Range and stability of cell fate determination in isolated sea urchin blastomeres

We have examined the developmental potential of blastomeres isolated from either the animal (mesomeres) or vegetal (macromeres-micromeres) half of 16-cell embryos of the sea urchin Lytechinus pictus. We have also examined the effects of two known vegetalizing agents on the development of isolated mesomeres; LiCl treatment and combination with micromeres, the small blastomeres found at the vegetal pole of the 16-cell embryo. The markers for differentiation used were both morphological (invaginations, spicules and pigment cells) and molecular (gut-specific alkaline phosphatase activity, and monoclonal antibodies against antigens specific for gut and oral ectoderm). Embryoids derived from isolated mesomeres expressed markers characteristic of vegetal differentiation only at very low levels. They did express an antigen characteristic of animal development, the oral ectoderm antigen, but with an altered pattern. Isolated macromere-micromere pairs expressed all markers characteristic of vegetal development, but did not express the marker characteristic of animal development. Increasing concentrations of LiCl caused isolated mesomeres to give rise to embryoids with an increasing tendency to express vegetal markers of differentiation, and it was found that expression of different vegetal markers begin to appear at different concentrations of LiCl. LiCl also caused the marker for oral ectoderm to be expressed in a more normal pattern. Combining micromeres with mesomeres also induced mesomere derivatives to differentiate in a vegetal manner. Micromeres were not completely effective in inducing a more normal pattern of expression of the marker for oral ectoderm. The treatment of isolated mesomeres with both LiCl and micromeres produces a synergistic effect resulting in embryoids expressing markers not induced by either treatment alone.     

Early inductive interactions are involved in restricting cell fates of mesomeres in sea urchin embryos

Isolated intact caps of animal blastomeres, obtained from either 8- or 16-cell embryos, differentiate as swollen ectodermal vesicles. These findings agree with earlier studies demonstrating that mesomeres contribute only to larval ectoderm during normal development. In contrast, we find that pairs of mesomeres isolated from 16-cell embryos can differentiate endodermal and mesenchymal cells in a substantial number of cases (23%). Thus, mesomeres have a greater developmental potential than is realized during normal development. Further results support hypotheses that graded distributions of morphogenetic determinants exist within these embryos, since the extent of differentiation of isolated mesomeres is related to the relative position of the third cleavage plane along the animal-vegetal axis. When the third cleavage plane is subequatorial and the resulting animal blastomeres inherit a fraction of the vegetal hemisphere, more cases (39%) differentiate endodermal and mesenchymal cell types. A significant number of mesomere pairs (9-14%), however, can still differentiate endodermal and mesenchymal cells when the mesomeres are formed within the animal hemisphere. Thus, putative vegetal morphogenetic determinants may extend into the animal hemisphere in some cases. Further results indicate a temporal restriction in the developmental potential of mesomeres or mesomere progenitor cells since their differentiative capability is greater if they are isolated earlier during development. Aggregates of isolated mesomere pairs also display a decreased developmental potential when compared to isolated mesomere pairs. These results suggest that associations with adjacent cells (vegetal cells as well as adjacent mesomeres) restrict the development of mesomeres between third and sixth cleavages.     

Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin.

The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease. In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates. The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases. The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development.     

Change in the adhesive properties of blastomeres during early cleavage stages in sea urchin embryo

Blastomeres of sea urchin embryo change their shape from spherical to columnar during the early cleavage stage. It is suspected that this cell shape change might be caused by the increase in the adhesiveness between blastomeres. By cell electrophoresis, it was found that the amount of negative cell surface charges decreased during the early cleavage stages, especially from the 32-cell stage. It was also found that blastomeres formed lobopodium-like protrusions if the embryos were dissociated in the presence of Ca2+. Interestingly, a decrease in negative cell surface charges and pseudopodia formation first occurred in the descendants of micromeres and then in mesomeres, and last in macromeres. By examining the morphology of cell aggregates derived from the isolated blastomeres of the 8-cell stage embryo, it was found that blastomeres derived from the animal hemisphere (mesomere lineage) increased their adhesiveness one cell cycle earlier than those of the vegetal hemisphere (macromere lineage). The timing of the initiation of close cell contact in the descendants of micro-, meso- and macromeres was estimated to be 16-, 32- and 60-cell stage, respectively. Conversely, the nucleus-to-cell-volume ratios, which are calculated from the diameters of the nucleus and cell, were about 0.1 when blastomeres became adhesive, irrespective of the lineage.     

A demonstration of cellular interactions during the formation of mesoderm and primordial germ cells in Pleurodeles waltlii

Animal, vegetal, dorsal and ventral blastomeres of eight-cell embryos of the urodele Pleurodeles waltlii were isolated and cultured for 15 days. The four animal blastomeres produced vesicles delimited by an irregularly shaped epidermis. In all other explants, the formation of mesodermal structures occurred, which can be interpreted as the result of inductive interaction, occurring during segmentation, between the ectodermal animal cap and vegetal yolk mass. Primordial germ cells (PGCs), which formed in 78% of cases when the presumptive ventral half to the embryo was cultured, occurred in only 48% of cases when the two ventral vegetal blastomeres were cultured alone. The absence of PGCs in the explants emanating from the four vegetal blastomeres is thought to have been due to inhibition of differentiation by notochord. This hypothesis has been confirmed by culture experiments in which the addition of presumptive chordomesoderm of young gastrulae prevented the differentiation of PGCs under conditions in which they are normally formed. These observations suggest that, in urodeles, PGCs do not arise from cells segregated as early as the eight-cell stage, but are the product of later inductive interaction between ectoderm and endoderm.     

Interactions of different vegetal cells with mesomeres during early stages of sea urchin development

It has been known from results obtained in the classical experiments on sea urchin embryos that cell isolation and transplantation showed extensive interactions between the early blastomeres and/or their descendants. In the experiments reported here a systematic reexamination of recombination of mesomeres and their progeny (which come from the animal hemisphere) with various vegetal cells derived from blastomeres of the 32- and 64-cell stage was carried out. Cells were marked with lineage tracers to follow which cell gave rise to what structures, and newly available molecular markers have been used to analyze different structures characteristic of regional differentiation. Large micromeres form spicules and induce gut and pigment cells in mesomeres, conforming to previous results. Small micromeres, a cell type not heretofore examined, gave rise to no recognizable structure and had very limited ability to evoke poorly differentiated gut tissue in mesomeres. Macromeres and their descendants, Veg 1 and Veg 2, form primarily what their normal fate dictated, though both did have some capacity to form spicules, presumably by formation from secondary mesenchyme. Macromeres and their descendants were not potent inducers of vegetal structures in animal cells, but they suppress the latent ability of mesomeres to form vegetal structures. The results lead us to propose that the significant interactions during normal development may be principally suppressive effects of mesomeres on one another and of adjacent vegetal cells on mesomeres.     

The fine structure of Comanthus japonica (Echinodermata: Crinoidea) from zygote through early gastrula

The fine structure of the early embryo of Comanthus has been described by scanning and transmission electron microscopy at approximately 20-min intervals from zygote (20 min) through early gastrula (260 min). In normally developing (and presumably monospermic) embryos, some non-fertilizing sperm were invariably trapped in the perivitelline space; this suggests that there is an effective block to polyspermy at the level of the plasma membrane. No trace of a hyaline layer is encountered in the pervitelline space. At first cleavage, which begins unilaterally at the animal pole, the contractile ring filaments are rather thick (50–150 Å) in comparison to those known for other marine invertebrates. From first cleavage through early gastrula, the lateral surfaces of the blastomeres are broadly adherent, and there is an intercellular material, presumably an adhesive, in the intercellular space. The blastocoel first appears during the four-cell stage. From the eight-cell stage through the start of gastrulation, only one opening, the vegetal pore, connects the blastocoel with the perivitelline space. Gastrulation begins at the 50–100-cell stage, while the vegetal pore is still open, and a clearly defined blastula stage is bypassed. Gastrulation is by a novel process, which I have called holoblastic involution. At gastrulation the eight most vegetal blastomeres, which encircle the vegetal pore, shoot out erect, unbranched filopodia for many microns through the blastocoel. The filopodia adhere to the blastocoelic surfaces of the animal blastomeres and contract, pulling the vegetal blastomeres into the blastocoel. The migrated vegetal blastomeres adhere to one another, forming the entoderm in the vegetal region of the embryo; the remaining blastomeres become the ectoderm. Soon after the completion of cell migration, the entodermal blastomeres appear to cast off their contractile microappendages and adhesive membranes into the blastocoel.     

Archenteron-Forming Capacity in Blastomeres Isolated from Eight-Cell Stage Embryos of the Starfish, Asterina pectinifera

Starfish blastomeres are reported to be totipotent up to the 8-cell stage. We reinvestigated the development of blastomeres of 8-cell stage embryos with a regular cubic shape consisting of two tiers of 4 blastomeres. On dissociation of the embryo by disrupting the fertilization membrane at the 8-cell stage, each of the 4 blastomeres of the vegetal hemisphere gave rise to an embryo that gastrulated, whereas blastomeres from the animal hemisphere did not. By injection of a cell lineage tracer into blastomeres of 8-cell stage embryos, we found that only those of the vegetal hemisphere formed cells constituting the archenteron. Next, we compressed 4-cell stage embryos along the animal-vegetal axis so that all the blastomeres in the 8-cell stage were in a single layer. When these 8 blastomeres were then dissociated, an average of 7 of them developed into gastrulae. By cell lineage analysis, all the blastomeres in single-layered embryos at the 8-cell stage were shown to have the capacity to form cells constituting an archenteron. Taken together, these findings indicate that the fate to form the archenteron is specified by a cytoplasmic factor(s) localized at the vegetal hemisphere, and that isolated blastomeres that have inherited this factor develop into gastrulae.     

Four-cell stage mouse blastomeres have different developmental properties

Blastomeres of the early mouse embryo are thought to be equivalent in their developmental properties at least until the eight-cell stage. However, the experiments that have led to this conclusion could not have taken into account either the spatial origin of individual blastomeres or the spatial allocation and fate of their progeny. We have therefore readdressed this issue having defined cell lineages in mouse embryos undergoing different patterns of cleavage in their second division cycle. This has enabled us to identify a major group of embryos in which we can predict not only the spatial origin of each given four-cell blastomeres, but also which region of the blastocyst is most likely to be occupied by its progeny. We show that a pattern of second cleavage divisions in which a meridional division is followed by one that is equatorial or oblique allows us to identify blastomeres that differ in their fate and in their developmental properties both from each other and from their cousins. We find that one of these four-cell stage blastomeres that inherits some vegetal membrane marked in the previous cleavage cycle tends to contribute to mural trophectoderm. The progeny of its sister tend to donate cells to part of the ICM lining the blastocyst cavity and its associated trophectoderm. Chimaeras made entirely of these equatorially or obliquely derived blastomeres show developmental abnormalities in both late preimplantation and early postimplantation development. By contrast, chimaeras made from four-cell stage blastomeres from early meridional divisions develop normally. The developmental defects of chimaeras made from the most vegetal blastomeres that result from later second cleavages are the most severe and following transplantation into foster mothers they fail to develop to term. However, when such individual four-cell blastomeres are surrounded by blastomeres from random positions, they are able to contribute to all embryonic lineages. In conclusion, this study shows that while all four-cell blastomeres can have full developmental potential, they differ in their individual developmental properties according to their origin in the embryo from as early as the four-cell stage.     

Differentiation of the four animal and the four vegetal blastomeres of the eight-cell-stage ofTriturus alpestris

Summary The 4 animal and 4 vegetal blastomeres of the eight-cell-stage ofTriturus alpestris were isolated and cultured for up to 12 days. Because of the difficulty of obtaining intact animal and vegetal blastomeres of the same embryo, we either cut off the vegetal blastomeres or sucked off the animal blastomeres. The culture of early embryonic amphibian cells is improved by the use of 50% Leibovitz-medium with added fetal calf serum providing a stable pH and optimal osmotic pressure.Isolated animal blastomeres differentiated to irregularly shaped ciliated epidermis. 30% of the cases showed small amounts of myotomes, notochord and neuroid cells in addition to irregular epidermis. The vegetal blastomeres formed trunk and tail structures but only 6% of all cases formed nearly complete head structures in addition.From the results we conclude that the vegetal blastomeres as well as the animal blastomeres of the eight-cell-stage are already determined as to their future fate. The possibility of partial regulation and the influence of asymmetric or irregular cleavage on the further development of isolated blastomeres is discussed.     

Animal-vegetal asymmetries influence the earliest steps in retina fate commitment in Xenopus.

An individual retina descends from a restricted and invariant group of nine animal blastomeres at the 32-cell stage. We tested which molecular signaling pathways are responsible for the competence of animal blastomeres to contribute to the retina. Inactivation of activin/Vg1 or fibroblast growth factor (FGF) signaling by expression of dominant-negative receptors does not prevent an animal blastomere from contributing to the retina. However, increasing bone morphogenetic protein (BMP) signaling in the retina-producing blastomeres significantly reduces their contribution. Conversely, reducing BMP signaling by expression of a dominant-negative BMP receptor or Noggin allows other animal blastomeres to contribute to the retina. Thus, the initial step in the retinal lineage is regulated by position within the BMP/Noggin field of epidermal versus neural induction. Vegetal tier blastomeres, in contrast, cannot contribute to the retina even when given access to the appropriate position and signaling fields by transplantation to the dorsal animal pole. We tested whether expression of molecules within the mesoderm inducing (activin, FGF), mesoderm-modifying (Wnt), or neural-inducing (BMP, Noggin) pathways impart a retinal fate on vegetal cell descendants. None of these, several of which induce secondary head structures, caused vegetal cells to contribute to retina. This was true even if the injected blastomeres were transplanted to the dorsal animal pole. Two pathways that specifically induce head tissues also were investigated. The simultaneous blockade of Wnt and BMP signaling, which results in the formation of a complete secondary axis with head and eyes, did not cause the vegetal clone to give rise to retina. However, Cerberus, a secreted protein that also induces an ectopic head with eyes, redirected vegetal progeny into the retina. These experiments indicate that vegetal blastomere incompetence to express a retinal fate is not due to a lack of components of known signaling pathways, but relies on a specific pathway of head induction.     

A Stereometric Analysis of Karyokinesis, Cytokinesis and Cell Arrangements during and following Fourth Cleavage Period in the Sea Urchin, Lytechinus variegatus

Fourth cleavage of the sea urchin embryo produces 16 blastomeres that are the starting point for analyses of cell lineages and bilateral symmetry. We used optical sectioning, scanning electron microscopy and analytical 3-D reconstructions to obtain stereo images of patterns of karyokinesis and cell arrangements between 4th and 6th cleavage. At 4th cleavage, 8 mesomeres result from a variant, oblique cleavage of the animal quartet with the mesomeres arranged in a staggered, offset pattern and not a planar ring. This oblique, non-radial cleavage pattern and polygonal packing of cells persists in the animal hemisphere throughout the cleavage period. Contrarily, at 4th cleavage, the 4 vegetal quartet nuclei migrate toward the vegetal pole during interphase; mitosis and cytokinesis are latitudinal and subequatorial. The 4 macromeres and 4 micromeres form before the animal quartet divides to produce a 12-cell stage. Subsequently, macromeres and their derivatives divide synchronously and radially through 8th cleavage according to the Sachs-Hertwig rule. At 5th cleavage, mesomeres and macromeres divide first; then the micromeres divide latitudinally and unequally to form the small and large micromeres. This temporal sequence produces 28-and 32-cell stages. At 6th cleavage, macromere and mesomere descendants divide synchronously before the 4 large micromeres divide parasynchronously to produce 56- and 60-cell stages.     

Dorsal and ventral cells of cleavage-stage Xenopus embryos show the same ability to induce notochord and somite formation

Cells in the dorsal marginal zone of the amphibian embryo acquire the potential for mesoderm formation during the first few hours following fertilization. An examination of those early cell interactions may therefore provide insight on the mechanisms important for organization of axial structures. The formation of mesoderm (notochord, somites, and pronephros) was studied by combining blastomeres from the animal pole region of Xenopus embryos (32- to 512-cell stages) with blastomeres from different regions of the vegetal hemisphere. The frequency of notochord and somite development was similar in combinations made with dorsal or ventral blastomeres, or with both. Our results show that during early cleavage stages the ventral half of the vegetal hemisphere has the potential to organize axial structures, a property previously believed to be limited to the dorsal region.     

Maternal information and localized maternal mRNAs in eggs and early embryos of the ascidian Halocynthia roretzi

Summary

The mosaic behavior of blastomeres isolated from ascidian embryos has been taken as evidence that localized ooplasmic factors (cytoplasmic determinants) specify tissue precursor cells during embryogenesis. Experiments involving the transfer of egg cytoplasm have revealed the presence and localization of various kinds of cytoplasmic determinants in eggs of Halocynthia roretzi. Three cell fates, epidermis, muscle and endoderm, are fixed by cytoplasmic determinants. The three kinds of tissue determinants move in different directions during ooplasmic segregation. Prior to the onset of the first cleavage the three kinds of determinants reside in egg regions that correspond to the future fate map of the embryo and then they are differentially partitioned into specific blastomeres. In addition to tissue-specific determinants, there is evidence suggesting that ascidian eggs contain localized cytoplasmic factors that are responsible for controlling the cleavage pattern and morphogenetic movements. Transplantation of posterior-vegetal egg cytoplasm to an anterior-vegetal position causes a reversal of the anterior-posterior polarity of the cleavage pattern. Localized cytoplasmic factors in the posterior-vegetal region are involved in the generation of a unique cleavage pattern. When vegetal pole cytoplasm is transplanted to the animal pole or equatorial position of the egg, ectopic gastrulation occurs at the site of transplantation. This finding supports the idea that vegetal pole cytoplasm specifies the site of gastrulation. Recently, we started a cDNA project to analyze maternal mRNAs. An arrayed cDNA library of fertilized eggs of H. roretzi was constructed, and more than 2000 clones have been partially sequenced so far. To estimate the proportion of the maternal mRNAs that are localized in the egg and embryo, 150 randomly selected clones were examined by in situ hybridization. We found eight mRNAs that are localized in the eight-cell embryo, of which three were localized to the myoplasm (a specific region of the egg cytoplasm that is partitioned into muscle-lineage blastomeres) of the egg, and then to the postplasm of cleavage-stage embryos. These results indicate that the proportion of localized messages is much higher than we expected. These localized maternal messages may be involved in the regulation of various developmental processes.     

Induction of ascidian peripheral neuron by vegetal blastomeres.

Ascidian tadpole larvae have a similar dorsal tubular nervous system as vertebrates. The induction of brain formation from a4.2-derived (a-line) cells requires signals from the A4.1-derived (A-line) cells. However, little is known about the mechanism underlying the development of the larval peripheral nervous system due to the lack of a suitable molecular marker. Gelsolin, an actin-binding protein, is specifically expressed in epidermal sensory neurons (ESNs) that mainly constitute the entire peripheral nervous system of the ascidian young tadpoles. Here, we address the role of cell interactions in the specification of ESNs using immunostaining with an anti-gelsolin antibody. Animal half (a4.2- and b4.2-derived) embryos did not give rise to any gelsolin-positive neurons, indicating that differentiation of ESNs requires signals from vegetal cells. Cell isolation experiments showed that A4.1 blastomeres induce gelsolin-positive neurons from a-line cells but not from b4.2-derived (b-line) cells. On the other hand, B4.1 blastomeres induce gelsolin-positive neurons both from b-line cells and a-line cells. This is in sharp contrast to the specification of brain cells which is not affected by the ablation of B4.1-derived (B-line) cells. Furthermore, basic fibroblast growth factor (bFGF) induced ESNs from the a-line cells and b-line cells in the absence of vegetal cells. Their competence to form ESNs was lost between the 110-cell stage and the neurula stage. Our results suggested that the specification of the a-line cells and b-line cells into ESNs is controlled by distinct inducing signals from the anterior and posterior vegetal blastomeres. ESNs in the trunk appear to be derived from the a8.26 blastomeres aligning on the edge of presumptive neural region where ascidian homologue of Pax3 is expressed. These findings highlight the close similarity of ascidian ESNs development with that of vertebrate placode and neural crest.     

Evolutionary modification of cell lineage in the direct-developing sea urchin Heliocidaris erythrogramma

The sea urchin Heliocidaris erythrogramma undergoes direct development, bypassing the usual echinoid pluteus larva. We present an analysis of cell lineage in H. erythrogramma as part of a definition of the mechanistic basis for this evolutionary change in developmental mode. Microinjection of fluoresceinated tracer dye and surface marking with vital dye are used to follow larval fates of 2-cell, 8-cell, and 16-cell blastomeres, and to examine axial specification. The animal-vegetal axis and adult dorsoventral axis are basically unmodified in H. erythrogramma. Animal cell fates are very similar to those of typically developing species; however, vegetal cell fates in H. erythrogramma are substantially altered. Radial differences exist among vegetal blastomere fates in the 8-cell embryo: dorsal vegetal blastomeres contribute proportionately more descendants to ectodermal and fewer to mesodermal fates, while ventral vegetal blastomeres have a complementary bias in fates. In addition, vegetal cell fates are more variable than in typical developers. There are no cells in H. erythrogramma with fates comparable to those of the micromeres and macromeres of typically developing echinoids. Instead, all vegetal cells in the 16-cell embryo can contribute progeny to ectoderm and gut. Alterations have thus arisen in cleavage patterns and timing of cell lineage partitioning during the evolution of direct development in H. erythrogramma.     

The BMP/CHORDIN antagonism controls sensory pigment cell specification and differentiation in the ascidian embryo

We have investigated the role of the bone morphogenetic protein (BMP) pathway during neural tissue formation in the ascidian embryo. The orthologue of the BMP antagonist, chordin, was isolated from the ascidian Halocynthia roretzi. While both the expression pattern and the phenotype observed by overexpressing chordin or BMPb (the dpp-subclass BMP) do not suggest a role for these factors in neural induction, BMP/CHORDIN antagonism was found to affect neural patterning. Overexpression of BMPb induced ectopic sensory pigment cells in the brain lineages that do not normally form pigment cells and suppressed pressure organ formation within the brain. Reciprocally, overexpressing chordin suppressed pigment cell formation and induced ectopic pressure organ. We show that pigment cell formation occurs in three steps. (1) During cleavage stages ectodermal cells are neuralized by a vegetal signal that can be substituted by bFGF. (2) At the early gastrula stage, BMPb secreted from the lateral nerve cord blastomeres induces those neuralized blastomeres in close proximity to adopt a pigment cell fate. (3) At the tailbud stage, among these pigment cell precursors, BMPb induces the differentiation of specifically the anterior type of pigment cell, the otolith; while posteriorly, CHORDIN suppresses BMP activity and allows ocellus differentiation.     

Nuclear migration and spindle formation in the fourth cleavage of sea urchin eggs under the influence of inhibitors.

Mitosis of sea urchin eggs was inhibited when exposed to 3 micrograms/ml aphidicolin from the 2-cell stage onwards. Nevertheless the nuclei migrated to the vegetal pole at the time of the fourth unequal division in control eggs. Two or four equal or unequal asters developed. Asters in proximity to the vegetal pole were always considerably smaller than those close to the center of the two blastomeres. In contrast to colchicine, cytokinesis but not migration of the nuclei in the vegetal half was prevented by treatments with 5 microM cytochalasin B or D. Various mitotic figures were formed in the vegetal blastomeres of eggs treated with 0.4 mM colchicine or 3 microM griseofulvin after the third cleavage. In some eggs a centrally localized monaster with chromosomes in sphere-like arrangement was formed in others a monopolar mitotic figure pushed the chromosomes in bowl-like arrangements to the most vegetal cortex. In anaphase one set of chromatids migrated to the monopole leaving the scattered sister-chromatids behind. The mechanism of migration of the nuclei and of chromosome arrangement in the metaphase plate is discussed.     

Direct Evidence for the Presence of Germ Cell Determinant in Vegetal Pole Cytoplasm of Xenopus laevis and in a Subcellular Fraction of It

To test for the presence of germ cell determinant in Xenopus embryos, vegetal pole cytoplasm containing the "germ plasm", or a subcellular fraction of it, was microinjected into single somatic blastomeres isolated from 32-cell embryos. Injected or non-injected (control) blastomeres were cultured in ³H-thymidine until normal control embryos reached the neurula stage. The labeled explants were then implanted into unlabeled host neurulae, which were allowed to develop to the tadpole stage. Labeled PGCs of explant origin in the genital ridges of the experimental tadpoles were examined by autoradiography.
Isolated blastomeres were injected with vegetal pole cytoplasm of 32-cell embryos or with a 20,000 g pellet made from vegetal pole cytoplasm of 2-cell embryos. Labeled PGCs were found in 7.6% and 2.3% of the experimental tadpoles, respectively. No labeled PGCs were found in the control tadpoles, except for one tadpole in the first experiment. These results strongly suggest that the vegetal pole cytoplasm and its subcellular fractions act as germ cell determinant.