Local Autonomy of Gastrulation Movements After Dorsal Lip Removal in Two Anuran Amphibians

J. Embryol. exp. Morph. Vol. 33, 1, pp. 147-157, 1975 147 Printed in Great Britain

Local autonomy of gastrulation movements after dorsal lip removal in two anuran amphibians

By JONATHAN COOKE1 From Division of Development Biology, National Institute for Medical Research, Mill Hill

SUMMARY The time-course of the remaining gastrulation movements has been investigated after removal of the dorsal lip, the presumptive foregut endoderm and the anterior mid-dorsal mesoderm as a plug-shaped mass of cells from beginning gastrulae of Xenopus laevis and Bombina orientalis. This embryonic region has been previously studied in its role as an organizer, when grafted into a host gastrula marginal zone. There is usually no effect of the removal of these cells, the first morphogenetically active ones, either upon rate of subsequent completion of the external aspects of gastrulation, or upon the internal evolution of the presumptive mesodermal mantle. This finding is discussed in connexion with results of a previous paper on pattern formation in early Xenopus development, since it may help to distinguish between possible types of explanation for those results.

INTRODUCTION A previous paper (Cooke, 1973 a) reported certain features of the results of implanting second organizer regions into the marginal zone of Xenopus blastulae and early gastrulae. The present paper begins to explore in greater detail the anatomy of gastrulation and the causal relationships between movements in parts of the embryo around the time of these organizer implantations. It reports the results of removing a mass of cells, consisting of the surroundings of the earliest visible organizer activity, together with the underlying bottle cells (Holtfreter, 1943), endoderm cells and presumptive anterior mid-dorsal mesoderm, from early stage-10 Xenopus (Nieuwkoop & Faber, 1956) and the equivalent stage in Bombina. This excision operation, the same as that reported previously, has been followed by detailed observation of the completion of gastrulation in comparison with individually paired, precisely synchronous controls. The rationale for including Bombina in the study was that due to the slower time-course of its gastrulation, a greater chance was offered for observation of possible retarding effects of the operation, since these might take some time to set in if they were of a physiological rather than a mechanical nature. 1 Author's address: Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, U.K. 148 J. COOKE Gastrulation involves the formation of bottle cells, and neuroectodermal spreading at the surface of the marginal zone, and the inrolling and animalward migration of presumptive mesoderm cells internally (Nieuwkoop & Florschutz, 1950; Lovtrup, 1965). These activities spread with time along an axis, the dorso- ventral, that corresponds with one of those for future pattern formation. Furthermore, when sites of the initially localized mid-dorsal activity are grafted to ventral regions of the margin, they can cause a similar spread of gastrulation movements in the surrounding cells as well as a new field of axial pattern formation subsequently (Cooke, 1972). It is thus likely that the time-course of gastrulation is originally co-ordinated and controlled by a gradient system identical with, or related to, that controlling the extent and proportions of the final pattern of cellular differentiation in the mesoderm. At some unknown early stage, this gradient system would then set, locally, the rate at which the physiology of cells evolved towards the initiation of those mechanical behaviours listed above as causing gastrulation. The question remains, however, as to how immediately cells can affect their neighbours during the actual progress of gastrulation. Are their interactions, along the dorso-ventral axis, at least partially of a mechanical and orientating or stretching nature, or are they solely physiological, reflecting the stability of a gradient system set up by earlier intercellular communication? If the latter, how stable is this gradient system? If a retarding effect were observed at any subsequent time during gastrulation, on removing the first mechanically active cells from the dorsal midline, it might have a mechanical explanation, showing that the process is controlled or 'led' mechanically from the dorsal lip area. Absence of such an effect would mean that control over the orderly dorso- ventral sequence of gastrulation is in fact of a 'physiological' character, and furthermore that by this time either the gradient or the cells' programmed response to it is sufficiently stable to avoid any effect of apex removal upon the schedule of morphogenetic movements. In fact, it is shown that there are normally no interactional effects of removing these first active cells, upon the subsequent cellular activities that complete the gastrulation process throughout the rest of the embryo. The consequences of this finding are discussed, for the interpretation of effects observed in the final patterns of cellular differentiation, when organizer implantation is combined in various ways with host organizer removal as described previously (Cooke, 1973 a).

MATERIALS AND METHODS Eggs were obtained from matings induced by subcutaneous injection of human chorionic gonadotropin ('Pregnyl', Organon Laboratories Ltd) for both Xenopus and Bombina. 250 i.u. per female and per male was satisfactory for the latter species. Groups of embryos were dejellied and demembranated manually at the late blastula stage (Nieuwkoop & Faber stage 8 in Xenopus) and stored Autonomy of gastrulation after dorsal lip removal 149

Dorsal ca. 1 mm (C)

Surface zone of marginal activity

Internal Line of progress D.L. of internal / > mesodcrmal mantle

Archenteron

Fig. 1. The excision operation. A, Vegetal surface view of stage-10 gastrula. B, Sagittal section. Heavy dashed boundaries indicate region removed, shown as an isolate in centre. C, Vegetal surface view of the healed embryo some 2h later (Xenopus) on completion of ventral blastoporal lips. D, Combined lateral surface view and sagittal section of same stage as C.

vegetal surface uppermost in black wax-bottomed dishes under two-thirds- strength Niu-Twitty solution (see Rugh, 1962) whose Ca2+ and Mg2+ concentration had been doubled by means of the chlorides, and the pH brought to 7-0 by HC1. This solution frequently provides perfect healing and morphogenesis after operations in the present material. By inspection at 15 min intervals, pairs of embryos were selected whose activity of the bottle-shaped cells (surface pigment gathering and dimpling) at the incipient dorsal lip had started syn- chronously within this margin of error. They were placed side by side, and the organizer region cleanly removed with tungsten needles from one of them. To control for a possible physiological effect of laying open the blastocoel to the culture fluid, a small tear was also made in the animal region to open the blastocoel in each embryo. Between \ h and 45 min after this operation, the strength of the solution was lowered to 0-1 with glass-distilled water to avoid the exogastrulation which often occurs without this. Observation, with camera 150 J. COOKE

Table 1. Time-course of completion of gastrulation movements after organizer excisions in Xenopus

Time to completion of marginal zone Yolk plug closure Experiment Pair no. activity, i.e. lips of blastopore to stage 12\ 1 0* -1 + 1 + 1 + 1 0 0 + 1 -1 -2 -1 -2 -1 0 + 1 + 1 0 + 2 + 1 + 1 3 -2 -1 (low -1 -2 temperature) + 2 + 2 0 + 2 0 -2 -1 0 * 0, 1 or 2 indicates synchrony, delay ( + ) or advance (-) of the time of completion of the stated phase of gastrulation in the experimental as compared with its initially synchronous control, the unit inter-observational period being 20 min. lucida drawing, was made at 10-20 min intervals thereafter for each synchronous pair. Between observations, pairs of embryos were lying on the laboratory bench, away from intense lighting and at an even temperature of between 21 and 23-5 °C (as between different experiments). On one occasion, Xenopus embryos were kept between observations in a cooled incubator between 16-5 and 17-5 °C. The group of cells removed was that described in previous papers (Cooke, 1972, 1973 a), laying open the blastocoel and leaving initially an embryo in which none of the marginal cells were visibly different from those of stage-9 blastulae. The operation is shown in Fig. 1. The wound, which was cleared of cell debris by gentle aspiration with a micropipette before the first observation, had usually closed off the blastocoel within 30 min in Xenopus and 1 h in Bombina, at which times the first new gastrulation activity was beginning. The excised plugs of cells were used as implants into other embryos, where in Xenopus they often caused complete secondary axes as described previously. Selected embryos were fixed for 10 min in 4 % formalin in two-thirds-strength Niu-Twitty, halfway through gastrulation, before examination of the internal course of mesoderm morphogenesis by bisecting in the frontal plane and then chipping away the endodermal blastocoel floor. Autonomy of gastrulation after dorsal lip removal 151

Table 2. Time-course of completion of gastrulation movements after organizer excisions in Bombina

Time to completion of marginal zone Yolk plug closure Experiment Pair no. activity, i.e. lips of blastopore to stage 12$ 11 0* +1 2 0 0 3 -1 0 4 -1 0 5 +1 +1 2 1 +1 +2 2 0 +1 3+1 0 4 -1 -1 5 -1 -2 3 1 +1 +1 2 0 +1 3 +2(35min) +2 4 +2(35min) +2 5 +2(25min) +2 * 0, 1 or 2 indicates synchrony, delay ( + ) or advance ( —) of the time of completion of the stated phase of gastrulation in the experimental as compared with its initially synchronous control, the unit inter-observational period being 20 min.

RESULTS Twelve pairs of Xenopus embryos, from three different egg-batches in separate experiments, were observed at laboratory temperature (21-23 °C), and four more pairs in another experiment at 16-17 °C. At the latter temperature, the total course of gastrulation between the operation and stage 12^ was pro- longed from the normal 4^- h to some 8 h. Pairs of Bombina embryos (15 from three different egg-batches) were observed only at laboratory temperatures where the equivalent morphogenetic stages last some 8f h. The results are presented in Tables 1 and 2, where (+1) and (-1) signify that in the experimental embryo completion of the annular zone of marginal cell activity (blastoporal lips), or reduction of the yolk plug to stage 12^ size, either followed (+) or preceded (-) that in the control by one inter-observation period. These unit periods were of about 10 min for the Xenopus experiments, and 20 min for Bombina. Apart from those of Bombina experiment 3, the values of 2 which appear in each set of results were in fact determined by extra observation to be more nearly a difference of one period than of two whole periods, so that the course of the gastrulation movements is seen to be rather precisely timed, and the relative degree of precision to be equivalent when the whole process takes longer. The variation in absolute gastrulation rate between experiments, due to varying laboratory temperature and, it is suspected, to characteristics of eggs 152 J. COOKE

ca. 2 mm

Fig. 2. The normal lack of timing effect after excision. Camera lucida of vegetal surfaces of four pairs of synchronous Bombina gastrulae, at various times beginning 1 h after organizer excision in the left of each pair. Short heavy arrows indicate mid-dorsal lines (i.e. early organizer sites), long fine arrows link the three successive observations of each pair. In the three left-hand pairs, gastrulation remained synchronous, whilst in the right-hand pair a delay arose and was maintained following organizer excision. laid by particular females, renders a population comparison of these meaning- less. For Xenopus, there is no evidence that the excision of the entire stage-10 dorsal lip, and its surroundings, influences at all the subsequent sequence of completion of the gastrulation movements. This holds true even when gastrulation is artificially slowed relative to the species norm by low temperature. In two out of three egg-batches of Bombina, the result was similar. In three out of the five pairs in the remaining experiment a delay close to 30 min was found in the operated member by blastoporal lip completion, and this delay was then maintained unchanged through yolk-plug closure. The laboratory temperature was then at its lowest during the experimental series, and due either to this, to the particular egg-batch characteristics, or to both, the total absolute time from stage (early 10) to (12|) was in these embryos somewhat over 9 h. In view of the decisive lack of an effect in the other experiments, where operated embryos are as likely as not to complete each gastrulation phase some minutes ahead of controls, this delay is probably significant. Autonomy of gastrulation after dorsal lip removal 153 Of the 16 experimental Xenopus, 13 remained uninfected until tail-bud stages, and of these 10 were normal whilst the other 3 showed deficient head patterns of types discussed previously (Cooke, 1973 a). All of the ten surviving Bombina experimental embryos showed qualitatively normal patterns at tail-bud stages, though two had small heads. Fig. 2 shows camera lucida drawings at four points during gastrulation in four pairs of Bombina embryos. Whenever final completion of well-marked stages in the process was essentially synchronous, as in the first three pairs shown, the intervening stages of spread of the lateral blastoporal lip showed no consistent trend of retardation or advance in experimentals as compared with their controls. This was easiest to confirm in Bombina, where gastrulation is itself more consistent in the sense that the lateral and ventral parts of the blastopore are formed by a rather regular progression of onset of mechanical activity at the surface. In Xenopus, although the lip is completed ventrally with great consistency between embryos, this is done in some egg-batches by a secondary zone of activity spreading to meet the first, dorsal one. The whole lip, apart from the organizer, often forms in a somewhat 'patchy' manner in this species. In the cases from the third Bombina experiment where a significant delay did occur, it appeared to be incurred at the start of the progression, i.e. during the hour after operation in which the spread of new movements actually started in. controls, and then to be maintained unchanged during the completion of the process. This delay may be related in some way to the unusually slow progression of gastrulation on this particular occasion (see above). Within 20 or 30 min after operations (or 1 h in Bombina) when cell debris overlying the wound was cleared away, the pit of the dorsal lip in the control was beginning to deepen. It could often be seen that the surroundings of the wound, now consisting only of uninjured cells, were performing mass invagination movements in time with those of the homologous area in the control, and distinct from the surface contraction accompanying healing after a wound elsewhere in the embryo. In several cases, these movements were seen whilst the cleaned wound was still just open through to the blastocoel beneath, and in these cases lines of invagination activity at the outer cell surface, the beginning extension of the blastoporal lips, were seen leaving the wound edge, having appeared since operation. This local autonomy with respect to early gastrulation movements is similar to that noted in other work (e.g. Spemann, 1938; Waddington, 1941). In five pairs of Bombina gastrulae dissected after 4 % formalin/saline fixation at the stage of ventral completion of the blastoporal lip, no evidence could be found that the stage of evolution of the internal mesoderm (Nieuwkoop & Florschutz, 1950; Lovtrup, 1965) had been altered by the excision operation. This sleeve, or ring of cells was distinguished by grey/brown, translucent appear- ance and homogeneous, small cell-size in this species. It rolls up from the 154 J. COOKE vegetal margin of the inner (sensorial) layer of neurectoderm to lie between this and the invaginating yolk mass, and was well formed at mid-lateral levels and just beginning in the mid-ventral region in all cases. It was not detectable as such in the latter region at the early gastrula stage 10 in this species. As visualized under x 50, after this admittedly inadequate fixation technique, this sleeve of cells is in distinct, though loose, mutual contact as a sheet with no detectable cell orientation in either the animal-vegetal or dorso-ventral axes of the embryo. Loose cellular structure and a slightly less deep invagination of the archenteron, mid-dorsally, were the only signs of the excision operation by this time, the amount of dorsal mesoderm appearing normal even though some of these cells are distinctly removed in the stage-10 operation. Thus there is some evidence that the removed cells may be replaced early on by spreading, but none that there is any mechanical orientating or attracting force to the dorsal midline in the normal mesoderm at this stage, which would be destroyed by the operation. DISCUSSION The results allow two conclusions. First, there is no evidence suggesting that either the external or the internal gastrulation activities are controlled in their dorso-ventral sequence by a transmission of mechanical traction forces that act across distances large compared with cells. Secondly, any physiological gradient underlying the time-course of gastrulation, or at least the local expression of an earlier response to the values of such a gradient by cells, is normally sufficiently stable by stage 10 to avoid any alteration of the programme upon removal of what is presumably the organizing region. There is other evidence, however (Cooke, 1972), that this organizer region, implanted sufficiently early into a blastula, can initiate a small field of gastrulation activity of its own in host cells. But the observations suggest that the erection of the gradient, and the cellular response to it, is slow in relation to the time- course of development as a whole in Xenopus. It is thus interesting to compare the lack of any immediate effect of apex removal, shown here, with the definite delaying effect that this operation has upon the much later pattern of differentiation within the late neurula (Cooke, 1973 a). It may be that the little understood force, which normally causes dorsal convergence of cells within the definitive mesodermal mantle at this latter stage, is missing or reduced after organizer excision, because cells originally adjacent to the excised mid-dorsal ones have not yet regulatively acquired their properties. This is a separate question from the one addressed in this paper, however, and will be investigated in the future. The present results tell us simply that during the few hours of gastrulation, following the removal of the first mechanically active cells which would have formed the anterior archenteron roof and dorsal midline of the mesoderm (Lovtrup, 1965), the balance of forces causing movement elsewhere in the embryo is not modified. Autonomy of gastrulation after dorsal lip removal 155 Preliminary observation of sectioned paraformaldehyde/glutaraldehyde-fixed material in this laboratory has confirmed the finding (Nieuwkoop & Florschutz, 1950) that the mesoderm in the Anura is internal throughout gastrulation. It also suggests, together with the dissection of gastrulating material described earlier, that presumptive mesoderm cells, before the late yolk-plug stages, migrate only parallel to the animal-vegetal (future antero-posterior) axis, and are not visibly stretched or orientated in the dorso-ventral plane at this time. This would render comprehensible the lack of mechanical interaction in this axis. In the one instance where an apparent delaying effect of the operation was observed, it is interesting that the absolute rate of development was at its slowest in the whole series of experiments. It may be that for a very few minutes after it is first visible, the organizer is indeed in the process of exerting a necessary effect on the cells lateral to it, and that on this one occasion, this effect was interrupted. In Xenopus, on which the pattern formation results discussed below were obtained, there is no evidence that the operation, as performed here, ever exerts a timing effect. In considering various current theories of the nature of cellular interactions controlling morphogenesis and the pattern of cellular differentiation (Crick, 1970; Wolpert, 1971; Wolpert, Clarke & Hornbruch, 1972; Lawrence, Crick & Munro, 1972), it is necessary to put these results beside those of another paper (Cooke, 1973 a), dealing with the establishment of double axial patterns by competing pairs of organizers under various conditions. They are among the few recent results that seem inconsistent with the theory that the actual cellular communication mediating pattern formation is via diffusion according to a concentration gradient only (though see Wilby & Webster, 1970). When a second organizer is implanted into a blastula of Xenopus at a wide angle from the host presumptive dorsal midline, a conspicuous asymmetry is typically observed in the sizes of the two axial mesodermal fields and their induced structures created in the host material. Since cell division is known to be essentially similar throughout the mesoderm at these stages, and plays no necessary role in morphogenesis around this time (Cooke, 19736), this result implies an asymmetry in the steepness, in space or on a per cell basis, of the gradients embodying positional information (Wolpert, 1971) for cellular differentiation, with mirror image polarity on either side of the boundary between the two fields. This asymmetry, whereby the grafted cells are at the head of a shorter, steeper gradient than that remaining due to the host's field, would not be troublesome for a diffusion theory of gradient control per se. However, it is maintained to a constant degree after implantations into hosts of a wide range of ages up to stage-10 gastrulae, and after post-operative development at widely differing rates due to experimentally varied ambient temperatures, suggesting that this position of the boundary between fields represents a stable state of some sort. It is obliterated, frequently giving equal 156 J. COOKE partition of the host mesoderm between the axes, under one experimental circumstance; that of the excision of the host's own group of cells, homologous with those used as an implant, but only when that group becomes visibly active some two or more hours after the implant has been healed in position elsewhere in the embryo. Two classes of explanation appeared possible for such a pattern of results. One of these supposed that the head-organizer region, which appears to initiate gastrulation, in fact leads to the formation of the definitive mesoderm by traction, and/or results in a stretching of the surface and deep cells of the marginal zone towards itself, due to some special adhesive-locomotory properties of its own cells. Thus, its removal, in delaying the process of gastrulation, was assumed to allow an organizer implanted elsewhere to continue and alter mechanically the balance between numbers of cells under its own influence and those remaining to the host field. From time-lapse studies on sea urchin embryos (Gustafson & Wolpert, 1967), where the mechanical activity initiating gastruJation can actually be seen, it would be plausible to expect this sort of interaction between pairs of early organizer regions in the present situation, where one of them is temporarily removed (i.e. until regulative restoration of its activity in neigh- bouring cells). Because we find, however, as shown here, that the local mechanical situation in more ventral parts of the gastrula remains unchanged following organizer excision (especially in Xenopus, the species on which the earlier experiments were done), it is very difficult to suppose that the boundary shifting results are due to mechanical competition between dorsal lips during gastrulation as such. Interactions between these local organizing regions, determining the relative position of the boundary between positional-information fields due to them, probably occur later, after gastrulation is completed in at least its external aspects. For the early morphogenetic movements, the host organizer seems to have no immediate controlling function for the cells around it, and the ventrally grafted one at most (i.e. when implanted early into a blastula) a local inducing one. The other class of explanation for the earlier results requires that organizer regions, in controlling the final gradients for positional information, affect cells around them in some other or additional way than by diffusion (restricted or otherwise) or mechanical stretching. The asymmetrical balance of these influences, coming from one recently established and one much older organizer, may then only be shifted to symmetry by the temporary removal of the special activity of cells at the host's own, more deeply established site. Another way of expressing such an idea is to say that one effect of a dominant region is to imbue individual cells elsewhere with a property that can be described as a polarity, or as vectorial, as opposed merely to causing a gradient in a scalar property whose slope automatically defines a polarity locally. The results of the present investigation do seem to mean that an adequate explanation for the pattern formation results would fall into this second class, Autonomy of gastrulation after dorsal lip removal 157 which challenges entirely diffusion-based models because of the normally stable maintenance of an asymmetry, under the influence of two organizers. Even if the symmetrical or asymmetrical distribution into morphogenetic fields within the mesoderm cell population is at some later time expressed mechanically, e.g. by cell migration towards each dorsal midline, it seems not to be caused mechanically. Future analysis will involve dissection and histology of paraformaldehyde/ glutaraldehyde fixed material at various stages following operations of the types discussed here and in the previous paper.

REFERENCES COOKE, J. (1972). Properties of the primary organisation field in the embryo of Xenopus laevis. I. Autonomy of cell behaviour at the site of initial organiser formation. /. Embryo/, exp. Morph. 28, 13-26. COOKE, J. (1973 a). Properties of the primary organisation field in the embryo of Xenopus laevis. V. Regulation after removal of the head organiser, in normal early gastrulae and in those already possessing a second implanted organiser. /. Embryol. exp. Morph. 30,283-300. COOKE, J. (1973/)). Properties of the primary organisation field in the embryo of Xenopus laevis. IV. Pattern formation and regulation following early inhibition of mitosis. /. Embryol. exp. Morph. 30, 49-62. CRICK, F. H. C. (1970). Diffusion in embryogenesis. Nature, Lond. 225, 420-422. GUSTAFSON, T. & WOLPERT, L. (1967). Cellular movement and contact in sea urchin morphogenesis. Biol. Rev. 42, 442-498. HOLTFRETER, J. (1943). A study of the mechanics of gastrulation. /. exp. Zool. 94, 261-318. LAWRENCE, P., CRICK, F. H. C. & MUNRO, M. (1972). A gradient of positional information in an insect Rhodnius. J. Cell Sci. 11, 815-853. LOVTRUP, S. (1965). Morphogenesis in the amphibian embryo: gastrulation and neurulation. Zoologica Gothoburgensia 1, 1-139. NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/Xenopus laevis (Daudin). Amsterdam: North Holland Publishing Company. NIEUWKOOP, P. D. & FLORSCHUTZ, P. A. (1950). Quelques caracteres speciaux de la gastrulation et de la neurulation de l'oeuf de Xenopus laevis, Daud. et de quelques autres anoures. Premiere partie. Etude descriptive. Archs Biol, Liege 61, 113-150.' RUGH, R. (1962). Experimental Embryology. Minnesota: Burgess Publ. Co. SPEMANN, H. (1938). Embryonic Development and Induction. Yale University Press. Reprinted 1967. New York: Hafner. WADDINGTON, C. H. (1941). Translocations of the organiser in the gastrula of Discoglossus. Proc. zool. Soc. Lond. A 111, 189-198. WILBY, O. K. & WEBSTER, G. (1970). Experimental studies on axial polarity in Hydra. J. Embryol. exp. Morph, 24, 595-613. WOLPERT, L. (1971). Positional information and pattern formation. Curr. Top. Devi Biol. 6, 183-223. WOLPERT, L., CLARKE, M. R. B. & HORNBRUCH, A. (1972). Positional signalling along Hydra. Nature, New Biol. 239, 101-105.

(Received 8 July 1974)