Jonathan Cooke

Summary

The idea that concentration gradients of crucial substances might control the pattern of development, even in the embryos of complex organisms, has been around for a long time, but mostly in obscure forms. Twenty five years ago clear, experimentally testable ideas about how such gradients might work were enunciated, and more recently the morphogen gradient principle was shown to underlie the beginnings of patterning in Drosophila. Is it also central to vertebrate development? Four recent papers raise experimentation to a new l e ~ e l ( l - ~ ) , while showing how difficult it might be to pin down the precise form of the mechanism.

Some background The first clearly documented use of the word ‘morphogen’ in speculation about mechanisms of biological development was probably by Turing(5), in the course of mathematical work on the speculative idea that substances in embryonic tissue might form concentration ‘landscapes’, either simple monot- onic gradients or sets of repeating peaks and troughs. Such landscapes could then create the overall pattern of develop- ment because of the concentration-dependent effects of the substances upon the course of cell differentiation. A monotonic gradient for one crucial substance (or perhaps in the concentration ratio between two such substances), con- trolling cells’ choices of developmental pathway, could indeed set the polarity and sequence of the domains in each dimen- sion of an embryo’s body plan. Turing himself was most inter- ested in the ways in which mutual feedback interactions, gov- erning the synthesis of his morphogens, might enable spontaneous generation and stabilisation of the gradients or other patterns. Certainly, some such spontaneous ‘symme- try-breaking’ system must be required to initiate pattern for- mation when, as for instance in higher vertebrate develop- ment, there is no evidence for structural pre-localisation in the fertilised egg of anything that might act as a ‘source’ for a dif- fusion-organised gradient. But in other embryo types, just such a pre-localised source appears to be involved. Thus in the insect blastoderm, a pre-localised mRNA is given in the highly structured egg@), and in the frog blastula, a less-well- understood mechanism of localised activation mediated by structural egg movements at fertilisation probably subserves a similar function(’). The current preoccupation is therefore more with morphogen identity and with the cellular mecha- nisms whereby actual substances, that are candidates for the role, might have their concentrations ‘interpreted’ by pluripo- tent cells to create pattern.

For vertebrate embryos, two key issues are: how fine- tuned could the differential responses by similar cells to the levels of any one morphogen possibly be, or in the real

world, how fine-tuned do they need to be? Operationally, between how many potential future states of specification can or do cells reliably ‘choose’, directly in relation to their positions within a morphogen gradient? Clearly, while we would like empirical information for real examples, the limits must be set by the available character of the signal transduc- tion machinery of the cells. Drosophila at the blastoderm stage, with its syncytial structure, can use transcription fac- tors directly in a morphogen role; that is, proteins produced after transcription and/or translation at one location can dif- fuse in order directly to influence genetic activity in other more-or-less distant ‘cells’. In truly cellularised developing systems things must be different, and for vertebrate embryos, morphogens are probably smallish secreted pro- teins (see below). It is currently difficult to imagine the engi- neering of signal transduction machinery that could underly reliable, but different, responses by early embryonic cells to several successive concentration ranges of an extracellular ligand. How well can the effective numbers of cell surface receptors or concentrations of presumed phosphorylated intermediary messengers, per cell nucleus, be controlled among the rapidly dividing, variable-sized blastomeres of the early frog embryo, for instance? The problem is certainly different from that involved in arranging high- and low-affinity binding sites for the same transcription factor within the con- trol regions of particular genes in the Drosophila blastoderm syncytium(*). In Drosophila the initiating gradient, in the homeobox transcription factor encoded by Bicoid, is appar- ently only the initiator of a hierarchical cascade of more localised graded gene expressions. Thus even the repeating pattern of gene activities that presages segmentation, with its quite regularly spaced and numerous boundaries, is finally accomplished without actually requiring that cells (in this case, cell nuclei) need ever choose between very many alternative states of gene activation at one time and using one mor~hogen(~). Is the vertebrate situation formally differ- ent from this, as well as necessarily using trans-cellular transduction machinery?

Induction of body parts in amphibian development In the frog blastula during the phase of primary pattern forma- tion, equatorial animal cap cells are induced by signals from the underlying yolky mass to form the definitive meso- and endoderm of the embryo, across which a ‘fate-map’ for the future body plan can be defined in relation to the position of cells(10). Peptide growth factors of the activin and fibroblast growth factor (FGF) families have been found to mimic closely these in vivo signals in in vitroassays, suggesting that (known or unknown) members of these families are involved in the pattern induction (reviewed in refs 11 -1 3). Nearly five years ago Green and Smith(14), working with purified recombi- nant activin in solution, proposed that responding blastula cells were able to distinguisii directly between multiple thresh- old levels of this signal, to enter one of five states of determi- nation, or body parts, that are normally produced in order across the fate map. These are: (1) ectoderm/presumptive epidermis (the default state, with at least no exogenously sup- plied activin, though see ref. 15); (2) ventrolateral mesoderm; (3) somite (identified as muscle differentiation); (4) notochord; and (5) a presumably more anterior meso-endodermal state identified by a strong ability of the cells themselves to induce neural tissue in naive ectoderm. In a subsequent paper(16), extending the work by studying interactive effects of the co- presence of basic FGF with the activin, the same laboratory proposed that a good approximation to the mesodermal fate map, as projected onto the embryo when mesoderm organi- sation is known to be occurring, can be reconstructed by assuming that cells acquire commitments directly from their positions within simple, superimposed gradations of two ‘mor- phogen’ inducing signals, the in vivo counterparts of activin and basic fibroblast growth factor (bFGF).

This system is indeed an attractive one for practical study of the morphogen principle, since a wealth of data now indi- cate that activin and bFGF closely simulate classes of in vivo inducer ligand, acting at least in part via the same receptor p a t h ~ a y s ( l ~ ~ ’ ~ ) . The authors disaggregated responsive ani- mal cap blastomeres in Ca2+;Mg2+-free saline medium, treated them with exact concentrations of the factors for a set time, then washed and reaggregated them for development until the equivalent of the neurula stage. Various levels of spatial organisation that might be important within whole blas- tulae, such as the proposed gradient in morphogen signal concentration itself, or one in responsiveness of the cells because of some pre-organisation of the induced tissue, were thus eliminated. The key result was that near-homogeneous allocation of cells to one or other of the above-mentioned types was seen in such reaggregates, in sharp relation to the original concentration of applied factors over a surprisingly narrow range. Thus, response was shifted to each successive new differentiation by a 1.5- to 2-fold concentration incre- ment. It is important to realise that the consequences of each inducer concentration were analysed in terms of markers of mature differentiated states, believed to be essentially irre- versible during normal development.

The image of primary body patterning in a vertebrate, suggested by the above work, is close to the most purebred form of the idea of positional information proposed by Lewis W~ lpe r t ( ’ ~ ) . This supposes pattern formation to result directly

from interpretation of the unique combination of concentra- tions of a few ‘morphogen’ signals existing at each point in embryonic tissue. Though proposed for the frog embryo in the apparent light of experimental results, the image came along in rather a revivalist spirit. By this time, both the work on the Drosophila blastoderm and also the theoretical problems in understanding highly multiple responses by similar cells to one intercellular signal, alluded to above, had engendered scepticism about this version of the positional information idea. It nevertheless has obvious merits of clarity and acces- sibility. So how near the truth of the mechanism is it likely to be?

Evidence that gradient interpretation is multi-step: inducers and cell suspensions Three recent studies by Wilson and Melton(’), Green et a/.(2) and Symes et a/.(3), all technical tours-de-force, use exten- sions of the disaggregation-activin-reaggregation method to conclude that in reality things are more complex. System properties in the form of downstream intercellular signalling, rather than the original cellular responses to the inducer directly, underlie the homogeneous and finely tuned states of differentiation observed in aggregates by the time they have reached the ‘neurula’ age. One of these paperd2) shares authorship with the original study, so that credit is due for that rather rare behaviour among real-world scientists, of actively and publicly revising one’s own recent and well-received con- clusions. But most ironically, an equally elegant study of the system by Gurdon et published almost synchronously, now claims to validate something closer to the ‘direct mor- phogen gradient interpretation’ model of five years ago, but within blastula tissue that remains intact throughout the experiment; thus essentially, within the embryo. While noth- ing can substitute for reading these remarkable but complex papers, their implications are worth considering together.

The new papers all use similar strategies. Blastula animal cap cell suspensions are treated with precise activin concen- trations and washed as before, but then the dose-response characteristics are compared for genes such as Noggin, Goosecoid, Brachyury and Xwnt-8, which, rather than being correlates of differentiations, appear much earlier in the response to induction and are more transient ‘geographical markers’. Such genes achieve localised regions of activation (sometimes more than one) within the fate map of the normal embryo during the hours of gastrulation. Their transcription is examined after a minimal time for response, for example at the equivalent of beginning gastrula stage, and then in repli- cate samples after development has reached the ‘neurula’ stage of the earlier ~ t u d i e s ( ’ ~ 3 ~ ~ ) . In addition, the results of passing the interval between induction and assay with the cells reaggregated, thus as at least a sort of tissue, are com- pared with those where they have remained dissociated. Despite potentially confusing differences of methodology, remarkably similar conclusions are reached, give-or-take an expression pattern detail or two (for instance in the case of the Noggin gene).

As examined in the older reaggregates, these gene tran- scripts exhibit rather sharp activin dose-response windows (in some cases two separate ones) or thresholds, an expected

finding in view of the differentiation of homogeneous ‘pattern- parts’ by aggregates of that age. But at short times after initial induction, these sharp responses are not seen; most levels of activin tested are found to turn on several of the transcripts to significant extents. The majority of the evolution of gene expression, required for the ultimately fine and homogeneous tuning of response to the original activin dose, requires inter- actions that have been allowed by cell re-aggregation. The very maintenance of transcription for some genes, after the early phase, requires the aggregated state. In certain instances, however, transition from the early to the late dose- response profile of a gene appears to need time alone, occur- ring even in continuing cell suspensions. This ‘focussing’ of the dose-response of a gene could occur in two ways. There may be an autoregulatory aspect to transcription such that only certain early levels result in longer-term stable activation. Alternatively the gene, while activated immediately by many inducer levels, may only remain active in cells where a partic- ular combination of other inducible genes is transcribed, a combination which implies a narrower range of initial inducer concentrations.

Each paper provides its distinctive addition to their com- mon story. Green et a/.(2) show that a geographical gradation in inducibility, whereby certain responses (those normally found dorso-anteriorly in the whole embryo) cannot be obtained from intact ventral animal cap tissue, is wiped out when cell suspensions are made. So this pre-regionalisation is no part of the multithreshold response shown by cell sus- pensions, even though it may add refinement to the pattern- ing mechanism in vivo. It must result from a ventrally centred, cell-socially maintained inhibitor or down-regulator of the response-spectrum. Another morphogen? It is important to realise that the early, apparently unfocussed gene activation spectrum has not been shown definitely to occur within indi- vidual cells. Alternatively, sharp but heterogeneous early responses in individual cells could go on to reach some con- sensus due to interaction upon reaggregation. That such may indeed be the case is further suggested by Wilson and Melton’s additional finding(’), which comes from mixing cell suspensions that have been washed after exposure to high, low or zero activin doses. They show convincingly that con- sensus, rather than dominance of one or other of the expected response categories, is the rule in longer-term development within such mixtures. In the limiting case, they can produce as the dominant gene expression pattern (thus, presumably, cell type), one that neither of the initiating inducer regimes would have given rise to.

The return to the whole embryo? To ask whether activin may organise pattern by a simple gra- dient principle in vivo, Gurdon and colleagues(4) have com- bined inducing cells with intact blastula tissue from the responsive animal cap region, as in the experiments that orig- inally gave rise to the concept of patterned induction of meso- derm and embryonic endoderm by the yolky vegetal cells(lO). They have used as the inducing component either cells of the vegetal mass or else other animal cap tissue, and in each case, have varied the source level of an activin signal by using donor embryos that had been loaded by injection with

various concentrations of activin mRNA at onset of cleavage. Recombinates where both ‘responding’ and inducing compo- nents are animal cap tissue seem particularly important, because then the RNA-injected component is more likely to be emitting activin signal alone in the first instance, rather than against a background of its normal endogenous in- ducers, or of others which some ‘autocrine’ activity of the activin has caused it to generate. The responding tissue com- ponent is lineage-labelled, and transcription of the frog Brachyury gene within it, analysed by in situ hybridisation, is used as a marker for an early induced state that is known not to occupy the position nearest to the inducer source in the normal pattern of development. Brachyury, while indeed not transcribed as near to the inducer source as are certain other markers such as Goosecoid (see below), marks much of the rest of the mesoderm at least transiently and so is not a strongly ‘positional’ gene at this stage. Nevertheless, it has enabled the authors to show that the higher the injected activin mRNA level, and thus the higher the signal level that can be assumed at the inducing/ responding junction in recombinates, then the further away into the responding tis- sue field lies the centre of a zone of Brachyury transcription, with response-competent tissue that does not express this gene but another one, Goosecoid, lying inbetween.

Ingenious controls are used to show that this patterning effect is truly a position-dependent gene activation, and not mediated by differential cell movement and/or cell division. The results undoubtedly form an impressive prima facie case for the involvement of an activin (or equivalent) concentration gradient achieved by passive diffusion in the embryo. In the authors’ view, their results also argue for rather direct ‘interpre- tation’ of the graded concentration of activin at each location, in terms of sharp induction thresholds for even the early response genes. This could lead on to tissue determinations by the mechanism originally proposed by Green and col- league~(~~1~6) before their recent results suggested more com- plexity. But this latter part of the case is not by any means closed. For instance, it is shown that Brachyury can be acti- vated in recombinates by signals passing through tissue that cannot meanwhile synthesise new protein. But the distancing effect on the location of activation of this gene and proximal replacement by a zone of a different gene, associated with the higher source concentrations of activin, is the result that might be diagnostic of direct one-step interpretation of the mor- phogen concentration. This has not yet been shown to occur as an early immediate effect, independent of protein synthesis throughout the tissue that gets patterned. It is thus possible that second order signalling is required for it. Furthermore, the patterns of gene activity in recombinates are observed at times claimed to correspond more to the ‘early’ timepoint, associ- ated with diffuse gene activation in the recent work on cell SUS-

pensions/reaggregates. But in the main body of the reported experiments, a time of 5-6 hours after tissue conjugation, with- out a need meanwhile to recover from cell dissociation, might indeed have allowed for considerable intercellular signalling in addition to formation of an activin gradient itself.

Conclusions There are cogent reasons for doubt that vertebrate primary

body patterning will turn out to rely upon direct reading of initial morphogen gradients to give five or more alternative cell states. A prior; difficulties in conceiving of transduction machinery for adequately reliable and fine-grained multi- threshhold responses to signals have been alluded to above, yet biology has a way of confounding theory-bound predic- tions. Among empirical observational reasons for doubting simplicity are the earlier-described ‘consensus’ effect, seen in mixed cell suspensions(l), and other experiments of Gurdon and his colleagues themselves, revealing a ‘community effect’(20) in the control of progress along, for instance, the muscle differentiation pathway by individual cells. It can be seen intuitively how either or both of these phenomena, that must involve further signalling, could mediate boundary- sharpening effects within a ‘pepper-and-salt‘ pattern that had been initiated by unfocussed but broadly incremental responses to a large-scale morphogen gradient.

Another property of vertebrate embryos, proportional scal- ing, also argues for a more cascade-like series of patterning signals, and furthermore, one that could not be mediated by local effects alone. Pattern is complete and the proportional extents of its parts are held constant, even when the develop- ment takes place in tissue fields of varied sizes in whole embryos. In experiments addressing this regulation in the frog blastula, the whole field of response-competent tissue, into which the in vivo equivalent of the activin signal is diffusing, is made abnormally large or abnormally small in exten@). The extents of notochord, somite muscle, lateral mesoderm and remaining ectoderm tissue finally differentiated are increased or decreased respectively, to remain in proportion (we do not know about the ‘early’ genes like Brachyuryand Goosecoidin these circumstances). This regulation takes place over a time when differential growth mechanisms can have no part in its explanation, and must involve shifts in the absolute positions of the final boundaries between zones of cell types, in relation to a constant-sized initiating morphogen source. Only some principle of formal negative feedback signalling, superim- posed on any simple morphogen gradient and its interpreta- tion, can readily account for it(22). The field of biological pat- tern formation thus continues to afford room for radical conjecture and controversy, to those who enjoy these things. But equally clearly, with each successive year from now on, we shall have more hard and elegantly obtained information on which to base our speculations and visions.

Acknowledgement I thank Dr Adam S. Wilkins, editor of BioEssays, for help in drafting this article.

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Jonathan Cooke is at the National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 I A A , UK.