Development of in vitro fertilized embryos of the polyclad flatworm,Hoploplana inquilina,following blastomere separation and deletion

Summary After in vitro fertilization of naked eggs of the polyclad turbellarian, Hoploplana inquilina, both cell separation experiments and deletions of specific blastomeres are possible. With these techniques one can analyze the developmental potential of isolated blastomeres and determine if the embryonic axes have been established at the four-cell stage in this primitive, equally-cleaving spiralian embryo. Two-cell separation experiments with development of both halves resulted in pairs of larvae 1) neither of which had an eye (29%), 2) both of which had one eye (19%), and 3) one of which was eyeless and the other was one-eyed (43%). Deletion of one blastomere at the four-cell stage resulted in 68% one-eyed, 28% two-eyed and 3% eyeless larvae. The one-eyed larvae were asymmetric with respect to eye position with more having right than left eyes. Abnormal or missing ventrolateral lobes occurred with deletion of any of the macromeres at four cells but were significantly more common when A or C rather than B or D was deleted. The experiments support the hypothesis that eye development is a consequence of cytoplasmic localization of both a specific eye precursor and an inducer which segregate independently of cleavage planes, and indicate that the embryonic axes have been determined at the four-cell stage.     

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.     

Experimental evidence for the origins of determinative development in the polyclad turbellarians

Cell-deletion experiments were carried out on the embryo of the polyclad turbellarian Hoploplana inquilina to examine further the nature of development in primitive spiralians. The polyclads are of particular interest because they provide a link between the regulative development of acoels and the determinative development of annelids and molluscs. Single blastomeres were deleted at the two- and four-cell stages by puncture through the eggshell membrane with tungsten needles. All deletions resulted in abnormal larvae with consistent characteristics representing half or three-quarter Müller's larvae. The number of larval eyes was a particularly useful character in revealing mosaicism. This study establishes the polyclad embryo as determinative, but with important cell interactions also occurring during early development, and provides evidence that mosaicism became associated with spiral cleavage in the quartet form during the evolution of the Turbellaria.     

On the evolution of central nervous systems: Implications from polyclad turbellarian neurobiology

The nervous system of the polyclad turbellarian Notoplana acticola consists of a series of nerve plexuses and a central ganglion, the brain. The brain contains a variety of cell types including multipolar heteropolar and bipolar neurons. These cell types are rare in other invertebrate ganglia. Individual neurons also contain a variety of different ion channels. both spiking and nonspiking neurons are found. Some neurons are multimodal interneurons. Habituation appears to be a postsynaptic phenomenon. Sensitization and long-term potentiation have not been demonstrated. Polyclads appear to represent a stage in the evolution of centralized nervous systems where much of the neuronal machinery underlying behavior occurs in the peripheral nervous system and the brain's main functions are the coordination and sequencing of peripherally placed reflexes. Even at this stage, however, the brain already contains cells that seem as advanced as those found in higher organisms.     

Ultrastructural studies of differentiation in the oocyte of the polyclad turbellarian,Prostheceraeus floridanus

Oocyte differentiation in the polyclad turbellarian Prostheceraeus floridanus has been examined to determine the nature of oogenesis in a primitive spiralian. The process has been divided into five stages. (1) The early oocyte: This stage is characterized by a large germinal vesicle surrounded by dense granular material associated with the nuclear pores and with mitochondria. (2) The vesicle stage: The endoplasmic reticulum is organized into sheets which often contain dense particles. Vesicles are found in clusters in the cytoplasm, some of which are revealed to be lysosomes by treatment with the Gomori acid phosphatase medium. (3) Cortical granule formation: Cortical granules are formed by the fusion of filled Golgi vasuoles which have been released from the Golgi saccules. The association between the endoplasmic reticulum and Golgi suggests that protein is synthesized in the ER and transferred to the Golgi where polysaccharides are added to form nascent cortical granules. (4) Yolk synthesis: After a large number of cortical granules are synthesized, yolk bodies appear. They originate as small membrane-bound vesicles containing flocculent material which subsequently increase in size and become more compact. Connections between the forming yolk bodies and the endoplasmic reticulum indicate that yolk synthesis occurs in the ER. (5) Mature egg: In the final stage, the cortical granules move to the periphery and yolk platelets and glycogen fill the egg. At no time is there any evidence of uptake of macromolecules at the oocyte surface. Except for occasional desmosomes between early oocytes, no membrane specialization or cell associations are seen throughout oogenesis. Each oocyte develops as an independent entity, a conclusion supported by the lack of an organized ovary.     

Asexual reproduction and the turbellarian archetype

The turbellarian archetype is widely believed to have been a hermaphrodite lacking asexual reproduction, and such asexual reproduction as is now seen in the Turbellaria (as paratomy and architomy) is generally assumed to have arisen secondarily several times independently. Asexual reproduction clearly prevails among the most primitive metazoans such as the placozoans, sponges, and radiates, however, and if the Platyhelminthes is indeed an early offshoot of bilaterian evolution, as some have claimed, then it is reasonable to expect asexual reproduction to be a primitive feature of the Turbellaria. Asexual reproduction by paratomy or architomy is found in all three main evolutionary lines of the Turbellaria and is most common among primitive groups such as the Catenulida and Macrostomida. The discovery of a new, apparently primitive marine genus of Macrostomida having paratomy widens the known incidence of asexual reproduction within that order. The presence of a muscle ring around the gut of several distantly related genera of the Macrostomida and similarities this ring shows with septa in the division plane of paratomizing species are evidence that paratomy was a feature of the stem species for this order — a feature only secondarily lost in most macrostomids — and suggest that asexual reproduction is a primitive feature for the Platyhelminthes as a whole.     

Initial morphological diversity as a criterion in deciphering turbellarian phylogeny

The most profound structural variety in morphofunctional systems and morphogenetic mechanisms, i.e. the highest morphological diversity, is observed in those groups where these systems and mechanisms are evolutionarily most primitive. Here, such variety can involve the basic body plan of a given phylum and the types of morphogenesis characteristic of it. This correlation provides a new criterion of evolutionary primitiveness, namely, the criterion of initial morphological diversity.The highest morphological diversity among turbellarian groups is observed in the order Acoela. Acoel turbellarians are archaic in most of their features, apparently being a group near the base of the turbellarian phylogenetic tree. Among other turbellarians there are a few groups that also are archaic in some few features (above all, the Catenulida), although on the whole they are more advanced than the Acoela. The Turbellaria as a whole is notable for its morphological diversity in comparison with other classes of the Scolecida.     

Development of cilia in embryos of the turbellarian Macrostomum

Electron microscopy of Macrostomum hystricinum raised in culture shows that ciliogenesis in the worm's epidermal blastomeres begins in embryos 39–41 h old with kinetosomal and de novo genesis of presumptive basal bodies, which are morphologically distinguishable from centrioles of the mitotic apparatus, and proceeds by the migration of basal bodies to the apical plasma membrane of the cells and their production there of ciliary axonemes by an age of 51–53 h when the bastomeres emerge between yolk cells on the embryo's surface. Ciliogenesis continues throughout development with the addition of cilia virtually one by one to the expanding epidermal cells' surfaces. At no time in ciliogenesis are stages seen that might show derivation of these multiciliated cells from the primitive monociliated cell type presumably present in the ancestors of the Turbellaria.     

Aspects of photoreceptor structure and phototactic behavior in Platyhelminthes,with particular reference to the symbiotic turbellarian Paravortex

Photoreceptor structure and function in the Platyhelminthes has traditionally been treated separately in the Turbellaria on one hand and the conventional parasitic classes on the other. In this paper, an attempt is made to bring together data from the literature and to highlight deficiencies and areas where a more integrated approach would be beneficial. This is done with particular reference to the endosymbiont genus Paravortex which belongs to the Turbellaria but which functionally has more similarities to the parasitic platyhelminths especially with regard to the host-finding requirement of the larval stages.     

Differentiation of the body wall musculature in Macrostomum hystricinum marinum and Hoploplana inquilina (Plathelminthes), as models for muscle development in lower Spiralia

Recent studies on the differentiation of the body wall musculature in a medicinal leech and in the free-living plathelminth Macrostomum hystricinum marinum, Beklemischev 1950 provide the first evidence of a complex developmental signalling pattern, possibly involving stem cells and the nervous system, in the organization of the muscle grid formed by developing myocytes. To enhance further our understanding of the ontogenetic and phylogenetic origin of such muscle grids, which consist of circular, longitudinal and diagonal muscle fibres, we have undertaken a study of muscle development in the polyclad flatworm Hoploplana inquilina Wheeler 1894 in collaboration with the Marine Biological Laboratory, Woods Hole. We have also continued our examination of the development of the body wall musculature in M. hystricinum. Both species were studied using rhodamine-phalloidin staining and transmission electron microscopy. Additional visualization of the fluorescent whole mount preparations was performed with confocal laser microscopy and digital image processing. The results of our investigation suggest that: (1) the mechanism of muscle development in H. inquilina supports the deeply rooted concept of bilateral symmetry (right and left longitudinal founder muscle), and (2) a first circular muscle in this species develops on the border between an anterior body unit and the main body; a caudalmost region is less obvious. The presence of a spiral muscle functioning as a circular muscle system of the head region points to a separate developmental mechanism for this region and the trunk. In contrast to H. inquilina, where the larval stage forces an intermediate restructuring of the musculature of the body wall before the adult body shape is finally developed, the formation of the body wall musculature of M. hystricinum already seems constrained by the adult body shape.     

Localization of mitochondrial ribosomal RNA on the chromatoid bodies of marine planarian polyclad embryos

Electron-dense cytoplasmic structures, referred to as chromatoid bodies, are observed in the somatic stem cells, called neoblasts, and germline cells in adult planarians. Although it has been revealed that the chromatoid bodies morphologically resemble germline granules in Drosophila and Xenopus embryos, what essential role it plays in the planarian has remained unclear. In the present study, to examine whether chromatoid bodies in planarian embryos are responsible for germline formation, the presence and behavior of chromatoid bodies during embryogenesis were examined. Mitochondrial large ribosomal RNA and mitochondrial small ribosomal RNA were used as candidate markers for components of the chromatoid body. Starting from the fertilized egg, extramitochondrial signals of both RNA (mtrRNA) were observed. At the ultrastructural level, mtrRNA were localized on the surface of the chromatoid bodies. At subsequent stages, the signals of mtrRNA were observed in certain restricted blastomeres that contribute to the formation of larval structures. The signals gradually decreased from the gastrula stage. These results suggest that the chromatoid bodies associated with mtrRNA in embryogenesis are not germline granules. The chromatoid bodies of blastomeres may be concerned with the toti- or pluripotency and cell differentiation as proposed in adult planarian neoblasts.     

An electron-microscopic study of syncytium formation during early embryonic development of the freshwater planarian Bdellocephala brunnea

To substantiate the assumption that the egg cell and blastomeres in planarian embryos influence surrounding yolk cells to form a syncytium, embryos at 1- to 8-cell stages were examined by electron microscopy. Within special areas of the endoplasmic reticulum both in the egg cell and in the blastomeres, a large number of vacuoles of various sizes formed and then disappeared at least four times over the period from egg-laying through the 8-cell stage as if their contents were being secreted. These activities diminished markedly at the 8-cell stage. Yolk cells surrounding the egg cell and blastomeres were aggregated in close contact with one another in a small clump shortly after egg-laying, and then, late in the 4-cell stage, became fused, forming a syncytium. The correlation between release of vacuoles by the egg cell and blastomeres and the formation of a syncytium by the yolk cells indicate that the cell fusion could be induced by a factor contained in the vacuoles.     

An electron microscope study of primary epidermis formation in freshwater planarian embryos

Early development in freshwater planarians is generally considered to be highly modified to the point of being unique. A careful examination by TEM, however, suggests that the primary epidermis (Skaer, 1965) is formed in a rather regular manner but is partially inverted with respect to the definitive body axes. After formation of the yolk cell syncytium, the blastomeres enclosed within it increase in number in the central area. Some of these blastomeres then move peripherally as a group and fuse to form another syncytium, the primordium of the primary epidermis. This primordium contacts the surface of the yolk-cell syncytium at the place where the primordium will subsequently flow out. The primordium spreads to the opposite pole through the spaces among the syncytial and non-syncytial yolk cell masses.     

Variation, plasticity and modularity in anuran development

Although anuran development is generally thought to be relatively conservative, a great deal of variation is evident when different species are compared. This report summarizes the results of comparative analyses of different aspects of anuran development. These include differences in sequence and timing of developmental events, the effects of genome size, and the effects of different life history strategies on anuran embryogenesis. The results show that anuran development is plastic at the evolutionary level, and many changes can occur in the developmental processes of anurans throughout their evolution. Changes are apparently rapid, and are as common as cladogenic events. This evolutionary plasticity can be attributed to the modular nature of anuran development. Different modules can shift relative to one another in time or in space, creating variations in the observed developmental patterns. However, shifts in modules can occur even without having a significant effect on the ultimate outcome of the process. I discuss the implications of the modular nature of development on the evolution of anuran development, and of the group in general.     

Ultrastructure of the frontal organ in Convoluta and Macrostomum spp.: significance for models of the turbellarian archetype

Present models of turbellarian evolution depict the organism with a frontal organ — a complex of glands whose necks emerge at the anterior tip of the body — and therefore imply that this organ is homologous throughout the Turbellaria. However, comparisons of representatives of the Acoela and Macrostomida, two putatively primitive orders of the Turbellaria, show that frontal organs in these two are not similar in ultrastructure or histochemistry. The acoel Convoluta pulchra had a prominent cluster of frontal mucous glands whose necks emerged together in a frontal pore at the exact apical pole of the organism, and an array of smaller glands of at least five other types opened at the anterior end, separately from and ventral to this pore. The frontal organs (Stirndrüsen) of two species of Macrostomum on the other hand, comprised an array of discretely emerging necks of at least two gland types including one with rhabdiform (rhammite) and one with globular mucous secretion granules neither of which emerge at the apical pole. In neither species did the organ appear to be sensory. Our findings indicate a low probability of homology between the frontal glands of the Acoela and Macrostomida.     

Comparison of the nervous system of the rhabdocoel Mesostoma ehrenbergii with that of the polyclad Notoplana acticola

The neuromuscular organization of the free-living marine polyclad Notoplana acticola (Boone) can be compared with that of the fresh water rhabdocoel Mesostoma ehrenbergii (Focke). These examples act as outgroups for each other, belonging to different clades of the Turbellaria and similarities between the two can be taken as synapomorphies to indicate features that might be primitive and general features of flatworm nervous systems. The fluorescent dye Lucifer Yellow was used to fill cells in the brains of the two species and the neuronal anatomies were photographed or drawn. Many different cell types have been found in both species. The predominant cell types in Mesostoma were heteropolar bipolar and heteropolar multipolar cells with isopolar bipolar and isopolar multipolar cells in much lower numbers; only a few unipolar cells were found. In Notoplana, many of the cells penetrated were also heteropolar bi- or multipolar cells. There were also neurons and architectural features that were morphologically very similar to each other, in the two animals, such as the BRA cells and commissural tracts. These shared features suggest that many of the peculiarities of polyclad nervous systems may be general flatworm features.     

A new species of freshwater turbellarian from Africa,predatory on mosquitoes: Mesostoma zariae n. sp. (Typhloplanoida)

A new species of the genus Mesostoma Ehrenberg 1935, M. zariae n.sp. is described and its relationship discussed. The new species belongs to the M. lingua species-group and is characterised by the presence of two kinds of prostate secretions and a pear-shaped penis papilla. It occurs in small standing or slowly running waters at Zaria, Nigeria. It has previously been proven (Mead 1978) that this species is predatory on the aquatic stages of mosquitoes.     

Ultrastructural aspects of nervous-system and statocyst morphogenesis during embryonic development of Convoluta psammophila (Turbellaria,Acoela)

The notion that statocysts originated from an infolding of ectoderm lined by ciliated sensory cells has been challenged with evidence of capsule-limited, non-ciliary statocysts in several independent phyla. Statocysts in turbellarians primitively lack cilia and are embedded within or closely adjoined to the cerebral ganglion; they are likely to be derived from nervous tissue. We investigated the development of the simple statocyst in an acoel turbellarian, a statocyst consisting of three cells. Observations of serial TEM sections of embryos at different stages of development support the hypothesis of an inner (non-epithelial) origin of the statocyst. First, a three-cell complex is delimited by a basal lamina; it then undergoes cavitation by swelling, autophagy, and fluid secretion. The statocyst becomes discernible within the precursor ganglion cells while they still contain yolk inclusions. The two outer (parietal) cells, enclosed together by a 10-nm-thick basal lamina, arrange themselves in an ovoid of about 10 µm diameter and surround the inner statolith-forming cell. The statolith is formed later within vacuoles of the statolith-forming cell.