The Development of Animal Cap Cells in Xenopus: the Effects of Environment on the Differentiation and the Migration of Grafted Ectodermal Cells

Development 101, 23-32 (1987) 23 Printed in Great Britain © The Company of Biologists Limited 1987

The development of animal cap cells in Xenopus: the effects of environment on the differentiation and the migration of grafted ectodermal cells

E. A. JONES and H. R. WOODLAND

MRC Animal Development Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

Summary

We have used blastocoel and vegetal pole grafts to stage 9 or earlier, or the host is stage 1(H or earlier, investigate the effect of environment on differen- the graft forms mesoderm, including striated muscle tiation and movement of animal pole cells of Xenopus. in the gut. This shows that muscle can develop in In the blastocoel of embryos earlier than stage 10, wholly the wrong environment, it suggests that the fragments of animal pole primarily form mesoderm. dorsal inductive signal from mesoderm is rather The cells are either integrated into normal host tissues general in the vegetal mass and suggests that dorsal or they organize a secondary posterior dorsal axis. If mesoderm development involves little subsequent either host or graft is later than stage 9 the graft adjustability. If the host is stage 11 or later, or the forms ectoderm and its cells all migrate into the host graft later than stage 9, the graft forms epidermis in ectoderm. Inner layer animal cells form sensorial the gut. This shows that the epidermal pathway of layer; outer cells move to the epidermis. Thus con- development is also insensitive to environment. siderable powers of appropriate movement are seen. In the vegetal pole no movement occurs. If the graft is Key words: Xenopus, animal cap cells, migration, graft.

Introduction isolated animal hemispheres only form epidermis (Holtfreter & Hamburger, 1955; Asashima & Grunz, In this paper, we describe experiments using mono- 1983; Slack, 1984; Jones & Woodland, 1986). It is clonal antibodies to epidermis and muscle-specific believed that this mesoderm is formed by the in- epitopes to investigate the migration, development ducing action of cells in the presumptive endoderm and subsequent differentiation of animal cap cells of on competent ectoderm, the latter being reported to Xenopus embryos when they are transplanted into be able to respond to this induction up to gastrulation unusual positions in the embryo. (Dale, Smith & Slack, 1985). In experimental tissue In Xenopus, the ectoderm is primarily derived from combinations, at least, the presumptive ectoderm the pigmented half of the embryo (Keller, 1975; may also form pharyngeal endoderm (Sudarwati & Cooke & Webber, 1985; Dale & Slack, 1987) though Nieuwkoop, 1971). Thus, in atypical sites, presump- even vegetal pole cells of the 32-cell embryo give tive epidermis might be expected to form mesoderm rise to a little ectoderm at high frequency (Heas- and anterior gut, in addition to the epidermis or man, Wylie, Hausen & Smith, 1984). The ectoderm nervous system that it normally forms. eventually produces two main components, epider- When single cells are placed in the blastocoel of a mis and nervous system, a process involving a number host embryo their descendants appear in a variety of of steps of commitment, first to ectoderm rather than tissues and the cells concerned apparently conform to mesoderm and subsequently to either epidermis or the differentiated state of their surroundings (Wylie, nervous system. Recent fate mapping shows that the Smith, Snape & Heasman, 1985; Wylie, Snape, Heas- animal cap region also forms much of the mesoderm man & Smith, 1987; Snape, Wylie, Smith & Heas- (Cooke & Webber, 1985; Dale & Slack, 1987) though man, 1987). Do they differentiate in accordance with 24 E. A. Jones and H. R. Woodland their surroundings or do they settle on a differen- staining with quinacrine. They exhibit intensely fluorescent tiation pathway and then move to the appropriate chromatin granules, which are absent from X. laevis site? Indeed, how much are migratory abilities of cells (Fig. 1A; Thi^baud, 1983). responsible for maintaining and achieving the three Blastocoel-grafted embryos were made by inserting rhodamine-labelled or X. borealis donor ectoderm into a small germ layer structure of the embryo? In this paper, we slit at the animal pole of demembranated embryos. Pieces show that ectodermal cells have considerable ability of ectoderm were approximately one eighth of an animal to migrate to their appropriate location in an embryo, cap in size. They were cultured in MBS [88mM-NaCl; but that this location is not necessary for them to form lmM-KCl; 24mM-NaHCO3; 15mM-Tris-HCl; 0-33 mM- epidermis. Similarly, muscle can develop in com- Ca(NO3)2; lmM-MgSO4; lmM-NaHCO,; 2mM-sodium pletely unusual surroundings, although mesbderm phosphate pH7-4; and 0-1 mM-Na2EDTA (Gurdon, 1977)] cells probably also have migratory abilities around to heal and then transferred into 1/10 MBS to gastrulate. the general blastocoel region. The picture that Vegetal pole grafts were achieved by grafting similar explants into gaps teased between vegetal pole cells or into emerges is first that the structure of the embryo is the holes left after removing whole vegetal pole blasto- probably maintained by sophisticated migratory abili- meres. All grafted embryos were healed in MBS. They ties in its constituent cells. Second, it seems that once were either maintained in this medium to produce exogas- certain major choices in differentiation pathways are trulae or transferred to 1/10 MBS to gastrulate normally. made, cells differentiate autonomously. Fixation, embedding, sectioning and staining with antibodies or simpler chemicals were as described by Jones & We have recently isolated two monoclonal anti- Woodland (1986). Fig. 1B,C shows the normal staining bodies that react specifically with the epidermis and pattern of the epidermal and muscle-specific antibodies on striated muscle of the amphibian embryo. The epider- stage-46 X. laevis embryos. mal marker reacts with all of the surface epidermal cells of the neurula, even though these cells may be as Results different as the cement gland and ciliated cells (Jones, 1985; Jones & Woodland, 1986). This antigen, which Migration and differentiation of epidermal cells in first appears in the stage-12^ late gastrula, is a major ectodermal sandwiches secreted molecule with a protein component, present In a normal embryo, the cells that form epidermis, in all superficial cells of the early neurula, except the the outermost layer of which binds 2F7.C7, bound the future central nervous system. It can be used as a embryo. The same is very largely true when a morula marker of the appearance of the epidermal pheno- or blastula explant of animal cap cells is cultured in type, even when cells do not gain the morphological saline, although in this case there is also a scattering characteristics of epidermis. For example, the marker of somewhat more lightly stained cells within the solid subsequently appears when cell division is blocked at ball of 'atypical epidermis' which forms (Jones & the mid-blastula stage, even though the cells become Woodland, 1986). Can highly pigmented ectodermal multicellular and disorganized (Jones & Woodland, cells differentiate into the strongly positive pheno- 1986). The muscle-specific marker is a monoclonal type in an internal position or is it essential that they antibody (5A3.B4) raised by immunizing Balb/c mice migrate to the cell surface before differentiating in with a homogenate of adult Xenopus muscle. It stains this way? striated muscle from stage 20 onwards and reacts with To find if this was so, we made a sandwich of two no other tissue type (Fig. 1). We also used a further animal caps from stage-9 blastulae and placed a muscle-specific antibody (Kinter & Brockes, 1985). smaller piece of animal cap from embryos of various All the antibodies used in this study stain X. laevis and stages in the centre (Fig. 2A). Implants were taken X. borealis in an identical way. from embryos between stage 3 and stage 9 (8-cell to late blastula) and placed in stage-9 tissue. In every case, greater than 20 in total, a high proportion of the implant, and all of its heavily pigmented cells, bound Methods 2F7.C7 strongly. However, the great majority did so without moving to the surface (Fig. 2B-D). An Embryo culture, manipulations and histology outside position is thus not necessary to form epider- Embryos were cultured and explants made as described by mis and migration to the surface of the explant does Jones & Woodland (1986). Ectodermal sandwich exper- not occur. However, the overall environment of the iments were made with ectodermal explants from X. explants is still ectodermal. borealis sandwiched between complete animal caps derived from two X. laevis stage-9 blastula and incubated. They Migration of ectodermal cells in blastocoel grafts were fixed when embryos synchronous with the implant had Since ectodermal cells do not migrate in the wholly reached stage 19. X. borealis cells were recognized by ectodermal environment of an ectodermal sandwich, Development of animal cap cells in Xenopus 25

we have tested the ability of ectodermal cells to migrate after grafting into different regions of the whole embryo. Initially, ectoderm was grafted into the blastocoel. Classically this operation was used as a test of the ability of the dorsal mesoderm to induce a secondary CNS, that is as a modification of the original Spemann and Mangold graft (Spemann & Mangold, 1924). As pointed out by Slack (1983), this kind of experiment introduces the graft into variable situations, with complex results, at least in terms of the overall tissue organization of the embryo. How- ever, we have grafted animal cap and not dorsal mesoderm, and primarily ask three very simple ques- tions: do the grafted cells migrate and, if so, what kind of tissue do the grafted cells enter and what differentiated phenotype do they display? Similar approaches have been used in Xenopus with single ectodermal and endodermal cells (Heasman et al. 1984; Wylie et al. 1985) and in axolotls with multiple disaggregated cells from a number of germ layers (Boucaut, 1974a,b). In mammals, the analogous tech- nique is to inject cells into the blastocyst (Gardner, 1985). When ectoderm from pregastrula embryos is grafted into the blastocoel of similar, but not necessarily identical, stages of embryos, secondary embryonic axes were often formed. Embryos with both normal and secondary embryonic axes were serially sectioned and the location of grafted cells and their differentiated phenotype recorded.

Tissue distribution of grafted cells When ectoderm, either from X. borealis or rhodamine-labelled X. laevis donor embryos, was taken prior to stage 10 and grafted into the blastocoel of X. laevis host embryos earlier than stage 10, it was mainly found in the mesoderm of the host, although some cells were also found in the ectoderm (Fig. 3A). No cells remained in the blastocoel. The grafted cells Fig. 1. Characterization of cellular and differentiation were usually integrated into all parts of the somitic markers on cryostat sections. (A) Grafted Xenopus borealis cells (arrowed) in the somites and nervous system and lateral plate mesoderm, though none were found of a host Xenopus laevis embryo showing the in the notochord. No grafted cells were ever seen in characteristic punctate nuclear pattern when stained with the endoderm, though experiments of Heasman et al. quinacrine. Host cells fluoresce diffusely. (1984), using single cells, show that ectodermal cells (B) Cryostat section through a stage-19 Xenopus laevis from these stages can be found in this location. embryo stained with the epidermal marker 2F7.C7. Only Grafted cells had the morphology typical of their the outer layer of the epidermis is stained. surroundings, which suggested that they had adopted (C) Longitudinal cryostat section through a stage-46 the appropriate differentiated phenotype. They were Xenopus laevis tadpole tail stained with the muscle tested with respect to two cell types, striated muscle marker 5A3.B4. The somites only are stained, showing a characteristic striated pattern. Abbreviations: a, and epidermis. As in control embryos, 2F7.C7 bound archenteron; ep, epidermal ectoderm; g, graft; ns, only to the outer ectodermal layer of the embryo, and nervous system; m, notochord; s, somitic muscle. Bar: this included some of the grafted cells. We identified (A,C)75^m; (B) 150^m. muscle cells using the two monoclonal reagents described in the methods. Grafted cells found in the myotomes reacted appropriately with these antibodies, even in cases where the embryos were quite 26 E. A. Jones and H. R. Woodland abnormal, proving that they had become differen- (Table 1). Thus, one can test the migratory capacity tiated as muscle (Fig. 3A). of cells that will later form ectoderm either by The tissue distribution in double-axis embryos was grafting stage-10 animal cap cells into a blastocoel of the same as in morphologically normal embryos, hosts at any stage, or by grafting pre-stage-10 cells except that in single-axis embryos grafted cells were into hosts at stage 10 or later. more frequently seen in lateral plate mesoderm or When the implanted donor ectoderm was derived epidermis. In double-axis embryos, grafted cells were from embryos at stage 10 or later, the final positions found more exclusively in the dorsal mesoderm. of the grafted cells were quite different. They all In this kind of experiment, the competence of the moved to the surface of the embryo and became animal cap cells to form mesoderm was lost at about incorporated into the ectoderm, where they formed stage 10 and 10i. The capacity of the host to induce either epidermis, including both the outer 2F7.C7- mesoderm formation also disappeared by stage 10 positive epidermal layer and the negative, inner, sensorial layer, or else they formed nervous system 2A (Fig. 3B). Often these grafts formed a blister, fold or pouch in the skin, usually ventrally, possibly simply because there were larger numbers of epidermal cells than usual in this region (Fig. 3C). However, the morphology of these regions was always typical of larval epidermis. These experiments suggest two conclusions. First, migration to the correct site (the outside of the embryo) is part of the ectodermal phenotype. Since the cells that form mesodermal tissues also become incorporated into normal/mesodermal structures, this conclusion also seems to be true of mesoderm, although the very frequent occurrence of secondary mesodermal axes suggests that this ability is limited. Second, animal cap cells are determined to form ectoderm by the early gastrula stage and have lost the ability to form mesoderm. However, this conclusion applies only to tissue fragment grafts into the blastocoel. We also tested the migration of the inner and outer ectodermal layers from donor embryos in blastocoel grafts. During normal development of Xenopus, the outer layer in non-neural regions predominantly forms epidermis, the inner layer forming the so-called

Fig. 2. Epidermal differentiation in ectodermal sandwiches. Ectodermal fragments were dissected from three different stage-9 embryos and the sandwich constructed as in A. The internal fragment was taken from X. borealis and the outer caps from X. laevis. Sandwiches were incubated until control embryos were stage 19, fixed and processed, as described in the methods. Sections were stained with 2F7.C7 and rhodamine-conjugated FITC RAM IgG as the second step antibody. Grafted X. borealis cells were identified by quinacrine staining. (B) Cryostat section through a representative graft containing region of a sandwich made from stage-9 embryos, illuminated to show antibody binding. The arrow indicates grafted 2F7.C7-positive cells. The arrowed region is enlarged to show antibody binding (C) and the punctate quinacrine staining diagnostic of graft-derived X. borealis cells. Abbreviations as in Fig. 1. Bar (B) 180fim; (C,D) 36^m. Development of animal cap cells in Xenopus 27

'sensorial layer'. In neural regions, both contribute to embryos were incorporated into mesoderm, epider- the developing nervous system. When grafted separ- mis and CNS. Both layers from stage-10 embryos ately into stage-9 hosts, both layers from stage-9 entered only epidermis and CNS, predominantly

Fig. 3. Tissue distribution of ectodermal cells grafted into the blastocoel. Small pieces of A", borealis ectoderm were inserted into the blastocoel of X. laevis host embryos and the grafted embryos allowed to develop to stage 25-28. The embryos were then fixed and processed as described in the Methods and stained with the muscle marker, 5A3.B4, or the epidermal marker 2F7.C7, and these antibodies were revealed with rhodamine RAM IgG and counterstained with quinacrine to identify the grafted X. borealis cells. Cells from stage-9 X. borealis grafted into stage-9 X. laevis hosts (A) were found mainly in the mesoderm, in this case the somites (arrowed), and stained strongly with the muscle marker. When ectoderm from stage-9 X. borealis was grafted into stage-10 hosts (B) grafted cells were found exclusively in the ectoderm and stained with 2F7.C7, when in the outer epidermal layer. Occasionally double epidermal folds or blisters were formed from this latter graft (C) which also showed the normal epidermal staining with 2F7.C7. D-F show quinacrine staining of the same regions, identifying X. borealis-derived grafted cells. Abbreviations as in Fig. 1. Bar: (A,B,D) 75^m; (C,E,F) 150^m. 28 E. A. Jones and H. R. Woodland

Table 1. Final destination of ectodermal cells grafted into the blastocoels of host embryos

Graft present in Number Secondary Donor stage Host stage analysed Ectoderm Mesoderm Endoderm axis

6i 9 3 3 3 0 2 9 9 9 9 8 0 6 9 inner 9 3 3 3 0 2 9 outer 9 3 3 3 0 1 9 10 5 5 0 0 0 10 9 2 2 0 0 0 10 inner 9 3 3 0 0 0 10 outer 9 3 3 0 0 0

returning to their original inner or outer locations in developing gut. No cells migrated away. All grafts the host embryos. from stage 6- to-9 embryos were found to express the muscle marker, indicating that the ectoderm had Migration is absent in cells grafted into the vegetal pole, but epidermal and muscle differentiation is been induced to form muscle (Fig. 4). None of these unaffected grafts expressed the epidermal marker, though some Perhaps the most unusual position for ectoderm to be of the grafted cells were negative with both anti- grafted is into the vegetal pole region of host em- bodies. The differentiation of grafted cells into noto- bryos. This region normally forms the internal border chord, assessed purely by morphological criteria, was of the gut. Can ectodermal cells migrate from such a not detected. In all grafts from stage 10 or later, the position and do they differentiate? The results of graft always expressed the epidermal marker (Fig. 5) these experiments are summarized in Table 2A,B. and did not express any muscle-specific determinants. The grafted embryos were stained sequentially with These results show that epidermal differentiation can the epidermal marker and then the muscle marker, take place in an environment as unusual as the centre since one of the likely outcomes of such a graft might of the gut. They also show that the extreme vegetal be its induction to form mesoderm. Table 2A shows pole of an embryo is capable of inducing dorsal the summary of results of grafting donor ectoderm mesoderm in competent ectoderm and that stage-10 from stage 6- to-12 embryos into the vegetal poles of ectoderm is no longer competent to respond to this hosts at stage 9 or 10. Grafted embryos gastrulated normally resulting in the graft being internalized into signal under the conditions of this graft. the gut region. The grafts were either found as Table 2B shows the results of a similar series of coherent tissue masses completely surrounded by experiments in which the stage of competent donor endoderm, or in regions bordering the lumen of the ectoderm was kept relatively constant, but the graft

Table 2. The differentiation of ectoderm in vegetal pole grafts Total number No with grafted cells No. with grafted cells Donor stage Host stage of embryos positive for epidermal marker positive for muscle marker

(A) The effect of varying donor stage 6 10 1 7 10 2 9 9 3 10 9 6 10-5 9 4 11 9 6 12 9 4

(B) The effect of varying host stage 9 7-5 2 9 8 2 9 9 2 7 10 2 8 10-5 2 9 11 2 Development of animal cap cells in Xenopus 29

Fig. 4. Determination of the ectoderm tested by grafting ectoderm into the vegetal pole of host blastulae; epidermal differentiation. Small pieces of donor X. laevis ectoderm were inserted into the vegetal pole region of host X. borealis embryos and allowed to develop until stage 25. Grafted embryos were fixed, embedded and stained as described before with 2F7.C7, rhodamine RAM IgG and quinacrine. A and B show the expression of the epidermal marker on cryostat sections of grafted cells on the archenteron wall following grafting from stage-9 and -10 donors, respectively, into stage-9 hosts. C and D show quinacrine staining of the same sections, identifying the graft, in this case, by the lack of X. borealis-speafic granules in the nuclei. Abbreviations as in Fig. 1. Bar: (A,B) 180jxm; (C,D) 15jxm.

was made into host embryos varying from stage 7 to stage 11. All grafts carried out into host embryos from stages 7 to 10i expressed the muscle marker and did not express the epidermal antigen. Those grafts carried out into stage-11 host embryos developed into balls of epidermis suggesting that the inductive stimulus is no longer present in the vegetal pole of stage-11 embryos. This shows that epidermal development, as defined by 2F7.C7 binding, can proceed in a wholly inappropriate environment. These experiments show that ectoderm grafted into the vegetal pole region of host embryos cannot Fig. 5. Determination of the ectoderm tested by grafting migrate from this position, but differentiates within into the vegetal pole of host blastulae. A graft from a the endoderm. The final differentiated cell type stage-9 X. borealis animal cap was made into the vegetal depends on the stage of development of both host and pole of a stage-9 X. laevis host embryo. The grafted donor tissue. If the ectoderm is still responsive to embryo was incubated until control embryos reached mesodermal induction and the endoderm still capable stage 30, fixed, embedded and stained with the muscle of producing the signal, then the graft differentiates marker 5A3.B4. Characteristic striated muscle is seen in as mesoderm and often quite a large proportion of the most of the graft. Abbreviations: e, endoderm; ism, induced somitic muscle. graft can be identified as apparently normal striated muscle. If either of these conditions is not fulfilled then the grafted cells differentiate into epidermis. 30 E. A. Jones and H. R. Woodland

Discussion (Boucaut, 1974a,b). They are also an intrinsic part of the single-cell transfers of late-stage animal cap cells Migratory abilities of embryonic ectodermal cells of Heasman et al. (1984), although in these exper- By placing the cells of the future ectoderm into the iments the differentiated state of the cells was not blastocoel of another embryo, we have been able to always tested with cell-type markers. test their migratory behaviour in later development. The results are clearest where the timing is arranged Migration of mesodermal cells so that the animal cap forms only ectoderm, rather Boucaut (1974/?) came to the conclusion that disag- than mesoderm. This is achieved by grafting animal gregated mesodermal cells when placed into the cap cells of any stages into an embryo at stage 10 or blastocoel of a recipient embryo had considerable later, when mesodermal induction does not ensue ability to organize themselves correctly within the (Table 1). Alternatively, unresponsive ectoderm mesoderm when injected into the blastocoel. Our from stage 10 or later may be placed in a blastocoel experiments would suggest that the same is true in at any stage (Table 1). By the tailbud tadpole stage, Xenopus. When fragments of animal caps are intro- the grafted cells are to be found in the epidermis, or duced into blastocoels under timing regimes where to a lesser extent the nervous system, of the host. they can and are induced to form mesoderm, we find Moreover, transplanted cells from the inner or outer that induced grafted cells are appropriately organized layers of the animal cap are predominantly found, into mesodermal tissues, although their fully differen- respectively, in the inner sensorial layer or in the tiated state can only be positively identified when the outer, epidermal layer, just as they are in normal grafts are incorporated into somites and the muscle- development. The appearance of a minority of inner specific monoclonal 5A3.B4 can be used. This situ- cells in the epidermal layer is consistent with the view ation mainly occurs in embryos displaying secondary that the scattered ciliated cells of the epidermis embryonic axes (61 % of grafts in inductive combi- originate in the inner layer (Billet & Courtenay, 1973; nations) when grafted cells are much more strongly Steinman, 1968; our unpublished observations). represented in dorsal mesoderm than in normal Thus ectodermal cells do find their way to their grafted embryos when the majority of grafted cells correct location in the embryo. Indeed, after grafting are in lateral plate and ventral mesoderm. A possible ectoderm into the blastocoel we never see cells interpretation of grafted embryos with secondary expressing the epidermal marker except in the epider- embryonic axes might lie in a reduced migratory mis. Since epidermis can differentiate in the endo- potential of dorsal mesoderm. If this were so, dorsal derm (see below), it seems that cells following this mesoderm formed would not move to the primary epidermal pathway find their correct location from dorsal region, but instead subverts gastrulation move- the blastocoel. In addition, we do not see negative ments and organizes a second embryonic axis from cells inside the sensorial layer. surrounding tissue. In contrast, ectodermal cells are These results contrast with the failure of the grafted both relatively inert at blastula and gastrula stages animal cells to move when surrounded by animal and more mobile. They might, consequently, have tissue as in a Holtfreter sandwich. In contrast, an longer in which to reach their appropriate positions explant that contains mesoderm shows proper organ- before they would upset development. However, ization of the ectoderm (data not shown). This since primary and secondary axes are properly organ- suggests that the normal inner components of the ized, and since mesoderm can differentiate in an embryo provide something necessary for the mi- unusual environment (see below), mesodermal cells gration of the cells. This could be extracellular can certainly organize themselves in the short range. matrix, positional cues as to the location of the graft, or a disaggregating environment, or a combination of The role of the environment in epidermal and these factors. mesodermal differentiation What is the role of this migration in normal When animal cap cells are placed in the vegetal pole development? The progeny of lineage-labelled ani- under circumstances where they are unresponsive to mal cells show very considerable scattering after mesodermal induction (post stage 104) or the host has gastrulation (Moody, 1987; Dale & Slack, 1987). This caused mesodermal induction (post stage 10£), they shows that the cells are naturally very mobile within invariably form epidermis in the walls of the gut or their germ layer. Our results suggest that the integrity within its tissue. This indicates first that neural of the layer is also actively maintained, to the extent inductive stimuli do not occur here and, second, that that a cell which becomes displaced as far as the once either the inductive stimulus or the competence blastocoel can still regain its appropriate location. to respond to mesodermal induction is lost, develop- Somewhat similar migratory abilities of the ectoderm ment into epidermis proceeds in a way that is not at were demonstrated by Boucaut in Pleurodeles waltl all upset by the bizarre environment. Development of animal cap cells in Xenopus 31

When mesodermal induction can occur, striated GURDON, J. B. (1977). Methods for nuclear muscle is always formed, even though this is not transplantation in amphibia. Methods Cell Biol. 16, normally found in the gut. Moreover, in normal 125-139. embryos, this cell interaction occurs with vegetal cells GURDON, J. B., BRENNAN, S., FAIRMANS, S. & MOHUN, T. in an entirely different location, that is at the dorsal J. (1984). Transcription of muscle-specific actin genes margin between vegetal and animal cells. Our results in early Xenopus development; nuclear transplantation indicate that dorsal inductive stimuli are present and cell dissociation. Cell 38, 691-700. generally through the vegetal mass, even at the HEASMAN, J., WYLIE, C. C, HAUSEN, P. & SMITH, J. C. extreme vegetal pole, and that once the stimulus to (1984). Fates and states of determination of single form muscle has occurred, the cells differentiate vegetal pole blastomers of Xenopus laevis. 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