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

23

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 toinvestigate the effect of environment on differen-tiation and movement of animal pole cells of Xenopus.In the blastocoel of embryos earlier than stage 10,fragments of animal pole primarily form mesoderm.The cells are either integrated into normal host tissuesor they organize a secondary posterior dorsal axis. Ifeither host or graft is later than stage 9 the graftforms ectoderm and its cells all migrate into the hostectoderm. Inner layer animal cells form sensoriallayer; outer cells move to the epidermis. Thus con-siderable powers of appropriate movement are seen.In the vegetal pole no movement occurs. If the graft is

stage 9 or earlier, or the host is stage 1(H or earlier,the graft forms mesoderm, including striated musclein the gut. This shows that muscle can develop inwholly the wrong environment, it suggests that thedorsal inductive signal from mesoderm is rathergeneral in the vegetal mass and suggests that dorsalmesoderm development involves little subsequentadjustability. If the host is stage 11 or later, or thegraft later than stage 9, the graft forms epidermis inthe gut. This shows that the epidermal pathway ofdevelopment is also insensitive to environment.

Key words: Xenopus, animal cap cells, migration, graft.

Introduction

In this paper, we describe experiments using mono-clonal antibodies to epidermis and muscle-specificepitopes to investigate the migration, developmentand subsequent differentiation of animal cap cells ofXenopus embryos when they are transplanted intounusual positions in the embryo.

In Xenopus, the ectoderm is primarily derived fromthe pigmented half of the embryo (Keller, 1975;Cooke & Webber, 1985; Dale & Slack, 1987) thougheven vegetal pole cells of the 32-cell embryo giverise to a little ectoderm at high frequency (Heas-man, Wylie, Hausen & Smith, 1984). The ectodermeventually produces two main components, epider-mis and nervous system, a process involving a numberof steps of commitment, first to ectoderm rather thanmesoderm and subsequently to either epidermis ornervous system. Recent fate mapping shows that theanimal cap region also forms much of the mesoderm(Cooke & Webber, 1985; Dale & Slack, 1987) though

isolated animal hemispheres only form epidermis(Holtfreter & Hamburger, 1955; Asashima & Grunz,1983; Slack, 1984; Jones & Woodland, 1986). It isbelieved that this mesoderm is formed by the in-ducing action of cells in the presumptive endodermon competent ectoderm, the latter being reported tobe able to respond to this induction up to gastrulation(Dale, Smith & Slack, 1985). In experimental tissuecombinations, at least, the presumptive ectodermmay also form pharyngeal endoderm (Sudarwati &Nieuwkoop, 1971). Thus, in atypical sites, presump-tive epidermis might be expected to form mesodermand anterior gut, in addition to the epidermis ornervous system that it normally forms.

When single cells are placed in the blastocoel of ahost embryo their descendants appear in a variety oftissues and the cells concerned apparently conform tothe differentiated state of their surroundings (Wylie,Smith, Snape & Heasman, 1985; Wylie, Snape, Heas-man & Smith, 1987; Snape, Wylie, Smith & Heas-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-tiation pathway and then move to the appropriatesite? Indeed, how much are migratory abilities of cellsresponsible for maintaining and achieving the threegerm layer structure of the embryo? In this paper, weshow that ectodermal cells have considerable abilityto migrate to their appropriate location in an embryo,but that this location is not necessary for them to formepidermis. Similarly, muscle can develop in com-pletely unusual surroundings, although mesbdermcells probably also have migratory abilities aroundthe general blastocoel region. The picture thatemerges is first that the structure of the embryo isprobably maintained by sophisticated migratory abili-ties in its constituent cells. Second, it seems that oncecertain major choices in differentiation pathways aremade, cells differentiate autonomously.

We have recently isolated two monoclonal anti-bodies that react specifically with the epidermis andstriated muscle of the amphibian embryo. The epider-mal marker reacts with all of the surface epidermalcells of the neurula, even though these cells may be asdifferent as the cement gland and ciliated cells (Jones,1985; Jones & Woodland, 1986). This antigen, whichfirst appears in the stage-12^ late gastrula, is a majorsecreted molecule with a protein component, presentin all superficial cells of the early neurula, except thefuture central nervous system. It can be used as amarker of the appearance of the epidermal pheno-type, even when cells do not gain the morphologicalcharacteristics of epidermis. For example, the markersubsequently appears when cell division is blocked atthe mid-blastula stage, even though the cells becomemulticellular and disorganized (Jones & Woodland,1986). The muscle-specific marker is a monoclonalantibody (5A3.B4) raised by immunizing Balb/c micewith a homogenate of adult Xenopus muscle. It stainsstriated muscle from stage 20 onwards and reacts withno other tissue type (Fig. 1). We also used a furthermuscle-specific antibody (Kinter & Brockes, 1985).All the antibodies used in this study stain X. laevis andX. borealis in an identical way.

Methods

Embryo culture, manipulations and histologyEmbryos were cultured and explants made as described byJones & Woodland (1986). Ectodermal sandwich exper-iments were made with ectodermal explants from X.borealis sandwiched between complete animal caps derivedfrom two X. laevis stage-9 blastula and incubated. Theywere fixed when embryos synchronous with the implant hadreached stage 19. X. borealis cells were recognized by

staining with quinacrine. They exhibit intensely fluorescentchromatin granules, which are absent from X. laevis(Fig. 1A; Thi^baud, 1983).

Blastocoel-grafted embryos were made by inserting rho-damine-labelled or X. borealis donor ectoderm into a smallslit at the animal pole of demembranated embryos. Piecesof ectoderm were approximately one eighth of an animalcap in size. They were cultured in MBS [88mM-NaCl;lmM-KCl; 24mM-NaHCO3; 15mM-Tris-HCl; 0-33 mM-Ca(NO3)2; lmM-MgSO4; lmM-NaHCO,; 2mM-sodiumphosphate pH7-4; and 0-1 mM-Na2EDTA (Gurdon, 1977)]to heal and then transferred into 1/10 MBS to gastrulate.Vegetal pole grafts were achieved by grafting similarexplants into gaps teased between vegetal pole cells or intothe holes left after removing whole vegetal pole blasto-meres. All grafted embryos were healed in MBS. Theywere either maintained in this medium to produce exogas-trulae or transferred to 1/10 MBS to gastrulate normally.

Fixation, embedding, sectioning and staining with anti-bodies or simpler chemicals were as described by Jones &Woodland (1986). Fig. 1B,C shows the normal stainingpattern of the epidermal and muscle-specific antibodies onstage-46 X. laevis embryos.

Results

Migration and differentiation of epidermal cells inectodermal sandwichesIn a normal embryo, the cells that form epidermis,the outermost layer of which binds 2F7.C7, bound theembryo. The same is very largely true when a morulaor blastula explant of animal cap cells is cultured insaline, although in this case there is also a scatteringof somewhat more lightly stained cells within the solidball of 'atypical epidermis' which forms (Jones &Woodland, 1986). Can highly pigmented ectodermalcells differentiate into the strongly positive pheno-type in an internal position or is it essential that theymigrate to the cell surface before differentiating inthis way?

To find if this was so, we made a sandwich of twoanimal caps from stage-9 blastulae and placed asmaller piece of animal cap from embryos of variousstages in the centre (Fig. 2A). Implants were takenfrom embryos between stage 3 and stage 9 (8-cell tolate blastula) and placed in stage-9 tissue. In everycase, greater than 20 in total, a high proportion of theimplant, and all of its heavily pigmented cells, bound2F7.C7 strongly. However, the great majority did sowithout moving to the surface (Fig. 2B-D). Anoutside position is thus not necessary to form epider-mis and migration to the surface of the explant doesnot occur. However, the overall environment of theexplants is still ectodermal.

Migration of ectodermal cells in blastocoel graftsSince ectodermal cells do not migrate in the whollyectodermal environment of an ectodermal sandwich,

Development of animal cap cells in Xenopus 25

Fig. 1. Characterization of cellular and differentiationmarkers on cryostat sections. (A) Grafted Xenopusborealis cells (arrowed) in the somites and nervous systemof a host Xenopus laevis embryo showing thecharacteristic punctate nuclear pattern when stained withquinacrine. Host cells fluoresce diffusely.(B) Cryostat section through a stage-19 Xenopus laevisembryo stained with the epidermal marker 2F7.C7. Onlythe outer layer of the epidermis is stained.(C) Longitudinal cryostat section through a stage-46Xenopus laevis tadpole tail stained with the musclemarker 5A3.B4. The somites only are stained, showing acharacteristic striated pattern. Abbreviations: a,archenteron; ep, epidermal ectoderm; g, graft; ns,nervous system; m, notochord; s, somitic muscle. Bar:(A,C)75^m; (B) 150^m.

we have tested the ability of ectodermal cells tomigrate after grafting into different regions of thewhole embryo. Initially, ectoderm was grafted intothe blastocoel. Classically this operation was used as atest of the ability of the dorsal mesoderm to induce asecondary CNS, that is as a modification of theoriginal Spemann and Mangold graft (Spemann &Mangold, 1924). As pointed out by Slack (1983), thiskind of experiment introduces the graft into variablesituations, with complex results, at least in terms ofthe overall tissue organization of the embryo. How-ever, we have grafted animal cap and not dorsalmesoderm, and primarily ask three very simple ques-tions: do the grafted cells migrate and, if so, whatkind of tissue do the grafted cells enter and whatdifferentiated phenotype do they display? Similarapproaches have been used in Xenopus with singleectodermal and endodermal cells (Heasman et al.1984; Wylie et al. 1985) and in axolotls with multipledisaggregated 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 isgrafted into the blastocoel of similar, but notnecessarily identical, stages of embryos, secondaryembryonic axes were often formed. Embryos withboth normal and secondary embryonic axes wereserially sectioned and the location of grafted cells andtheir differentiated phenotype recorded.

Tissue distribution of grafted cellsWhen ectoderm, either from X. borealis or rhoda-mine-labelled X. laevis donor embryos, was takenprior to stage 10 and grafted into the blastocoel ofX. laevis host embryos earlier than stage 10, it wasmainly found in the mesoderm of the host, althoughsome cells were also found in the ectoderm (Fig. 3A).No cells remained in the blastocoel. The grafted cellswere usually integrated into all parts of the somiticand lateral plate mesoderm, though none were foundin the notochord. No grafted cells were ever seen inthe endoderm, though experiments of Heasman et al.(1984), using single cells, show that ectodermal cellsfrom these stages can be found in this location.

Grafted cells had the morphology typical of theirsurroundings, which suggested that they had adoptedthe appropriate differentiated phenotype. They weretested with respect to two cell types, striated muscleand epidermis. As in control embryos, 2F7.C7 boundonly to the outer ectodermal layer of the embryo, andthis included some of the grafted cells. We identifiedmuscle cells using the two monoclonal reagents de-scribed in the methods. Grafted cells found in themyotomes reacted appropriately with these anti-bodies, even in cases where the embryos were quite

26 E. A. Jones and H. R. Woodland

abnormal, proving that they had become differen-tiated as muscle (Fig. 3A).

The tissue distribution in double-axis embryos wasthe same as in morphologically normal embryos,except that in single-axis embryos grafted cells weremore frequently seen in lateral plate mesoderm orepidermis. In double-axis embryos, grafted cells werefound more exclusively in the dorsal mesoderm.

In this kind of experiment, the competence of theanimal cap cells to form mesoderm was lost at aboutstage 10 and 10i. The capacity of the host to inducemesoderm formation also disappeared by stage 10

2A

(Table 1). Thus, one can test the migratory capacityof cells that will later form ectoderm either bygrafting stage-10 animal cap cells into a blastocoel ofhosts at any stage, or by grafting pre-stage-10 cellsinto hosts at stage 10 or later.

When the implanted donor ectoderm was derivedfrom embryos at stage 10 or later, the final positionsof the grafted cells were quite different. They allmoved to the surface of the embryo and becameincorporated into the ectoderm, where they formedeither epidermis, including both the outer 2F7.C7-positive epidermal layer and the negative, inner,sensorial layer, or else they formed nervous system(Fig. 3B). Often these grafts formed a blister, fold orpouch in the skin, usually ventrally, possibly simplybecause there were larger numbers of epidermal cellsthan usual in this region (Fig. 3C). However, themorphology of these regions was always typical oflarval epidermis.

These experiments suggest two conclusions. First,migration to the correct site (the outside of theembryo) is part of the ectodermal phenotype. Sincethe cells that form mesodermal tissues also becomeincorporated into normal/mesodermal structures,this conclusion also seems to be true of mesoderm,although the very frequent occurrence of secondarymesodermal axes suggests that this ability is limited.Second, animal cap cells are determined to formectoderm by the early gastrula stage and have lost theability to form mesoderm. However, this conclusionapplies only to tissue fragment grafts into the blasto-coel.

We also tested the migration of the inner and outerectodermal layers from donor embryos in blastocoelgrafts. During normal development of Xenopus,the outer layer in non-neural regions predominantlyforms epidermis, the inner layer forming the so-called

Fig. 2. Epidermal differentiation in ectodermalsandwiches. Ectodermal fragments were dissected fromthree different stage-9 embryos and the sandwichconstructed as in A. The internal fragment was takenfrom X. borealis and the outer caps from X. laevis.Sandwiches were incubated until control embryos werestage 19, fixed and processed, as described in themethods. Sections were stained with 2F7.C7 andrhodamine-conjugated FITC RAM IgG as the secondstep antibody. Grafted X. borealis cells were identified byquinacrine staining. (B) Cryostat section through arepresentative graft containing region of a sandwich madefrom stage-9 embryos, illuminated to show antibodybinding. The arrow indicates grafted 2F7.C7-positivecells. The arrowed region is enlarged to show antibodybinding (C) and the punctate quinacrine stainingdiagnostic of graft-derived X. borealis cells. Abbreviationsas 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 tothe developing nervous system. When grafted separ-ately into stage-9 hosts, both layers from stage-9

embryos were incorporated into mesoderm, epider-mis and CNS. Both layers from stage-10 embryosentered only epidermis and CNS, predominantly

Fig. 3. Tissue distribution of ectodermal cells grafted into the blastocoel. Small pieces of A", borealis ectoderm wereinserted into the blastocoel of X. laevis host embryos and the grafted embryos allowed to develop to stage 25-28. Theembryos were then fixed and processed as described in the Methods and stained with the muscle marker, 5A3.B4, orthe epidermal marker 2F7.C7, and these antibodies were revealed with rhodamine RAM IgG and counterstained withquinacrine 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 theectoderm and stained with 2F7.C7, when in the outer epidermal layer. Occasionally double epidermal folds or blisterswere formed from this latter graft (C) which also showed the normal epidermal staining with 2F7.C7. D-F showquinacrine 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

Donor stage

6i

99 inner9 outer

9

1010 inner10 outer

Host stage

9

999

10

999

Numberanalysed

3

933

5

233

Ectoderm

3

933

5

233

Graft present

Mesoderm

3

833

0

000

in

Endoderm

0

000

0

000

Secondaryaxis

2

621

0

000

returning to their original inner or outer locations inthe host embryos.

Migration is absent in cells grafted into the vegetalpole, but epidermal and muscle differentiation isunaffected

Perhaps the most unusual position for ectoderm to begrafted is into the vegetal pole region of host em-bryos. This region normally forms the internal borderof the gut. Can ectodermal cells migrate from such aposition and do they differentiate? The results ofthese experiments are summarized in Table 2A,B.The grafted embryos were stained sequentially withthe epidermal marker and then the muscle marker,since one of the likely outcomes of such a graft mightbe its induction to form mesoderm. Table 2A showsthe summary of results of grafting donor ectodermfrom stage 6- to-12 embryos into the vegetal poles ofhosts at stage 9 or 10. Grafted embryos gastrulatednormally resulting in the graft being internalized intothe gut region. The grafts were either found ascoherent tissue masses completely surrounded byendoderm, or in regions bordering the lumen of the

developing gut. No cells migrated away. All graftsfrom stage 6- to-9 embryos were found to express themuscle marker, indicating that the ectoderm hadbeen induced to form muscle (Fig. 4). None of thesegrafts expressed the epidermal marker, though someof the grafted cells were negative with both anti-bodies. The differentiation of grafted cells into noto-chord, assessed purely by morphological criteria, wasnot detected. In all grafts from stage 10 or later, thegraft always expressed the epidermal marker (Fig. 5)and did not express any muscle-specific determinants.These results show that epidermal differentiation cantake place in an environment as unusual as the centreof the gut. They also show that the extreme vegetalpole of an embryo is capable of inducing dorsalmesoderm in competent ectoderm and that stage-10ectoderm is no longer competent to respond to thissignal under the conditions of this graft.

Table 2B shows the results of a similar series ofexperiments in which the stage of competent donorectoderm was kept relatively constant, but the graft

Table 2. The differentiation of ectoderm in vegetal pole grafts

Total number No with grafted cellsDonor stage Host stage of embryos positive for epidermal marker

No. with grafted cellspositive for muscle marker

(A) The effect679

1010-51112

(B) The effect

999789

of varying donor stage101099999

of varying host stage

7-589

1010-511

1236464

222222

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

Fig. 5. Determination of the ectoderm tested by graftinginto the vegetal pole of host blastulae. A graft from astage-9 X. borealis animal cap was made into the vegetalpole of a stage-9 X. laevis host embryo. The graftedembryo was incubated until control embryos reachedstage 30, fixed, embedded and stained with the musclemarker 5A3.B4. Characteristic striated muscle is seen inmost of the graft. Abbreviations: e, endoderm; ism,induced somitic muscle.

was made into host embryos varying from stage 7 tostage 11. All grafts carried out into host embryos fromstages 7 to 10i expressed the muscle marker and didnot express the epidermal antigen. Those graftscarried out into stage-11 host embryos developed intoballs of epidermis suggesting that the inductive stimu-lus is no longer present in the vegetal pole of stage-11embryos. This shows that epidermal development, asdefined by 2F7.C7 binding, can proceed in a whollyinappropriate environment.

These experiments show that ectoderm grafted intothe vegetal pole region of host embryos cannotmigrate from this position, but differentiates withinthe endoderm. The final differentiated cell typedepends on the stage of development of both host anddonor tissue. If the ectoderm is still responsive tomesodermal induction and the endoderm still capableof producing the signal, then the graft differentiatesas mesoderm and often quite a large proportion of thegraft can be identified as apparently normal striatedmuscle. If either of these conditions is not fulfilledthen the grafted cells differentiate into epidermis.

30 E. A. Jones and H. R. Woodland

Discussion

Migratory abilities of embryonic ectodermal cellsBy placing the cells of the future ectoderm into theblastocoel of another embryo, we have been able totest their migratory behaviour in later development.The results are clearest where the timing is arrangedso that the animal cap forms only ectoderm, ratherthan mesoderm. This is achieved by grafting animalcap cells of any stages into an embryo at stage 10 orlater, when mesodermal induction does not ensue(Table 1). Alternatively, unresponsive ectodermfrom stage 10 or later may be placed in a blastocoelat any stage (Table 1). By the tailbud tadpole stage,the grafted cells are to be found in the epidermis, orto a lesser extent the nervous system, of the host.Moreover, transplanted cells from the inner or outerlayers of the animal cap are predominantly found,respectively, in the inner sensorial layer or in theouter, epidermal layer, just as they are in normaldevelopment. The appearance of a minority of innercells in the epidermal layer is consistent with the viewthat the scattered ciliated cells of the epidermisoriginate in the inner layer (Billet & Courtenay, 1973;Steinman, 1968; our unpublished observations).

Thus ectodermal cells do find their way to theircorrect location in the embryo. Indeed, after graftingectoderm into the blastocoel we never see cellsexpressing the epidermal marker except in the epider-mis. Since epidermis can differentiate in the endo-derm (see below), it seems that cells following thisepidermal pathway find their correct location fromthe blastocoel. In addition, we do not see negativecells inside the sensorial layer.

These results contrast with the failure of the graftedanimal cells to move when surrounded by animaltissue as in a Holtfreter sandwich. In contrast, anexplant that contains mesoderm shows proper organ-ization of the ectoderm (data not shown). Thissuggests that the normal inner components of theembryo provide something necessary for the mi-gration of the cells. This could be extracellularmatrix, positional cues as to the location of the graft,or a disaggregating environment, or a combination ofthese factors.

What is the role of this migration in normaldevelopment? The progeny of lineage-labelled ani-mal cells show very considerable scattering aftergastrulation (Moody, 1987; Dale & Slack, 1987). Thisshows that the cells are naturally very mobile withintheir germ layer. Our results suggest that the integrityof the layer is also actively maintained, to the extentthat a cell which becomes displaced as far as theblastocoel can still regain its appropriate location.Somewhat similar migratory abilities of the ectodermwere demonstrated by Boucaut in Pleurodeles waltl

(Boucaut, 1974a,b). They are also an intrinsic part ofthe single-cell transfers of late-stage animal cap cellsof Heasman et al. (1984), although in these exper-iments the differentiated state of the cells was notalways tested with cell-type markers.

Migration of mesodermal cellsBoucaut (1974/?) came to the conclusion that disag-gregated mesodermal cells when placed into theblastocoel of a recipient embryo had considerableability to organize themselves correctly within themesoderm when injected into the blastocoel. Ourexperiments would suggest that the same is true inXenopus. When fragments of animal caps are intro-duced into blastocoels under timing regimes wherethey can and are induced to form mesoderm, we findthat induced grafted cells are appropriately organizedinto mesodermal tissues, although their fully differen-tiated state can only be positively identified when thegrafts are incorporated into somites and the muscle-specific monoclonal 5A3.B4 can be used. This situ-ation mainly occurs in embryos displaying secondaryembryonic axes (61 % of grafts in inductive combi-nations) when grafted cells are much more stronglyrepresented in dorsal mesoderm than in normalgrafted embryos when the majority of grafted cellsare in lateral plate and ventral mesoderm. A possibleinterpretation of grafted embryos with secondaryembryonic axes might lie in a reduced migratorypotential of dorsal mesoderm. If this were so, dorsalmesoderm formed would not move to the primarydorsal region, but instead subverts gastrulation move-ments and organizes a second embryonic axis fromsurrounding tissue. In contrast, ectodermal cells areboth relatively inert at blastula and gastrula stagesand more mobile. They might, consequently, havelonger in which to reach their appropriate positionsbefore they would upset development. However,since primary and secondary axes are properly organ-ized, and since mesoderm can differentiate in anunusual environment (see below), mesodermal cellscan certainly organize themselves in the short range.

The role of the environment in epidermal andmesodermal differentiationWhen animal cap cells are placed in the vegetal poleunder circumstances where they are unresponsive tomesodermal induction (post stage 104) or the host hascaused mesodermal induction (post stage 10£), theyinvariably form epidermis in the walls of the gut orwithin its tissue. This indicates first that neuralinductive stimuli do not occur here and, second, thatonce either the inductive stimulus or the competenceto respond to mesodermal induction is lost, develop-ment into epidermis proceeds in a way that is not atall upset by the bizarre environment.

Development of animal cap cells in Xenopus 31

When mesodermal induction can occur, striatedmuscle is always formed, even though this is notnormally found in the gut. Moreover, in normalembryos, this cell interaction occurs with vegetal cellsin an entirely different location, that is at the dorsalmargin between vegetal and animal cells. Our resultsindicate that dorsal inductive stimuli are presentgenerally through the vegetal mass, even at theextreme vegetal pole, and that once the stimulus toform muscle has occurred, the cells differentiatewithout reference to their environment. This corre-lates with the fact that blastula cells can differentiateinto muscle when disaggregated (Gurdon, Brennan,Fairmans & Mohun, 1984; Sargent, Jamrich &Dawid, 1986). It also fits with the observation thatdorsal mesoderm can change the fate of more vegetalregions, but is not itself influenced (Slack & Forman,1980). All of these observations support the idea thata certain number of major steps in commitment canbe made in early development and that for thesesubsequent reference to the environment is not made.

This work was funded by the Medical Research Council.The authors acknowledge the clerical assistance of Mrs LenSchofield and the technical assistance of P. Day.

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