gastrulation and larval pattern in xenopus after blastocoelic injection of a xenopus-derived...

DEVELOPMENTAL BIOLOGY 131,383-400 (1989)

Gastrulation and Larval Pattern in Xenopus after Blastocoelic Injection of a Xenopus-Derived Inducing Factor: Experiments Testing

Models for the Normal Organization of Mesoderm

JONATHAN COOKE AND J . C. SMITH

Laboratory of Embryogenesis, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

Accepted October ~, 1988

When a Xenopus XTC cell-derived mesoderm-indueing factor (MIF) is injected into the blastocoel of Xe~mpus embryos before gastrulation, they develop almost normally until jus t af ter the onset of mesoderm involution at the internal blastoporal lip. Cells from the ent ire lining of the blastocoel roof and inner marginal zone then undergo a synchronous, sudden change of contact and a r rangement which resembles the t ransformat ion undergone by normal mesoderm at its t ime of involution a t the vegetal edge of the marginal zone. We describe a dose-dependent spectrum of subsequent abnormalit ies in gastrulat ion and, in cases where gastrulat ion partially recovers, in the result ing larval pat tern. Because of such recovery, embryos injected with widely different doses may appear equally abnormal at the early gastrula stage but very different by control larval stages. Ext ra spinocaudal axial patterns, in the area of ectopie mesoderm, are seen after MIF doses tha t jus t permit recovery of gastrulat iom The sudden cellular t ransformat ion corresponding to involution, in the ectopically specified mesoderm, spreads throughout the animal cap within 15 min in individuals, a t a t ime significantly later than the earliest normal t ransformat ion in the marginal zone. No systematic al terat ion could, however, be detected in its timing, in relation to a 250-fold range of injected MIF concentration or a 3.5-hr difference in t ime of injection. The severity of the effects on final embryonic pa t tern is largely independent of the blastular stage of injections. Spli t t ing of the total injected dose into two, separated by 2 to 3 h r of blastular development, reveals tha t the degree of effect on gastrulat ion and pat terning depends only upon the highest experienced concentration at any time before response. When fibroblast growth factor (bFGF), a different effective mesoderm inducer, is similarly injected, a s imilar abnormal cell behavior and ectopie mesoderm formation are seen, but beginning only a t m idgas t ru l a r s tages some 1.5 h r beyond t h a t charac te r i s t i c of XTC-MIF. The findings are in t roduced and discussed in te rms of models for the natural organization of the t ime course of gastrulat ion and mesodermal pattern. �9 1989 Academic Press, Inc.

INTRODUCTION

�9 Xenopus XTC cell culture supernatant contains a sol- uble protein that is a highly effective inducer of mesoderm, including tissue of dorsal axial character, from amphib ian b las tu la r an imal cap cells (XTC-MIF) (Smith, 1987; Symes and Smith, 1987; Cooke et aL, 1987). The factor has been purified and partially characterized, is likely to be a member of the TGFfl-related fam- ily of secreted proteins, and is effective in the low picomolar concentration range (Smith et al., 1988). The above publications have concentrated on those features of the response that encourage the belief that XTC-MIF is or closely resembles a normal signal for mesoderm formation (see Nieuwkoop, 1977; Gurdon et aL, 1985; Warner and Gurdon, 1987), though definitive evidence for this is lacking to date. The factor probably initiates mesoderm as a s ta te of specification per se (Slack, 1983), while subsequent formation of particular mesodermal tissue types perhaps involves a cascade of further intercellular stimuli (e.g., Symes et aL, 1988). There is, however, already evidence that an in situ gradient of concentration in the molecule, if present in the embryo,

might orientate the dorsoventral aspects of normal mesodermal pattern (Cooke et aL, 1987; Smith et aL, 1988).

The whole animal hemisphere of the blastula is competent to form mesoderm, and we presume the normal relatively restricted origin of mesoderm from the equatorial (marginal) region to be related to the origin of an initiating signal in the vegetal region (e.g., Gimlich and Gerhart, 1984), in conjunction with some as yet un- known regulating signal of the sort that ensures pro- portionality in biological patterns generally (Wolpert, 1971; see Cooke, 1985, in relation to Xenopus development). It is nevertheless possible to initiate ectopic mesoderm specification, throughout the blastocoel wall, by injecting MIF at p regas t ru la r stages. Such experiments, followed by anatomical analysis, can comple- ment work at the cell and molecular levels in helping us understand how the organization of in situ inductive events leads to the regulated spatial pa t te rn of the body.

We have studied the effects on the whole Xenopus embryo, at all subsequent stages up to the larval, of

383 0012-1606/89 $3.00 Copyright �9 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

384 DEVELOPMENTAL BIOLOGY VOLUME 131, 1989

irrigating the blastocoel with a wide range of XTC-MIF concentrations at a range of blastula stages that probably spans much of the period for normal mesoderm induction. Embryos that have developed ectopic mesoderm alongside or instead of their own such tissue are potentially informative in assessing particular models for normal mesodermal patterning, especially if the timing and concentration of the experimental, ectopic signal have been varied widely within the extended period of competence to respond to it. We describe two aspects of the effects observed, which we believe are relevant to thinking about the natural organization of mesoderm, and then discuss in detail just those observations which we have used in our own thinking.

First, we extend and illustrate in more detail the observations on alteration of the course of gastrulation, and subsequent reduction in larval body pattern, mentioned in a paper surveying all the biological properties of XTC-MIF (Cooke et al., 1987). Especially when taken together with the work of Keller and his colleagues (Keller, 1976, 1986; Keller and Schoenwolf, 1977; Keller and Danilchik, 1988), these observations can help us understand the mechanical organization of gastrulation and certain aspects of pattern formation within normally induced mesoderm.

Second, on the basis of the general observation about timing of ectopic cellular changes in injected embryos, we have performed a series of more detailed experiments aimed at testing certain simple hypotheses for the origin of anteroposterior patterning in normally induced mesoderm. These experiments capitalize on the fact that amphibian gastrulation (probably like that of all ve r t eb ra tes ) i s a highly spatially and temporally organized process. They consist of observing closely the exact developmental time at which the rapid sequence of cell behavior occurs that corresponds to the normal process of involution, but within the ectopically specified mes0derm. We call this change of behavior the "mesoderm transformation." We find that the exact time point of the ectopic transformation is essentially invariant with the concentration and timing of the ectopic MIF signal. In contrast, normal transformation of mesoderm is progressive and spatially organized in the marginal zone, occurring over several hours.

Since we use these observat ions as exper imenta l challenges to broad models for the natural organization of gastrulation and the body plan, it is appropriate first to review briefly the evidence that justifies t reat ing normal variation in the time of mesoderm transformation during development as an expression of some preliminary patterning across this tissue. By the onset of gas t ru la t ion there undoub ted ly exists a fa te map, whereby cells' positions within the marginal zone in normal development significantly predict their posi-

t ions wi th in the mesoderm of the final body (e.g., Keller, 1976). The mediolateral sequence of cell types and also the presumptive head-to-tail axial sequence are related to latitude or height of preinvolution mesoderm, and thus to distance from the initiating source of induction. We present below some observations that support previous ones from this and other laboratories (Cooke, 1979, and especially Keller, 1984, 1986) in suggesting that the times at which local groups of cells perform the activities that we call involution into the mesodermal mantle are also ordered, in a systematic sequence that relates to the pregastrulation fate map. The tissue is recruited into a head-to-tail sequence, over some hours, by the progressive involvement in transformation (and thus involution) of cells that were situated ever further from the original endomesodermal boundary in the blastula. These correlations reflect a qui te detai led o rches t r a t ion of graded proper t ies , whereby each locali ty cont r ibutes on its own time schedule to the active shape change that drives gastrulation. Observations on a range of experimentally disturbed embryos have shown that these time schedules for morphogenetic activity are autonomous and fixed by gastrulation, and reflect broad specifications, within the tissue, for the particular parts of a normal body plan that cells will construct (Cooke, 1985, 1987). The absolute time of onset and then the duration of each embryo's gastrulation movements, in relation to those of normal siblings, is predictive of the parts of a normal body plan that its mesoderm will make.

Some gene products that very probably have primary roles in encoding axial positional specificity appear to be synthesized only at postinvolution stages of mesoderm development in ve r tebra tes (e.g., Condie and Harland 1987; Gaunt, 1987, 1988). The positional organization in preinvoluted tissue, that is the subject of the present experiments, may well be of a physiological nature that does not as yet involve differential gene activity (see Gerhart, 1980). But this preorganization is very probably a prerequisite for the immediately postgas- trular, s table regionalization in mesoderm that has been documented in this and in other vertebrate embryos (e.g., Kieny et al., 1972; Snow, 1981). Such "preorganization" of the newly specified mesoderm could be underway by stage 8, when the movements of epiboly begin to concentrate it into the marginal zone (see later), and is certainly advanced and autonomous, in the dorsal region at least, by the onset of the schedule of involution at stage 9§ to 10. Moderate-sized pieces of the preinvoluted tissue, reorientated in situ or hetero- grafted at the latter stages, retain autonomous, polarized schedules of involution activity corresponding to local parts of the whole spatiotemporal pattern of gastrulation (Cooke, 1972; Keller, 1984).

COOKE AND S~,IITH Gastrulation a~zd Larval Pattern in Xe,zop~s 385

In the present work we have assumed that the timing of mesoderm "transformation" indicates its axial positional value; the place that it would have been specified to occupy in a normal body plan. A measure of independent validation for the deduction of mesoderm's character from its schedule of mechanical behavior is provided by the observation, presented below, that mesoderm ectopically produced after injection of a different mesoderm-inducing factor, bFGF, which is not expected to be of dorsal axial type, does indeed transform at a time in gastrulation very different from that due to XTC- MIF. In the discussion we return to certain possible models as to how an in situ signal equivalent to XTC- MIF might be utilized in the normal preorganization process, and conclude that none of these corresponds with the real mechanism in light of the results presented. It is therefore proposed that early patterning of the marginal zone is achieved by the addition of component signals that have been initiated by an original activator or activators of mesoderm formation. XTC- MIF remains a strong candidate for an early, activating component in this mechanism, and its source may be asymmetrically arranged in the egg's vegetal region.

MATERIALS AND METHODS

Preparation of Morphogen Dialysate

Partially purified MIF was obtained as described by Cooke etaL (1987). It was dialyzed for 3 hr at 4~ in a microdialysis apparatus against 1000 vol of 67% NAM (a balanced amphibian saline) (Slack, 1984), pH 8.0. Bo- vine serum albumin fraction V was added to 0.5 mg/ml before dialysis to minimize protein losses. This preparation was active at a dilution of 500- to 1000-fold in the animal cap isolate assay for mesodermal induction (Smith, 1987). A control dialysate prepared from 30X concentrated medium from culture of XL cells (non- MIF-secreting) was wi thout any of the effects to be described and allowed normal development when injected, even though it contained a much higher diver- sity and total concentration of trace proteins. Accord- ingly, it was used as a diluent to achieve the required range of MIF concentrations in blastocoelic injections of constant volume, and as the injection for control embryos. The single dialysate preparation for the work reported here was frozen as 5-~1 aliquots at -70~ and each aliquot was rethawed only once to 4~ and used over 3 days or less. The pure protein, which is now partially characterized and reproduces all the properties of cruder preparations, induces axial mesoderm in cultured animal caps at picomolar concentrations, i.e., <1 ng/ml. A dialysate as above but containing only 0.5 mg/ml BSA and this protein at about 2 pg/ml (i.e., active at 1/5000) was used in the timing comparison

experiment involving a 250-fold range of blastocoel concentrations.

Preparation, Injection, and Onward C~dture of Embryos

Batches of synchronously developing eggs, of uniform size and pigmentation type from each female frog for each experiment, were obtained by in vitro fertilization according to normal procedures. Embryos were dejel- lied at stages 6 and 7 (Nieuwkoop and Faber, 1967) by gentle swirling and passage through a wide-mouthed pipet in 2% cysteine HC1 adjusted to pH 7.9 with NaOH, washed in 50% NAM, and kept for injection in a layer of 5% Ficoll (Sigma, 70,000 mol wt) in 10% NAM. They were then orientated animal pole uppermost in fitting wells in a Teflon plate and left to develop to the required stage. Under these conditions blastocoels are well developed and regular-shaped even at the earlier, larger-celled stages when blastocoel volume is smaller.

Groups of embryos received a uniform injection with set dilutions of the MIF dialysate, through the vitelline membrane at the animal pole using a finely tapered micropipet of about 20-pm tip diameter and a c o m : pletely oil-filled pressure system driven by an Agla mi- crometer. Dissection and measurement with a microm- eter eyepiece sugges ts tha t blastocoel volumes at around stage 8 in different egg batches are between 300 and 500 nl, and might double during development from stages 7+ to 9+. The observations described first in this paper follow injection at blastula stage 8, with an injection volume of 250 nl, although we know that there is little change in final effect when a particular concentration is delivered anywhere from stages 7 to 9+. Fol- lowing injection over about 10 sec, the embryo swells visibly and then relaxes more slowly as the volume is restored by elastic tension and leakage between cell borders at sites around the animal cap. Similar injection into a 500-nl drop of saline-containing yolk parti- cles reveals rapid and complete mixing, so we assume blastocoel lining surfaces to experience a homogeneous concentration of MIF in the minutes following injection.

Sibling blastulae in the precise timing experiments were given injection volumes of 200 nl (stage 7.5) or 300 nl (stage 9). Differences in the graded effects on development already described are only jus t appreciable after synchronous injection to give a 2-fold difference of ectopic MIF concentration in the sensitive middle part of the dose range. The 1.5-fold difference of volume injected according to stage was thus considered to represent an equivalent dose to cells lining the blastocoel.

Embryos were removed to 10% NAM alone, 20 min after injection, and any showing damage other than a small herniation of animal pole tissue at the membrane

386 D E V E L O P M E N T A L B I O L O G Y VOLUI~IE 131, 1 9 8 9

puncture site were discarded. Onward culture was always side by side a t uni form 19 or 20~ for the synchronously developing samples of each experiment.

Fixation and Inter~al Examination of Embryos

Embryos at gastrula stages were fixed in modified �89 strength Karnovsky fixative (Ito and Karnovsky, 1968), resin embedded, sectioned at 2 pm, and stained with toluidine blue. Addition of 0.1% picric acid to the fix, and use of TAAB "4 part" resin (TAAB Laboratories, Ltd.) embedding procedure, has been found to improve the histology of this material though it has proved im- possible to obtain a complete and reliable series of fix- ations for these very delicate changes in cell behavior. In addition, embryos at these stages were explored by bisection of subsamples of five synchronous siblings with tungsten needles, in an approximately sagi t ta l plane a few minutes after the start of fixation in 1% potassium dichromate, 2% glacial acetic acid in dis- tilled water. This allows good visualization of the layer structure and cell behavior under the high power dis- secting microscope. It could be clearly seen, in such individual embryos, whether or not the transformation and delamination of inner animal cap cells to form a new, abnormal mesodermal layer had occurred or were occurring. The progress, normal or otherwise, of endogenous mesoderm transformation could also be recorded, as could such features as the epibolic redistribution of animal cap cell numbers by the onset of normal gastrulation, the convergence of axial type ectopic mesoderm from stage 10.5 onward (superficially, a movement the reverse of epiboly), and the abnormal invasion onto mesodermised blastocoel wall by endoderm. Develop- ment was followed between stages 9+ and (where neces- sary as in the case of bFGF injection) 11.5. The times taken to transit the gastrula stages at 19~ were noted

�9 in'certain egg batches. The timed specimens were embedded in TAAB resin and cut parasagittally at 2 pm as a record of the natural time course for the sequence of mesoderm transformation.

The pattern of differentiation in larvae at stage 32 was examined by double wax embedding, sectioning at 7 pm, and staining with the Feulgen/light green/orange G procedure, as well as by indirect immunofluorescence on polyethylene distearate wax sections using a rabbit antiserum to Xenopus larval/adult globins (Cooke and Smith, 1987), a monoclonal antibody recognizing muscle (12/101--courtesy of J. Brockes, MRC), and one recognizing amphibian central nervous system (aNS 269-- courtesy of Elizabeth Jones, University of Warwick).

The gradations observed in the abnormal macro- Scopic appearance by control axial larval stages, following graded blastocoelic doses of the factor, enabled the severity of the effect in particular samples to be com-

pared in a semiquantitative way (see observations and Fig. 4).

OBSERVATIONS

The Ab~mrmal Course of Gastrzdation

The first dramatic abnormality observed following MIF injection occurs short ly but significantly after onset of the sequence of involution of the normal mesoderm. We describe it here in more detail than was recorded in Cooke et al. (1987), to facilitate the understanding of the entire sequence of abnormal development. There is a sudden change in the affinity properties and surface motility of a large fraction of the cells lining the entire animal hemisphere, i.e., from the presumptive deep ectoderm and normal mesoderm (Keller, 1976). These cells go through a brief (<20 min)

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FIG. 1. The onset of abnormal morphogenesis early in gastrulation. Tracings from montaged 2-pm plastic sections stained with toluidine blue. (A) Longitudinal section near the dorsal midline of stage 10.5 normal gastrulation. The overall shape of the embryo is distorted but the cellular behavior of the layers is well seen. External lip near internal origin of mesoderm is to the left (arrow), and sharp interface of endoderm and blastocoel wall ahead of anter iormost mesoderm is to the right. Future posterior end is to the left, and dorsal is a t the top. (B) As in (A), but from an embryo af ter an injection into the blastocoel of MIF (0.01 units, stage 8+). Orderly behavior change and involution of mesoderm at the left is no longer apparent, while the ectopic mesoderm has already formed a separate tissue, leaving a surface monolayer of remaining ectodermal cells in the wall of the blastocoel to the right. Endoderm is beginning its disorderly advance onto the ectopic mesoderm. Scale bar equals approx 130 pro. Ecto- derm, pale grey; mesoderm, dark grey; endoderm, stippled.

COOKE AND SMITH Gastrulation and Larval Pattern in Xenopus 387

phase of loosening themselves from their previous in- active, relatively close-packed conformation, to emerge and separate from their layer of origin. They then sit as a new tissue of loosely joined cells upon the basal surface of what remains of tha t layer (Figs. 1 and 2). This behavior resembles that of normal newly involuted mesoderm that is first seen at the vegetal end of the marginal zone beneath the floor of the blastocoel, and which is indeed meanwhile forming in these embryos (Fig. la). But whereas normal involution is localized and is undergone sequentially by cells derived from progressively higher levels of the marginal zone as time proceeds, the abnormal ectopic version occurs substantially everywhere at once. A new, ectopic tissue layer, in a condition corresponding to postinvolution mesoderm, is

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FIG. 2. The effect of MIF on gastrular organization by midgastru- lation. A normal gastrula (A) and one arrested by MIF injection (0.015 units, stage 9) (B) at stage 11.5. The embryos are shown in longitudinaI section, with the original site of the blastopore ring at the upper left and the dorsal meridian at the top and right. In the abnormal case, the arrest of blastopore closure, the disorganization of the marginal zone, and the contraction of the animal pole region due to convergence of its ectopic mesoderm are clearly seen. Scale bar equals approx 450 pm. Germ layers are shaded as in Fig. 1.

thus created within minutes to line the entire blastocoelic roof and wall. There is no possibility of its coher- ent migration since, in contrast to the normal situation, none of the suitable substrate (i.e., the remaining basal ectodermal surface) is unoccupied. Figures 1 and 2 show thin-sectional appearances of normal and blastular MIF-treated embryos during early and middle gastrula stages, some 30 and 90 min after the delamination of the ectopic mesoderm that occurs during stage 10.

Dissection of newly fixed embryos reveals a subtle earlier abnormality in blastulae that have received the early, relatively high dose injections of MIF only (e.g., 0.02 units or more at stage 7+). The normal movements of epiboly, whereby the marginal zone achieves more cell layers at the expense of a reduction in cell layers near the animal pole by late blastula stages, is often absent or diminished. The entire animal cap remains of uniform s t ruc ture averaging around three cells in depth, giving the blastocoel a more central location in these embryos. This observation implies that the normal equatorial accumulation of tissue is due to actively driven thinning of those parts of the animal cap that have not been respecified as mesoderm (see Ke l le r , 1986). In MIF-injected cases the entire deep animal region can a t ta in this specification so tha t the active thinning is switched off.

Once the dramatic departure from normality has occurred during stage 10, the processes of external blastoporal lip advance, archenteron formation, and orderly extension of involuted mesoderm are permanent ly (high MIF doses) or t empora r i ly (low MIF doses) halted. The ectopically induced mesoderm will not act as a substrate for migration, or allow convergent extension upon itself, of other mesoderm. There is thus no advance of a mantle of involuted mesoderm under tha t which is still approaching its own time point of involution (see Keller, 1986; Keller and Danilchik, 1988), and the advance of any "endogenous" mesoderm that had already involuted is halted by the ectopic tissue (Figs. lb and 2b). Shortly after this, during control stages 10.5 to 11, the tissue of the endodermal mass begins to change configuration to invade the entire face of ectopic mesoderm, thus eliminating the blastocoel. An abnormal new cavity forms within the endoderm. This change is seen in progress in Figs. lb and 2b, and complete in Fig. 3b. Normally, the cup-shaped anterior face of the endoderm comigrates with the anterior edge of the mesodermal mantle until finally the blastocoel remains as a small anteroventral space roofed with epidermis that has never been lined by mcsoderm. The general nature of this halt to gastrulation has also been depicted schematically in Cooke et al. (1987) (Fig. 3).

An additional, dose-dependent factor concerns the proportion of the animal cap cells that has been meso-

38S DEVELOPMENTAL BIOLOGY VOLUME 131, 1989

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dermized via the ectopic route. At all doses above 0.001 units MIF injected, this is substantial, and enough for a complete new tissue layer to be formed at the time of transformation. After higher dose injections, however, it is so massive as to effectively eliminate the reservoir of presumptive normal mesoderm, high in the marginal zone, that would have involuted at later stages in the normal sequence. Compare the monolayer of ectodermal cells remaining to the right in Fig. lb with the thick wall of the normal embryo (albeit exaggerated due to slightly tangential plane of section) in Fig. la.

Figure 3 shows thin sections of embryos at the control late gastrula stage 12.5. It illustrates differences between experimental individuals that develop by these stages, dependent upon the blastocoelic MIF concentration originally given (see legend). One variation is in the postinvolution morphogenetic behavior of the ectopic mesoderm, now situated between the wrongly migrated endoderm and the more or less depleted ectoderm around the presumptive anterior end. After the lower dose inject ions this ectopic mesoderm may become multilayered but remains of open, spongy texture. The animal cap of such embryos remains relaxed in external appearance, with little tendency to contract its area as a neural plate does. A puckered surface appearance may be due to inner cells having been transformed and thus lost to the new, mesodermal layer. After higher doses, however, convergence and tighter intercellular packing to give columnar cells do develop in ectopic mesoderm, overlaid by strong loci of pigment gathering and co- lumnarization in the ectoderm. We assume these cellular behaviors to represent adoption of dorsal axial character, and the associated occurrence of neural induction (see later).

The second dose-dependent variation illustrated in Fig. 3 is in the degree to which the schedule of normal gastrulation is taken up again after its initial arrest. Above about 0.025 units MIF this scarcely occurs, and embryos mechanically unable to gastrulate may nevertheless be kept until control larval stages after which their epidermal differentiation fails and they disinte- grate. After progressively lower doses, significant recovery of normal involution activity, with lip advance or even archenteron invagination, occurs progressively sooner within the normal time schedule for completion of that process. Thus, despite total arrest during the first hour or more of the control schedule, mesodermal involut ion and migra t ion on an in ternal in ter face around a closing yolk plug may have attained a largely normal appearance by stage 12 or 13. Correlation with the final larval pat terns (see below) reveals that the later the resumption of such normal gastrulation activity, the more exclusively posterior is the only well-organized part of the body that results. Even at the lowest doses, tha t allow an otherwise largely normal axial pattern to form, there is a region filled with ectopic mesoderm replacing the normal position of the blasto: coel remnant anteriorly.

It is important to emphasize that embryos never re- normalize by an altogether belated, "catch-up" gastrulation. The results fit with those of all other experimental alterations of Xenopus body pattern formation (see Cooke, 1985; Cooke and Webber, 1985), in suggesting that this recovered normal mesoderm contributes to the plan of the body according to its intrinsic physiological "age" at the t ime of its active performance of the t rans format ion tha t is involution, within a normal spread of such "ages."

FIG. 3. Variable recovery of mesoderm transformat ion in the marginal zone, and appearance near the animal pole, a t late gast rula stage of MIF-injected embryos. (A-C) Low power photomicrographs of sibling embryos in horizontal longitudinal section at stage 12.5. (D-H) Higher power views of regions within them. (A) The normal appearance. The advance of the blastopore toward closure is seen, as is the organized progressive t ransformat ion of mesoderm, which migrates between a thick (cell rich) ectoderm and the endoderm. The f ront end of the embryo (right) and the roof of the blastocoel r emnan t are seen to be still free of mesoderm. The lateral corners of the archenteron are apparent posteriorly. (B) An embryo af ter a dose of blastocoelic MIF tha t permanent ly prohibits gastrulat ion (0.04 units) delivered at stage 9. Mesoderm formation by normal progressive t ransformat ion and lip formation remain at a standstill . There is massive abnormally derived mesoderm between a thin ectoderm and the endoderm throughout. The blastocoel cavity is obliterated and there is no archenteron, but volume is maintained by a massive new vegetal or "posterior" cavity in the endoderm. (C) A fur ther embryo af ter a low dose of MIF (0.004 units) delivered a t stage 9. Such an embryo will go on to form a body with various of the features i l lustrated in Figs. 4 and 5. Although ectopic mesoderm is seen throughout the space between ectoderm and endoderm, and endoderm has migrated to obli terate the blastocoel, blastopore closure and normal t ransformat ion of " la ter" mesoderm are seen to have resumed. (D) The blastopore region of same embryo as in (A) still shows massive t ransformat ion of mesoderm to be occurring on lateral meridians (same orientat ion as in (A)). (E) Region from the marginal zone of the embryo in (B). This is the only region in this embryo at which anything resembling an external lip and normal mesoderm t ransformat ion is seen. Note tha t the ectoderm contains fewer deep cells than tha t in (D). (F), (G), and (H) are shown rotated 90 ~ counter- clockwise is relation to the low power views. (F) An animal pole region from the embryo in (B) where the massive ectopic mesoderm is beginning convergence. The remaining ectoderm is almost a monolayer, and its basal interface with the mesoderm appears to be disorganized a t this part icular site. (G) The blastopore region in (C) shows a recovered appearance and considerable mesoderm transformation, but note reduced populations of deep ectodermal layer and thus of "normal" mesoderm. (H) An animal pole region from the embryo in (C). Note tha t eetopic mesoderm is much less massive, and ectoderm thicker, than tha t in (F). Scale bar equals approx 650 #m for (A-C), and 300 #m for (D-H).


The Ab~mrmal Pattern o f the Larval Body

A two hundred fifty-fold range of MIF concentrations has been employed altogether in the various parts of this study, although it is only across the lower 20-fold part of this range that any recovery of gastrulation to give partial larval patterns is seen. Figure 4a shows a group of such larval forms, and Fig. 4b illustrates a complete representative series of external appearances which can be given arbi trary grades, much as has been done for forms tha t follow uv i r rad ia t ion of eggs (Scharf and Gerhart , 1983) or lithium t rea tmen t of early blastulae (Cooke and Smith, 1988). These are illustrated here, with their grades, because such semiquantitative t reatment enables samples to be compared for severity of effects in analytical experiments (see later). Typical values for the amounts of MIF activity injected to cause each grade, in previously defined units (Cooke et aL, 1987), are given in the figure legend. One

unit of activity is now known to correspond to less than 0.5 ng of pure protein (Smith et al., 1988).

Figure 5 shows histological features in larval bodies after various grades of "recovered" development. There is progressive loss of the anterior end of the mesoderm pat tern and endoderm archi tecture with increasing dose. Following the higher permissive doses the anteriorly situated ectopic mesoderm is liable to differentiate as somite muscle, though never notochord (Cooke et aL, 1987). Even after the lowest doses where brain parts and head endo-mesodermal structure are still formed, these are compressed and ill-differentiated (Fig. 5d). This may be because of the tack of a suitable substrate for the spread of an "endogenous" prechordal mesoderm that does in fact involute normally, rather than because of a direct effect of the ectopic inductive stimulus in the blastocoel. Delay of the injection until stage 10+, which delays ectopic transformation until stage 10.5 when the normal prechordal material has migrated

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FIG. 4. The external appearance and graded severity of abnormal larval forms af ter blastular MIF injection. (A) A group of synchronous siblings at control axial larval stages. Control (grade 0) is at the top, and individuals of increasingly disturbed morphology are arranged from above to below, with two tha t have been permanent ly arrested at the bottom (grade 5). (B) Drawings from camera lucida sketches of typical members of the series, used in quant i ta t ion of the effect of XTC-MIF on development. All MIF injections were done prior to stage 9. The grade number is given next to each individual. The control (grade 0) shows the anatomy tha t is usually apparent to external inspection. In grade 1, seen af ter injection of 0.002-0.003 units of MIF (see Cooke et aL, 1987), the hear t is underdeveloped or absent and there is no blood formed. Head parts are present but bent down and often reduced in size. Anter ior somites are poorly organized. In grade 2 (0.005-0.006 uni ts MIF), the nervous system pat tern is incomplete anteriorly and the neural tube is often reduced, opened, and joined by a second axial formation in the anteroventral region. In grade 3 (0.008-0.01 units) yolk plug closure is incomplete and there is no axial s t ructure visible except an unexpanded tailbud. The epidermis is wrinkled anteriorly and thin or monolayered elsewhere. In grade 4 (0.01-0.02 units) most of the epidermis is wrinkled and underlain by solid eetopie mesoderm, but significant posterior axis formation has occurred around the r im of the exposed yolk mass. In grade 5 (above ca. 0.02 units MIF) there has been no recovery of gastrulation, and a contracted cap of monolayered ectoderm and eetopic mesoderm sits on top of everted yolky endoderm. Note tha t in the experiments on t iming of ectopic mesoderm transformation, the dose range explored is expanded above this threshold. Within tha t upper dose range, a range of properties of the ectopic mesoderm is seen by graf t ing (e.g., Cooke et aL, 1987). Scale bar equals 4 mm approx, in drawings in (B).

COOKE AND SMITH Gastrulatiou a~zd Lart'al Pattern in Xenopus 391

, + o ,+

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Scale FIG. 5. Structural features of larval body pat terns developing af ter low-dose I~IIF injections (0.005 units, stage 8+). (A) Transverse section

through anteroventral region of a body tha t shows accessory axial s t ructure derived from ectopic mesoderm. Stained with DAPI for arrangement of nuclei. Note tha t a ridge of mesoderm with irregular cellular s tructure underlies a tubular formation connected with the ectoderm. (B) The irregular cellular s t ructure is revealed by appropriate immunofluorescence of a neighboring section of tha t in (A) to be a s t r ip of disorganized somite muscle (12/101 antibody, see Materials and hlethods). Such axial formations do not segment in the dimension normal to the photomicrograph, but they and the tubular s tructures are elongated (see Fig. 4). (C) On another such sectioned formation, the tubular s t ructure is revealed to be neural by immunofluorescence (aNS269 antibody, see hiaterials and Methods). (D) Grazing section through the downbent anter ior end of a body after a low MIF dose. Feulgen/ l ight green/orange G wax histology. Pharynx (ph), brain ventricle (br), base of eyecup (e), notochord (nc), and anter ior somite (s) appear in the section, but the normally large hear t and pericardial cavities are absent. The ventral reflexion of the headpar ts is related to this and to the absence of any blastocoel-derived cavity free of mesoderm. (E) Transverse section a t pronephric level of the normal stage 32 body, showing a highly organized mesoderm and bilayered epidermis, n, notochord; s, somite; pn, pronephros, lpl, lateral plate. (F) Similar sectional level through a low-medium dose "MIF" body. Apar t from the massive notochord, the mesoderm appears disorganized and indistinct in its layer structure. The epidermis is monolayered only, and an abnormal neural formation appears ventrally. Scale bar equals approx 250 pm for (A-C) and 750 ttm for (D-F).

well, allows much better development of head pattern at these low doses.

Beginning at the low end of the dose range, blood, heart, and (in the much more severe instance) proneph-

ros are lost from the pattern of differentiation in that order. The absence of blood in even minimally affected larvae was confirmed by immunocytochemistry for glo- bin-containing tissue on sections of 15 individuals at


stage 40 (Cooke and Smith, 1987). In none were any erythrocytes or hemopoietic tissue detected, even where heart and endothelial spaces were present. Hemoglo- bin-free larvae with beating hearts can be observed when almost no other pattern disruption is apparent. Notochord and parachordal or unorganized somite tissues are the last elements to be lost from the original mesodermal pattern, and can be found as a thick mantle surrounding a featureless core of endoderm in examples (grade 4) that have only just recovered gastrulation. Effects in ectoderm-derived structures are the progressive impoverishment of the cell numbers available for formation of epidermis and neural plate. The former is often abnormally stretched or even single- rather than double-layered in larvae of grade 2 and above, while in those of grade 3 and especially grade 4, any neural ru- diment is clearly unable to undergo proper morphogenesis.

Figure 4 shows that the simple progression of the grades of pattern impairment is complicated, at doses that just permit gastrulation and at slightly lower ones, by significantly organized accessory tail or "spinocaudal" axial patterns. These are of a characteristic kind, whereby the associated neural tube or plate runs into the embryo's main neuraxis anteriorly at an approximately hindbrain level of pattern, with more rostral pattern being absent. The following of development in fix-dissections reveals the mesoderm of these accessory axes to have been organized from the ectopic population lining what would have been the blastocoel remnant in an anteroventral position. They form from the examples of such mesoderm that s tar t to behave in axially organized fashion at control late gastrula stages (see previous section). Figures 5a-5c show the tissue compo- sition of such structures, verified by immunofluorescence for somite muscle and Xenopus nervous system. They never contain notochord and, in contrast with second axes obtained after "Spemann grafting" of MIF- induced tissue to a host (Cooke et al., 1987), their ridge of somite tissue is never truly segmented. Their organization and production of a tail is, however, unmistakable. They are not seen in control-injected specimens, and their formation is not obviously centered on the original pipet penetration site. Their presence does not form par t of the grading system because, al though common, their incidence is highly dependent upon individual egg batch.

Exact Timing of Transformation in Ectopic Mesoderm in Relation to Concentration, Blastular Stage of DelivelT, and Molecular Identity of the Injected Inducer

As outlined in the Introduction, the relative time of a mesoderm cell's transformation, within gastrulation as

a whole, seems to be diagnostic of mesoderm prespeci- fled for different overall locations within the body. To check the time sequence of normal mesoderm involution, we examined gastrulae in semithin longitudinal sections after Karnovsky fixation. These gastrulae had been developing at 19~ in a batch that was meanwhile the subject of one of the timing experiments on ectopic, MIF-induced "involution" that are presented below. The timing relationships are as follows. Dorsal and lateral prechordal material begins involution about 11 hr after fertilization, before stage 10 onset, and proceeds for 45 min to 1 hr before the first "axial" mesoderm involution begins. The latter is more obviously sequen- t ially organized around a narrow zone of behavior change or "internal lip," with convergent extension occurring immediately after involution from stage 10.5. The progressive invagination of an archenteron then occurs because this axial mesodermal tissue grips the suprablastoporal endoderm that overlies it while per- forming the involution and extension sequence (see Keller et aL, 1985; Keller, 1986; Keller and Danilchik, 1988, for fuller explanation). Figure 6a shows the involution sequence of the normal dorsal axial mesoderm to be still in progress up to small yolk plug stages, more than 2 hr after the onset of involution of dorsal axial mesoderm. Only after this is the posterior ectoderm seen to have thinned completely by contr ibut ing its deeper layers of cells to the sequence of involution via the "inner blastoporal lip." This mesoderm can be said to be that which will convergently extend in an organized way to give the notochord and juxtanotochordal portions of all the somites (Cooke and Webber, 1985; Keller et al., 1985). Natural transformation in mesoderm from lateroventral sectors begins some 30 min after that of axial mesoderm, thus more than an hour after that of prechordal material. Figilre 6b shows that its time course too is protracted, continuing to an appreciable extent up to stage 13, perhaps 5 hr after the point of MIF-induced transformation (see also Cooke, 1979; Keller, 1976).

Figure 7 shows camera lucida outline dra~vings of fixed late blastulae and gast rulae bisected approximately in the saggital plane. Cellular textures of particular regions, i.e., the approximate numbers and dispo- sitions of cells, are also indicated for certain cases. It is seen that apart from the lack of epiboly and thus the equatorial position of the new dorsal lip after some early blastular MIF injections, experimental embryos at the point of ectopic mesoderm transformation are rather similar in appearance and Nieuwkoop and Faber stage, over a variety of concentrations and times of de- livery of MIF. This observation confirms that the history of its induction is "stored" by the responding tissue for from one to several hours, until the appropriate

COOKE AND SI~IITH Gastrulation and Larval Patter,: in Xeq2opus 393

A

T 1

TO T 1

C

\

FIG. 6. The extended time course of the normal craniocaudal sequence of mesoderm transformat ion in Xenopu~ (A) The extent of mesoderm transformat ion in the normal dorsal midline at stage 11.5, traced from a toluidine blue-stained thin section near the sagit tal plane. The cellular detail does not reproduce adequately at the required size for an entire photomontage of the mesoderm. This is a t least 3 h r af ter the onset of t ransformat ion of prechordal mesoderm a t stage 9-10, and 2.5 af ter the t ime at which synchronous t ransformation of ectopic, MIF-induced mesoderm occurs regardless of dose and blastular stage of MIF injection. Appreciable axial tissue of the normal blastopore lip is yet to pass through the t ransformat ion process posteriorly. (B) Photomicrograph of the blastoporal lip of a stage 13 embryo, oblique section in the paraxial region. Undoubted t ransformation of mesoderm, of lateroventral character, is still occurring 4.5 hr la ter than the t ime of MIF-induced ectopic t ransformation. (C) Schematic views of the dorsal marginal zone before (left) and af te r (right) the entire mesoderm-transformation sequence. Lines To, TI, etc. represent "isochrons" of the average times at which cells in each p regas t ru l a r posit ion undergo t r a n s f o r m a t i o n while progress ing through the " inner blastoporal lip," and then their relative anteroposterior positions in the final axial mesoderm. Cells a t isochron 0

general period for the expression of involution behavior (see Symes and Smith, 1987). The goal of the experiments we report next was to ascertain whether, on a finer time scale within gastrulation itself, the time of t r ans fo rma t ion of ectopic mesoderm never theless varies as a function of the prior timing and concentration of the XTC-MIF signal.

We first ascertained the minimum physiologically required time for this expression of the response, by injecting groups of midstage 10 gastrulae with various moderate doses of MIF. Isolated ectoderm of this stage is known to be still competent to respond to the molecule (Smith, 1987), but would by this time be already "due" to be executing transformation within the whole embryo if respecified as mesoderm. There would be no call to store the history of having been induced, but rather one to execute new behavior as soon as possible. Dissection of these embryos revealed in all cases tha t ectopic transformation occurred during stage 10.5, between 25 and 35 min after injection. The usual sequence of abnormal blastocoel roof appearance was observed, even though the normal involution at the vege.tal end of the marginal zone was by then more advanced than in our other observations. We may take this as indicating tha t in all the following experiments, t ransforming mesoderm was responding in its own "clock" time and was not rate limited by the possible response time, as the late blastular injections preceded the transformation seen by at least an hour.

Table i documents the results of two experiments, on different batches of matched, synchronous sibling blastulae, where four samples have been compared with respect to the exact timing of ectopic transformation. Two further such experiments gave the same result. Two samples had received a high and a low ectopic MIF concentration in the stage 7+ blastocoel, a t least 4 h r before the onset of natural mesoderm involution, and the other two, the same two concentrations but at stage 9, rather less than an hour before that onset. The concentrations differed by 15-fold in each experiment, 'but were staggered so tha t a total 50-fold range was inves-

gain the most a n t e r i o r posi t ion in the axis, whi le cells of la te isochrons may be largely from positions tha t were lateral a t the out- set (not represented here). Because of in terdigi ta t ion of mater ia l from more lateral positions with tha t nearer the midline, the conser- vation of preinvolution order in definitive mesoderm is statist ical only. Thus the whole zone of multilayered preinvoluted tissue from stage 10, across which preorganization is required, may subtend litt le more than 10 cell d iameters in surface view from vegetal to animal limits. The asterisk marks the position of the anter ior end of the archenteron before and af ter its invagination. Mesoderm tha t has undergone involution ( t ransformat ion) behavior is shown stippled, while the noninvoluting marginal zone (future baseplate of the nervous system) is shown black.


, . . . . . , . . - . - . . . . . .

G

,~.~ / e c t

I

Scale FIG. 7. Medial sections of fix-dissected embryos, af ter injection with control dialysates and with mesoderm-inducing factors. Drawings are

from camera lucida, dur ing early stages of the control gastrulat ion schedule, af ter injections into the blastocoel at various pregastrular stages. Insets indicate the cellular s tructures of regions of the blastocoel roof and walls. (A, B) Stage 10- , jus t before any MIF-induced ectopic t ransformat ion would occur. (A) Injected with MIF (0.015 units) during stage 9, shows the appearance normal for the stage. (B) Injected 3 hr earlier a t stage 7+ (0.015 units), lacks the normal th inning of the pole region and concentration of the deep animal cap cells into the marginal zone. Epiboly is thus prevented, leaving the site of the beginning dorsal lip abnormally near the equator. (C) The normal mid-stage 10 appearance seen in an individual tha t has not yet changed af ter a moderate (0.008 units) but late (stage 9) dose of ectopic MIF. (D-F) The appearance at stage 10+ in three MIF-injected individuals half an hour older than in (C). (D) Received a higher, early dose (0.02 units, stage 7+). Note massiveness of ectopic mesoderm-and near-monolayercd remaining ectoderm, and high lip position as in (B). (E) Received a high but late dose (0.03 units, stage 9§ so t ha t presumptive mesoderm collected to the marginal zone as normally, but diversion to the ectopic, synchronous form of mesoderm development is massive throughout the animal cap. (F) Received a dosage only a tenth of tha t for (D) and (E), which allows normal epiboly regardless of injection time. The ectopic mesoderm is made from a much smaller proportion of the animal cap cells. (G) The normal appearance maintained unti l stage 11+ af ter blastular injection of bFGF (0.5 ng, stage 8). Migration of the normal mesodermal mant le and the ectodermal character of the blastocoel roof are undisturbed. (H) The appearance at the normal stage 11.5. Note extent of arehenteron and the sharp anter ior l imit of the endoderm mass internally�9 (I, J) Two bFGF-injected examples (0.5 ng, stage 8) synchronous with (H), showing abnormal mesodermization of the remaining blastocoel roof and invasion upon it of endoderm together with hal t or reversal of invagination, dl, external dorsal lip; n, normally t ransformed mesoderm; ect, ectopically t ransformed mesoderm. Scale bar equals I mm.

COOKE AND SM1TH Gasirulation and Larval Pattern in Xenopus 395

T A B L E 1 TIME OF ECTOPIC ~IESODERM TRANSFORMATION IN RELATION TO MIF

CONCENTRATION AND ~LASTULAR INJECTION TIME

stage injected

st 7+ st 7+ st 9 st 9

Experiment 1

Injection volume (nl) 200 15 300 22 Time of sampling

st 9 + 50 rains 0 0 0 (1) st 9 + 70 min 0 (1) (3) (1)

1 st 9 + 90 rain (1) (4) 5 (2)

4 3 st 9 + 110 min 5 (1) 5 5

4 s t 9 + 130 min 5 5 5 5

Index of larval abnormality a 3.2 3.0

E x p e r i m e n t 2

In jec t ion vo lume (nl) 150 10 220 15 T i m e of s a m p l i n g

s t 9 + 45 min 0 0 0 0 s t 9 + 65 min 1 0 (2) 3

2 st 9 + 85 min '(1) (1) (1) (2)

2 4 4 3 st 9 + 105 rain 5 (1) 4 (3)

4 4 2 st 9 + 125 min 5 5 5 5

Index of larval abnormality ~ 1.9 1.7

Nota Entries show the number of embryos, in a sample of five, in which transformation is occurring (parenthesis around number) or has occurred (no parenthesis). In the entries in the table, subsamples of five gastrulae were withdrawn from each sample and fix-dissected at 20-min intervals start ing at the time stated in the first row. The transformation refers to formation of the cohesive new tissue layer. In each experiment 3 to 3.5 hr, at 19-20~ intervened between the injections at Stage 7+ and those at stage 9. The larger doses used at the later stage were to compensate for the estimated increase in blastocoel volume.

The index was scored on populations of 25 larva from the same female frogs' eggs, injected at the stated dose and stage, but from a separate in vitro fertilized batch. For scoring system see Fig. 4.

tigated. This range corresponds to tha t studied previously (Cooke et aL, 1987). Its upper, major par t satu- rates the scale of whole embryo effects as it does not permit any gastrulation, but remains of interest because it gives differential propert ies to graf t s f rom donors ' animal pole regions as organizers of second body axes. In a fifth experiment, a separate dialysate of the newly available pure XTC-MIF protein (Smith et al., 1988) was used to create a 3-point, 250-fold dose comparison (0.002-0.032-0.5 units per embryo), between samples all injected at stage 8. The result was again the same.

In no experiment was a systematic difference of t iming in the ectopic t r ans fo rma t ion observed between treated samples. The times chosen for injection span much of the period during which natural induction of mesoderm must be occurring, though they may miss its earliest part. The great concentration range was to take account of the evidence for an incremental response, in terms of the tissue types and morphogenesis seen in animal cap isolates, over at least a 100-fold concentration range of the inducing molecule. The salient conclusion is clearly tha t neither MIF concentration itself nor the time of its first presence between the early and late blastula stages can affect significantly the t ransformation time of the mesoderm induced. The variations observed in this t ransformation time, between individuals within experiments, between the egg batches of different experiments, and in relation to precise Nieuwkoop and Faber stage 10 are very limited on the extended time scale of the natural involution sequence of gastrulation (see above). The true s tar t of gastrulation may be said to be the involution of the thin, spreading prechordal and lateral head mesoderm, usually visible in the fix-dissections before stage 10, the time of external lip indentation. XTC-MIF induced ectopic t ransformat ion postdates, typically by 30-40 min, the first t ransformation in that mesoderm. It is characteristically followed by cell behavior resembling the postinvolution convergence that is performed only by the axial region and not by the prechordal region. The best estimate is that it is synchronous with the onset of the prolonged involution sequence of the normal dorsal axial, convergently ex- tending mesoderm.

In at least three of the experiments the lower dose injection was one where embryos went on to show the gradable types of deficient larval morphogenesis. It is revealed (Table 1) tha t while these later effects are in- tensely dose dependent as already shown, they are little if a t all influenced by the p regas t ru la r stage of the ectopic inducer signal.

Table 2 shows the results of a somewhat different experiment, in which three samples have been compared. One has been injected with a single MIF dose at the midblastula stage 8, in the sensitive par t of the concentration range for comparison of effects on morphogenesis (around form 3, Fig. 4). The second has been injected with half the first dose at the same stage. The third sample has been injected twice, a t stages 7+ and 9, 3 hr apart, with the two injections together giving the equivalent of the higher single dose. Two other such experiments gave a result similar to tha t shown. There is no suggestion, in either mesodermal t ransformation time or subsequent grade of morphogenesis, of any potent iat ing effect of a part icular signal intensity tha t precedes another, similar one by a significant interval


TABLE 2 TIME OF ECTOPIG MESODERM RECRUITMENT AND DEGREE OF LARVAL ABNORMALITY AFTER SINGLE AND SPLIT DOSES OF BLASTOCOELIG MIF

Stage injected

s t 8 s t 8 s t 7 + / s t 9

Injection volume (nl) Time of sampling

st 9 + 60 min st 9 + 80 min st 9 + 100 min

s t 9 + 120 min s t 9 + 140 min

Index of larval a b n o r m a l i t y b

15 30 10/20 ~

0 0 o 0 (1) (1) (1) (1) (1) 4 3 4 5 5 5 5 5 5 1.8 2.9 1.9

~ Stage 7+ and Stage 9 were 3 h r apa r t at 20~ in this experiment. b For other notes see Table 1.

within the normal period of competence to respond. It seems simply to be the highest concentration of MIF encountered, regardless of pregastrular timing, tha t matters. A similar conclusion emerged from two experiments which compared samples injected with a low early dose, with a later higher dose, or with both of these. Those receiving both injections were clearly as affected as those receiving the second only, and much more so than those receiving the first only (data not shown). There is thus no "protective" effect on cells, or

subversion of their ability to record and respond to a particular level of signal, caused by an earlier, lower level of signal.

Figure 7 includes representations of normal and experimental embryos at mid- to late-gastrula stages. These would be redundant for the XTC-MIF-related in- vestigations, having been illustrated already, but are relevant in view of the comparative effect of injecting basic fibroblast growth factor (bFGF). This molecule also induces mesoderm formation in cultured Xenopus animal cap tissue (Slack et al., 1987; Kimelman and Kirschner, 1987), but inductions differ in character from that typically produced by XTC-MIF in being pre- dominantly of nonaxial type without extreme degrees of convergent extension. Once the appropriate stages of normal gastrulation are reached, the transformation of mesoderm that accompanies its involution sequence in the ventral sector is just as distinctive as that on the dorsal side, although the spontaneous convergent extension does not follow (see Figure 2a, also Keller and Danilchik, 1988). In two exper iments , we injected groups of synchronous siblings at stage 8.5 with either bFGF in 250 nl of our control dialysate (final blastocoel concentration, 0.5 to 1 pg/ml) or a similar injection to give a concentration of XTC-MIF near the middle of our large employed range (corresponding to perhaps 30 to 60 ng/ml). Injected FGF had been found in pilot tests to

cause a syndrome of temporary halt in gastrulation, and loss of epidermis, that was reminiscent of though not identical to that in our MIF-treated whole: embryos. The many-fold excess concentrations used, over those required for response in isolated competent ectoderm, are in keeping with our estimate tha t a 50-fold excess in XTC-MIF concentration is required to cause ectopic axial mesoderm in whole embryos, over that required in tl~e in vitro animal cap assay (Smith, 1987). As seen from Figs. 6g-6j, bFGF-injected gastrulae remained in- distinguishable from controls until about 1.5 hr after the onset of abnormality in their MIF-injected siblings. The latter, at control stages 11 to 11.5, had then reached the advanced stage of abnormal endoderm encroach- ment onto their ectopic mesoderm and blastoporal lip regression. Undoubted abnormality did then set in, in the bFGF cases, with thickening and delamination of a cell population having mesodermal properties from the entire inner blastocoel wall. Almost simultaneously, abnormal forward invasion of the entire endoderm surface onto this area began, occluding the blastocoel and apparent ly destabil izing archenteron invagination since this regressed.

Ectopic mesoderm induced by bFGF in these experiments thus began transformation after a major part of the dorsal sequence and a considerable part of the lateroventral one. Its preliminary positional specification would thus have been as neither axial nor anterior, but this result must be viewed with caution as the particular molecule used was of heterologous, mammal ian origin.

DISCUSSION

The Effects of XTC-MIF upon Gastrtdation and Larval Pattern

An early blastular MIF injection can prevent the normal accumulation of cells into the deep layers of the marginal zone that thins the animal pole region during epiboly. This accumulation, which is quite extensive and involves cells from well up in the original animal hemisphere, would therefore seem to be the first mechanical sign that marginal tissue has recorded inductive stimuli and is respecified, even though it has yet to begin its sequence of more dramat ic t rans format ion . Keller (1986) speculates that the thinning of the pole region might be an actively driven process. If so, induction might act normally by switching it off in marginal re- glens.

The abnormality that becomes obvious at gastrulation, after the presence of MIF free in the blastocoel, is generally what we would expect if the molecule's action closely resembles that of a normal initiator of mesoderm formation by transcellular signalling. A variable but large fraction of the cell population lining the blastocoel wall has become respecified as mesoderm, but in


a way that lacks spatial pat tern by comparison with the normal inductive sequence in the inner marginal zone. Keller and associates have best described and explained the changes of cell affinity and behavior that create the normal arrangement during gastrulation (Keller, 1984, 1986; Keller and Scheenwolf, 1977). The perversion of these events seen in the present embryos can readily be understood as stemming from an ectopic, synchronous change to mesodermal character throughout the blastocoel lining. This is followed by predictable migra- tory/adhesive responses on the part of endoderm, and by paralysis of the normal expansion and relative movement at the anterior edge of any mesoderm specified and involuted via the normal mechanisms. Two major consequences of this are on the mechanics of subsequent morphogenetic movements and the extent and organization of any mesoderm that can still be formed via the normal mechanism. Epibolic expansion of the outer shell of the gastrula, archenteron invagination, and yolk plug closure, all driven by mechanical activity of newly involuted normal mesoderm, are halted, while endoderm invades the ectopic mesodermal area to oblit- erate the blastocoel. Then, following MIF doses of less than about 0.02 units per embryo, there is a dose-graded recovery of the latter par t of the normal schedule of mesoderm transformation, and thus of gastrulation. At intermediate doses that allow this recovered postgas- trular development, but that give ectopic mesoderm of "axial" character, the lat ter is frequently the site of formation of a poorly organized but unmistakable partial axial pattern, with associated neural structure. The "normally" induced mesoderm is variably reduced in cell population and lacking in anterior and ventral pattern elements. In the present state of our understanding there can be no obvious hypotheses about the mechanism whereby the natural gastrulat ion sequence recovers a f t e r total abrogation of its initial parts. What- ever the physiological nature of this recovery, we can see it in the renewal of active mesodermal involution round a "lip" in gastrulae whose future anterior regions are already abnormal (Fig. 3), as well as in final larval pattern.

We believe that the lining of the animal cap is exposed to a homogeneous concentration of injected MIF during its initial response to the signal, whereas the normal initiating signal is almost certainly local, followed by gradat ion , diffusion, a n d / o r p ropaga t ion across tissue. The incidence of undoubted spatial patterning in the ectopic mesoderm is therefore a strong indicator of the self-organizing propensities of a field of dorsal axial mesoderm. We believe that systems em- bodying self-activating or autocatalytic modes of cellular activity, coupled to later production of signals that can inhibit or transform the earlier activity, are good candidate mechanisms for the prpduction of positional

gradations giving biological patterns, especially if the intercellular signals involved have particular effective "diffusion ranges" (Gierer and Meinhardt, 1972; Mein- hardt, 1982). XTC-MIF appears to act like an initiating, activating component in such a signalling system for mesodermal pattern, rather than like a specific induc- tor of one tissue type. A characteristic of such systems when computer simulated is the instability that leads to spontaneous, if aberrant, patterning following the (bio- logically abnormal) initial condition of a spatially homogeneous level of activation.

The differing mechanical and, finally, histogenetic characters of ectopic mesoderm after receipt of different injected concentrations of MIF does suggest that its in situ equivalent could be more than just a nonspecific activator of mesoderm formation. Along with results from explant culture (Smith, 1987; Smith et aL, 1988), it suggests a natura l role in organizing at least some aspects of pattern; those that are expressed as the final mediolateral sequence of tissues. Further evidence for this is the ordered disappearance from the body patterns of larvae of blood and then heart, with increasing MIF dose at the low end of the range. This is in accord with the normal positions of origin of these pattern elements, relatively far from that of dorsal axial mesoderm in the fate map, and also with the phenomeneon known as dorsalization of nonaxial by axial mesodermal tissue, seen in mixed explant cultures and after heterotopic grafting operations on gastrula stage material (Slack and Forman, 1980; Smith and Slack, 1983). Experience of XTC-MIF concentrations above particular thresholds seems to prohibit the allocation of mesoderm cells to the development of particular, relatively "ventral" structures. If during normal development, a rather s teep in situ gradation of the molecule's release existed around the meridians of the blastula/gastrula (i.e., in relation to distance from the position of sperm ent ry- -Gerhar t et aL, 1981) this could act as a positional signal for medial-to-lateral mesoderm character.

Experiments on Precise Timing of Ectopic Transformation: Models for the Normal Preorganization of Anteroposterior Mesodemnal Sequence

Both theory and experiment are even less advanced as regards initiation and control of the anteroposterior or craniocaudal aspect of,vertebrate body pattern than the mediolateral sequence. In the Introduction we have given the evidence for the belief that something under- lying anteroposterior sequence in mesoderm is significantly preorganized by onset of gastrulation, and in a way that itself organizes gastrulat ion mechanically. The main aim of the experiments reported in the second part of this paper has been to see whether, using XTCo


MIF, we can'gain clues as to what the organizing principles during the period of induction might be.

Because preinvoluted mesoderm exists as a "belt" of tissue compressed in what will become the long dimension of the body, much exchange of neighbors necessar- ily occurs at the single cell level during the involution movements. Positions of cells within the already mesodermal preinvoluted t i s sue- - the i r vegetal- to-animal order in the deep marginal zone--must nevertheless be significantly related to their anteroposterior order in the mesodermal mantle and the final body. It is reason- able to propose tha t preinvoluted axial mesodermal cells are significantly spatially ranked with respect to some physiological variable; a variable that is of great importance to the organization of a normal body plan by ordering the mechanical activities of involution and then the sequence of axial positional character in post- involuted tissue.

Published evidence that the mesoderm terri tory is significantly preorganized at gastrulation comes from other vertebrate groups as well as amphibians. Such early stability of positional character is principally expressed in mesoderm by what has been termed its non- equivalence (Lewis and Wolpert, 1976), i.e., the autonomous development of regional ly specific s t ruc tures after heterotopic grafting, or culture of isolated part- embryos (see Kieny et aL, 1972, for birds, and Snow, 1981, for mammals). In anuran amphibians, the culture of isolates made so as to contain only the ectoderm and all or par t of the preinvoluted mesoderm results in well-organized body axes or parts thereof, minus endoderm (Cooke, in preparation; Keller et aL, 1985). A body of evidence f romgraf t ing operations indicates that the autonomous performance of parts of the prolonged, polarized sequence of the normal involution movements is the first expression of body-posi t ional charac te r in Xe~opus mesoderm. In this species with its precipitate early phases of embryogenesis, the "clock" on which this sequence is organized is set in train in the first cell cycle and thus marches closely parallel in cofertilized siblings. We would propose that gastrulation events in other vertebrate types are really equally organized, but orchestrated by a clock that is activated somewhat later in "cleavage" and at a somewhat variable time. Hence the relative lack of synchrony among gastrulating individuals in relat ion to fer t i l izat ion in, say, birds or mammals.

Observation in this laboratory shows the sequence of normal dorsal mesoderm involution, from the prechordal region up to its close during stage 12, to occupy nearly 4 hr at the temperatures obtained in the experimental study (see also Keller, 1976). Figure 6c indicates schematically how the time-graded involution schedule seems to correlate with the body plan. The episode of behavior change to be seen beneath the dorsal part of

the blastopore at any one stage, in involuting mesoderm, resembles closely the episode undergone' during about 15 rain by mesoderm that has been specified ectopically after XTC-MIF injection. But the timing of this "ectopic" episode can hardly be altered detectably by varying the concentration of our inducing signal as much as 250-fold, or by adjust ing its t ime of onset within what appears to be a major par t of the natural period of ectodermal competence to be induced. In addition, an earlier MIF signal seems to be without any priming, enhancing, or desensitizing effect upon the response level to a subsequent one. It seems that at least across blastula stages, the intensity or character of the response to a spatially homogeneous induction by the factor (including the complex effects upon gastrular and later anatomy), depends only upon maximum experienced concentration. Moreover, while the histological character (notochordal, somitogenic, etc.) of mesoderm produced is highly dependent on concentration in this way, the anteroposterior positional character is not. This, on the basis of its timing of transformation within gastrulati0n, corresponds always to the anteriormost zone of the dorsal axial region.

The above observations seem to rule out of further consideration certain classes of simple model mechanism for the natural organization of anteroposterior values within mesoderm at these early stages. One such mechanism might be a gradient of concentration with respect to the in situ counterpart of MIF, decreasing with distance from the vegetal source, i.e., with height in the marginal zone. The relatively narrow extracellu- lar spaces for diffusion within the tissue, and perhaps progressive sequestration or inactivation of the molecule by the cells, could form an effective medium for such a gradient. Perceived concentration could then be interpreted directly to give the graded property from which anteroposterior organization starts . A second possible mechanism would be the active propagation of the MIF signal (see Cooke et al., 1987), but with an appreciable accumulating delay due to the response and relay time for individual cells. If cells meanwhile had access to an invariant common "clock" in each embryo as they are believed to at these stages, the intrinsic "age" of competent cells on first receipt of inducing signal would advance systematically with distance from the vegetal initiating source. Such age on first signal receipt could then constitute local positional information. Neither of these models reflects the real organization in view of the experimental evidence presented. Other patterning principles might utilize a phenome- non whereby cells whose receptors for a MIF-like signal have al ready been tr iggered make a potent ia ted response to a later round of occupancy of receptors, or make some modulated or down-regulated response. The main positional factor would then be not absolute time


of signal receipt, bug rather the variable length of time across which cells at different positions actively receive signal. Those nearer the ini t iat ing source, receiving signal molecules over a longer period than those more distant, would on this model become progressively more activated or otherwise modified. Our results offer no support for such ideas either.

The first-involuting mesoderm of lateroventral character appears to transform later than mesoderm induced by ectopic XTC-MIF, even when the latter is injected at the lowest concentrations that induce significant tissue. The mediolateral or dorsoventral dimension of natural organization may thus involve another initiating molecule of different character, rather than just a declining gradient of one initiator around the meridians of the marginal zone as suggested earlier. The syn- :lrome due to uv irradiation of the egg, in which meso- ]erm induced all around the gastrula is of lateroventral type but rather normal in quanti ty (Malacinski et aL, 1977; Scharf and Gerhart, 1983; Cooke and Smith, 1987), is perhaps further evidence in support of this. bFGF iSlack et al., 1987; Kimelman and Kirschner, 1987) will nduce mesoderm of principally nonaxial type from �9 .ompetent Xenopus ectoderm, and it or a related mole- :ule might seem a candidate for such a second initiating nductor, bFGF of mammalian origin, injected into the Yenopus blastocoel, induces mesoderm that t ransforms )ehaviorally at stages of the closing yolk plug, suggest- ng a somewhat posterior as well as nonaxial position vithin the normal sequence of gastrulation. The real fistological character of the majori ty of tissue that ex- libits mesodermal behavior, induced by this molecule, las not to our knowledge been established. It is too soon :o predict whether in two molecules identical to or :losely related to XTC-MIF and a Xenopus FGF, respec- :ively, we have the two components of the normal ini- :iating system that is laid out around the embryo.

Further models for first organization of anteropos- :erior position in axial mesoderm, which must now be '.onsidered, involve the coding of position by combina- ions of the levels of various signals at each location. In ~uch an organization, there would be a spatially polar- zed cascade of signals, perhaps initiated by the secre- ion of one acting like XTC-MIF, but in which respond- ng cells went on to produce their own modulating, in- fibitory, etc. signaling contributions. The "polarity," vith anteriormost specification occurring at the vegetal :ndo-mesodermal boundary, would again be set by the :ource of the initiating signal. The evidence for such ~ignals in the embryo that modulate and inhibit cells' 'esponses to MIF is considerable and diverse, but all of t is currently indirect. It lies mostly outside the scope d this discussion and is to be reviewed elsewhere Cooke, 1989). Here, one point only will be mentioned, :oncerning the origin in the normal pattern of the very

first involuting, prechordal mesoderm which alone has the ability to organize an anteriorly complete second axial plan when used as a graft to the ventral marginal zone of a host embryo. It will be remembered that it is this mesoderm alone which transforms, in involution, ahead of the time of mesoderm ectopically induced by blastocoelic XTC-MIF in the whole embryo. In keeping with this observation, XTC-induced ectopic mesoderm in its embryo of origin will differentiate as somite, but never as prechordal s t ructure or, indeed, the dorsal midline tissue notoehord. Yet upon transplantation of small blocks of such tissue, only an hour or less after receipt of inducing stimulus, to hosts' ventral midlines, they can indeed go on to organize axial mesodermal patterns that include these lat ter parts (Cooke et aL, 1987). This may imply that there is no distinct in situ signal molecule, in the normal generation of mesodermal pattern, to account for the prechordal and dorsal extremes of structure. We are reminded again of the activation/inhibition, reaction-diffusion class of theo- retical model for the biological pattern referred to earlier. This predicts that although self-organization of polarized patterns may occur under the abnormal condition of large areas of initially homogeneous "activation" (e.g., our blastocoel injections), such patterns m a y not be able to include an area of the normally highest activation of all, because the homogeneous field of in- hibitory signal that is evoked has no "sink" available in the system into which to diffuse to lower its concentration. For extended explanation and discussion of the properties of such hypothetical systems, see Gierer and Meinhardt (1972) and Meinhardt (1982).

Whatever the physiological nature of these first, preliminary codings for body position in embryonic tissue turns out to be, they are particularly fascinating in that they involve an aspect of cellular machinery that is unfamiliar and, as yet, quite obscure, the presence of what can be termed a "clock" that registers absolute progression of some kind since (in Xenopus) fertilization, and the capacity to vary the time of a particular new cel lular ac t iv i ty wi th in the schedule of t h a t "clock." The pursuit of the nature of such temporal organization in embryonic cells is one of the deepest challenges for developmental biologists.

We thank Drs. Jeremy Brockes, of the Ludwig Inst i tute for Cancer Research, London, and Elizabeth Jones, of the University of Warwick, UK, for gifts of monoclonal antibodies specific for Xenopus somitic muscle and central nervous tissue, respectively, bFGF was a gift from Dr. John Heath, Depar tment of Biochemistry, University of Oxford, UK. We thank Marylin Brennan for typing, and are especially grate- ful to John Gerhar t for devoted help with the presentat ion of the work.

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