Jo A. M. van den Biggelaar

Spatial aspects of cell interactions involved in the determination of dorsoventral polarity in equally cleaving gastropods and regulative abilities of their embryos, as studied by micromere deletions inLymnaea andPatella

SummaryIn equally cleaving gastropods, the interactions between micromeres and macromeres involved in the determination of the mesentoblast mother cell (3D) were studied by changing the spatial arrangement of the micromeres by deleting one first quartet micromere or its progeny. A fixed relation was found between the deletion site and the place of origin of 3D; therefore, a fixed relation also exists in the configuration of the three remaining first quartet micromeres and the 3D. These results argue against the possibility that the animal-vegetal interactions do not choose between macromeres, but only permit the expression of a choice already made in another way and at another moment. The results are consistent with a stochastic model in which accidental differences between the macromeres in the number or extent of contacts with first quartet micromeres play a discriminating role during micromere — macromere interactions, that lead to 3D determination.Embryos which lack a given first quartet micromere show a total absence of regulation in the larval head pattern; only the cephalic plates show regulative abilities while forming the adult head structures. Therefore, in later stages new activating and restraining factors seem to play a part in the head development.

Dye-coupling between blastomeres in early embryos ofPatella vulgata (mollusca, gastropoda): Its relevance for cell determination

SummaryDuring the early development of the molluscPatella, the dorsoventral axis is established after the fifth cleavage due to direct interaction between the animal micromeres and one of the vegetal macromeres. This vegetal macromere is thereby induced to become the mesentoblast mother cell (3D). In this study we have examined intercellular communication in earlyPatella embryos by monitoring the transfer of the fluorescent dye, Lucifer Yellow, upon iontophoretic injection into blastomeres between the second and sixth cleavage. Up to the fifth cleavage dye transfer is detectable neither inin toto embryos nor in serial sections. Shortly after the fifth cleavage dye-coupling between blastomeres becomes apparent. This occurs approximately 40 min before the interaction between animal micromeres and the future mesentoblast mother cell. Inspection of serially sectioned embryos after dye-iontophoresis in either animal micromeres or in the central macromere 3D showed the absence of direct dye-transfer between these cells at the stage of interaction. The reduced rate of dye-transfer from the 3D macromere to its dorsal neighbour 2d2 suggests a bilateral symmetrical transfer pattern, the axis of which corresponds with the dorsoventral axis at the sixth cleavage. Cell deletion experiments demonstrated that the establishment of dye-coupling between the vegetal macromeres occurs independently of the interaction between animal and vegetal blastomeres.

Brefeldin A or monensin inhibits the 3D organizer in gastropod, polyplacophoran, and scaphopod molluscs

In molluscs, the 3D vegetal blastomere acts as a developmental signaling center, or organizer, and is required to establish bilateral symmetry in the embryo. 3D is similar to organizing centers in other metazoans, but detailed comparisons are difficult, in part because its organizing function is poorly understood. To elucidate 3D function in a standardized fashion, we used monensin and brefeldin A (BFA) to rapidly and reversibly interfere with protein processing and secretion, thereby inhibiting the signaling interactions that underlie its specification and patterning. In the gastropods, Patella vulgata and Lymnaea stagnalis, the polyplacophoran, Mopalia muscosa, and the scaphopod, Antalis entalis, treatments initiated before the organizer-dependent onset of bilateral cleavage resulted in radialization of subsequent development. In radialized P. vulgata, L. stagnalis, and M. muscosa, organizer specification was blocked, and embryos failed to make the transition to bilateral cleavage. In all four species, the subsequent body plan was radially symmetric and was similarly organized about a novel aboral–oral axis. Our results demonstrate that brefeldin A (BFA) and monensin can be used to inhibit 3D’s organizing function in a comparative fashion and that, at least in M. muscosa, the organizer-dependent developmental architecture of the embryo predicts subsequent patterns of morphogenetic movements in gastrulation and, ultimately, the layout of the adult body plan.

Progressive Restrictions in Gap Junctional Communication during Development

One of the most fascinating challenges in developmental biology is to elucidate the mechanisms that control the organization of the body plan during embryonic development. In this regard intercellular transduction of signals is of particular importance for a coordinated pattern of cell differentiation. Because of their ability to allow the exchange of ions and small molecules up to a molecular weight of about 1200 D (Simpson et al., 1977), gap junctions have been proposed as a putative pathway for the intercellular transfer of developmentally important signals (Furshpan and Potter, 1968). In most of the embryos so far studied gap junctions appear in the early stages of development, when important developmental decisions take place (see Caveney, 1985; Guthrie, 1987 for reviews). More striking results have shown that differences in junctional communication may be found between embryonic tissues with different developmental programmes. The changes of functional gap junctional communication between cells with divergent developmental fates suggest an involvement of gap junctional-mediated signal transduction in the specialization and organization of the different domains of the embryo (Van den Biggelaar, 1988).

The presence of an extracellular matrix between cells involved in the determination of the mesoderm bands in embryos ofPatella vulgata (Mollusca, gastropoda)

SummaryIn 32-cell stage embryos ofPatella vulgata one of the macromeres contacts the animal micromeres, and as a result is induced to differentiate into the stem cell of the mesodermal cell line. In this study we show the presence of an extracellular matrix (ECM) between these two interacting cell types. The ECM appears to be formed by the micromeres during the 32-cell stage. Staining experiments with alcian blue and tannic acid indicate that in contains glycoconjugates, possibly in the form of proteoglycans. The characteristics of the ECM were examined further by fluorescein isothiocyanate (FITC)-lectin labelling. Of 17 lectins tested, concanavalin A (ConA), succinyl-ConA, LCH-B (Lens culinaris) and PEA (Pisum sativum) showed a positive labelling of the ECM. These results are in accordance with the electron microscopic data. The appearance of the ECM at this specific stage and place suggests that it might play an important role in the induction of the mesodermal cell line.

Brefeldin A and monensin inhibit the D quadrant organizer in the polychaete annelids Arctonoe vittata and Serpula columbiana

SUMMARY The D quadrant organizer is a developmental signaling center that is localized to the vegetal D quadrant in different spiral‐cleaving lophotrochozoan embryos and may be homologous to axial organizing regions in other metazoans. Patterning by this organizing center creates a secondary developmental axis and is required for the transition from spiral to bilateral cleavage and later establishment of the adult body plan. Organizer specification in equal‐cleaving embryos is thought to involve inductive interactions between opposing animal and vegetal blastomeres. To date, experimental demonstration of this interaction has been limited to molluscs and nemerteans. Here, we examine three families of equal‐cleaving polychaete annelids for evidence of animal–vegetal contact. We find that contact is present in the polynoid, Arctonoe vittata, but is absent in the serpulid, Serpula columbiana, and in the oweniid, Oweniia fusiformis. To interfere with cell signaling during the period predicted for organizer specification and patterning in A. vittata and S. columbiana, we use two general inhibitors of protein processing and secretion: Brefeldin A (BFA) and monensin. In A. vittata, we detail subsequent embryonic and larval adult development and show that treatment with either chemical results in radialization of the embryo and subsequent body plan. Radialized larvae differentiate many larval and adult structures despite the loss of bilateral symmetry but do so in either a radially symmetric or four‐fold radially symmetric fashion. Our results suggest that the D quadrant organizer is functionally conserved in equal‐cleaving polychaetes, but that details of its specification, induction, and patterning have diverged relative to other spiral‐cleaving phyla.

Spiral cleavage and cell position contribute to the specification of the dorsoventral axis in the embryo of the archaeogastropod Haliotis tuberculata

In the embryo of Haliotis tuberculata spiral cleavage induces size differences between the quadrants in the 4‐cell embryo. These size differences, together with the formation of compact cell configurations, induce asymmetrical positions of equivalent cells in the 8‐ and 16‐cell embryo. The asymmetries in size and position influence the final specification of the dorsoventral asymmetry in the 32‐cell embryo, as well as formation of the mesentoblast.

Gap Junctional Communication and Cell Cycle Duration in the Early Molluscan Embryo

The most remarkable aspect of the early molluscan development is the extremely regular succession and pattern of cleavages. Without any variance the embryos of the same species exactly reproduce the same cell pattern. Initially, all blastomeres are in phase, and divide synchronously. After a species specific number of simultaneous division cycles, the cells in the different lineages become out of phase and an in time and space well defined program of cell divisions is executed. In this chapter the possible influence of intercellular communication on the duration of the cell cycle in early molluscan development will be discussed. Although extracellular factors may also influence progress through the cell cycle, these will be left out of consideration.