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.     

The effects of altering the position of cleavage planes on the process of localization of developmental potential in ctenophores.

During the transition from the four- to the eight-cell stage in ctenophore embryos, each blastomere produces one daughter cell with the potential to form comb plate cilia and one daughter cell that does not have this potential. If the second cleavage in a two-cell embryo is blocked, at the next cleavage these embryos frequently form four blastomeres which have the configuration of the blastomeres in a normal eight-cell embryo. At this division there is also a segregation of comb plate-forming potential. By compressing a two-cell embryo in a plane perpendicular to the first plane of cleavage it is possible to produce a four-cell blastomere configuration that is identical to that produced following the inhibition of the second cleavage. However, under these circumstances the segregation of comb plate potential does not occur. These results suggest that the appropriate plane of cleavage must take place for a given cleavage cycle, in order for localizations of developmental potential to be properly positioned within blastomeres.     

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.     

Macromere cell fates during sea urchin development.

This paper examines the cell lineage relationships and cell fates in embryos of the sea urchin Strongylocentrotus purpuratus leading to the various cell types derived from the definitive vegetal plate territory or the veg2 tier of cells. These cell types are gut, pigment cells, basal cells and coelomic pouches. They are cell types that constitute embryonic structures through cellular migration or rearrangement unlike the relatively non-motile ectoderm cell types. For this analysis, we use previous knowledge of lineage to assign macromeres to one of four types: VOM, the oral macromere; VAM, the aboral macromere, right and left VLM, the lateral macromeres. Each of the four macromeres contributes progeny to all of the cell types that descend from the definitive vegetal plate. Thus in the gut each macromere contributes to the esophagus, stomach and intestine, and the stripe of labeled cells descendant from a macromere reflects the re-arrangement of cells that occurs during archenteron elongation. Pigment cell contributions exhibit no consistent pattern among the four macromeres, and are haphazardly distributed throughout the ectoderm. Gut and pigment cell contributions are thus radially symmetrical. In contrast, the VOM blastomere contributes to both of the coelomic pouches while the other three macromeres contribute to only one or the other pouch. The total of the macromere contribution amounts to 60% of the cells constituting the coelomic pouches.     

Inductive interactions and embryonic equivalence groups in a basal metazoan, the ctenophore Mnemiopsis leidyi

Ctenophores undergo locomotion via the metachronal beating of eight longitudinally arrayed rows of comb plate cilia. These cilia are normally derived from two embryonic lineages, which include both daughters of the four e1 micromeres (e11 and e12) and a single daughter of the four m1 micromeres (the m12 micromeres). Although the e1 lineage is established autonomously, the m1 lineage requires an inductive interaction from the e1 lineage to contribute to comb plate formation. Successive removal of the e1 progeny at later stages of development indicates that this interaction takes place after the 32-cell stage and likely proceeds over a prolonged period of development. Normally, the e1, cell lies in closest proximity to the m12 cell that generates comb plate cilia; however, either of the e1 daughters (e11 or e12) is capable of emitting the signal required for m1 descendants to form comb plates. Previous cell lineage analyses indicate that the two e1 daughters generate the same suite of cell fates. On the other hand, the m1 daughters (m11 and m12) normally give rise to different cell fates. Reciprocal m1 daughter deletions show that in the absence of one daughter, the other cell can generate all the cell types normally formed by the missing cell. Together, these findings demonstrate that the two m1 daughters (m11 and m12) represent an embryonic equivalence group or field and that differences in the fates of the two m1 daughters are normally controlled by cell-cell interactions. These combined properties of ctenophore development, including the utilization of deterministic cleavage divisions, inductive interactions, and the establishment of embryonic fields or equivalence groups, are remarkably similar to those present in the development of various bilaterian metazoans.     

SPECIES SPECIFIC PATTERN OF CILIOGENESIS IN DEVELOPING SEA URCHIN EMBRYOS

The events of cell division and ciliogenesis in individual blastomeres of developing embryos of the sea urchins Temnopleurus toreumaticus and Hemicentrotus pulcherrimus were followed with a Nomarski differential interference microscope. The number of cell divisions before initiation of ciliogenesis was determined with respect to species. In T. toreumaticus , ciliogenesis began about 4 hr after fertilization at 25°C. The sequence of ciliogenesis was as follows: cilia appeared first on smaller micromeres, followed in order by blastomeres derived from larger micromeres, those from mesomeres and finally those derived from macromeres. Blastomeres originating from mesomeres, macromeres, larger micromeres and smaller micromeres had completed the 8th, 9th, 7th and 5th divisions respectively, before they generated cilia.
In H. pulcherrimus , embryos started to form cilia about 9 hr after fertilization at 18°C. Cilia appeared first on blastomeres of mesomere origin and, then on those of macromere origin. Before initiation of ciliogenesis, descendants of mesomeres and macromeres completed 9 and 10 rounds of cell division. Descendants of larger micromeres and the majority of cells derived from smaller micromeres did not acquired cilia even when the embryo began to rotate within the fertilization membrane. At this stage, the former had completed the 6th division and the latter the 8th division. Cell counts of blastomeres per embryo at the blastula stage also supported this observation.     

Development of dorsoventral polarity and mesentoblast determination in Patella vulgata

In Patella vulgata the 32-cell stage represents a pause in the mitotic activity prior to the differentiation of the mesentoblast mother cell 3D. At the onset of this stage, the embryo is radially symmetrical. Nevertheless, the plane of bilateral symmetry is indicated as it passes through the macromeres forming the vegetal cross-furrow. From the early beginning of the 32-cell stage, all four macromeres intrude far into the interior and touch the centrally radiating cells of the first quartet of micromeres. The two cross-furrow forming macromeres (3B and 3D) intrude the farthest and come into contact with the greatest number of micromeres. Finally, the contacts are extended significantly and maintained with only one of these macromeres. From that moment, this cell can be called the macromere 3D and the dorsoventral axis is determined. The evolution of the internal cell contacts between the micromeres of the first quartet and the macromeres indicates an essential role of the former in the determination of one of the latter as the mesentoblast mother cell, and thus in the determination of dorsoventral polarity.     

F-Actin is a marker of dorsal induction in earlyPatella embryos

Summary The dorsal-ventral axis inPatella vulgata embryos is established at the 32-cell stage by an inductive interaction between the animal micromeres and one vegetal macromere. This vegetal macromere, once induced, is called the 3D macromere, and marks the future dorsal side of the embryo. We examined the pattern of filamentous (F) actin in such embryos using fluorescent phalloidin and found that this dorsal 3D macromere contains more F-actin than the remainder of the cells. In addition, only one of its two daughter cells, i.e. the 4D macromere, retains this higher density. In embryos in which the establishment of the dorsal-ventral axis has been experimentally inhibited via treatment with monensin, such differences in F-actin were not found. These results suggest that the appearance of an increased density of F-actin in the dorsal 3D and 4D macromeres of normal embryos requires the inductive interactions that establish the dorsal-ventral axis. We therefore conclude that F-actin is an early marker for dorsal induction in thePatella embryo.     

Multiple inductive signals are involved in the development of the ctenophore Mnemiopsis leidyi.

Ctenophores possess eight longitudinally arrayed rows of comb plate cilia. Previous intracellular cell lineage analysis has shown that these comb rows are derived from two embryonic lineages, both daughters of the four e(1) micromeres (e(11) and e(12)) and a single daughter of the four m(1) micromeres (the m(12) micromeres). Although isolated e(1) micromeres will spontaneously generate comb plates, cell deletion experiments have shown that no comb plates appear during embryogenesis following the removal of e(1) descendents. Thus, the m(1) lineage requires the inductive interaction of the e(1) lineage to contribute to comb plate formation. Here we show that, although m(12) cells are normally the only m(1) derivatives to contribute to comb plate formation, m(11) cells are capable of generating comb plates in the absence m(12) cells. The reason that m(11) cells do not normally make comb rows may be attributable either to their more remote location relative to critical signaling centers (e.g., e(1) descendants) or to inhibitory signals that may be provided by other nearby cells such as sister cells m(12). In addition, we show that the signals provided by the e(1) lineage are not sufficient for m(1)-derived comb plate formation. Signals provided by endomesodermal progeny of either the E or the M lineages (the 3E or 2M macromeres) are also required.     

Microinjection of lectins, hyaluronidase, and hyaluronate fragments interferes with cleavage delay and mesoderm induction in embryos of Patella vulgata

In embryos of Patella vulgata at the 32-cell stage, one of the four vegetally located macromeres makes contacts with overlying animal micromeres. As a result, this macromere (designated 3D) divides significantly later than the other macromeres and forms the mesodermal stem cell 4d. Shortly before and during this interaction two types of extracellular matrix are present: a basal lamina-like layer on the tips of the micromeres and a loose fibrillar meshwork in the blastocoel. In this paper we examine the role of the matrix in cleavage delay and mesoderm determination. The microinjection of extracellular matrix-binding lectins, or of hyaluronidase, or of decasaccharide fragments of hyaluronate into the blastocoel results in embryos in which either no or two macromeres are delayed in cleavage and are presumably determined as mesodermal stem cells. We suggest that the fibrillar meshwork is needed for macromere elongation toward the micromeres and that the basal lamina-like layer is involved in the determination process itself.     

Intracellular fate mapping in a basal metazoan, the ctenophore Mnemiopsis leidyi, reveals the origins of mesoderm and the existence of indeterminate cell lineages.

Ctenophores are marine invertebrates that develop rapidly and directly into juvenile adults. They are likely to be the simplest metazoans possessing definitive muscle cells and are possibly the sister group to the Bilateria. All ctenophore embryos display a highly stereotyped, phylum-specific pattern of development in which every cell can be identified by its lineage history. We generated a cell lineage fate map for Mnemiopsis leidyi by injecting fluorescent lineage tracers into individual blastomeres up through the 60-cell stage. The adult ctenophore body plan is composed of four nearly identical quadrants organized along the oral-aboral axis. Each of the four quadrants is derived largely from one cell of the four-cell-stage embryo. At the eight-cell stage each quadrant contains a single E ("end") and M ("middle") blastomere. Subsequently, micromeres are formed first at the aboral pole and later at the oral pole. The ctene rows, apical organ, and tentacle apparatus are complex structures that are generated by both E and M blastomere lineages from all four quadrants. All muscle cells are derived from micromeres born at the oral pole of endomesodermal precursors (2M and 3E macromeres). While the development of the four quadrants is similar, diagonally opposed quadrants share more similarities than adjacent quadrants. Adult ctenophores possess two diagonally opposed endodermal anal canals that open at the base of the apical organ. These two structures are derived from the two diagonally opposed 2M/ macromeres. The two opposing 2M/ macromeres generated a unique set of circumpharyngeal muscle cells, but do not contribute to the anal canals. No other lineages displayed such diagonal asymmetries. Clones from each blastomere yielded regular, but not completely invariant patterns of descendents. Ectodermal descendents normally, but not always, remained within their corresponding quadrants. On the other hand, endodermal and mesodermal progeny dispersed throughout the body. The variability in the exact complements of adult structures, along with previously published cell deletion experiments, demonstrates that cell interactions are required for normal cell fate determination. Ctenophore embryos, like those of many bilaterian phyla (e.g., spiralians, nematodes, and echinoids), display a highly stereotyped cleavage program in which some, but not all, blastomeres are determined at the time of their birth. The results suggest that mesodermal tissues originally evolved from endoderm tissue.     

Dorsoventral polarity and mesentoblast determination as concomitant results of cellular interactions in the mollusk Patella vulgata.

In mollusks with an equal four-cell stage, dorsoventral polarity becomes noticeable in the interval between the formation of the third and fourth quartet of micromeres, i.e., between the fifth and sixth cleavage. One of the two macromeres at the vegetal cross-furrow then partly withdraws from the surface and becomes located more toward the center of the embryonic cell mass than the other three macromeres. Only this specific macromere (3D) contacts the micromeres of the animal pole, divides with a delay, and develops into the stem cell of the mesentoblast (4d). After suppression of the normal contacts between micromeres and macromeres either by dissociation of the embryos or by deletion of first quartet cells, the normal differentiation of the macromeres fails to appear. By deleting a decreasing number of first quartet cells, an increasing percentage of embryos shows the normal differentiation pattern. Deletion of one of the cross-furrow macromeres does not preclude formation of the mesentoblast, which then originates by differentiation of an other macromere. It is concluded that initially the embryo is radially symmetrical and that the four quadrants have identical developmental capacities; mesentoblast differentiation from one macromere is induced through the contacts of the first quartet cells and that single macromere.     

Cellular distribution of RNA populations in 16-cell stage embryos of the sand dollar, Dendraster excentricus

RNA was extracted from pure preparations of micromeres and meso-plus macromeres isolated from 16-cell stage embryos of Dendraster excentricus. Molecular hybridization-competition experiments disclosed that the binding of 16-cell stage labeled RNA to denatured sperm DNA was competed equally well by micromere RNA, meso-plus macromere RNA, total 16-cell RNA and unfertilized egg RNA, indicating the egg-type populations were distributed almost equally in the different blastomeres. In contrast, experiments with ³H-RNA extracted from micromeres obtained from pulse-labeled 16-cell stage embryos showed qualitative differences when unfertilized egg RNA and total 16-cell stage RNA were used as competitors. Such differences in RNA populations could not be detected in ³H-RNA isolated from the meso-plus macromere fraction.     

Protistan epibionts of the ctenophore Mnemiopsis mccradyi Mayer

Mnemiopsis mccradyi, a common coastal ctenophore, was observed to bear two distinct, exclusive assemblages of protistan epibionts. The mobiline peritrich, Trichodina ctenophorii (Estes et al., 1997), and small Flabellula-like gymnamoebae inhabited only the surface of the comb plates. By contrast, small Vexillifera-like gymnamoebae and large Protoodinium-like dinoflagellates were found on the ectoderm. The relationship of the epimicrobial protists with their host varied from possible mutualism (vexilliferids) to commensalism (trichodinids) to parasitism (flabellulids and protoodinids). Trichodinids may benefit from comb plate attachment by enhanced food capture. Although they did not obviously impair comb plate beating, they did distort the surface and appear to produce fissures in the comb plate surface, which could provide inroads for more severe comb plate damage by amoebae. By contrast, when flabellulid amoebae occurred in very high surface densities (up to 5000 mm^–2), they clearly damaged comb plates by eroding the surface. Where flabellulid pseudopodia invaded the comb plate, we observed local degradation of comb plate cilia, as evidenced by central pair disorientation and plasma membrane perturbation and overt phagocytosis of comb plate cilia. Ectodermal vexilliferids, which occurred at much lower densities, did not appear to have any degradative impact on the ctenophore. By contrast, clusters of ectodermal protoodinids were found in localized depressions most likely caused by invasive phagocytosis. The impact of the protistan assemblages on ctenophore populations is unclear, but under conditions of severe infestation they might depress ctenophore population density.     

Fractionation of Micromeres,Mesomeres, and Macromeres of 16-cell Stage Sea Urchin Embryos by Elutriation*

A protocol was developed to fractionate micromeres, mesomeres and macromeres of 16-cell stage sea urchin embryos by elutriation. The purities of these fractions were 99%, 93%, and 90%, and their recoveries were 75%, 31%, and 42%, respectively. Using this method, several hundred milligrams of each blastomere type were obtainable from a single-pair mating. On culture, micromeres formed spicules in the presence of horse serum, mesomeres developed into ciliated ectodermal vesicles and macromeres formed gastrula-like or exogastrula-like embryoids with spicules. To analyze the different structures characteristic of the blastomere lineage, we examined the expressions of marker genes. Cells of the micromere lineage expressed the primary mesenchyme-specific SM50 gene exclusively, those of the mesomere lineage expressed the ectoderm-specific arylsulfatase gene strongly, and SM50 and the endoderm-specific Endo 16 genes weakly, whereas those of the macromere lineage expressed all three marker genes. These results indicate that blastomeres fractionated by elutriation were equivalent to those isolated by hand under a microscope with respect to development and gene expression.     

A morphometric comparison of dissimilar early development in sibling species of Platynereis (Annelida,Polychaeta)

Summary Early development of Platynereis massiliensis was studied in serial sections of fixed embryos and in living or fixed embryos whose nuclei had been made visible with a fluorescent label. The unfertilized egg is an ellipsoid with three axes of differing length. The longest axis corresponds to the dorsoventral axis of the developing embryo. Egg volume is ten times that in the sibling species, P. dumerilii, mainly due to increased yolk content. The timing and spatial pattern of cleavage were observed from first cleavage to the 62-cell stage. Volumes of the blastomeres, their nuclei, their yolk-free cytoplasm and their yolk were determined from serial sections up to the 29-cell stage. In the P. massiliensis embryo, cell cycles are on average 3.7 times longer than in P. dumerilii; volume proportions among the blastomeres also differ and the macromeres containing the bulk of yolk are particularly large, but otherwise the cleavage patterns, differential segregation of yolk and yolk-free cytoplasm, and the histogenetic fates of the blastomeres are the same as in P. dumerilii. This equivalence of cell lineage and of cytoplasmic segregation mechanisms in both species, maintained in spite of the different appearance of the embryos, suggests functional importance of and selective constraint on these developmental features. The relatively accelerated divisions of the 2d cell line in P. massiliensis may be interpreted as the precocious development of cell lines which give rise to adult structures. Several structures, obviously functional in developing P. dumerilii, have lost their function in P. massiliensis: the egg contains few cortical granules, giving rise to only a moderate egg jelly layer in the zygote; prototroch cells develop cilia, but the heavy embryo is unable to swim; the larva develops three pairs of parapodia but, unlike the corresponding stage in P. dumerilii, is not capable of coordinate locomotion. This loss of motility is related to the brooding habit of the species developing inside the parental tube and is explained as the result of a switch from pelagic to benthic, protected reproduction in P. massiliensis. Offprint requests to: A.W.C. Dorresteijn     

Specification of secondary mesenchyme-derived cells in relation to the dorso-ventral axis in sea urchin blastulae

To learn how the dorso-ventral (DV) axis of sea urchin embryos affects the specification processes of secondary mesenchyme cells (SMC), a fluorescent dye was injected into one of the macromeres of 16-cell stage embryos, and the number of each type of labeled SMC was examined at the prism stage. A large number of labeled pigment cells was observed in embryos in which the progeny of the labeled macromere were distributed in the dorsal part of the embryo. In contrast, labeled pigment cells were scarcely noticed when the descendants of the labeled macromere occupied the ventral part. In such embryos, free mesenchyme cells (probably blastocoelar cells) were predominantly labeled. CH3COONa treatment, which is known to increase the number of pigment cells, canceled such patterned specification of pigment cells and blastocoelar cells along the DV axis. Pigment cells were also derived from the ventral blastomere in the treated embryo. In contrast, a similar number of coelomic pouch cells was derived from the labeled macromere, irrespective of the position of its descendants along the DV axis. After examination of the arrangement of blastomeres in late cleavage stage embryos, it was determined that 17-20 veg2-derived cells encircled the cluster of micromere descendants after the 9th cleavage. From this number and the numbers of SMC-derived cells in later stage embryos, it was suggested that the most vegetally positioned veg2 descendants at approximately the 9th cleavage were preferentially specified to pigment and blastocoelar cell lineages. The obtained results also suggested the existence of undescribed types of SMC scattered in the blastocoele.     

Evolutionary implications of the mode of D quadrant specification in coelomates with spiral cleavage

In annelids, molluscs, echiurans and sipunculids the establishment of the dorsal-ventral axis of the embryo is associated with D quadrant specification during embryogenesis. This specification occurs in two ways in these phyla. One mechanism specifies the D quadrant via the shunting of a set of cytoplasmic determinants located at the vegetal pole of the egg to one blastomere of the four cell stage embryo. In this case, at the first two cleavages of embryogenesis there is an unequal distribution of cytoplasm, generating one macromere which is larger than the others at the four cell stage. The D quadrant can also be specified by a contact mediated inductive interaction between one of the macromeres at the vegetal pole with micromeres at the animal pole of the embryo. This mechanism operates at a later stage of development than the cytoplasmic localization mechanism and is associated with a pattern of cleavage in which the first two cleavages are equal. An analysis of the phylogenetic relationships within these phyla indicates that the taxa which determine the D quadrant at an early cleavage stage by cytoplasmic localization tend to be derived and lack a larval stage or have larvae with adult characters. Those taxa where the D quadrant is specified by induction include the ancestral groups although some derived groups also use this mechanism. The pulmonate mollusc Lymnaea uses an inductive mechanism for specifying the D quadrant. In these embryos each of the four vegetal macromeres has the potential of becoming the D macromere; however under normal circumstances one of the two vegetal crossfurrow macromeres almost invariably becomes the D quadrant. Experiments are described here in which the size of one of the blastomeres of the four cell stage Lymnaea embryo is increased; this macromere invariably becomes the D quadrant. These experiments suggest that developmental change in relative blastomere size during the first two cleavages in spiralian embryos that normally cleave equally may have provided a route that has led to the establishment of the cytoplasmic localization mechanism of D quadrant formation.     

The ‘organizing’ role of the D quadrant in an equal-cleaving spiralian,Lymnaea stagnalis as studied by UV laser deletion of macromeres at intervals between third and fourth quartet formation

Summary

In the spiralian embryos studied which display unequal-cleavage at the first two cleavages (either by a polar lobe or an asymmetric cleavage mechanism) the D quadrant is determined at the four cell stage by an unequal segregation of cytoplasmic stuffs. The normal formation of eyes, foot, and shell by overlying micromeres in these forms requires the inductive interaction with the D quadrant before the formation of the third quartet of micromeres. In equal-cleaving spiralians the D quadrant (3D macromere) becomes determined as a result of inductive interactions with first quartet derivatives (animal-vegetal interaction) sometime after the production of the third quartet of micromeres. This paper investigates the exact timing of D quadrant determination and the inductive role of third-order macromeres on the development of micromere derived structures in an equal-cleaving spiralian. Deletions of third-order macromeres, and their derivatives, were performed without rupturing the egg capsule membrane of the Lymnaea embryo with a UV laser microbeam. Virtually normal snails were produced when the 3A, 3B, 3C, or 4D macromere was irradiated. Juvenile snails lacking all mesodermal structures but possessing eyes, foot, and shell were obtained when the mesentoblast (4d) or its progenitor (3D) were deleted. Furthermore, ‘mesoderm-less’ snails were produced by deleting one of the two possible 3D candidates (cross furrow macromeres) as early as 20 min after third quartet formation. These results indicate that the 3D macromere begins to become determined at, or soon after, animal-vegetal interaction; before the 3D macromere becomes visibly distinguishable from the 3B macromere. The results also demonstrate that normal pattern formation in the overlying micromeres does not require the ‘prolonged’ interaction with an asymmetrically positioned 3D macromere. Possible adhesive differences between the 3D macromere and the remaining three macromeres are also revealed.