INTRODUCTION

Gastrulation movements morphologically transform theamphibian embryo from a ball of cells into an elongatedtadpole with distinct dorsal-ventral and anterior-posterior axes.In addition, during this time, many tissue and phenotype spec-ifications occur, and many region-specific regulatory genes arefirst expressed, e.g.,

Xlim-1, fork head, and goosecoid (Tairaet al., 1992; Dirksen and Jamrich, 1992; Blumberg et al.,1991). Identification of the developmental history of the cellsoccupying the various regions of the gastrula is fundamentalfor understanding the role of these genes and the upstreamevents that lead to their region-specific expression. Currently,we know the developmental fate of the early cleavage stageblastomeres (Hirose and Jacobson, 1979; Jacobson and Hirose,1981; Jacobson, 1983; Dale and Slack, 1987; Moody, 1987a,b;Moody and Kline, 1990), and there is a detailed fate map ofthe early gastrula (Keller, 1975, 1976). However, there are fewdata that relate these two maps; we do not know where the blas-tomere clones are located in the gastrula. In this study, thelocations of the blastomere clones prior to and during gastru-lation were mapped so that the existing early and late mapscould be integrated and the blastomere progenitors of regions

of the gastrula that express unique gene products could be iden-tified. Since many experiments use the cleavage stage embryofor targeting foreign gene products, and rely on changes in fatefor the interpretation of gene function, the comprehensive mapof blastomere clones at blastula and gastrula stages will allowinvestigators to target correct progenitors for gene misexpres-sion, ‘knockout,’ and dominant negative experiments. Thisstudy demonstrates that some clones move prior to gastrula-tion, such that important developmental regions of the embryochange in composition over time. For example, the animal capis used to assay the inductive capacity of exogenous moleculesor ectopically expressed gene products (e.g., Slack et al., 1987;Kimelman and Kirschner, 1987; Smith, 1987; Rosa et al.,1988; Sokol and Melton, 1992; Christian et al., 1992). Inves-tigators have used animal caps of various sizes and stages,which can lead to different results (see Dawid, 1991). Ourstudy demonstrates that animal caps of different sizes atdifferent stages in fact contain different clones. Anotherimportant developmental region is Spemann’s Organizer, theinducer of the nervous system. This region has been identifiedin the stage 10 embryo (Keller, 1976), but investigatorsdisagree on the identity of the cleavage stage progenitors(Gimlich, 1986; Takasaki, 1987; Masho, 1988). It often has

1179Development 120, 1179-1189 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

Recent investigations into the roles of early regulatorygenes, especially those resulting from mesoderm inductionor first expressed in the gastrula, reveal a need to elucidatethe developmental history of the cells in which their tran-scripts are expressed. Although fates both of the early blas-tomeres and of regions of the gastrula have been mapped,the relationship between the two sets of fate maps is notclear and the clonal origin of the regions of the stage 10embryo are not known. We mapped the positions of eachblastomere clone during several late blastula and earlygastrula stages to show where and when these clones move.We found that the dorsal animal clone (A1) begins to moveaway from the animal pole at stage 8, and the dorsal animalmarginal clone (B1) leaves the animal cap by stage 9. Theventral animal clones (A4 and B4) spread into the dorsalanimal cap region as the dorsal clones recede. At stage 10,

the ventral animal clones extend across the entire dorsalanimal cap. These changes in the blastomere constituentsof the animal cap during epiboly may contribute to thechanging capacity of the cap to respond to inductive growthfactors. Pregastrulation movements of clones also result inthe B1 clone occupying the vegetal marginal zone tobecome the primary progenitor of the dorsal lip of theblastopore (Spemann’s Organizer). This report providesthe fundamental descriptions of clone locations during theimportant periods of axis formation, mesoderm inductionand neural induction. These will be useful for the correcttargeting of genetic manipulations of early regulatoryevents.

Key words: fate map, epiboly, dorsal lip, cell lineage, animal capassays.

SUMMARY

The cleavage stage origin of Spemann’s Organizer: analysis of the

movements of blastomere clones before and during gastrulation in Xenopus

Daniel V. Bauer1, Sen Huang2 and Sally A. Moody2,*1Department of Anatomy and Cell Biology, University of Virginia 2Department of Anatomy and Neuroscience Program, The George Washington University Medical Center, 2300 I Street, NWWashington, DC 20037, USA

*Author for correspondence

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been assumed that Spemann’s Organizer region arises from thevegetal hemisphere of the cleavage stage embryo (see reviewElinson and Kao, 1989) but, in fact, our initial studies (Hainskiand Moody, 1992) and those of others (Takasaki, 1987; Masho,1988) show that most of the dorsal blastopore lip actuallyarises from an animal hemisphere blastomere. In the presentstudy, we detail the blastomeres (from 16- and 32-cellembryos) that contribute to the Organizer region, as well as thelateral and ventral lips, and provide a developmental history ofthe pregastrulation movements of these important clones.

MATERIALS AND METHODS

Embryos were obtained from natural matings of adult frogs that hadbeen induced to mate with chorionic gonadotropin (Sigma). Fertilizedeggs were dejellied and selected for lineage dye injections as detailedin previous reports (Moody, 1987a,b). Only embryos with stereotypedcleavage furrows (Fig. 1) were used in order to label consistently thesame progenitor in all embryos. Embryos were held in Steinberg’ssolution until they reached the 16- or 32-cell stage.To study clonesderived from the 16-cell embryo, each of the eight different blas-tomeres (Fig. 1A) was injected with 1 nl of 5% horseradish peroxi-dase (HRP, Boeringer-Mannheim). To study clones derived from the32-cell embryo, two neighboring blastomeres were injected, one with1 nl of 0.5% Texas Red-dextran-amine (TRDA, Molecular Probes)and the other with 2 nl of 0.5% fluorescein-dextran-amine (FDA,Molecular Probes). Only the midline blastomeres of the three animal-most tiers were examined (Fig. 1B,C). For easier reading, the nomen-clature of Nakamura (Fig. 1C) is used in the text when referring to32-cell blastomeres. However, Jacobson’s nomenclature is illustratedin Fig. 1B so that reference can be made to mother cells (Fig. 1A) andto the 32-cell fate map of Moody (1987b).

Injected embryos were raised in Steinberg’s solution, the fluores-cent ones in the dark, at room temperature. Embryos were fixed, atintervals from stage 7 to stage 13 (Nieuwkoop and Faber, 1967), in4% paraformaldehyde in 0.1 M P04 buffer (pH 7.4). Most of the HRP-labeled embryos were processed as whole mounts. They were washed,reacted in 3,3

′-diaminobenzidine (Sigma), dehydrated, cut in half andembedded in clear plastic (Eukitt, Calibrated Instruments, Inc.). Mostof the fluorescently labeled embryos were sectioned at 17 µm with acryostat, washed and coverslipped with Tris/glycerol. For stages 7-10, the animal pole was identified by determining the center of theblastocoel, from its dorsal/ventral and left/right walls, and projectinga line to the surface.

To quantify the extent of movement of the B1 clone between stages

8 and 9, video images of sagittal sections were measured with aHamamatsu Argus-10 image processor. Sections were chosen fromeach embryo (four at stage 8 and five at stage 9) that contained thelargest area of the labeled B1 clone. The circumference of the tissuesection was divided into 360° of arc. The animal-most and vegetal-most boundaries of the B1 clone were measured relative to a linearprojection of the floor of the blastocoel to the surface. These bound-aries were expressed in degrees of arc.

RESULTS

The positions of clones change before gastrulationAlthough the appearance of the dorsal lip of the blastopore atstage 10 is commonly used as the indicator of the onset of gas-trulation, Xenopus blastula cells become motile at stage 8(Newport and Kirschner, 1982), clones have intermixed bythree cell diameters by stage 9 (Wetts and Fraser, 1989) andcellular events indicative of gastrulation movements beginnearly an hour before stage 10 (Keller, 1978). We investigatedboth the movements of each blastomere clone and the mixingbetween clones before stage 10 to determine whether thesemovements reorganize the clones prior to the invagination atthe dorsal blastopore lip.

The stage 7 clones were in the original position and wedgeshape of the injected blastomere (Figs 2,3). Clones of animalblastomeres interdigitated along their edges with neighboringunlabeled cells, especially at the marginal zone border of theclone (Fig. 3). The D1.1 clone was the most intermixed (Fig.3B), especially with the contralateral D1.1 clone (Fig. 4).Clones of vegetal blastomeres had little mixing at the borders(Figs 2,3). At stage 8 some clones began to shift positions. TheA4 clone extended a few cell diameters across the geometricanimal pole into the dorsal area, while the animal-most descen-dants of A1 receded one or two cell diameters away from theanimal pole (Figs 5,6B). In addition, all of the clones mixedalong their borders with neighboring clones at a depth of oneor two cell diameters (Figs 5,6B).

At stage 9, several of the clones had moved from theiroriginal positions. The ventral animal clones (A4, B4) pushedtoward the dorsal side, over the animal pole (Figs 6C, 7). Inthe blastocoel roof, these clones overlapped one another indifferent layers, superficial or deep. In some cases, B4-derived

D. V. Bauer, S. Huang and S. A. Moody

Fig. 1. The location and nomenclature of blastomeres used in this study. (A) 16-cell embryo (Hirose and Jacobson, 1979). (B) 32-cell embryolabeled with the nomenclature of Jacobson and Hirose (1981). (C) 32-cell embryo labeled with the nomenclature of Nakamura and Kishiyama(1971).

1181Blastomere clones during gastrulation

cells occupied the superficial layer over the A4-derived cellslining the blastocoel; in other cases, the positions of the cloneswere reversed. The dorsal animal clones moved vegetallytoward the marginal zone (Figs 6C, 8). The animal-mostdescendants of A1 were nearly 45° distant from that pole andthe vegetal-most descendants were within two to three celldiameters of the blastocoel floor (Fig. 6C). The B1 cloneoccupied the dorsal marginal zone (Fig. 8); its rostral extent

was about five cell diameters animal to the floor of the blasto-coel. The C1 clone was compressed toward the vegetal pole,and most of its constituents had left the dorsal marginal zone(Fig. 8). In addition, as described by Wetts and Fraser (1989)for dorsal animal clones, mixing at the boundaries between

Fig. 2. Camera-lucida drawings of labeled surface cells (stippled),members of clones of 16-cell blastomeres, at stage 7. Each cloneretains the shape and position of its progenitor blastomere, withinterdigitation of cells at the edge of the clone. (A) D1.1, animalview, dorsal to the top. (B) D1.2, animal view, dorsal to the top.(C) D2.1, vegetal view, dorsal to the top. (D) V2.1, view of ventralmidline, animal to the right.

Fig. 3. Camera-lucida drawings of labeled members of 16-cell clones(stippled) in parasagittal (A), sagittal (B,D) and coronal (C) sectionsof stage 7 embryos. (A) The D1.2 clone mixes with neighboringclones by one or two cell diameters at the marginal zone edge.(B) The D1.1 clone contains numerous unlabeled cells 3- to 6-celldiameters from the edge of the clone. This is especially notable at theanimal cap border. (C) The D2.1 clone, viewed from just below thefloor of the blastocoel, mixes by one cell diameter with neighboringclones at several sites. (D) The V2.1 clone barely interdigitates withthe clone of its lateral neighbor.

Fig. 4. At stage 7, mixing of the D1.1 clone is primarily with its contralateral neighbor. One D1.1 blastomere was labeled with FDA (green),the contralateral D1.1 blastomere was labeled with TRDA (red). Transverse section, dorsal is to the top. Bar equals 200 µm in all colorphotomicrographs.Fig. 5. At stage 8, descendants of A4 (red) were found one or two cell diameters on the dorsal side of the animal pole (line), while the animal-most descendants of A1 (green) no longer reached the animal pole. Sagittal section, dorsal is to the right. Fig. 7. At stage 9, the A4 clone (green) has moved into the dorsal part of the animal cap. Its deep cells underlie more superficial cells of the B4clone (red) in the blastocoel roof. Sagittal section, dorsal is to the left, animal pole is marked by line. Fig. 8. At stage 9 both the B1 clone (red) and the C1 clone (green) have moved into the vegetal marginal zone. A few descendants of C1 can befound three or four cell diameters within the clone of B1. Parasagittal section, dorsal is to the right. Dotted line indicates level of the blastocoel.Fig. 9. At stage 10, the A4 clone (green) has spread over most of the blastocoel roof. The B4 clone (red) extends from the animal cap to thenoninvoluting marginal zone (solid arrow), contributing some deep cells to the involuting mesoderm (open arrow). Sagittal section, dorsal is inthe lower right corner. Animal pole is marked by line. Fig. 10. At stage 11, the clones of A1 (green) and B1 (red) are distinct in preinvolution mesoderm, but mix as they approach the site ofinvagination (arrow) and enter the postinvolution mesoderm. Sagittal section, dorsal is to the top. Fig. 11. At stage 10, the B1 clone (red) occupies the dorsal blastopore lip to the site of invagination (arrow). The C1 clone (green) stretchesfrom the yolk plug to the floor of the blastocoel. Figs 12, 13. Two examples of stage 10 embryos in which the B1 clone (red) is the primary contributor to the marginal zone of the dorsalblastopore lip, but members of the C1 clone (green) also make a small contribution. Arrows mark the site of invagination. Orientation is thesame as in Fig. 11.Fig. 14. At stage 12.5, the A1 clone (green) has reached the blastopore lip (arrow) and contributes extensively to the entire neural ectoderm.The B1 clone (red) extends throughout the anterior/posterior extent of both neural and mesodermal layers. a, anterior; p, posterior. Sagittalsection dorsal is to the top.Fig. 15. At stage 12, the A4 clone (green) and the B4 clone (red) extend across the ventral epidermis from anterior (a) to posterior (p). Theventral blastopore lip is at the upper left edge of the micrograph (arrow). The clones intermix extensively in the posterior half of the deep layer.Sagittal section dorsal is to the top.

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clones occurred at a depth of three to four cell diameters (Figs6C, 7, 8).

During gastrulation, the main bodies of the clones continuedto change positions as involution proceeded. At stage 10, thespread of ventral animal clones (A4, B4) over the blastocoel roofwas extensive (Figs 6D, 9) and, at the midline, they coveredabout three-quarters of the roof. At stage 11, the A4 clonestretched across nearly the entire blastocoel roof, the B4 cloneextended from the middle of the blastocoel roof into the prein-volution mesoderm close to the ventral lip, and the C4 clone wasinvoluting (Fig. 6E). On the dorsal side, the animal clones (A1,B1) at stage 10 had condensed to fill the dorsal marginal zoneand the blastopore lip (Figs 6D, 11-13). At stage 11, membersof the A1 and B1 clones had involuted (Fig. 10), and membersof the C1 and D1 clones that had formed the dorsal floor of the

blastocoel were pulled toward the animal pole by the leadingedge of the involuting mesoderm (Fig. 6E). At stage 12, thesedescendants of C1 and D1 formed the floor of the archenteronand the prechordal mesoderm of the head (Fig. 6F).

During gastrulation, the blastomere clones were still recog-nizable as discrete masses. Surface cells were mostly contigu-ous with members of their own clone, but deep cells began tomix extensively with neighboring clones (Fig. 6D-F). The deepcells of the dorsal clones mixed as they crowded toward thedorsal lip of the blastopore and involuted. In the stage 11blastopore lip, for example, the descendants of A1 and B1 werewell circumscribed in the preinvolution mesoderm but thor-oughly mixed in the postinvolution mesoderm (Fig. 10). By theend of gastrulation, each descendant of A1 and B1 was withinthree cell diameters of a descendant of both A1 and B1 (Fig.

D. V. Bauer, S. Huang and S. A. Moody

Figs 4-5, 7-15. Legends on p. 1181

1183Blastomere clones during gastrulation

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14). Deep cells of the ventral clones mixed as they spread overthe embryo, and involuted at the ventral lip of the blastopore(Figs 6E-F, 9, 15).

Which clones make up the animal cap?The movements of epiboly and perhaps an asymmetricexpansion of the blastocoel reorganize the relative positions ofthe blastomere clones, such that the constituents of the animalcap gradually change. Since the animal cap is used extensivelyin mesoderm induction assays, we detailed the clonal compo-sition of these caps at different stages. At stages 7 and 8, eachanimal blastomere occupied nearly its original position withrespect to the blastocoel (Figs 2, 3, 6B). However, at stage 8some dorsal animal clones (D1.2 and A1) had receded slightlyfrom the geometric pole, and some ventral animal clones (A4and V1.2) had moved dorsolaterally a few cell diameters overthe animal pole (Figs 5, 6B). Between stage 8 and 9, the B1clone moved to the vegetal part of the dorsal marginal zone,and had very few descendants in the animal cap (Figs 6C, 8).At stage 8, the mean rostral border of the B1 clone was 24°animal to the blastocoel floor (n=4) and the mean caudal borderwas 4° vegetal to that border. At stage 9 the mean rostral borderof the B1 clone was 9° animal to the blastocoel floor (n=5) andthe mean caudal border was 26° vegetal to that border. At stage9, the A1 clone had moved to the original position of B1, andthe A4 clone stretched across the animal pole into the dorsalanimal cap (Figs 6C, 7). At stage 10, the B1 clone no longercontributed to the animal cap (Figs 6D, 11). The animal portionof the dorsal marginal zone was occupied by descendants ofA1 (Fig. 6D). The A4 and B4 clones constituted nearly three-quarters of the animal cap at the midline (Fig. 9).

Which blastomere clones contribute to theblastopore lip?Dorsal LipAt early stage 10, the D1.1 clone constituted the majority ofthe dorsal blastopore lip (Fig. 16). The lateral edges of the lipconsisted of descendants of D2.2. Descendants of D2.1 usuallyoccupied the yolk plug ventral to the lip, but occasionally someof its descendants were located on the dorsal side of the lip.Labeling B1 and C1 in the same 32-cell embryo revealed thatdescendants of B1 populated the dorsal surface of the lip, aswell as the deep cells at the site of invagination (Figs 6D, 11),while the descendants of C1 populated the yolk plug side ofthe lip. Most bottle cells derived from C1. Although themajority of the stage 10 dorsal lip consisted of descendants ofB1, the proportion of contributing C1 descendants varied fromone embryo to the next (cf. Figs 11, 12, 13). However, in allcases, the site of invagination was less than three cell diametersaway from the interface between the B1 and C1 clones.

At stage 11, the D1.1 clone populated the midline dorsal lip,and descendants of D1.2 and D2.2 populated the more lateralregions (Fig. 16); the deeper layers also contained scatteredcells from D2.1. It is notable that the D1.2 clone invaded thevegetal hemisphere by this stage, separating the D1.1 and D2.2clones. Both daughters of D1.1 contributed to the stage 11dorsal lip. The A1 clone did not reach the dorsal lip on thesurface, but deep descendants extended into the preinvolutionand postinvolution mesoderm (Fig. 10). While the B1 cloneformed most of the postinvolution mesoderm and archenteronroof (Figs 6E, 10), we estimated that between 3 and 12% of

the descendants of C1 also contributed to these structures (Fig.6E).

At stage 12, the clones derived from the three dorsal blas-tomeres (D1.1, D1.2, D2.2) were compressed into the dorsalthird of the blastopore lip (Fig. 16). D1.1 and D1.2 clonesformed stripes in the developing neural plate, parallel to theanterior-posterior axis, and extended through the lip to con-tribute to mesoderm and archenteron roof. The superficialdescendants of A1 just reached the blastopore lip with a fewdescendants intermixed with the B1 clone in the region of invo-lution (Fig. 14). The B1 clone extended from the region ofinvolution across the archenteron roof and throughout thedorsal axial mesoderm (Fig. 14). With the reduction in the sizeof the blastopore, only a few descendants of D2.1 remained inthe yolk plug. Because the region of involution lies along thedorsal side of the D2.1 clone, its descendants lined the archen-teron floor (Fig. 6F).

Lateral lipAt early stage 10, the lateral edges of the lip consisted ofdescendants of D2.2 (Fig. 16). At stage 11, the lateral lip wascomposed mostly of a mixture from the lateral vegetal clones(D2.2 and V2.2) with a few cells from V1.2. In about half ofthe specimens, the surface cells of the V1.2 clone were at thelip and, in the rest, they were a few cell diameters away. In allcases, the deep members of the V1.2 clone were in the prein-volution mesoderm at the lip, but in only a few specimens hadlabeled cells involuted. At stage 12, the surface descendants ofD2.2 occupied a relatively small patch in the lateral blastoporelip, while the surface descendants of V1.2 had spread to occupynearly a quarter of the lateral surface, as viewed from theblastopore (Fig. 16).

Ventral lipAt stage 11, the ventral lip consisted of a mixture of cellsderived from V2.2 and V2.1 (Fig. 16). Only the tier-3 daughterof V2.1 (C4) contributed to the involuting zone; the tier-4daughter (D4) formed only the yolk plug (Fig. 6E). At stage12, the ventral lip consisted mostly of descendants of V2.1 andV2.2, the clones of which extended in both superficial and deeplayers from the marginal zone through the lip. In addition, thesurface descendants of V1.1 almost reached the site of involu-tion, intermixing with surface cells from V2.1. Only the deepdescendants of the vegetal daughter of V1.1 (B4) contributedcells to the involuting mesoderm (Fig. 15).

DISCUSSION

Xenopus embryos and animal caps isolated from them arecommonly used to study the induction of fate changes bygrowth factors or genetic manipulations. Understanding thepotential of a tissue in situ or in vitro requires full knowledgeof its origin and the fates of its progenitors, especially sincesome fates are determined prior to these manipulations (e.g.,Takasaki, 1987; Kageura, 1990; Gallagher et al., 1991). Ourstudy was designed to investigate the movements of the blas-tomere clones between the morula and gastrulation, to evaluatethe consequences of these movements for the ultimate fates ofthe blastomere clones, and to determine the identities of theblastomere progenitors of the animal cap and blastopore lip.

D. V. Bauer, S. Huang and S. A. Moody

1185Blastomere clones during gastrulation

We found that clones move prior to gastrulation, such that theconstituents of the animal cap and dorsal marginal zone changebetween stages 8 and 10. These data are important for inter-preting animal cap assays and fate changes resulting fromgenetic and other cellular manipulations.

Contributions of the blastomeres to the stage 10fate mapEach blastomere of the cleavage stage embryo contributes towidely diverse tissues in the tailbud stage (Dale and Slack,1987; Moody, 1987a,b). Because the tissues of the tailbudembryo are represented by discrete territories in stage 10embryos (Keller, 1975, 1976), we mapped the positions of theblastomere clones onto these territories (Table 1). For themidline clones, the 32-cell data are presented and, for thelateral clones, the 16-cell data are presented. Table 1 showswhere the clones are located at stage 10, in general and usingKeller’s specific regional descriptions, what these regions arefated to become (Keller, 1975, 1976), and what these blas-tomeres are fated to become (Moody, 1987a,b). This compar-ison establishes the concordance of the fate maps derived fromcleavage stage embryos with those derived from the earlygastrula. It provides much more phenotypic detail to someregions of Keller’s map (e.g., ectodermal specializations of the

head, neural crest derivatives and branchial arches), it demon-strates that Keller’s maps cannot be superimposed over theoriginal blastomeres (Fig. 6A), and it clarifies the process bywhich blastomere clones move through gastrulation to achievetheir eventual fates.

Pregastrulation movements change the positions ofthe blastomere clonesStudy of the pregastrula using cinemicrographic techniquesindicates that the apical surfaces of superficial cells in theanimal and marginal regions expand between stages 7 and 9(Keller, 1978). After stage 9, dorsal marginal cells expand evenmore rapidly, extending the marginal zone vegetally toward theforming blastopore invagination. As a consequence of thesepregastrulation movements, blastomere clones occupy verydifferent space in the embryo at stage 10 than they did atcleavage stages. With the formation of the blastocoel and themovements of epiboly, each blastomere clone changes shapeand position to occupy a characteristic region of the gastrulathat is quite different from its original position (Fig. 6; Table1). These changes in the positions of blastomere clones werenot predicted by the cinemicrography. For example, it appearedthat the expansion of the animal cap was uniform (Fig. 5A;Keller, 1978), but our data show shifts of specific groups of

Fig. 16. Camera-lucida drawings of the positions of 16-cell clones in the vegetal hemisphere during gastrulation. In the top row clones of D2.2,V2.1, D1.2, and V1.1 are shown. In the bottom row clones of D1.1, D2.1, V2.2, and V1.2 are shown. At stage 10 (left), the four vegetal clonesare in the positions of the original blastomeres. In addition D1.1 has moved from the animal hemisphere to form the dorsal lip of the blastopore(bottom, stippled). Note that cells from the D2.2 clone form the lateral edge of the lip (top, stippled) and that a few cells from the D2.1 cloneare in the dorsal lip (bottom, white). At stage 11 (middle), the two lateral animal clones (D1.2, top; V1.2, bottom) have extended into thevegetal hemisphere and reached the blastopore lip. The D1.1 clone has undergone convergent extension to become a narrow midline stripe. Atstage 12 (right), the dorsal clones have narrowed due to convergent extension and the ventral clones have spread over a larger surface area. Asmall portion of the V1.1 clone (top, black) approaches the ventral lip at the midline, but does not involute.

1186 D. V. Bauer, S. Huang and S. A. Moody

Table 1. Positions of 16- and 32-cell clones in the stage 10 gastrula and their fatesBlastomere Position of clone at stage 10 Stage 10 fate map (Keller) Tail bud fate map of clones (Moody)

A1 dorsal animal cap: anterior epidermal area head epidermis cement gland, olfactory, lens, cranial ganglia, (D1.1.1) epidermis

anteror neural area brain brain, neural crest, retina

dorsal marginal zone: middle, posterior neural area hindbrain, spinal cord hindbrain, spinal cordsuprablastoporal endoderm archenteron roof archenteron roof of hindgutdorsal preinvolution mesoderm notochord notochorddorsal involuted mesoderm head mesoderm dorsal head mesenchyme, branchial arches

pharyngeal endoderm pharynx, foregutsomites head somites, central trunk somites

B1 dorsal marginal zone: anterior, middle, posterior neural area brain and spinal cord retina, brain, spinal cord

(D1.1.2) dorsal blastoporal lip: suprablastoporal endoderm anterior, middle archenteron roof archenteron roofdorsal preinvolution mesoderm notochord notochorddorsal involuted mesoderm head mesoderm dorsal head mesenchyme, branchial arches

pharyngeal endoderm pharynx, foregutsomites head somites, central trunk somites

C1 yolk plug: subblastoporal endoderm anterior, middle archenteron floor hindgut(D2.1.2) bottle cells anterior archenteron roof liver

dorsal blastoporal lip: dorsal involuted mesoderm head mesoderm dorsal head mesenchyme, branchial archesheart heartnotochord, somites notochord, somitespharyngeal endoderm pharynx, foregut

D1.2 lateral animal cap: anterior, middle epidermal areas head, trunk epidermis cement gland, olfactory, lens, otocyst, epidermis

lateral anterior, middle, posterior dorsal brain and spinal cord dorsal brain, dorsal and intermediate neural areas spinal cord, cranial ganglia, neural crest part of

branchial arches

lateral marginal zone: suprablastoporal endoderm posterior archenteron roof archenteron rooflateral preinvolution mesoderm somites head somites, central trunk somites

D2.2 lateral marginal zone: middle epidermal area dorsal trunk epidermis dorsal trunk epidermismiddle, posterior neural areas hindbrain and spinal cord dorsal, intermediate hindbrain and spinal cord

lateral blastoporal lip: suprablastoporal endoderm posterior, middle archenteron roof archenteron

yolk plug: lateral preinvolution mesoderm somites somiteslateral involuted mesoderm heart, somites, lateral mesoderm heart, somites, lateral plate, nephrotomesubblastoporal endoderm middle archenteron floor hindgut

A4 animal cap: anterior, middle epidermal area head and trunk epidermis head and trunk epidermis, cranial ganglia, neural (V1.1.1) crest, neural crest part of branchial arches

ventral marginal zone: ventral preinvolution mesoderm somites, lateral mesoderm somites, lateral plate, nephrotome

B4 animal cap: middle, posterior epidermal area trunk epidermis trunk epidermis, neural crest

(V1.1.2) ventral marginal zone: ventral preinvolution mesoderm somites, lateral mesoderm somites, lateral plate, nephrotome

C4 ventral marginal zone: posterior epidermal area trunk epidermis trunk epidermis, neural crest(V2.1.2) posterior neural area dorsal spinal cord dorsal spinal cord

subblastoporal endoderm posterior archenteron floor hindgut

suprablastoporal endoderm posterior archenteron roof archenteron roofventral preinvolution mesoderm somites, lateral mesoderm somites, lateral plateventral involuted mesoderm lateral mesoderm lateral plate, nephrotome

V1.2 lateral animal cap: anterior, middle, posterior epidermal head and trunk epidermis head and trunk epidermis, cranial ganglia,areas middle, posterior neural areas dorsal hindbrain, spinal cord dorsal hindbrain, spinal cord, neural crest,

neural crest part of branchial arches

lateral marginal zone: lateral preinvolution mesoderm somites, lateral plate somites, lateral plate, nephrotome

V2.2 yolk plug: subblastoporal endoderm middle, posterior archenteron floor hindgut

ventral marginal zone: suprablastoporal endoderm posterior archenteron roof archenteron roofposterior epidermal area trunk epidermis trunk epidermis, neural crestposterior neural area posterior spinal cord posterior spinal cordlateral preinvolution mesoderm somites, lateral plate somites, lateral platelateral involuted mesoderm lateral plate lateral plate, nephrotome

The locations of the blastomere clones at stage 10 were described according to the regions defined by Keller (1975, 1976). The fates of these regions (Keller,1975, 1976) and the observed phenotypes of the blastomeres clones in the tailbud embryo (Moody, 1987a,b) are compared. It should be noted that as aconsequence of mixing between clones, as described in the Results section, each clone also makes minor contributions to neighboring regions not listed in thistable.

1187Blastomere clones during gastrulation

cells (clones A4 and B4) from ventral to dorsal between stages8 and 10, and then the spread of these groups from animal toventral between stages 10 and 12. The expansion of the apicesof superficial cells also did not predict that groups of cells (bothdeep and superficial members of the clone) would move enmasse from animal to dorsal marginal zone regions.

A feature of these clonal movements is the mixing of cellsbetween clones. In zebrafish, whose blastomere clones do nothave predictable fates and can contribute to all regions of theembryo (Kimmel and Law, 1985), the clones mix freely duringgastrulation and descendants of distant clones come in contactwith one another (Warga and Kimmel, 1990). However, inXenopus, whose blastomere clones are predictable and region-ally discrete (Dale and Slack, 1987; Moody, 1987a,b), theclones mix only along their borders until just before involu-tion. Wetts and Fraser (1989) showed that, at stage 9, dorsalanimal clones interdigitated by about three cell diameters. Weshow that this intermixing begins as early as stage 7, prior tothe onset of cellular motility (Newport and Kirschner, 1982).Mixing occurs more in animal clones than vegetal clones andmore in the marginal zone. The earliest and most extensivemixing occurs across the dorsal animal midline, which predictsthe bilateral origin of forebrain structures (Jacobson andHirose, 1978; Huang and Moody, 1992, 1993). Mixing thatoccurs prior to stage 8 probably results from the interdigitationof cells during mitoses and permissiveness to mixing at clonalboundaries.

During epiboly the mixing of clones increases. Superficiallayers remain nearly coherent, with a few interposing cellsfrom other clones. This is consistent with the cinemicrographicobservation that during epiboly cells from the superficial anddeep layers do not mix (Keller, 1978). In contrast, cells fromdifferent clones in the deep layers mix extensively. In the deeplayers of the blastocoel roof, this mixing is accomplished byclones sliding past one another and cells intercalating (Keller,1980). In the deep layers of the marginal zone, the mixing isaccomplished by the radial intercalation of cells in the prein-volution zone and the mediolateral intercalation during invo-lution (Wilson and Keller, 1991) These movements causethorough mixing of the dorsal midline clones along the antero-posterior axis. For example, the clone of A1, which involutesafter the clone of B1, extends across the entire anterior-posterior extent of the dorsal axial tissues and is coextensivewith descendants of B1 through all but the most anteriorsensory epithelium (Fig. 14). Even after gastrulation a slowprogressive mixing of clones continues, at least in the nervoussystem (Wetts and Fraser, 1989).

Changes in animal cap competence may result fromchanges in clonal constituentsIsolated Xenopus animal caps form only atypical epidermis inculture, but can be induced to form mesoderm (Sudarwati andNieuwkoop, 1971). Thus, they are used in a standard assay totest the effectiveness of factors to induce mesoderm. However,investigators have used large or small animal caps from variousstages, sometimes with different results (Dawid, 1991). Forexample, it has been suggested that different laboratoriesobserved different abilities of Xwnt-8 to induce mesodermbecause of differences in explant size (Christian et al., 1992)or stage of isolation (Sokol, 1993). While it has been proposedthat these differences are due to the diffusion from the vegetal

hemisphere to the animal pole of an endogenous signal (Sokol,1993), an alternative hypothesis is that the constituents of theanimal cap change during pregastrula stages. Although thestage 10 fate maps show that the animal cap produces onlyectodermal derivatives (Keller, 1975), the normal fates of stage8 or 9 animal caps in the intact embryo have not been mapped.Since the 16- and 32-cell fate maps show that the animal capblastomeres (tiers-1 and -2) contribute to all three germ layers,and transplantation studies show that dorsal animal blas-tomeres are determined to produce dorsal mesodermal tissues(Takasaki, 1987; Kageura, 1990; Gallagher et al., 1991), it ispossible that nonectodermal cells are within the animal cap ofthe blastula and leave it by stage 10. These proposed changesin the constituents of the animal caps could result in changesin their competence to respond to manipulations.

In fact, our data demonstrate that the clones comprising theanimal cap change after the midblastula transition. The stage8 animal cap, whether large (including the entire blastocoelroof) or small (including only cells within 45° of the animalpole), contains the entire clone of B1. Previous experimentsindicate that this blastomere can autonomously differentiate toform dorsal mesoderm (Takasaki, 1987; Kageura, 1990;Gallagher et al., 1991) and that it contains dorsal information(Elinson and Kao, 1989; Hainski and Moody, 1992). Further-more, B1 is the primary progenitor of Spemann’s Organizerand a major progenitor of the notochord (Dale and Slack, 1987;Moody, 1987b). The presence of the B1 clone, whose dorsalmesoderm fate seems determined before stage 8, probably is asignificant factor in the state of competence of the stage 8animal cap. For example, the presence of some B1 descendantsin the stage 8 cap may account for the ability of activin toinduce dorsal mesoderm only from dorsal halves of the cap(Sokol and Melton, 1991).

At stage 9, the B1 clone has moved vegetally into themarginal zone. Consequently, the small stage 9 animal capcontains primarily the descendants of A4, B4 and A1. Thelarge stage 9 animal cap, however, still includes some descen-dants of B1. This may result in differences in inducibilitybetween large and small stage 9 animal caps (e.g., Christian etal., 1992; Sokol, 1993). In addition, the movement of B1 outof the animal cap correlates with the observation thatmesoderm does not form in stage 8 or large stage 9 animal capsisolated from embryos injected with exogenous Xwnt-8 mRNA(Sokol, 1993). Perhaps the presence of the B1 clone inhibitsthe Xwnt-8 signal.

By stage 10 the B1 clone occupies the dorsal lip of theblastopore, so that a large animal cap includes only neuroec-todermal (A1) and epidermal (A4, B4) progenitors, while asmall cap includes only epidermal descendants of A4 and B4.Interestingly, the competence of the animal cap to formmesoderm ceases at this stage (Jones and Woodland, 1987),when all the blastomere clones known to produce mesodermhave exited from the animal cap. Thus, the different con-stituents of the animal cap at different stages may stronglyinfluence the competence of the cap to be induced, and itsresponsiveness to the presence of exogenous gene products.

The major progenitor of the dorsal lip of theblastopore is an animal blastomereThe dorsal lip of the blastopore is the Organizer of thedorsoventral axis of the embryo (Spemann and Mangold, 1924)

1188

and recently novel gene transcripts have been localized to thisregion (Blumberg et al., 1991; Dirksen and Jamrich, 1992;Taira et al., 1992). If the progenitor of the dorsal lip is to betargeted for genetic manipulation, one needs to specificallyidentify that progenitor. Generalizing the 32-cell fate map toresemble the stage 10 fate map has led to the common assump-tion that the dorsal lip arises from C1, the tier-3 dorsal midlinecell. Although one lineage tracing study reported that the siteof invagination is in the cleavage furrow between the C1 andD1 blastomeres (Gimlich, 1986), others have placed it close tothe cleavage furrow between C1 and B1 (Masho, 1988;Takasaki, 1987), with the B1 clone forming most of the prein-volution mesoderm and the C1 clone forming most of the bottlecells (Takasaki, 1987). By observing many embryos in whichboth B1 and C1 were labeled, we concur that the clone of B1is the primary contributor to the dorsal lip. Although C1 doeshave descendants in this region, especially the bottle cells, theywere never the major constituents. The proportion of C1descendants in the dorsal lip varied from embryo to embryo(Figs 11-13), probably due to variability in the location of thethird cleavage furrow (see also Masho, 1988). Thus, themajority of evidence indicates that the Organizer developsmostly from a blastomere in the animal hemisphere and that,if one wants to target Spemann’s Organizer for genetic manip-ulations, B1 is the best candidate blastomere. Furthermore, ourstudy demonstrates that descendants of B1 orient and drive theinitial mediolateral intercalation of cells at the dorsal lip.Recent work by Shih and Keller (1992a,b) shows that thesuprablastoporal endoderm can organize mesodermal behaviorand induce dorsal axis formation, while immediately adjacentinvoluting marginal zone drives mediolateral intercalation. B1is the primary contributor to both of these regions (Table 1).

Targeting manipulations to the most appropriateblastomere progenitorTwo important techniques for testing the function of presumedregulatory genes are to express the gene in an inappropriatelocation or to block the function of the endogenous product ofthe gene in its native location. Several studies have shown thatthe site of injection of mRNAs at morula stages can determinethe effectiveness of the genetic manipulation. By detailing themovements of blastomere clones prior to gastrulation andrelating the 32-cell and stage 10 fate maps, the effectivenessof targeted genes may be improved. For example, Steinbesseret al. (1993) recently expressed goosecoid mRNA in a tier-3blastomere of a ventralized embryo without achievingcomplete rescue of the dorsal axis. The data herein demonstratethat B1, a tier-2 blastomere, normally produces most ofSpemann’s Organizer, which is the normal site of goosecoidexpression (Cho et al., 1991). Perhaps if goosecoid mRNA isexpressed in tier-2 cells the dorsal axis would be rescued com-pletely.

Understanding the movements of clones through gastrula-tion also may help explain the results of manipulations on amechanical versus genetic basis. For example, Xwnt-8 mRNA(Smith and Harland, 1991), noggin mRNA (Smith andHarland, 1992) and a native RNA extracted from dorsal blas-tomeres (Hainski and Moody, 1992) are more effective atrescuing the dorsal axis of ventralized embryos or inducing asecondary axis when injected into ventral vegetal, rather thananimal cells. It seems unlikely that injections of exogenous

mRNA into ventral animal cells fail to effect changes in axialfate due to a lack in a signalling pathway or a lack of compe-tence, because ventral animal cells transplanted dorsally canmake axial structures (Huang and Moody, 1993), and in culturecan be induced to produce dorsal mesoderm (Hainski, 1992).What is notable from this study of gastrulation movements isthat the ventral animal clone (V1.1) does not involute. If a blas-tomere is competent, but its clone physically cannot ingress,there is little chance that an axis can be organized byexogenous gene products. This idea is supported by the obser-vations that dorsal animal blastomeres can organize secondarydorsal axes when transplanted to ventral tier-3 or tier-4positions (Gallagher et al., 1991), but rarely do so when trans-planted to ventral tier-1 and tier-2 positions (Kageura, 1990;Huang and Moody, 1993).

We wish to thank Mrs. Lianhua Yang for her excellent technicalassistance. This work was supported by NIH grant NS23158.

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(Accepted 18 January 1994)