, can lead to full pattern in such eggs (Gimlich and Gerhart, 1984).

Possible mechanisms for cellular interpretation of the early positional system, leading to specified states

It can readily be seen that the behaviour just described for the positional system in this amphibian egg is very different from that which must characterise the system with equivalent function in many other vertebrate types. There, the body plan is set up in a blastodisc or its equivalent, after a prolonged period of cleavage plus cell mixing that causes a quite indefinite distribu- tion of the individual egg's original structure. We would expect the more dynamic, 'diffusion-mediated' mechanisms that have been modelled to better represent the system that organises such embryos. The cellular mechanism which then interprets local values of the spatial variables, we might expect to involve systematic modification of nuclei, some-

how 'priming' them for specific sequences of future transcriptional activity in relation to their position within the system. This mechanism is much more likely to be universal, or at least highly conserved, across large parts of the metazoan king- dom, whereas it is understandable that the strategy of early spatial organisation should be evolution- arily labile and adapted to different egg types, environments for early development, etc. Attempts to investigate the early (nuclear?) system for positional interpretation, using the particular ad- vantages of the amphibian embryo (large size, accessibility and well-understood phenomenology of development), are thus a worthwhile addition to the exciting advances that are currently being made elsewhere using molecular genetic analysis.

Both in normal Xenopus development and in that of individual isolates or UV-irradiated whole eggs, the absolute time (at each ambient tempera- ture) after fertilisation, at which the new cell behaviours of gastrulation begin on each meridian of the egg material, is closely correlated with the positional contribution to a normal pattern that is made by the mesoderm there (Cooke and Webber, 1985a, b; Cooke, 1985b). In other words, the schedule (onset time and duration) of gastrulation movements made by any exPerimental embryo, in relation to the schedule being pursued by synchro- nously fertilised controls, is a predictor of the extent and boundaries of the fraction of the whole body-pattern that it will finally produce. This cor- relation may exist merely because local setting of the developmental rate is an intimate reflection of the state of positional specification from early stages. Alternatively, a setting of the rate of some process in relation to other measures of elapsed time during development may actually be a part of the specification machinery. In Xenopus the onset of zygotic transcriptional activity is well organised, probably spatially patterned (Boterenbrood et al., 1983), and time-controlled by a mechanism that is experimentally separable from that which controls other parts of the programme, such as onset of the gastrulation movements (Newport and Kirschner, 1982). Onset of transcription can be experimentally manipulated, but the patterns achieved by blastulae in which this has been done are not recorded. The possibility thus exists to test a work-

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ing hypothesis that the timing of systematic changes in chromatin structure etc., in relation to the progress of other processes whose rate has been locally set by the positional variable in the cleaving egg, is instrumental in converting position within the embryo into a preliminary state of nuclear specification. In insect (Foe and Alberts, 1983) and in at least some other types of vertebrate early development (Eyal-Giladi, 1984) the 'open- ing up' of the chromatin for transcriptional activity appears organised to occur at comparable stages in terms of the cleavage schedule.

The rather stable state of the positional specification system, revealed by 4-cell stage isolates in Xenopus, is reminiscent of the state of certain insect embryos at syncytial or newly cellularised blastoderm stages (Sander, 1975). Here also, separation by ligature into two halves leads to a range of results from 'mosaic' development of partial segmental patterns according to fate to the omis- sion of considerable pattern especially to one side of the ligature site (the 'gap phenomenon' as in Fig. 3b). In Drosophila, at the same or slightly later developmental stages in terms of the number of elapsed cell cycles, transcripts from a family of genetic loci known to be involved in establishing the regional pattern along the body axis are synthesised actively but in spatially restricted ways in the blastoderm (McGinnis et al., 1984). These transcripts share a strongly conserved region (the ' homoeo box') which is recognisable in small numbers of copies in genomes of several animal types, including vertebrates, that share the need to re- gionalise a segmented body axis. Just before these stages of development, which involve the first de- tectable programme of zygotic mRNA generally, sets of proteins have been identified immunologi- cally in both Drosophila and Xenopus as passing into the nuclei from the cytoplasm (see, e.g. De- quin et al., 1984). Transcripts that possess the conserved homoeo box in Xenopus include one already abundant in the mature oocyte and thus probably translated and used as protein during morula/blastula stages (Muller et al., 1984). The sequence and conserved nature of the homoeo box suggest coding for a protein domain that binds DNA or other conserved protein domains in a specific manner, and it is possible that when the

protein products of the transcripts are identified and can be probed for, representatives of them will turn out to be present in the egg in both species, performing a function that can begin before the first round of embryonic mRNA achieves expres- sion as protein.

Whether or not the 'homoeo box' proteins turn out to be central parts of it, we can imagine an apparatus whereby nuclei are differentially primed or set up for future genomic activity according to locally perceived values of the positional system in the egg. The dynamics of the latter have been the subject of this article. If the appropriate settings for future chromatin activity are made by combi- natorial patterns of presence or absence in the nuclei of a small family of proteins, this could be patterned in relation to the early structural events of Xenopus egg activation in either of two ways. Those events might create a series of spatial localisations or true plasms in the egg structure, containing specific combinations of the proteins, or they might create a gradation in some cellular variable (structural or metabolic) that is interpreted by recruitment of specific subsets of the proteins into nuclei in different regions, with the whole set of relevant proteins being initially present throughout the vegetal zone of the egg. We have seen that the Xenopus specification system, despite its early inception and durability, does not behave like a series of egg plasms that correspond in a one-to-one manner with the relative extents and positions of differentiations (see Fig. 4b). The capacity of re-orientation in gravity to 'rescue' the specification system after its impairment by UV- irradiation also speaks strongly against the idea of specific plasms (Scharf and Gerhart, 1980). The second arrangement, a graded signal coupled with a (nuclear) interpretation apparatus, would appear to fit our understanding of the system much better and corresponds in principle with the idea of positional information (Wolpert, 1971), even though the graded variable can hardly be a diffus- ing morphogen.

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