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.     

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.     

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.     

MAPK signaling by the D quadrant embryonic organizer of the mollusc Ilyanassa obsoleta

Classical experiments performed on the embryo of the mollusc Ilyanassa obsoleta demonstrate that the 3D macromere acts as an embryonic organizer, by signaling to other cells and inducing them to assume the correct pattern of cell fates. We have discovered that MAP kinase signaling is activated in the cells that require the signal from 3D for normal differentiation. Preventing specification of the D quadrant lineage by removing the polar lobe disrupts the pattern of MAPK activation, as does ablation of the 3D macromere itself. Blocking MAPK activation with the MAP Kinase inhibitor U0126 produces larvae that differentiate the same limited complement of tissues as D quadrant deletions. Our results suggest that the MAP Kinase signaling cascade transduces the inductive signal from 3D and specifies cell fate among the cells that receive the signal.     

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.     

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.     

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.     

The role of MAPK signaling in patterning and establishing axial symmetry in the gastropod Haliotis asinina

Gastropods are members of the Spiralia, a diverse group of invertebrates that share a common early developmental program, which includes spiral cleavage and a larval trochophore stage. The spiral cleavage program results in the division of the embryo into four quadrants. Specification of the dorsal (D) quadrant is intimately linked with body plan organization and in equally cleaving gastropods occurs when one of the vegetal macromeres makes contact with overlying micromeres and receives an inductive signal that activates a MAPK signaling cascade. Following the induction of the 3D macromere, the embryo begins to gastrulate and assumes a bilateral cleavage pattern. Here we inhibit MAPK activation in 3D with U0126 and examine its effect on the formation and patterning of the trochophore, using a suite of territory-specific markers. The head (pretrochal) region appears to maintain quadri-radial symmetry in U0126-treated embryos, supporting a role for MAPK signaling in 3D in establishing dorsoventral polarity in this region. Posterior (posttrochal) structures - larval musculature, shell and foot - fail to develop in MAPK inhibited trochophores. Inhibition of 3D specification by an alternative method - monensin treatment - yields similar abnormal trochophores. However, genes that are normally expressed in the ectodermal structures (shell and foot) are detected in U0126- and monensin-perturbed larvae in patterns that suggest that this region has latent dorsoventral polarity that is manifested even in the absence of D quadrant specification.     

Widespread RNA segregation in a spiralian embryo

Asymmetric cell divisions are a crucial mode of cell fate specification in multicellular organisms, but their relative contribution to early embryonic patterning varies among taxa. In the embryo of the mollusc Ilyanassa, most of the early cell divisions are overtly asymmetric. During Ilyanassa early cleavage, mRNAs for several conserved developmental patterning genes localize to interphase centrosomes, and then during division they move to a portion of the cortex that will be inherited by one daughter cell. Here we report an unbiased survey of RNA localization in the Ilyanassa embryo, and examine the overall patterns of centrosomal localization during early development. We find that 3-4% of RNAs are specifically localized to centrosomes during early development, and the remainder are either ubiquitously distributed throughout the cytoplasm or weakly enriched on centrosomes compared with levels in the cytoplasm. We observe centrosomal localization of RNAs in all cells from zygote through the fifth cleavage cycle, and asymmetric RNA segregation in all divisions after the four-cell stage. Remarkably, each specifically localized message is found on centrosomes in a unique subset of cells during early cleavages, and most are found in unique sets of cells at the 24-cell stage. Several specifically localized RNAs are homologous to developmental regulatory proteins in other embryos. These results demonstrate that the mechanisms of localization and segregation are extraordinarily intricate in this system, and suggest that these events are involved in cell fate specification across all lineages in the early Ilyanassa embryo. We propose that greater reliance on segregation of determinants in early cleavage increases constraint on cleavage patterns in molluscs and other spiralian groups.     

Cell-cell interactions determine the dorsoventral axis in embryos of an equally cleaving opisthobranch mollusc

Dorsoventral polarity in molluscan embryos can arise by two distinct mechanisms, where the mechanism employed is strongly correlated with the cleavage pattern of the early embryo. In species with unequal cleavage, the dorsal lineage, or "D quadrant", is determined in a cell-autonomous manner by the inheritance of cytoplasmic determinants. However, in gastropod molluscs with equal cleavage, cell-cell interactions are required to specify the fate of the dorsal blastomere. During the fifth cleavage interval in equally cleaving embryos, one of the vegetal macromeres makes exclusive contacts with the animal micromeres, and this macromere will give rise to the mesodermal precursor cell at the next division, thereby identifying the dorsal quadrant. This study examines D-quadrant determination in an equally cleaving species from a group of previously uninvestigated gastropods, the subclass Opisthobranchia. Blastomere ablation experiments were performed on embryos of Haminoea callidegenita to (i) determine the developmental potential of macromeres before and after fifth cleavage, and (ii) examine the role of micromere-macromere interactions in the establishment of bilateral symmetry. The results suggest that the macromeres are developmentally equivalent prior to fifth cleavage, but become nonequivalent soon afterward. The dorsoventral axis corresponds to the displacement of the micromeres over one macromere early in the fifth cleavage interval. This unusual cellular topology is hypothesized to result from constraints imposed on micromere-macromere interactions in an embryo that develops from a large egg and forms a stereoblastula (no cleavage cavity). Ablation of the entire first quarter of micromeres results in embryos which remain radially symmetrical in the vegetal hemisphere, indicating that micromere-macromere interactions are required for the elaboration of bilateral symmetry properties. Therefore, inductive interactions between cells may represent a general strategy for dorsoventral axis determination in equally cleaving gastropods.     

D quadrant specification in the leech Helobdella: Actomyosin contractility controls the unequal cleavage of the CD blastomere

The unequal division of the CD blastomere at second cleavage is critical in establishing the second embryonic axis in the leech Helobdella, as in other unequally cleaving spiralians. When CD divides, the larger D and smaller C blastomeres arise invariantly on the left and right sides of the embryo, respectively. Here we show that stereotyped cellular dynamics, including the formation of an intercellular blastocoel, culminate in a morphological left-right asymmetry in the 2-cell embryo, which precedes cytokinesis and predicts the chirality of the second cleavage. In contrast to the unequal first cleavage, the unequal second cleavage does not result from down-regulation of one centrosome, nor from an asymmetry within the spindle itself. Instead, the unequal cleavage of the CD cell entails a symmetric mitotic apparatus moving and anisotropically growing rightward in an actomyosin-dependent process. Our data reveal that mechanisms controlling the establishment of the D quadrant differ fundamentally even among the monophyletic clitellate annelids. Thus, while the homologous spiral cleavage pattern is highly conserved in this clade, it has diverged significantly at the level of cell biological mechanisms. This combination of operational conservation and mechanistic divergence begins to explain how the spiral cleavage program has remained so refractory to change while, paradoxically, accommodating numerous modifications throughout evolution.     

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.     

High-resolution fate map of the snail Crepidula fornicata: the origins of ciliary bands, nervous system, and muscular elements

The littorinimorph gastropod Crepidula fornicata shows a spiralian cleavage pattern and has been the subject of studies in experimental embryology, cell lineage, and the organization of the larval nervous system. To investigate the contribution of early blastomeres to the veliger larva, we used intracellular cell lineage tracers in combination with high-resolution confocal imaging. This study corroborates many features derived from other spiralian fate maps (such as the origins of the hindgut and mesoderm from the 4d mesentoblast), but also yields new findings, particularly with respect to the origins of internal structures, such as the nervous system and musculature that have never been described in detail. The ectomesoderm in C. fornicata is mainly formed by micromeres of the 3rd quartet (principally 3a and 3b), which presumably represents a plesiomorphic condition for molluscs. The larval central nervous system is mainly formed by the micromeres of the 1st and 2nd quartet, of which 1a, 1c, and 1d form the anterior apical ganglion and nerve tracks to the foot and velum, and 2b and 2d form the visceral loop and the mantle cell. Our study shows that both first and second velar ciliary bands are generated by the same cells that form the prototroch in other spiralians and apparently bear no homology to the metatroch found in annelids.     

Secondary embryonic axis formation by transplantation of D quadrant micromeres in an oligochaete annelid

Among spiral cleaving embryos (e.g. mollusks and annelids), it has long been known that one blastomere at the four-cell stage, the D cell, and its direct descendants play an important role in axial pattern formation. Various studies have suggested that the D quadrant acts as the organizer of the embryonic axes in annelids, although this has never been demonstrated directly. Here we show that D quadrant micromeres (2d and 4d) of the oligochaete annelid Tubifex tubifex are essential for embryonic axis formation. When 2d and 4d were ablated the embryo developed into a rounded cell mass covered with an epithelial cell sheet. To examine whether 2d and 4d are sufficient for axis formation they were transplanted to an ectopic position in an otherwise intact embryo. The reconstituted embryo formed a secondary embryonic axis with a duplicated head and/or tail. Cell lineage analyses showed that neuroectoderm and mesoderm along the secondary axis were derived from the transplanted D quadrant micromeres and not from the host embryo. However, endodermal tissue along the secondary axis originated from the host embryo. Interestingly, when either 2d or 4d was transplanted separately to host embryos, the reconstituted embryos failed to form a secondary axis, suggesting that both 2d and 4d are required for secondary axis formation. Thus, the Tubifex D quadrant micromeres have the ability to organize axis formation, but they lack the ability to induce neuroectodermal tissues, a characteristic common to chordate primary embryonic organizers.     

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.     

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.     

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.