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

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, NW Washington, DC 20037, USA *Author for correspondence

SUMMARY

Recent investigations into the roles of early regulatory the ventral animal clones extend across the entire dorsal genes, especially those resulting from mesoderm induction animal cap. These changes in the blastomere constituents or first expressed in the gastrula, reveal a need to elucidate of the animal cap during epiboly may contribute to the the developmental history of the cells in which their tran- changing capacity of the cap to respond to inductive growth scripts are expressed. Although fates both of the early blas- factors. Pregastrulation movements of clones also result in tomeres and of regions of the gastrula have been mapped, the B1 clone occupying the vegetal marginal zone to the relationship between the two sets of fate maps is not become the primary progenitor of the dorsal lip of the clear and the clonal origin of the regions of the stage 10 blastopore (Spemann’s Organizer). This report provides embryo are not known. We mapped the positions of each the fundamental descriptions of clone locations during the blastomere clone during several late blastula and early important periods of axis formation, mesoderm induction gastrula stages to show where and when these clones move. and neural induction. These will be useful for the correct We found that the dorsal animal clone (A1) begins to move targeting of genetic manipulations of early regulatory away from the animal pole at stage 8, and the dorsal animal events. marginal clone (B1) leaves the animal cap by stage 9. The ventral animal clones (A4 and B4) spread into the dorsal Key words: fate map, epiboly, dorsal lip, cell lineage, animal cap animal cap region as the dorsal clones recede. At stage 10, assays.

INTRODUCTION of the gastrula that express unique gene products could be iden- tified. Since many experiments use the cleavage stage embryo Gastrulation movements morphologically transform the for targeting foreign gene products, and rely on changes in fate amphibian embryo from a ball of cells into an elongated for the interpretation of gene function, the comprehensive map tadpole with distinct dorsal-ventral and anterior-posterior axes. of blastomere clones at blastula and gastrula stages will allow In addition, during this time, many tissue and phenotype spec- investigators to target correct progenitors for gene misexpres- ifications occur, and many region-specific regulatory genes are sion, ‘knockout,’ and dominant negative experiments. This first expressed, e.g., Xlim-1, fork head, and goosecoid (Taira study demonstrates that some clones move prior to gastrula- et al., 1992; Dirksen and Jamrich, 1992; Blumberg et al., tion, such that important developmental regions of the embryo 1991). Identification of the developmental history of the cells change in composition over time. For example, the animal cap occupying the various regions of the gastrula is fundamental is used to assay the inductive capacity of exogenous molecules for understanding the role of these genes and the upstream or ectopically expressed gene products (e.g., Slack et al., 1987; events that lead to their region-specific expression. Currently, Kimelman and Kirschner, 1987; Smith, 1987; Rosa et al., we know the developmental fate of the early cleavage stage 1988; Sokol and Melton, 1992; Christian et al., 1992). Inves- blastomeres (Hirose and Jacobson, 1979; Jacobson and Hirose, tigators have used animal caps of various sizes and stages, 1981; Jacobson, 1983; Dale and Slack, 1987; Moody, 1987a,b; which can lead to different results (see Dawid, 1991). Our Moody and Kline, 1990), and there is a detailed fate map of study demonstrates that animal caps of different sizes at the early gastrula (Keller, 1975, 1976). However, there are few different stages in fact contain different clones. Another data that relate these two maps; we do not know where the blas- important developmental region is Spemann’s Organizer, the tomere clones are located in the gastrula. In this study, the inducer of the nervous system. This region has been identified locations of the blastomere clones prior to and during gastru- in the stage 10 embryo (Keller, 1976), but investigators lation were mapped so that the existing early and late maps disagree on the identity of the cleavage stage progenitors could be integrated and the blastomere progenitors of regions (Gimlich, 1986; Takasaki, 1987; Masho, 1988). It often has

1180 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 embryo labeled with the nomenclature of Jacobson and Hirose (1981). (C) 32-cell embryo labeled with the nomenclature of Nakamura and Kishiyama (1971). been assumed that Spemann’s Organizer region arises from the 8 and 9, video images of sagittal sections were measured with a vegetal hemisphere of the cleavage stage embryo (see review Hamamatsu Argus-10 image processor. Sections were chosen from Elinson and Kao, 1989) but, in fact, our initial studies (Hainski each embryo (four at stage 8 and five at stage 9) that contained the and Moody, 1992) and those of others (Takasaki, 1987; Masho, largest area of the labeled B1 clone. The circumference of the tissue 1988) show that most of the dorsal blastopore lip actually section was divided into 360¡ of arc. The animal-most and vegetal- arises from an animal hemisphere blastomere. In the present most boundaries of the B1 clone were measured relative to a linear projection of the floor of the blastocoel to the surface. These bound- study, we detail the blastomeres (from 16- and 32-cell aries were expressed in degrees of arc. embryos) that contribute to the Organizer region, as well as the lateral and ventral lips, and provide a developmental history of the pregastrulation movements of these important clones. RESULTS

MATERIALS AND METHODS The positions of clones change before gastrulation Although the appearance of the dorsal lip of the blastopore at Embryos were obtained from natural matings of adult frogs that had stage 10 is commonly used as the indicator of the onset of gas- been induced to mate with chorionic gonadotropin (Sigma). Fertilized trulation, Xenopus blastula cells become motile at stage 8 eggs were dejellied and selected for lineage dye injections as detailed (Newport and Kirschner, 1982), clones have intermixed by in previous reports (Moody, 1987a,b). Only embryos with stereotyped three cell diameters by stage 9 (Wetts and Fraser, 1989) and cleavage furrows (Fig. 1) were used in order to label consistently the same progenitor in all embryos. Embryos were held in Steinberg’s cellular events indicative of gastrulation movements begin solution until they reached the 16- or 32-cell stage.To study clones nearly an hour before stage 10 (Keller, 1978). We investigated derived from the 16-cell embryo, each of the eight different blas- both the movements of each blastomere clone and the mixing tomeres (Fig. 1A) was injected with 1 nl of 5% horseradish peroxi- between clones before stage 10 to determine whether these dase (HRP, Boeringer-Mannheim). To study clones derived from the movements reorganize the clones prior to the invagination at 32-cell embryo, two neighboring blastomeres were injected, one with the dorsal blastopore lip. 1 nl of 0.5% Texas Red-dextran-amine (TRDA, Molecular Probes) The stage 7 clones were in the original position and wedge and the other with 2 nl of 0.5% fluorescein-dextran-amine (FDA, shape of the injected blastomere (Figs 2,3). Clones of animal Molecular Probes). Only the midline blastomeres of the three animal- blastomeres interdigitated along their edges with neighboring most tiers were examined (Fig. 1B,C). For easier reading, the nomen- unlabeled cells, especially at the marginal zone border of the clature of Nakamura (Fig. 1C) is used in the text when referring to 32-cell blastomeres. However, Jacobson’s nomenclature is illustrated clone (Fig. 3). The D1.1 clone was the most intermixed (Fig. in Fig. 1B so that reference can be made to mother cells (Fig. 1A) and 3B), especially with the contralateral D1.1 clone (Fig. 4). to the 32-cell fate map of Moody (1987b). Clones of vegetal blastomeres had little mixing at the borders Injected embryos were raised in Steinberg’s solution, the fluores- (Figs 2,3). At stage 8 some clones began to shift positions. The cent ones in the dark, at room temperature. Embryos were fixed, at A4 clone extended a few cell diameters across the geometric intervals from stage 7 to stage 13 (Nieuwkoop and Faber, 1967), in animal pole into the dorsal area, while the animal-most descen- 4% paraformaldehyde in 0.1 M P04 buffer (pH 7.4). Most of the HRP- dants of A1 receded one or two cell diameters away from the labeled embryos were processed as whole mounts. They were washed, animal pole (Figs 5,6B). In addition, all of the clones mixed ′ reacted in 3,3 -diaminobenzidine (Sigma), dehydrated, cut in half and along their borders with neighboring clones at a depth of one embedded in clear plastic (Eukitt, Calibrated Instruments, Inc.). Most or two cell diameters (Figs 5,6B). of the fluorescently labeled embryos were sectioned at 17 µm with a cryostat, washed and coverslipped with Tris/glycerol. For stages 7- At stage 9, several of the clones had moved from their 10, the animal pole was identified by determining the center of the original positions. The ventral animal clones (A4, B4) pushed blastocoel, from its dorsal/ventral and left/right walls, and projecting toward the dorsal side, over the animal pole (Figs 6C, 7). In a line to the surface. the blastocoel roof, these clones overlapped one another in To quantify the extent of movement of the B1 clone between stages different layers, superficial or deep. In some cases, B4-derived Blastomere clones during gastrulation 1181

Fig. 3. Camera-lucida drawings of labeled members of 16-cell clones Fig. 2. Camera-lucida drawings of labeled surface cells (stippled), (stippled) in parasagittal (A), sagittal (B,D) and coronal (C) sections members of clones of 16-cell blastomeres, at stage 7. Each clone of stage 7 embryos. (A) The D1.2 clone mixes with neighboring retains the shape and position of its progenitor blastomere, with clones by one or two cell diameters at the marginal zone edge. interdigitation of cells at the edge of the clone. (A) D1.1, animal (B) The D1.1 clone contains numerous unlabeled cells 3- to 6-cell view, dorsal to the top. (B) D1.2, animal view, dorsal to the top. diameters from the edge of the clone. This is especially notable at the (C) D2.1, vegetal view, dorsal to the top. (D) V2.1, view of ventral animal cap border. (C) The D2.1 clone, viewed from just below the midline, animal to the right. floor of the blastocoel, mixes by one cell diameter with neighboring clones at several sites. (D) The V2.1 clone barely interdigitates with the clone of its lateral neighbor. cells occupied the superficial layer over the A4-derived cells lining the blastocoel; in other cases, the positions of the clones were reversed. The dorsal animal clones moved vegetally toward the marginal zone (Figs 6C, 8). The animal-most was about five cell diameters animal to the floor of the blasto- descendants of A1 were nearly 45¡ distant from that pole and coel. The C1 clone was compressed toward the vegetal pole, the vegetal-most descendants were within two to three cell and most of its constituents had left the dorsal marginal zone diameters of the blastocoel floor (Fig. 6C). The B1 clone (Fig. 8). In addition, as described by Wetts and Fraser (1989) occupied the dorsal marginal zone (Fig. 8); its rostral extent for dorsal animal clones, mixing at the boundaries between

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 color photomicrographs. 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 B4 clone (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 be found 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 the noninvoluting marginal zone (solid arrow), contributing some deep cells to the involuting mesoderm (open arrow). Sagittal section, dorsal is in the 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 of invagination (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) stretches from 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 dorsal blastopore lip, but members of the C1 clone (green) also make a small contribution. Arrows mark the site of invagination. Orientation is the same 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. Sagittal section 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). The ventral 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. 1182 D. V. Bauer, S. Huang and S. A. Moody clones occurred at a depth of three to four cell diameters (Figs blastocoel were pulled toward the animal pole by the leading 6C, 7, 8). edge of the involuting mesoderm (Fig. 6E). At stage 12, these During gastrulation, the main bodies of the clones continued descendants of C1 and D1 formed the floor of the archenteron to change positions as involution proceeded. At stage 10, the and the prechordal mesoderm of the head (Fig. 6F). spread of ventral animal clones (A4, B4) over the blastocoel roof During gastrulation, the blastomere clones were still recog- was extensive (Figs 6D, 9) and, at the midline, they covered nizable as discrete masses. Surface cells were mostly contigu- about three-quarters of the roof. At stage 11, the A4 clone ous with members of their own clone, but deep cells began to stretched across nearly the entire blastocoel roof, the B4 clone mix extensively with neighboring clones (Fig. 6D-F). The deep extended from the middle of the blastocoel roof into the prein- cells of the dorsal clones mixed as they crowded toward the volution mesoderm close to the ventral lip, and the C4 clone was dorsal lip of the blastopore and involuted. In the stage 11 involuting (Fig. 6E). On the dorsal side, the animal clones (A1, blastopore lip, for example, the descendants of A1 and B1 were B1) at stage 10 had condensed to fill the dorsal marginal zone well circumscribed in the preinvolution mesoderm but thor- and the blastopore lip (Figs 6D, 11-13). At stage 11, members oughly mixed in the postinvolution mesoderm (Fig. 10). By the of the A1 and B1 clones had involuted (Fig. 10), and members end of gastrulation, each descendant of A1 and B1 was within of the C1 and D1 clones that had formed the dorsal floor of the three cell diameters of a descendant of both A1 and B1 (Fig.

Figs 4-5, 7-15. Legends on p. 1181 Blastomere clones during gastrulation 1183 C F composite summaries of at least three embryos per blastomere pair. Diagrams are oriented as in Fig. 1. Clones are represented by the following colors: lilac, C4; orange, B4; green, A4; yellow, A1; red, B1; blue, C1; purple, D1. Illustrations in B-F are based on midsagittal photomicrographs by Hausen and Riebesell (1991). E B ANIMAL VEGETAL A Summary diagrams illustrating the locations of clones derived from midline D Fig. 6. blastomeres of the 32-cell embryo (A) at stage 8 (B), 9 (C), 10 (D), 11 (E), and stage 12.5-13 (F). Data were derived from tissue sections in which two adjacent blastomere clones were labeled with different lineage dyes. These illustrations are 1184 D. V. Bauer, S. Huang and S. A. Moody

14). Deep cells of the ventral clones mixed as they spread over the descendants of C1 also contributed to these structures (Fig. the embryo, and involuted at the ventral lip of the blastopore 6E). (Figs 6E-F, 9, 15). At stage 12, the clones derived from the three dorsal blas- tomeres (D1.1, D1.2, D2.2) were compressed into the dorsal Which clones make up the animal cap? third of the blastopore lip (Fig. 16). D1.1 and D1.2 clones The movements of epiboly and perhaps an asymmetric formed stripes in the developing neural plate, parallel to the expansion of the blastocoel reorganize the relative positions of anterior-posterior axis, and extended through the lip to con- the blastomere clones, such that the constituents of the animal tribute to mesoderm and archenteron roof. The superficial cap gradually change. Since the animal cap is used extensively descendants of A1 just reached the blastopore lip with a few in mesoderm induction assays, we detailed the clonal compo- descendants intermixed with the B1 clone in the region of invo- sition of these caps at different stages. At stages 7 and 8, each lution (Fig. 14). The B1 clone extended from the region of animal blastomere occupied nearly its original position with involution across the archenteron roof and throughout the respect to the blastocoel (Figs 2, 3, 6B). However, at stage 8 dorsal axial mesoderm (Fig. 14). With the reduction in the size some dorsal animal clones (D1.2 and A1) had receded slightly of the blastopore, only a few descendants of D2.1 remained in from the geometric pole, and some ventral animal clones (A4 the yolk plug. Because the region of involution lies along the and V1.2) had moved dorsolaterally a few cell diameters over dorsal side of the D2.1 clone, its descendants lined the archen- the animal pole (Figs 5, 6B). Between stage 8 and 9, the B1 teron floor (Fig. 6F). clone moved to the vegetal part of the dorsal marginal zone, and had very few descendants in the animal cap (Figs 6C, 8). Lateral lip At stage 8, the mean rostral border of the B1 clone was 24¡ At early stage 10, the lateral edges of the lip consisted of animal to the blastocoel floor (n=4) and the mean caudal border descendants of D2.2 (Fig. 16). At stage 11, the lateral lip was was 4¡ vegetal to that border. At stage 9 the mean rostral border composed mostly of a mixture from the lateral vegetal clones of the B1 clone was 9¡ animal to the blastocoel floor (n=5) and (D2.2 and V2.2) with a few cells from V1.2. In about half of the mean caudal border was 26¡ vegetal to that border. At stage the specimens, the surface cells of the V1.2 clone were at the 9, the A1 clone had moved to the original position of B1, and lip and, in the rest, they were a few cell diameters away. In all the A4 clone stretched across the animal pole into the dorsal cases, the deep members of the V1.2 clone were in the prein- animal cap (Figs 6C, 7). At stage 10, the B1 clone no longer volution mesoderm at the lip, but in only a few specimens had contributed to the animal cap (Figs 6D, 11). The animal portion labeled cells involuted. At stage 12, the surface descendants of of the dorsal marginal zone was occupied by descendants of D2.2 occupied a relatively small patch in the lateral blastopore A1 (Fig. 6D). The A4 and B4 clones constituted nearly three- lip, while the surface descendants of V1.2 had spread to occupy quarters of the animal cap at the midline (Fig. 9). nearly a quarter of the lateral surface, as viewed from the blastopore (Fig. 16). Which blastomere clones contribute to the blastopore lip? Ventral lip Dorsal Lip At stage 11, the ventral lip consisted of a mixture of cells At early stage 10, the D1.1 clone constituted the majority of derived from V2.2 and V2.1 (Fig. 16). Only the tier-3 daughter the dorsal blastopore lip (Fig. 16). The lateral edges of the lip of V2.1 (C4) contributed to the involuting zone; the tier-4 consisted of descendants of D2.2. Descendants of D2.1 usually daughter (D4) formed only the yolk plug (Fig. 6E). At stage occupied the yolk plug ventral to the lip, but occasionally some 12, the ventral lip consisted mostly of descendants of V2.1 and of its descendants were located on the dorsal side of the lip. V2.2, the clones of which extended in both superficial and deep Labeling B1 and C1 in the same 32-cell embryo revealed that layers from the marginal zone through the lip. In addition, the descendants of B1 populated the dorsal surface of the lip, as surface descendants of V1.1 almost reached the site of involu- well as the deep cells at the site of invagination (Figs 6D, 11), tion, intermixing with surface cells from V2.1. Only the deep while the descendants of C1 populated the yolk plug side of descendants of the vegetal daughter of V1.1 (B4) contributed the lip. Most bottle cells derived from C1. Although the cells to the involuting mesoderm (Fig. 15). majority of the stage 10 dorsal lip consisted of descendants of B1, the proportion of contributing C1 descendants varied from one embryo to the next (cf. Figs 11, 12, 13). However, in all DISCUSSION cases, the site of invagination was less than three cell diameters away from the interface between the B1 and C1 clones. Xenopus embryos and animal caps isolated from them are At stage 11, the D1.1 clone populated the midline dorsal lip, commonly used to study the induction of fate changes by and descendants of D1.2 and D2.2 populated the more lateral growth factors or genetic manipulations. Understanding the regions (Fig. 16); the deeper layers also contained scattered potential of a tissue in situ or in vitro requires full knowledge cells from D2.1. It is notable that the D1.2 clone invaded the of its origin and the fates of its progenitors, especially since vegetal hemisphere by this stage, separating the D1.1 and D2.2 some fates are determined prior to these manipulations (e.g., clones. Both daughters of D1.1 contributed to the stage 11 Takasaki, 1987; Kageura, 1990; Gallagher et al., 1991). Our dorsal lip. The A1 clone did not reach the dorsal lip on the study was designed to investigate the movements of the blas- surface, but deep descendants extended into the preinvolution tomere clones between the morula and gastrulation, to evaluate and postinvolution mesoderm (Fig. 10). While the B1 clone the consequences of these movements for the ultimate fates of formed most of the postinvolution mesoderm and archenteron the blastomere clones, and to determine the identities of the roof (Figs 6E, 10), we estimated that between 3 and 12% of blastomere progenitors of the animal cap and blastopore lip. Blastomere clones during gastrulation 1185

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 clones are 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 clone are in the dorsal lip (bottom, white). At stage 11 (middle), the two lateral animal clones (D1.2, top; V1.2, bottom) have extended into the vegetal hemisphere and reached the blastopore lip. The D1.1 clone has undergone convergent extension to become a narrow midline stripe. At stage 12 (right), the dorsal clones have narrowed due to convergent extension and the ventral clones have spread over a larger surface area. A small portion of the V1.1 clone (top, black) approaches the ventral lip at the midline, but does not involute.

We found that clones move prior to gastrulation, such that the head, neural crest derivatives and branchial arches), it demon- constituents of the animal cap and dorsal marginal zone change strates that Keller’s maps cannot be superimposed over the between stages 8 and 10. These data are important for inter- original blastomeres (Fig. 6A), and it clarifies the process by preting animal cap assays and fate changes resulting from which blastomere clones move through gastrulation to achieve genetic and other cellular manipulations. their eventual fates. Contributions of the blastomeres to the stage 10 Pregastrulation movements change the positions of fate map the blastomere clones Each blastomere of the cleavage stage embryo contributes to Study of the pregastrula using cinemicrographic techniques widely diverse tissues in the tailbud stage (Dale and Slack, indicates that the apical surfaces of superficial cells in the 1987; Moody, 1987a,b). Because the tissues of the tailbud animal and marginal regions expand between stages 7 and 9 embryo are represented by discrete territories in stage 10 (Keller, 1978). After stage 9, dorsal marginal cells expand even embryos (Keller, 1975, 1976), we mapped the positions of the more rapidly, extending the marginal zone vegetally toward the blastomere clones onto these territories (Table 1). For the forming blastopore invagination. As a consequence of these midline clones, the 32-cell data are presented and, for the pregastrulation movements, blastomere clones occupy very lateral clones, the 16-cell data are presented. Table 1 shows different space in the embryo at stage 10 than they did at where the clones are located at stage 10, in general and using cleavage stages. With the formation of the blastocoel and the Keller’s specific regional descriptions, what these regions are movements of epiboly, each blastomere clone changes shape fated to become (Keller, 1975, 1976), and what these blas- and position to occupy a characteristic region of the gastrula tomeres are fated to become (Moody, 1987a,b). This compar- that is quite different from its original position (Fig. 6; Table ison establishes the concordance of the fate maps derived from 1). These changes in the positions of blastomere clones were cleavage stage embryos with those derived from the early not predicted by the cinemicrography. For example, it appeared gastrula. It provides much more phenotypic detail to some that the expansion of the animal cap was uniform (Fig. 5A; regions of Keller’s map (e.g., ectodermal specializations of the Keller, 1978), but our data show shifts of specific groups of 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 fates Blastomere 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 cord suprablastoporal endoderm archenteron roof archenteron roof of hindgut dorsal preinvolution mesoderm notochord notochord dorsal involuted mesoderm head mesoderm dorsal head mesenchyme, branchial arches pharyngeal endoderm pharynx, foregut somites 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 roof dorsal preinvolution mesoderm notochord notochord dorsal involuted mesoderm head mesoderm dorsal head mesenchyme, branchial arches pharyngeal endoderm pharynx, foregut somites 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 arches heart heart notochord, somites notochord, somites pharyngeal 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 roof lateral preinvolution mesoderm somites head somites, central trunk somites D2.2 lateral marginal zone: middle epidermal area dorsal trunk epidermis dorsal trunk epidermis middle, 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 somites lateral involuted mesoderm heart, somites, lateral mesoderm heart, somites, lateral plate, nephrotome subblastoporal 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 roof ventral preinvolution mesoderm somites, lateral mesoderm somites, lateral plate ventral 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 roof posterior epidermal area trunk epidermis trunk epidermis, neural crest posterior neural area posterior spinal cord posterior spinal cord lateral preinvolution mesoderm somites, lateral plate somites, lateral plate lateral 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 a consequence of mixing between clones, as described in the Results section, each clone also makes minor contributions to neighboring regions not listed in this table. Blastomere clones during gastrulation 1187 cells (clones A4 and B4) from ventral to dorsal between stages hemisphere to the animal pole of an endogenous signal (Sokol, 8 and 10, and then the spread of these groups from animal to 1993), an alternative hypothesis is that the constituents of the ventral between stages 10 and 12. The expansion of the apices animal cap change during pregastrula stages. Although the of superficial cells also did not predict that groups of cells (both stage 10 fate maps show that the animal cap produces only deep and superficial members of the clone) would move en ectodermal derivatives (Keller, 1975), the normal fates of stage masse from animal to dorsal marginal zone regions. 8 or 9 animal caps in the intact embryo have not been mapped. A feature of these clonal movements is the mixing of cells Since the 16- and 32-cell fate maps show that the animal cap between clones. In zebrafish, whose blastomere clones do not blastomeres (tiers-1 and -2) contribute to all three germ layers, have predictable fates and can contribute to all regions of the and transplantation studies show that dorsal animal blas- embryo (Kimmel and Law, 1985), the clones mix freely during tomeres are determined to produce dorsal mesodermal tissues gastrulation and descendants of distant clones come in contact (Takasaki, 1987; Kageura, 1990; Gallagher et al., 1991), it is with one another (Warga and Kimmel, 1990). However, in possible that nonectodermal cells are within the animal cap of Xenopus, whose blastomere clones are predictable and region- the blastula and leave it by stage 10. These proposed changes ally discrete (Dale and Slack, 1987; Moody, 1987a,b), the in the constituents of the animal caps could result in changes clones mix only along their borders until just before involu- in their competence to respond to manipulations. tion. Wetts and Fraser (1989) showed that, at stage 9, dorsal In fact, our data demonstrate that the clones comprising the animal clones interdigitated by about three cell diameters. We animal cap change after the midblastula transition. The stage show that this intermixing begins as early as stage 7, prior to 8 animal cap, whether large (including the entire blastocoel the onset of cellular motility (Newport and Kirschner, 1982). roof) or small (including only cells within 45¡ of the animal Mixing occurs more in animal clones than vegetal clones and pole), contains the entire clone of B1. Previous experiments more in the marginal zone. The earliest and most extensive indicate that this blastomere can autonomously differentiate to mixing occurs across the dorsal animal midline, which predicts form dorsal mesoderm (Takasaki, 1987; Kageura, 1990; the bilateral origin of forebrain structures (Jacobson and Gallagher et al., 1991) and that it contains dorsal information Hirose, 1978; Huang and Moody, 1992, 1993). Mixing that (Elinson and Kao, 1989; Hainski and Moody, 1992). Further- occurs prior to stage 8 probably results from the interdigitation more, B1 is the primary progenitor of Spemann’s Organizer of cells during mitoses and permissiveness to mixing at clonal and a major progenitor of the notochord (Dale and Slack, 1987; boundaries. Moody, 1987b). The presence of the B1 clone, whose dorsal During epiboly the mixing of clones increases. Superficial mesoderm fate seems determined before stage 8, probably is a layers remain nearly coherent, with a few interposing cells significant factor in the state of competence of the stage 8 from other clones. This is consistent with the cinemicrographic animal cap. For example, the presence of some B1 descendants observation that during epiboly cells from the superficial and in the stage 8 cap may account for the ability of activin to deep layers do not mix (Keller, 1978). In contrast, cells from induce dorsal mesoderm only from dorsal halves of the cap different clones in the deep layers mix extensively. In the deep (Sokol and Melton, 1991). layers of the blastocoel roof, this mixing is accomplished by At stage 9, the B1 clone has moved vegetally into the clones sliding past one another and cells intercalating (Keller, marginal zone. Consequently, the small stage 9 animal cap 1980). In the deep layers of the marginal zone, the mixing is contains primarily the descendants of A4, B4 and A1. The accomplished by the radial intercalation of cells in the prein- large stage 9 animal cap, however, still includes some descen- volution zone and the mediolateral intercalation during invo- dants of B1. This may result in differences in inducibility lution (Wilson and Keller, 1991) These movements cause between large and small stage 9 animal caps (e.g., Christian et thorough mixing of the dorsal midline clones along the antero- al., 1992; Sokol, 1993). In addition, the movement of B1 out posterior axis. For example, the clone of A1, which involutes of the animal cap correlates with the observation that after the clone of B1, extends across the entire anterior- mesoderm does not form in stage 8 or large stage 9 animal caps posterior extent of the dorsal axial tissues and is coextensive isolated from embryos injected with exogenous Xwnt-8 mRNA with descendants of B1 through all but the most anterior (Sokol, 1993). Perhaps the presence of the B1 clone inhibits sensory epithelium (Fig. 14). Even after gastrulation a slow the Xwnt-8 signal. progressive mixing of clones continues, at least in the nervous By stage 10 the B1 clone occupies the dorsal lip of the system (Wetts and Fraser, 1989). blastopore, so that a large animal cap includes only neuroec- todermal (A1) and epidermal (A4, B4) progenitors, while a Changes in animal cap competence may result from small cap includes only epidermal descendants of A4 and B4. changes in clonal constituents Interestingly, the competence of the animal cap to form Isolated Xenopus animal caps form only atypical epidermis in mesoderm ceases at this stage (Jones and Woodland, 1987), culture, but can be induced to form mesoderm (Sudarwati and when all the blastomere clones known to produce mesoderm Nieuwkoop, 1971). Thus, they are used in a standard assay to have exited from the animal cap. Thus, the different con- test the effectiveness of factors to induce mesoderm. However, stituents of the animal cap at different stages may strongly investigators have used large or small animal caps from various influence the competence of the cap to be induced, and its stages, sometimes with different results (Dawid, 1991). For responsiveness to the presence of exogenous gene products. example, it has been suggested that different laboratories observed different abilities of Xwnt-8 to induce mesoderm The major progenitor of the dorsal lip of the because of differences in explant size (Christian et al., 1992) blastopore is an animal blastomere or stage of isolation (Sokol, 1993). While it has been proposed The dorsal lip of the blastopore is the Organizer of the that these differences are due to the diffusion from the vegetal dorsoventral axis of the embryo (Spemann and Mangold, 1924) 1188 D. V. Bauer, S. Huang and S. A. Moody and recently novel gene transcripts have been localized to this mRNA into ventral animal cells fail to effect changes in axial region (Blumberg et al., 1991; Dirksen and Jamrich, 1992; fate due to a lack in a signalling pathway or a lack of compe- Taira et al., 1992). If the progenitor of the dorsal lip is to be tence, because ventral animal cells transplanted dorsally can targeted for genetic manipulation, one needs to specifically make axial structures (Huang and Moody, 1993), and in culture identify that progenitor. Generalizing the 32-cell fate map to can be induced to produce dorsal mesoderm (Hainski, 1992). resemble the stage 10 fate map has led to the common assump- What is notable from this study of gastrulation movements is tion that the dorsal lip arises from C1, the tier-3 dorsal midline that the ventral animal clone (V1.1) does not involute. If a blas- cell. Although one lineage tracing study reported that the site tomere is competent, but its clone physically cannot ingress, of invagination is in the cleavage furrow between the C1 and there is little chance that an axis can be organized by D1 blastomeres (Gimlich, 1986), others have placed it close to exogenous gene products. This idea is supported by the obser- the cleavage furrow between C1 and B1 (Masho, 1988; vations that dorsal animal blastomeres can organize secondary Takasaki, 1987), with the B1 clone forming most of the prein- dorsal axes when transplanted to ventral tier-3 or tier-4 volution mesoderm and the C1 clone forming most of the bottle positions (Gallagher et al., 1991), but rarely do so when trans- cells (Takasaki, 1987). By observing many embryos in which planted to ventral tier-1 and tier-2 positions (Kageura, 1990; both B1 and C1 were labeled, we concur that the clone of B1 Huang and Moody, 1993). is the primary contributor to the dorsal lip. Although C1 does have descendants in this region, especially the bottle cells, they We wish to thank Mrs. Lianhua Yang for her excellent technical were never the major constituents. The proportion of C1 assistance. This work was supported by NIH grant NS23158. descendants in the dorsal lip varied from embryo to embryo (Figs 11-13), probably due to variability in the location of the REFERENCES third cleavage furrow (see also Masho, 1988). Thus, the majority of evidence indicates that the Organizer develops Blumberg, B., Wright, C. V. E., De Robertis, E. M. and Cho, K. W. Y. (1991). Organizer-specific homeobox genes in Xenopus laevis embryos. mostly from a blastomere in the animal hemisphere and that, Science 253, 194-196. if one wants to target Spemann’s Organizer for genetic manip- Cho, K. W. Y., Blumberg, B., Steinbesser, H. and De Robertis, E. M. ulations, B1 is the best candidate blastomere. Furthermore, our (1991). 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