Analysis of ciliary band formation in the mollusc Ilyanassa obsoleta

Two primary ciliary bands, the prototroch and metatroch, are required for locomotion and in the feeding larvae of many spiralians. The metatroch has been reported to have different cellular origins in the molluscs Crepidula fornicata and Ilyanassa obsoleta, as well as in the annelid Polygordius lacteus, consistent with multiple independent origins of the spiralian metatroch. Here, we describe in further detail the cell lineage of the ciliary bands in the gastropod mollusc I. obsoleta using intracellular lineage tracing and the expression of an acetylated tubulin antigen that serves as a marker for ciliated cells. We find that the I. obsoleta metatroch is formed primarily by third quartet derivatives as well as a small number of second quartet derivatives. These results differ from the described metatrochal lineage in the mollusc C. fornicata that derives solely from the second quartet or the metatrochal lineage in the annelid P. lacteus that derives solely from the third quartet. The present study adds to a growing body of literature concerning the evolution of the metatroch and the plasticity of cell fates in homologous micromeres in spiralian embryos.     

MAPK activation and the specification of the D quadrant in the gastropod mollusc, Crepidula fornicata

Embryos of the gastropod snail Crepidula fornicata exhibit a typical spiral cleavage pattern. Although a small polar lobe is formed at the first and second cleavage divisions, the embryo of C. fornicata exhibits a mode of development similar to that of equal-cleaving spiralians in which the D quadrant is conditionally specified by inductive interactions involving the derivatives of the first quartet micromeres. This study demonstrates that mitogen activated protein kinases, MAPK, are initially activated in the progeny of the first quartet micromeres, just prior to the birth of the third quartet (e.g., late during the 16-cell and subsequently during the 20-cell stages). Afterwards, MAPK is activated in 3D just prior to the 24-cell stage, transiently in 4d and finally in a subset of animal micromeres immediately following those stages. This pattern of MAPK activation differs from that reported for other spiralians. Using an inhibitor of MAPK kinase (MEK), we demonstrated that activated MAPK is required for the specification of the 3D macromere, during the late 16-cell through early 24-cell stages. This corresponds to the interval when the progeny of the first quartet micromeres specify the D quadrant macromere. Activated MAPK is not required in 3D later during the 24-cell stage or in the embryonic organizer, 4d, for its normal activity. Likewise, activated MAPK is not required in the animal micromeres during subsequent stages of development. Additional experiments suggest that the polar lobe, though not required for normal development, may play a role in restricting the activation of MAPK and biasing the specification of the 3D macromere.     

Cell specification and the role of the polar lobe in the gastropod mollusc Crepidula fornicata

A small polar lobe forms at the first and second cleavage divisions in the gastropod mollusc Crepidula fornicata. These lobes normally fuse with the blastomeres that give rise to the D quadrant at the two- and four-cell stages (cells ultimately generating the 4d mesentoblast and D quadrant organizer). Significantly, removal of the small polar lobe had no noticeable effect on subsequent development of the veliger larva. The behavior of the polar lobe and characteristic early cell shape changes involving protrusion of the 3D macromere at the 24-cell suggest that the D quadrant is specified prior to the sixth cleavage division. On the other hand, blastomere deletion experiments indicate that the D quadrant is not determined until the time of formation of the 4d blastomere (mesentoblast). In fact, embryos can undergo regulation to form normal-appearing larvae if the prospective D blastomere or 3D macromere is removed. Removal of the 4d mesentoblast leads to highly disorganized, radial development. Removal of the first quartet micromeres at the 8-cell stage also leads to the development of radialized larvae. These findings indicate that the embryos of C. fornicata follow the mode of development exhibited by equal-cleaving spiralians, which involves conditional specification of the D quadrant organizer via inductive interactions, presumably from the first quartet micromeres.     

Fate maps of the first quartet micromeres in the gastropod Ilyanassa obsoleta.

Cell fate specification in the gastropod mollusc Ilyanassa obsoleta involves both cell autonomous and inductive mechanisms, which depend on determinants localized first in the polar lobe and then in the D quadrant of the embryo. A complete cell lineage is lacking for this embryo and is essential for a critical interpretation of previous experimental results and an analysis of the mechanisms at the molecular level. Lineages of the first quartet micromeres were followed using Lucifer Yellow dextran as a tracer. The tracer was injected into individual first quartet micromeres using iontophoresis and patterns of fluorescence were analyzed in the larva after 8 days of development. Fluorescence was limited to head structures, including eyes, tentacles and velum. Structures on the left side were derived from 1a and 1d micromeres; 1a gave rise to the left eye, including the lens. Right side structures were derived from the 1c micromere and 1b contributed to the apical plate between the eyes and symmetrically to both sides of the velum. First quartet lineage data are compared with results from previous cell ablation experiments and with lineage data from other species.     

The morphogenetic significance of the first quartet micromeres for the development of the snailBithynia tentaculata

Summary The significance of the first quartet of micromeres for the morphogenesis ofBithynia — a polar lobe-forming gastropod-has been studied by deletion experiments. After removal of the whole first quartet at the 8-cell stage a dorsoventrally organized veliger larva is formed. Apparently, an interaction between the animal micromeres and a vegetal macromere, which is essential for the origin of a dorsoventral organization in equally cleaving gastropods, is not required in polar lobe forming eggs. It is concluded that in these eggs dorsoventrality is determined by segregation of the polar lobe. The embryos, in which the first quartet has been removed, never develop head structures. This indicates that the capacity to form head structures is restricted to the first quartet of micromeres. Deletion of a specific first quartet micromere (1a, 1b, 1c or 1d) showed, however, that the individual cells of this quartet are not strictly determined right from their origin. Frequently regulative development was observed after removal of individual first quartet cells.     

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.     

Conserved mechanism of dorsoventral axis determination in equal-cleaving spiralians

Many members of the spiralian phyla (i.e., annelids, echiurans, vestimentiferans, molluscs, sipunculids, nemerteans, polyclad turbellarians, gnathostomulids, mesozoans) exhibit early, equal cleavage divisions. In the case of the equal-cleaving molluscs, animal-vegetal inductive interactions between the derivatives of the first quartet micromeres and the vegetal macromeres specify which macromere becomes the 3D cell during the interval between fifth and sixth cleavage. The 3D macromere serves as a dorsal organizer and gives rise to the 4d mesentoblast. Even though it has been argued that this situation represents the ancestral condition among the Spiralia, these inductive events have only been documented in equal-cleaving molluscs. Embryos of the nemertean Cerebratulus lacteus also undergo equal, spiral cleavage, and the fate map of these embryos is similar to that of other spiralians. The role of animal first quartet micromeres in the establishment of the dorsal (D) cell quadrant was examined in C. lacteus by removing specific combinations of micromeres at the eight-cell stage. To follow the development of various cell quadrants, one quadrant was labeled with DiI at the four-cell stage, and specific first quartet micromeres were removed from discrete positions relative to the location of the labeled quadrant. The results indicate that the first quartet is required for normal development, as removal of all four micromeres prevented dorsoventral axis formation. In most cases, when either one or two adjacent first quartet micromeres were removed from one side of the embryo, the cell quadrant on the opposite side, with its macromere centered under the greatest number of the remaining animal micromeres, ultimately became the D quadrant. Twins containing duplicated dorsoventral axes were generated by removal of two opposing first quartet micromeres. Thus, any cell quadrant can become the D quadrant, and the dorsoventral axis is established after the eight-cell stage. While it is not yet clear exactly when key inductive interactions take place that establish the D quadrant in C. lacteus, contacts between the progeny of animal micromeres and vegetal macromeres are established during the interval between the fifth and sixth round of cleavage divisions (i.e., 32- to 64-cell stages). These findings argue that this mechanism of cell and axis determination has been conserved among equal-cleaving spiralians.     

The embryonic value of the micromeres in Ilyanassa obsoleta,as determined by deletion experiments. III. The third quartet cells and the mesentoblast cell, 4d

Summary

Each of the third quartet micromeres and the mesentoblast of the fourth quartet was removed and the effects on larval development analyzed. Removal of 3a resulted in a reduction in size of the left velar lobe. Removal of 3b resulted in a moderate reduction in size of the right velar lobe. Removal of 3c resulted in the absence of the right half of the foot and usually the right statocyst. Removal of 3d resulted in the absence of the left half of the foot and the left statocyst. Removal of both 3c and 3d resulted in the absence of the foot in most cases. Removal of the mesentoblast, 4d, resulted in the absence of the intestine, heart and larval kidney and various deficiencies of the midgut. On the basis of deletion experiments, each third quartet micromere and the mesentoblast is judged to have a specific embryonic value. The generally good development of the main ectodermal derivatives of the body following removal of the mesentoblast do not suggest any role for it or its derivatives as the primary organizer of the body axis and form.     

Progress in nemertean biology: development and phylogeny

This paper reviews progress in developmental biology and phylogenyof the Nemertea, a common but poorly studied spiralian taxonof considerable ecological and evolutionary significance. Analysesof reproductive biology (including calcium dynamics during fertilizationand oocyte maturation), larval morphology and development anddevelopmental genetics have significantly extended our knowledgeof spiralian developmental biology. Developmental genetics studieshave in addition provided characters useful for reconstructingmetazoan phylogeny. Reinvestigation of the cell lineage of Cerebratuluslacteus using fluorescent tracers revealed that endomesodermforms from the 4d cell as in other spiralians and that ectomesodermis derived from the 3a and 3b cells as in annelids, echiuransand molluscs. Studies examining blastomere specification showthat cell fates are established precociously in direct developersand later in indirect developers. Morphological characters usedto estimate the phylogenetic position of nemerteans are criticallyre-evaluated, and cladistic analyses of morphology reveal thatconflicting hypotheses of nemertean relationships result becauseof different provisional homology statements. Analyses thatinclude disputed homology statements (1, gliointerstitial cellsystem 2, coelomic circulatory system) suggest that nemerteansform the sister taxon to the coelomate spiralian taxa ratherthan the sister taxon to Platyhelminthes. Analyses of smallsubunit rRNA (18S rDNA) sequences alone or in combination withmorphological characters support the inclusion of the nemerteansin a spiralian coelomate clade nested within a more inclusivelophotrochozoan clade. Ongoing evaluation of nemertean relationshipswith mitochondrial gene rearrangements and other molecular charactersis discussed.     

What studies of turbellarian embryos can tell us about the evolution of development mechanisms

In spiralian embryos determination of the axes of bilateral symmetry is associated with D quadrant specification. This can occur late through equal cleavage and cell interactions (conditional specification) or by the four-cell stage through unequal cleavage and cytoplasmic localization (autonomous specification). Freeman & Lundelius (1992) suggest that in spiralian coelomates the former method is ancestral and the latter derived, with evolutionary pressure to shorten metamorphosis resulting in early D quadrant determination through unequal cleavage and appearance of adult features in the larvae. Because of the key phylogenetic position of the turbellarian platyhelminthes, understanding the method of axis specification in this group is important in evaluating the hypothesis. Polyclad development, with equal quartet spiral cleavage, is believed to represent the most primitive condition among living turbellarians and has been examined experimentally in Hoploplana inquilina. Blastomere deletions at the two and four-cell stage produce larvae that are abnormal in morphology and symmetry, indicating that early development is not regulative, and also establish that the embryo does not have an invariant cell lineage. Deletions of micromeres and macromeres at the eight-cell stage indicate that cell interactions are involved in dorso-ventral axis determination, with cross-furrow macromeres playing a more significant role than non-cross-furrow cells. The results support the idea that conditional specification is the primitive developmental mode that characterized the common ancestor of the turbellarians and spiralian coelomates. Evolutionary trends in development in polyclads and other turbellarian orders are discussed.     

The role of animal-vegetal interaction with respect to the determination of dorsoventral polarity in the equal-cleaving spiralian,Lymnaea palustris

Summary Spirally cleaving embryos in which the first two cleavages generate four equal-sized blastomeres remain radially symmetrical along their animal-vegetal axis until the interval between third and fourth quartet formation. At this time animal micromeres and vegetal macromeres contact each other as they elongate and occlude the central, fluid-filled cleavage cavity. The overlying micromeres focus their contacts onto one of the four macromeres, the presumptive 3D macromere, as it elongates to a central position within the embryo. We tested the hypothesis that this animal-vegetal interaction was causally involved in the determination of the symmetry properties in both the animal and vegetal hemispheres by reversibly inhibiting animal-vegetal contact at the 24 cell stage with cytochalasin-B. Embryos remained hollow throughout the treatment period and animal-vegetal interaction did not occur. After treatment, blastomere elongation occurred but no D quadrant macromere appeared and the vegetal hemisphere remained radialized. On the basis of cleavage and ciliation patterns of first quartet derivatives, treated embryos remained fully or partially radialized, showing a strong tendancy to develop as ventral quadrants. These results show that the quadrants of this equal-cleaving spiralian are not definitively determined until after the 24 cell stage and that animal-vegetal interaction is required for D quadrant determination. The mechanisms of symmetrization in the animal and vegetal hemispheres of equal-cleaving spiralians is also discussed.     

Quantitative analysis of cellular differentiation during early embryogenesis ofPlatynereis dumerilii

Summary As in many spiralian embryos with unequal cleavage, cleavage inPlatynereis follows an invariant pattern. Preceding each cleavage the cytoplasm is reorganized, allowing the spiral cleavage mode to produce cells with different cytoplasmic composition. The fertilized egg undergoes a dramatic ooplasmic segregation after the completion of the cortical reaction. As a consequence, a plug of clear cytoplasm becomes located at the animal pole. Once the four quadrants of the embryo have been established, the cleavage sequence of the D quadrant differs clearly from that of the other three quadrants. The results presented here suggest that differential distribution of the clear cytoplasm governs this sequence. The first quartet of micromeres, which will form the ectoderm and the cerebral ganglia of the head, is clearly bilaterally symmetrical from the onset of the third cleavage. Dorsoventral polarity and bilateral symmetry in the ectoderm of the trunk is expressed most markedly by the dorsal location of the large 2d cell, whose rapid proliferation is bilaterally symmetrical with respect to the median plane. As a result of this proliferation it comes to fill most of the posttrochal region (ectoderm, three pairs of anlagen for the setal sacs, and the ventral plate which forms the nerve cord). The other micromeres contribute only a minor portion of the ventral ectoderm and are involved in the formation of the stomodaeum. The mesentoblast, 4d, i.e. the stem cell of the primary mesoderm, forms at the sixth cleavage, also in a position on the dorsal mid-line. The daughter cells, which arise from 4d by strictly bilaterally symmetrical cleavage, form the mesodermal germ bands, which lie beneath the ectoderm. The trochoblasts are formed by asynchronously cleaving founder cells, but further cleavages in these cells are synchronous. This suggests that cell-cell interaction is involved in the development of this alleged mosaic embryo.     

Evolutionary modification of specification for the endomesoderm in the direct developing echinoid <Emphasis Type="Italic">Peronella japonica</Emphasis>: loss of the endomesoderm-inducing signal originating from micromeres

We investigated the inductive signals originating from the vegetal blastomeres of embryos of the sand dollar Peronella japonica, which is the only direct developing echinoid species that forms micromeres. To investigate the inductive signals, three different kinds of experimental embryos were produced: micromere-less embryos, in which all micromeres were removed at the 16-cell stage; chimeric embryos produced by an animal cap (eight mesomeres) recombined with a micromere quartet isolated from a 16-cell stage embryo; and chimeric embryos produced by an animal cap recombined with a macromere-derived layer, the veg1 or veg2 layer, isolated from a 64-cell stage embryo. Novel findings obtained from this study of the development of these embryos are as follows. Micromeres lack signals for endomesoderm specification, but are the origin of a signal establishing the oral–aboral (O–Ab) axis. Some non-micromere blastomeres, as well as micromeres, have the potential to form larval skeletons. Macromere descendants have endomesoderm-inducing potential. Based on these results, we propose the following scenario for the first step in the evolution of direct development in echinoids: micromeres lost the ability to send a signal endomesoderm induction so that the archenteron was formed autonomously by macromere descendants. The micromeres retained the ability to form larval spicules and to establish the O–Ab axis.     

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.     

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 MAPK cascade in equally cleaving spiralian embryos

Spiralian development is shared by several protostome phyla and characterized by regularities in early cleavage, fate map, and larva. Experimental evidence from multiple spiralian species implicates cells in the D quadrant lineage as the organizer of future axial development of the embryo. However, the mechanisms by which the D quadrant is specified differ between species with equal and unequal spiral cleavage. Equally cleaving mollusc embryos establish the D quadrant via cell-cell interactions between the micromeres and macromeres at the 24- to 36-cell stage. In unequally cleaving embryos, the D quadrant is established at the 4-cell stage via asymmetries in the first 2 cell divisions. We have begun to explore the molecular mechanisms of D quadrant patterning in spiralians. Previously, we showed that, in the unequally cleaving embryo of the mollusc Ilyanassa obsoleta, the MAPK pathway is activated and functionally required in 3D and also in the micromeres known to require a signal from 3D. Here, we examine the role of MAPK signaling in 4 spiralians with equal cleavage. In 3 equally cleaving molluscs, the chiton Chaetopleura, the limpet Tectura, and the snail Lymnaea, the MAPK pathway is activated in the 3D cell but not in the overlying micromeres. In the equally cleaving embryo of the polychaete annelid Hydroides, MAPK activation was not detected in the 3D macromere but was observed in one of its daughter cells, 4d. In addition, inhibiting Tectura MAPK activation disrupts differentiation of 3D and cells induced by it, supporting a functional role for MAPK in axis specification in equally cleaving spiralians. Thus, MAPK signaling may have a conserved role in the D quadrant organizer cell 3D in molluscs. However, there have been at least 2 evolutionary changes in the activation of the MAPK pathway during spiralian evolution. MAPK function in the Ilyanassa micromeres is a recent cooption and, since the divergence of annelids and molluscs, there has been a shift in onset of MAPK activation between 3D and 4d. We propose that this latter shift correlates with a change in the timing of specification of the secondary embryonic axis.     

Embryonic origins of cells in the leech Helobdella triserialis

To ascertain the embryonic origins of the cells in various tissues of the leech Helobdella triserialis, horseradish peroxidase (HRP) was injected as a cell lineage tracer into all identified blastomeres of the early embryo in turn, except for a few of the micromeres, and the resulting distribution of HRP-labeled cells was then examined in the late embryo. In this way it was found that in every body segment a topographically characteristic set of neurons in the ganglion and body wall and a characteristic territory of the epidermis is derived from each of the four paired ectodermal teloblasts N, O/P, O/P, and Q, whereas the muscles, nephridia, and connective tissue, as well as a few presumptive neurons in each segmental ganglion, are derived from the paired mesodermal teloblast, M. Each topographically characteristic, segmentally iterated set of neurons descended from a given teloblast is designated as a kinship group. However, the prostomial (nonsegmental) epidermis and the neurons of the supraesophageal ganglion were found to be derived from the a, b, c, and d micromere quartet to which the A, B, C, and D blastomeres give rise at the dorsal pole of the embryo. The superficial epithelium of the provisional integument, which covers the surface of the embryo midway through development and is sloughed off at the time of body closure, was found to be derived from the a, b, c, and d micromere quartet, as well as from other micromeres produced in the course of teloblast formation. The contractile fibers of the provisional integument were found to be derived from the paired M teloblast. These results demonstrate that development of the leech embryo proceeds according to a highly stereotyped pattern, in the sense that a particular identifiable blastomere of the early embryo regularly gives rise to a particular set of cells of the adult (or provisional embryonic) tissues.     

β-catenin and early development in the gastropod, Crepidula fornicata

This study describes the early expression and function of β-catenin in the gastropod, Crepidula fornicata. In other bilaterians β-catenin functions in cell adhesion, gastrulation, and cell signaling, which is related to the establishment of the dorso-ventral axis and mesendoderm. Here, we studied the distribution of β-catenin mRNA and protein in C. fornicata via whole mount in situ hybridization and by expressing GFP-tagged β-catenin in vivo. During early cleavage, β-catenin mRNA and protein appear to be broadly localized to all cells in the early embryo. The mRNA tends to be concentrated at inter-phase centrosomes in these cells. At later stages, the mRNA is predominantly in the vegetal macromeres, and subsequently in the rudiment of the hindgut, stomodeum, and velar lobes. Expression of full-length GFP-tagged protein suggests that there is no active mechanism to degrade β-catenin within cells of the early embryos prior to the 25-cell stage. However, by the second day of development, when the fourth quartet micromeres have formed, β-catenin becomes selectively stabilized in the progeny of the 4d mesentoblast (e.g., ML and MR and their daughters) and is missing from most other blastomeres, including vegetal macromeres. Over the next 2 days of development, during subsequent divisions of 4d, β-catenin protein becomes progressively degraded, along the proximo-distal axes, within the progeny of the paired mesendodermal bands. The cells located at the tips of the mesodermal bands (2?mL2 and 2?mR2) are the last to contain this protein, which is no longer detected after 4 days of development. In animals like C. fornicata, which undergo a spiral cleavage program (e.g., molluscs, annelids, nemerteans, and polyclad flatworms), the mesentoblast or 4d cell represents the progenitor of endomesoderm (forming hindgut, internal and external kidneys, and various muscles). Therefore, the selective stabilization of β-catenin in the progeny of 4d in C. fornicata is consistent with arguments that a basic, ancestral role of β-catenin lies in the formation of endomesodermal fates. Experiments using a truncated β-catenin clone show that the regions located in the C-terminus, distal to the 11th armadillo repeat, are required for normal stabilization/degradation of β-catenin protein within the embryo. Microinjection of translation blocking β-catenin morpholinos into zygotes led to the down-regulation of β-catenin expression. This resulted in the subsequent failure of gastrulation, but did not interfere with the formation and early cleavage of 4d, although there were no discernable differentiated cell fates in these defective embryos. These results are compared with those obtained in other metazoans.