Regional specification during embryogenesis in the inarticulate brachiopod Discinisca.

The process of embryogenesis is described for the inarticulate brachiopod Discinisca strigata of the family Discinidae. A fate map has been constructed for the early embryo. The animal half of the egg forms the dorsal ectoderm of the apical and mantle lobes. The vegetal half forms mesoderm and endoderm and is the site of gastrulation; it also forms the ectoderm of the ventral regions of the apical and mantle lobes of the larva. The plane of the first cleavage goes through the animal-vegetal axis of the egg along the future plane of bilateral symmetry of the larva. The timing of regional specification in these embryos was examined by isolating animal, vegetal, or lateral regions at different times from the 2-cell stage through gastrulation. Animal halves isolated at the 8-cell and blastula stages formed an epithelial vesicle and did not gastrulate. When these halves were isolated from blastulae they formed the cell types typical of apical and mantle lobes. Vegetal halves isolated at all stages gastrulated and formed a more or less normal larva; the only defect these larvae had was the lack of an apical tuft, which normally forms from cells at the animal pole of the embryo. When lateral isolates were created at all developmental stages, these halves gastrulated. Cuts which separated presumptive anterior and posterior regions generated isolates at the 4-cell and blastula stages that formed essentially normal larvae; however, at the midgastrula stage these halves formed primarily anterior or posterior structures indicating that regional specification had taken place along the anterior-posterior axis. The plane of the first cleavage, which predicts the plane of bilateral symmetry, can be shifted by either changing the cleavage pattern that generates the bilateral 16-cell blastomere configuration or by isolating embryo halves prior to, or during, the 16-cell stage. These results indicate that while the plane of the first cleavage predicts the axis of bilateral symmetry, the axis is not established until the fourth cleavage. The development of Discinisca is compared to development in the inarticulate brachiopod Glottidia of the family Lingulidae and to Phoronis in the phylum Phoronida.     

The bases for and timing of regional specification during larval development in Phoronis

A fate map has been constructed for Phoronis vancouverensis. The animal pole of the egg gives rise to the apical plate in the hood of the actinotroch larva. The vegetal pole of the egg marks the site of gastrulation. During the initiation of gastrulation the cells of the animal pole of the embryo are directly opposite those at the vegetal pole of the embryo. The plane of the first cleavage always goes through the animal-vegetal pole of the egg. In about 70% of the cases the plane of the first cleavage is perpendicular to the future anterior-posterior axis of the actinotroch larva; in the remaining cases the plane of the first cleavage is either oblique with reference to, or occurs along, the future anterior-posterior axis of the larva. Following gastrulation catecholamine-containing cells first make their appearance in the apical plate and gut cells first produce esterase. The timing of regional specification in these embryos has been examined by isolating animal or vegetal, anterior or posterior, or lateral regions at different time periods between the initiation of cleavage and gastrulation and examining their ability to differentiate. Animal halves isolated from early cleavage through late blastula stages do not gastrulate and do not form catecholamine-containing cells. When animal halves are isolated with endoderm during gastrulation, they differentiate catecholamine-containing cells. Vegetal halves isolated at the 8- to 16-cell stage gastrulate and form normal actinotroch larvae with esterase-positive gut and catecholamine-containing apical plate cells. When this same region is isolated at blastula stages it does not gastrulate and does not differentiate these cell types. Vegetal halves isolated during gastrulation subsequently form esterase-positive gut cells, but they do not form catecholamine-containing apical plate cells. When presumptive anterior, posterior, or lateral halves are isolated from early cleavage through blastula stages, each half forms a normal actinotroch larva. Lateral halves isolated during gastrulation also form normal larvae. Anterior halves isolated during late gastrulation differentiate only the anterior end of the actinotroch larva. These isolates have a hood with catecholamine-containing apical plate cells and the first part of an esterase-positive gut but lack the anlagen of the intestine and protonephridia. Posterior halves isolated during late gastrulation differentiate only the posterior end of the actinotroch which lacks a hood with catecholamine-containing cells but has an esterase-positive gut, protonephridia, and the anlagen of the intestine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Patterns of junctional communication during development of the early amphibian embryo

Cell-cell communication through gap junctions was examined in Xenopus laevis embryos between the 16-cell and early blastula stages using Lucifer Yellow, Fluorescein, lead EDTA and dicyanoargentate as probes of junctional permeability. Injections were made into cells whose position was identified with respect to the primary cleavage axis and the grey crescent. FITC dextrans revealed cytoplasmic bridges between the injected cell and its sister only. In the animal pole at the 16-cell stage at the future dorsal side of the embryo, Lucifer Yellow was frequently and extensively transferred between cells through gap junctions. At the future ventral side gap junctional transfer of Lucifer Yellow was significantly less frequent and less extensive. The asymmetry of transfer between future dorsal and ventral sides of the animal pole was more marked at the 32-cell stage. In the vegetal pole also at the 32-cell stage, a dorsoventral difference in junctional permeability to Lucifer Yellow was observed. At the 64-cell stage the transfer of Lucifer Yellow was relatively frequent between cells lying in the same radial segment in the animal pole; transfer into cells outside each segment was infrequent, except at the grey crescent. At the 128-cell stage, Lucifer transfer between future dorsal or future ventral cells in the equatorial region was infrequent. A high incidence of transfer was restored at the future dorsal side at the 256-cell stage. At the 32-cell stage, fluorescein was infrequently transferred between animal pole cells although lead EDTA moved from cell to cell with high, comparable frequency in future dorsal and ventral regions. Dicyanoargentate always transferred extensively, both at the 32- and 64-cell stages. Treatment of embryos with methylamine raised intracellular pH by 0.15 units, increased the electrical conductance of the gap junction and produced a 10-fold increase in the frequency of Lucifer Yellow transfer through gap junctions in future ventral regions of the animal pole at the 32-cell stage.     

A study of cell interactions involved in Pleurodeles waltlii epidermal differentiation

Summary A polyclonal antibody (SP-2) has been produced, which recognizes antigens expressed in epidermal cells of Pleurodeles waltlii embryos. The antigens appear first at the end of gastrulation in the external surface of the embryo and are selectively expressed in ectodermally derived epidermal structures. Ectodermal commitment was investigated using cell cultures and blastocoel graft experiments. The four animal blastomeres of the 8-cell stage as well as the animal cap explants of the early gastrula stage cultured in vitro differentiate into epidermis, and SP-2 antigens are expressed. The expression of SP-2-defined antigens is inhibited both in vivo and in vitro by the inductive interaction of chordomesoderm. Once dissociated, ectodermal cells do not react with SP-2. Conversely, the aggregation of ectodermal cells may restore the expression of SP-2 antigens. Transplantation of animal cap explants or isolated ectodermal cells into the blastocoel of a host embryo at the early gastrula stage shows that only cells integrated into the epidermis express the marker antigens. When vegetal cells were dissociated from donor embryos before the mid-blastula stage and implanted into the blastocoel of host embryos at the early gastrula stage, their progeny were found in all germ layers, cells that were found in the host epidermis were stained with SP-2, whereas those contributing to mesoderm and endoderm were not. Thus the acquisition of cell polarity in epidermal differentiation and the organization of cells into epithelial structures are essential for SP-2-defined antigen expression.     

Fate map for the 32-cell stage of Xenopus laevis

A complete fate map has been produced for the 32-cell stage of Xenopus laevis. Embryos with a regular cleavage pattern were selected and individual blastomeres were injected with the lineage label fluorescein-dextran-amine (FDA). The spatial location of the clones was deduced from three-dimensional (3D) reconstructions of later stages and the volume of each tissue colonized by labelled cells in each tissue was measured. The results from 107 cases were pooled to give a fate map which shows the fate of each blastomere in terms of tissue types, the composition of each tissue by blastomere, the location of each prospective region on the embryo and the fate of each blastomere in terms of spatial localization. Morphogenetic movements up to stage 10 (early gastrula) were assessed by carrying out a number of orthotopic grafts at blastula and gastrula stages using donor embryos uniformly labelled with FDA. Although there is a regular topographic projection from the 32-cell stage this varies a little between individuals because of variability of positions of cleavage planes and because of short-range cell mixing during gastrulation. The cell mixing means that the topographic projection fails for anteroposterior segments of the dorsal axial structures and it is not possible to include short segments of notochord or neural tube or individual somites on the pregastrulation fate map.     

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.     

Nuclear localization of beta-catenin in vegetal pole cells during early embryogenesis of the starfish Asterina pectinifera

Recently, beta-catenin has been reported to control the expression of morphogenetic genes through the Wnt signaling pathway in invertebrate embryogenesis. In this study, the distribution pattern of beta-catenin during starfish embryogenesis was investigated using immunohistochemistry. In 16-cell stage embryos, beta-catenin began to accumulate in some nuclei at the vegetal pole. During the early cleavage stage, the cells expressing nuclear beta-catenin increased in number in the vegetal pole region of the embryos, and the beta-catenin signal increased in intensity in each nucleus. At the blastula stage, signal for beta-catenin was also found in the cytoplasm of the cells with nuclear beta-catenin. At the vegetal plate stage, almost all vegetal plate cells expressed beta-catenin in both the nucleus and cytoplasm. When the embryos developed to early gastrulae, cells with nuclear beta-catenin were restricted to the archenteron tip, and the signal gradually faded in later stages. The localization and temporal change of beta-catenin expression suggests that beta-catenin has a pivotal role in archenteron formation in starfish embryos.     

Determinants of Early Amphibian Development

The animal-vegetal organization of the amphibian egg may originatefrom the axis of organelles and cytoskeletal elements establishedin the oocyte as it divides from the oogonium. Along this axis,cytoplasmic materials are localized during oogenesis: yolk platelets,for example, are translocated toward the vegetal pole, increasingtheir amount and size in that region. In the first cell cycleafter fertilization, the egg cortex rotates 30° relativeto the cytoplasmic core, modifying animal-vegetal organization.The direction of this rotation, biased by the point of spermentry, defines the site of development of anatomical structuresof the dorsal midline of the embryo. As its immediate effect,rotation activates the cytoplasm of a subregion of the vegetalhemisphere, causing cells cleaved from this subregion to bemore effective than other vegetal parts in inducing marginalzone cells to initiate gastrulation movements. The most stronglyinduced part of the marginal zone begins gastrulation first(the dorsal lip of the blastopore) and proceeds through a seriesof cell interactions leading to its determination as the anteriordorsal mesoderm of the embryo. If these cell movements are inhibitedin the gastrula stage, or if vegetal induction is inhibitedin the blastula stage, or if cortical rotation is inhibitedin the first cell cycle after fertilization, the embryo alwaysfails to develop dorsal structures of the anterior end of itsbody axis; the more inhibition, the more posterior is the levelof truncation, until a radial ventralized embryo develops, derivedfrom the animal-vegetal organization of the oocyte.     

Cleavage pattern,gastrulation, and neurulation in the appendicularian, <Emphasis Type="Italic">Oikopleura dioica</Emphasis>

The appendicularian, Oikopleura dioica is a chordate. Its life cycle is extremely short—approximately 5 days—and its tadpole shape with a beating tail is retained throughout entire life. The tadpole hatches after 3 h of development at 20°C. Here, we describe the cleavage pattern and morphogenetic cell movements during gastrulation and neurulation. Cleavage showed an invariant pattern. It is basically bilateral but also shows various minor left–right asymmetries starting from the four-cell stage. We observed two rounds of unequal cleavage of the posterior-vegetal B-line cells at the posterior pole. The nature of the unequal cleavages is reminiscent of those in ascidian embryos and suggests the presence of a centrosome-attracting body, a special subcellular structure at the posterior pole. The representation of the cell division pattern in this report will aid the identification of each cell, a prerequisite for clarifying the gene expression patterns in early embryos. Gastrulation started as early as the 32-cell stage and progressed in three phases. By the end of the second phase at the 64-cell stage, every vegetal cell had ingressed into the embryo, and animal cells had covered the entire embryo by epiboly. There was no archenteron formation. In the anterior region, eight A-line cells were aligned as a 2 × 4 array along the anterior–posterior axis and become internalized during the 64-cell stage. This process was considered to correspond to neurulation. The simple and accelerated development of Oikopleura, nevertheless giving rise to a conserved chordate body plan, is advantageous for studying developmental mechanisms using molecular and genetic approaches and makes this animal the simplest model organism in the phylum Chordata. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The cnidarian and the canon: the role of Wnt/beta-catenin signaling in the evolution of metazoan embryos

In a recent publication, Wikramanayake and colleagues have implicated the canonical Wnt/beta-catenin signaling pathway as a mediator of axial polarity and germ-layer specification in embryos of the cnidarian Nematostella. In this anthozoan, beta-catenin is localized in nuclei of blastomeres in one region of the 16- to 32-cell embryo whose descendants subsequently form the entoderm of the embryo. They claim that the pattern of nuclear localization is significant for two reasons: (1) when nuclear localization of beta-catenin was inhibited, gastrulation does not occur, and (2) when localization of beta-catenin took place in all cells of the pregastrula embryo, the number of entodermal cells increases. Since the Wnt/beta-catenin signaling pathway also plays a role in establishing axial polarity and specifying endoderm and mesoderm in a number of bilaterians, Wikramanayake et al. imply that this developmental mechanism is an evolutionary inheritance from a radially symmetrical ancestor. Some of the gaps in the current evidence, which must be filled to evaluate their interpretation, are discussed.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development

Recent studies suggest early (preimplantation) events might be important in the development of polarity in mammalian embryos. We report here lineage tracing experiments with green fluorescent protein showing that cells located either near to or opposite the polar body at the 8-cell stage of the mouse embryo retain their same relative positions in the blastocyst. Thus they come to lie on either end of an axis of symmetry of the blastocyst that has recently been shown to correlate with the anterior-posterior axis of the postimplantation embryo (see R. J. Weber, R. A. Pedersen, F. Wianny, M. J. Evans and M. Zernicka-Goetz (1999). Development 126, 5591-5598). The embryonic axes of the mouse can therefore be related to the position of the polar body at the 8-cell stage, and by implication, to the animal-vegetal axis of the zygote. However, we also show that chimeric embryos constructed from 2-cell stage blastomeres from which the animal or the vegetal poles have been removed can develop into normal blastocysts and become fertile adult mice. This is also true of chimeras composed of animal or vegetal pole cells derived through normal cleavage to the 8-cell stage. We discuss that although polarity of the postimplantation embryo can be traced back to the 8-cell stage and in turn to the organisation of the egg, it is not absolutely fixed by this time.     

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.     

Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus

We examined the timing and mechanisms of mesodermal and neural determination in Cynops , using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops .     

Asymmetric Segregation and Polarized Redistribution of Pole Plasm During Early Cleavages in the Tubifex Embryo: Role of Actin Networks and Mitotic Apparatus

In the precleavage zygote of Tubifex , pole plasm, which is yolk-free cytoplasm, is located at the animal and vegetal poles. The present study describes the fate and localization pattern of the pole plasms in embryonic development of Tubifex . The process of pole plasm localization during cleavage stages is comprised of three steps. The first step is asymmetric segregation which results in bipolar localization of pole plasm masses in the D-cell of the 4-cell embryo. The spatial organization of pole plasm at this stage depends on F-actin but not on microtubules. The second step is the redistribution of the vegetal pole plasm toward the animal pole and its unification with the animal pole plasm. These give rise to localization of unified pole plasm at the animal side (i.e. future dorsal side of the embryo) of the D-quadrant. The polarized redistribution is sensitive to colchicine and topographically related to the mitotic apparatus located at the animal pole of the D-cell. Electron microscopy shows the association with astral microtubules of constituents of pole plasm, suggesting the involvement of astral microtubules in cytoplasmic movement which gives rise to redistribution. In addition, centrifuge experiments suggest that the directional information for this polarized redistribution may be provided by some cytoplasmic organizations which are resistant to centrifugal force. The last step of the localization process is partitioning of unified pole plasm into two micromeres 2d and 4d. The spatial organization of pole plasm at this stage depends on microtubules but not on F-actin.     

Differential Protein Distribution Related to Dorsoventral Polarity in Pleurodeles waltl Cleaving Egg

The molecular basis for the initial specification of dorsoventral polarity in the Amphibian egg prior to the mid-blastula transition still remains an open question. Regional differences in the protein pattern of Pleurodeles egg were investigated during early cleavage (8- and 512-cell stages, prior to the mid-blastula transition). Animal-dorsal, animal-ventral, vegetal-dorsal and vegetal-ventral quarters were separated and proteins were analyzed by 2D-electrophoresis. The comparison of acidic protein patterns from dorsal and ventral quarters revealed differences between vegetal cells but no difference was detected between animal cells. One protein (p11, 30 kDa) was characterized in the dorsal side as early as the 8-cell st. and two dorsal spots were detected at the 512-cell st. (p11 and p5, 65 kDa). Similarly one protein (p7b, 46 kDa) appears to be ventral-specific from the 8-cell st. The p11 spot was shown to appear in ventral cells as a consequence of a dorsalizing LiCl-treatment at the 32-cell stage. Conversely, p11 disappeared from dorsal cells and p5 did not appear at 512-cell stage after UV-irradiation of the uncleaved egg, which results in the expression of the ventral-specific protein p7b in the dorsal part of the egg. Therefore differential protein expression is already present at very early cleavage stages. Its significance needs further investigation.     

ANALYSIS OF CELL PROLIFERATION DURING EARLY EMBRYOGENESIS

Cell proliferation was examined during early embryogenesis of the newt ( Triturus pyrrhogaster ) by various methods. After the two-cell stage, at 23°C, the blastomere (cell) number per whole embryo increased logarithmically until the mid-blastula stage (for about 19 hr) and the rate of increase slowed down in and after the late blastula stage. On the other hand, the synchronous cleavage of the blastomeres at the animal pole continued for 18 hr until the twelfth cleavage (mid-blastula) and the transition from synchronous to asynchronous division occurred abruptly at and after the thirteenth cell division (late blastula). The study also showed that the presumptive neuro-ectoderm consisted mainly of cells of the fifteenth generation (G-15) at the onset of gastrulation (pigment stage).
The present study suggested that the number of ectodermal cells of the early gastrula (stage 12a) nearly doubled during gastrulation at the presumptive neuro-ectoderm. This means that most of the ectodermal cells are in G-16 at the end of gastrulation. On the other hand, both mitotic activity and the rate of cell increase gradually diminished during gastrulation in the ectoderms of both the presumptive neural and epidermal regions, and there are evidently significant differences in both activities between the neuro-ectoderm and the epidermal ectoderm after stage 13b: the epidermal ectoderm showed greater decrease in the rate of both mitotic activity and cell proliferation than the neuro-ectoderm.
These facts suggested that, whether the ectodermal cells will differentiate into neural cells or epidermal cells is determined during G-15 or G-16 in normal primary induction.     

The oral-aboral axis of a sea urchin embryo is specified by first cleavage

Several lines of evidence suggest that the oral-aboral axis in Strongylocentrotus purpuratus embryos is specified at or before the 8-cell stage. Were the oral-aboral axis specified independently of the first cleavage plane, then a random association of this plane with the blastomeres of the four embryo quadrants in the oral-aboral plane (viz. oral, aboral, right and left) would be expected. Lineage tracer dye injection into one blastomere at the 2-cell stage and observation of the resultant labeling patterns demonstrates instead a strongly nonrandom association. In at least ninety percent of cases, the progeny of the aboral blastomeres are associated with those of the left lateral blastomeres and the progeny of the oral blastomeres with the right lateral ones, respectively. Thus, ninety percent of the time the oral pole of the future oral-aboral axis lies 45 degrees clockwise from the first cleavage plane as viewed from the animal pole. The nonrandom association of blastomeres after labeling of the 2-cell stage implies that there is a mechanistic relation between axis specification and the positioning of the first cleavage plane.     

Experimental reversal of the normal dorsal-ventral timing of blastopore formation does not reverse axis polarity in Xenopus laevis embryos

During gastrulation in Xenopus laevis, the dorsal lip of the blastopore normally appears before the ventral lip. Metabolic gradient models propose that the dorsal lip develops from the region of highest metabolic activity and somehow dominates other regions to prevent them from becoming dorsal. To test these ideas, I applied a temperature gradient of 12 degrees C across the embryo. Localized heating of the prospective ventral vegetal region from early in the first cleavage period until gastrulation causes the blastopore lip to form first by 2 hr at the prospective ventral meridian rather than at the prospective dorsal meridian. Despite this reversal of the timing of blastopore formation, gastrulation is completed, and the neural plate forms at its usual position on the prospective dorsal meridian. This demonstrates that the earliest gastrulating regions of the blastopore do not necessarily become dorsal, nor do they inhibit dorsal development by other regions. It is unlikely that axis polarity is based on regional differences in energy metabolism.