Germ layer specification and axial patterning in the embryonic development of the freshwater planarian Schmidtea polychroa

Although patterning during regeneration in adult planarians has been studied extensively, very little is known about how the initial planarian body plan arises during embryogenesis. Herein, we analyze the process of embryo patterning in the species Schmidtea polychroa by comparing the expression of genes involved in the establishment of the metazoan body plan. Planarians present a derived ectolecithic spiralian development characterized by dispersed cleavage within a yolk syncytium and an early transient embryo capable of feeding on the maternally supplied yolk cells. During this stage of development, we only found evidence of canonical Wnt pathway, mostly associated with the development of its transient pharynx. At these stages, genes involved in gastrulation (snail) and germ layer determination (foxA and twist) are specifically expressed in migrating blastomeres and those giving rise to the temporary gut and pharyngeal muscle. After yolk ingestion, the embryo expresses core components of the canonical Wnt pathway and the BMP pathway, suggesting that the definitive axial identities are established late. These data support the division of planarian development into two separate morphogenetic stages: a highly divergent gastrulation stage, which segregates the three germ layers and establishes the primary organization of the feeding embryo; and subsequent metamorphosis, based on totipotent blastomeres, which establishes the definitive adult body plan using mechanisms that are similar to those used during regeneration and homeostasis in the adult.     

The embryonic development of the temnocephalid flatworms Craspedella pedum and Diceratocephala boschmai

We have analyzed the embryonic development of the temnocephalid flatworms Craspedella pedum and Diceratocephala boschmai, using a combination of fuchsin-labeled whole-mount preparation, histology, and transmission electron microscopy. Following the staging system recently introduced for another flatworm species (Mesostoma lingua), we can distinguish eight morphologically defined stages. Temnocephalids produce eggs of the neoophoran type in which a small oocyte is surrounded by a layer of yolk cells. Cleavage takes place in the center of the yolk mass (stages 1-2) and results in an irregular, multilayered disc of mesenchymal cells that moves to the future ventral egg pole (stage 3). Organ primordia, including those of the brain, pharynx, male genital apparatus, sucker, and epidermis "crystallize" within this disc without undergoing gastrulation movements (stage 4). An invagination of the epidermal primordium pushes the embryo back into the center of the yolk ("embryonic invagination"). As a result, organogenesis begins while the embryo is invaginated (stage 5). The brain differentiates into an outer cortex of cell bodies that surround a central neuropile. Precursor cells of the epidermis, pharynx, and protonephridia become organized into epithelia. During stage 6, the embryonic primordium everts back to the surface, where organogenesis and cell differentiation continues. Epidermal cells fuse into a syncytium that expands around the yolk. Myoblasts initially do not spread out in the way epidermal cells do; they remain concentrated in two narrow, longitudinal bands that extend along the sides of the embryo. Three pairs of axon tracts extending posteriorly from the brain follow the bands of myoblasts. Stages 7 and 8 are characterized by the appearance of eye pigmentation, brain condensation, and the formation of tentacles and a sucker that bud out from the epidermis of the anterior and posterior end, respectively. Comparison of morphogenesis in temnocephalids with observations in other flatworm taxa suggests a phylotypic stage for this phylum of invertebrates.     

The embryonic development of the flatworm <Emphasis Type="Italic">Macrostomum</Emphasis> sp.

Macrostomid flatworms represent a group of basal bilaterians with primitive developmental and morphological characteristics. The species Macrostomum sp., raised under laboratory conditions, has a short generation time of about 2–3 weeks and produces a large number of eggs year round. Using live observation, histology, electron microscopy and immunohistochemistry we have carried out a developmental analysis of Macrostomum sp. Cleavage (stages 1–2) of this species follows a modified spiral pattern and results in a solid embryonic primordium surrounded by an external yolk layer. During stage 3, cells at the anterior and lateral periphery of the embryo evolve into the somatic primordium which gives rise to the body wall and nervous system. Cells in the center form the large yolk-rich gut primordium. During stage 4, the brain primordium and the pharynx primordium appear as symmetric densities anterior-ventrally within the somatic primordium. Organ differentiation commences during stage 5 when the neurons of the brain primordium extend axons that form a central neuropile, and the outer cell layer of the somatic primordium turns into a ciliated epidermal epithelium. Cilia also appear in the lumen of the pharynx primordium, in the protonephridial system and, slightly later, in the lumen of the gut. Ultrastructurally, these differentiating cells show the hallmarks of platyhelminth epithelia, with a pronounced apical assembly of microfilaments (terminal web) inserting at the zonula adherens, and a wide band of septate junctions underneath the zonula. Terminal web and zonula adherens are particularly well observed in the epidermis. During stage 6, the somatic primordium extends around the surface dorsally and ventrally to form a complete body wall. Muscle precursors extend myofilaments that are organized into a highly regular orthogonal network of circular, diagonal and longitudinal fibers. Neurons of the brain primordium differentiate a commissural neuropile that extends a single pair of ventro-lateral nerve trunks (the main longitudinal cords) posteriorly. The primordial pharynx lumen fuses with the ventral epidermis anteriorly and the gut posteriorly, thereby generating a continuous digestive tract. The embryo adopts its final shape during stages 7 and 8, characterized by the morphallactic lengthening of the body into a U-shaped form and the condensation of the nervous system.Edited by J. Campos-Ortega     

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.     

Early embryonic development,including germ-disk stage,in the theridiid spider Achaearanea japonica (Bös. et Str.)

Early embryonic development, from the first cleavage to the germ-disk stage, in the theridiid spider Achaearanea japonica was examined by light and electron microscopy. The eggs are syncytial during the first four cleavages, and then invaginations of cell membranes fuse to generate the blastomeres at the sixteen-nucleus stage. The cleavage pattern is a modified type of total cleavage. It appears that radial bundles of microtubules that radiate from the perinuclear cytoplasm may participate in the migration of cleavage nuclei for the formation of the blastoderm. The large yolk granules are sequestered by cell membranes from the blastomeres or blastoderm cells into the interior of the embryo together with various organelles and glycogen granules. Most of the blastoderm cells converge in the upper hemisphere to form the germ disk, whereas a few cells remain in the lower hemisphere. The embryo at the germ-disk stage contains many spherical germ-disk cells. Almost no large yolk granules are found in these cells, but the flat remaining cells each contain several large yolk granules. These remaining cells may preserve a flat shape to cover the surface of the embryo that does not include the germ disk. © 1995 Wiley-Liss, Inc.     

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.     

Pre-gastrula expression of zebrafish extraembryonic genes

Background  

Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL).     

Structural Changes Associated with Resistance of Soybean to Heterodera glycines

Subcellular responses to infection by Race 3 of Heterodera glycines in susceptible (''Lee'') and resistant (''Forrest'' and ''Bedford'') soybean cultivars were compared. Syncytial formation, initiated in susceptible as well as resistant soybean cultivars, was characterized by wall perforations, dense cytoplasm, and increased endoplasmic reticulum, In susceptible plants, syncytia developed continuously until nematode maturity. This included hypertrophy of nuclei, increase of rough endoplasmic reticulum in early stages of infection, and formation of wall ingrowths at a late stage of infection. In the resistant reaction in Forrest, a necrotic layer surrounded syncytium component cells demarcating them from surrounding normal cells and leading to syncytial necrosis. Wall appositions were prominently formed near the necrotic layer, and the cytoplasm of the syncytium component cells was extremely condensed. The whole syncytium became necrotic at a late stage of infection. Bedford had nuclear degeneration prior to cytoplasmic degradation. Chromatin was often scattered throughout the syncytial cytoplasm. Finally the whole syncytium became degenerated with plasmalemma completely detached from the syncytial cell walls. The differences in resistant responses reflect a difference in genetic composition of the soybean cultivars tested.     

Comparative approach to developmental analysis: the case of the dalyellid flatworm, Gieysztoria superba

Dalyellida represents a taxon of small rhabdocoel flatworms that occur in freshwater habitats all over the world. Combining histology and electron microscopy we have analyzed the embryonic development of a new dalyellid species, Gieysztoria superba, in order to obtain more comparative data pertaining to morphogenesis and organogenesis in flatworms. We have used a morphological staging system that we recently introduced for another rhabdocoel, Mesostoma lingua (Younossi-Hartenstein et al., 2000). Our data show that in many fundamental respects, such as the irregular cleavage, mesenchymal embryonic primordium, and lack of gastrulation movements, Gieysztoria is highly similar to Mesostoma. During cleavage (stages 1 and 2) the embryo is located in the center of the egg where it is surrounded by a layer of yolk cells. Cleavage leads up to a solid, disc shaped cell cluster. During stage 3, the embryo migrates to the ventral side of the egg and acquires bilateral symmetry. Stages 4/5 sees the emergence of the first organ primordia, the brain, epidermis and pharynx. A peculiar invagination of the epidermal layer pushes the embryo back into the center of the yolk ("embryonic invagination"). Organogenesis takes place during stages 5 and 6 while the embryo is invaginated. A junctional complex, consisting initially of small septate junctions, followed later by a more apically located zonula adherens, is formed in all epithelial tissues, including epidermis, protonephridia, and pharynx. During late stages (6-8), Gieysztoria embryos evert back to the surface where the epidermal primordium expands and grows around the yolk to close dorsally. During this phase of development cytodifferentiation of the different organ systems takes place. Stage 7 is characterized by the appearance of eye pigmentation, brain condensation and spindle shaped myocytes. Stage 8 describes the fully dorsally closed and differentiated embryo. In comparison to other rhabdocoels, including Mesostoma, Gieysztoria exhibits a precocious differentiation of an intestinal epithelium and male genital apparatus. In Mesostoma, these structures are formed post hatching.     

An ultrastructural study of alimentary tract development in the cercariae of Prosorhynchoides borealis (Digenea,Bucephalidae)

Podvyaznaya I. 2011. An ultrastructural study of alimentary tract development in the cercariae of Prosorhynchoides borealis (Digenea, Bucephalidae). —Acta Zoologica (Stockholm) 92 : 170–178. The development of digestive system in Prosorhynchoides borealis cercariae was studied using transmission electron microscopy. The foregut and caecum primordia arise in early cercarial embryos as two adjoining cellular cords. The primordial pharynx appears as a cluster of myoblasts in the mid‐part of the foregut primordium whose proximal end abuts onto the ventral embryonic tegument. Later, a lumen develops within the gut primordia and their component cells form the embryonic cellular epithelium with an essentially similar structure in the foregut and caecal regions. Subsequently, the foregut epithelial cells merge to form a syncytium. This process proceeds asynchronously and the most proximal foregut area remains cellular for the longest time. The syncytial lining of the foregut establishes syncytial connections with secretory cytons differentiating in the surrounding parenchyma. These cytons produce secretory granules, which are transported through cytoplasmic connections to the foregut syncytium. Before cercariae reach maturity, their foregut epithelium becomes anucleate and continuous with the external tegument. By the end of cercarial development, numerous short lamellae appear on the luminal surface of the caecal epithelium. The caecal cells become involved in secretory activity as indicated by the presence of Golgi‐derived secretory bodies in their cytoplasm.     

Cleavage in vivo and in culture in the Dasyurid marsupial Antechinus stuartii (macleay)

Cleavage in the brown marsupial mouse, Antechinus stuartii, from the zygote to the unilaminar blastocyst, was observed in vivo and in culture and in sections of embryos. The first three divisions were meridional and passed from the yolk pole to the opposite pole. Deutoplasmolysis, resulting in a distinct yolk mass, occurred during the first two divisions. Prior to the third and fourth divisions, the blastomeres elongated and flattened against the zona pellucida. The fourth division was latitudinal and resulted in two histologically distinct rings of eight blastomeres which were at first rounded and then became flattened against the zona. Further divisions and flattening of the blastomeres resulted in a complete unilaminar blastocyst by the time the blastocyst numbered 22 to 30 cells. Some expansion, causing compression of the zona and mucoid layers, occurred before completion of the blastocyst, but most expansion occurred once the blastocyst was complete. No histological differences could be detected between the blastomeres at this stage. Embryos were successfully cultured from the zygote to the rounded four-cell stage and from the flattened four-cell stage to the completed unilaminar blastocyst of around 32 cells. Total estimated cleavage times were slower in culture than in vivo, but the relative lengths of time for each division were approximately the same.     

Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8-16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast),which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm--yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.     

Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L.

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8?C16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast), which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm-yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.     

Mesoderm formation by isolated and cultivated 8-cell stage blastomeres of the teleost, Leucopsarion ptersii (shiro-uo).

Isolation of cleavage-stage blastomeres and the study of their developmental potential has been used extensively for analyzing the mechanisms of embryogenesis in vertebrates, including amphibians and echinoderms. We devised a method to isolate 8-cell stage blastomeres in the teleost, shiro-uo, by utilizing its unique cleavage pattern of the horizontal 3rd cleavage plane. Removal of all the upper blastomeres at the 8-cell stage allowed almost normal embryogenesis from the remaining lower blastomeres and yolk cell mass. Isolated upper or lower blastomeres formed vesicles and spherical bodies, which later showed morphological changes during cultivation. Mesoderm formation was detected not only in the cultivated lower blastomeres or whole blastomeres but also in the upper blastomeres isolated from the yolk cell mass at the 8-cell stage, although at a lower frequency than the lower blastomeres. These results indicated the presence of very early signaling for mesoderm induction, which is independent from the currently postulated signals from the yolk syncytial layer at later stages. This also indicated non-equivalence or differentiation of the blastomeres from the very early cleavage stage in teleost embryos.     

Three‐dimensional fate mapping of larval tissues through metamorphosis in the glass sponge Oopsacas minuta

The tissue of glass sponges (Class Hexactinellida) is unique among metazoans in being largely syncytial, a state that arises during early embryogenesis when blastomeres fuse. In addition, hexactinellids are one of only two poriferan groups that already have clearly formed flagellated chambers as larvae. The fate of the larval chambers and of other tissues during metamorphosis is unknown. One species of hexactinellid, Oopsacas minuta, is found in submarine caves in the Mediterranean and is reproductive year round, which facilitates developmental studies; however, describing metamorphosis has been a challenge because the syncytial nature of the tissue makes it difficult to trace the fates using conventional cell tracking markers. We used three‐dimensional models to map the fate of larval tissues of O. minuta through metamorphosis and provide the first detailed account of larval tissue reorganization at metamorphosis of a glass sponge larva. Larvae settle on their anterior swimming pole or on one side. The multiciliated cells that formed a belt around the larva are discarded during the first stage of metamorphosis. We found that larval flagellated chambers are retained throughout metamorphosis and become the kernels of the first pumping chambers of the juvenile sponge. As larvae of O. minuta settle, larval chambers are enlarged by syncytial tissues containing yolk inclusions. Lipid inclusions at the basal attachment site gradually became smaller during the six weeks of our study. In O. minuta, the flagellated chambers that differentiate in the larva become the post‐metamorphic flagellated chambers, which corroborate the view that internalization of these chambers during embryogenesis is a process that resembles gastrulation processes in other animals.     

Endosperm differentiation in barley wild-type and sex mutants

The cereal endosperm is a storage organ consisting of the central starchy endosperm surrounded by the aleurone layer. In barley, endosperm development is subdivisible into four main stages, i.e. the syncytial (I), the cellularization (II), the differentiation (III) and the maturation stage (IV). During stage I, a multinucleate syncytium is formed, which in stage II develops into the undifferentiated cellular endosperm. During stage III the cells of the endosperm differentiate into two types of aleurone cells (peripheral and modified) and three different starchy endosperm cell types (irregular, prismatic and subaleurone). To elucidate the ontogenetic relationship between the endosperm tissues, the phenotypes of sex (shrunken endosperm mutants expressing xenia) mutant endosperms were studied. These mutants can be classified into two groups, i.e. those in which development is arrested at one of the four wild-type stages described above, and those with abnormal development with new organizational patterns in the endosperm or with novel cell types. Based on these studies, it is suggested that the two endosperm halves represent cell lines derived from the two daughter nuclei of the primary endosperm nucleus, and that the prismatic starchy endosperm cells arise from a peripheral endosperm meristematic activity during stage III. Finally, a model for the main molecular events underlying the morphogenetic processes is discussed.     

An ultrastructural analysis of the dispersed cell phase during development of the annual fish,Cynolebias

Cell ultrastructure was investigated during the dispersion phase of development in the annual fish Cynolebias. Three cellular populations encompass the yolk mass during dispersion, namely, 1) the yolk syncytial layer (YSL) or periblast, which lies directly over the surface of the yolk; 2) the deep blastomeres of the blastoderm, which engage in morphogenetic movements on the surface of the YSL and beneath the enveloping layer prior to forming the future embryo; and 3) the enveloping layer (EVL) of the blastoderm, which is a cohesive epithelium that forms the outermost cell layer of the blastoderm. Deep blastomeres contain numerous mitochondria and scattered glycogen rosettes that appear to function in the utilization of energy reserves. These cells also possess surface extensions such as filopodia and ruffles. Numerous microfilaments running parallel to the plasma membrane occur in cell extensions and in the cortical cytoplasm of neighboring blastomeres. In bleb-like extensions such as ruffles, microfilamentous stress fibers run parallel to the plane of the plasma membrane and prevent cellular organelles from entering the hyaline cap of the ruffle. Deep blastomeres also have basal projections that contain glycogen as well as pits in the basal membrane. Blastomeres move about using the YSL as a substrate. The YSL possesses specializations for nutrient uptake, storage, and transport such as numerous multivesicular bodies and large amounts of glycogen. Glycogen, in the rosette form, occurs in extraordinary amounts, virtually occluding the cytoplasm. Glycogen reserves are postulated to serve as an energy source during diapause. Glycogen is sometimes contained within villous projections that extend from the apical surface of the YSL. This configuration suggests the possibility of glycogen transport to the overlying deep blastomeres. Specializations of the EVL include apical tight junctions and basal lateral zonulae adherentes that interdigitate with those of adjacent EVL cells. The EVL serves as an impermeable membrane that protects the developing egg from the vicissitudes of its environment.     

Embryogenesis in mouth-breeding cichlids (Osteichthyes,Teleostei) structure and fate of the enveloping layer

The eggs of African mouth-brooders are of unusual size and shape. Studying their development may help to more clearly understand epiboly, gastrulation, and the relation between enveloping layer (periderm) and epidermis. When epiboly has progressed over just one fifth of the yolk mass, the germ ring and embryonic shield are already well established. Behind the germ ring very few deep cells are present at this early stage of epiboly, except in the embryonic shield. When the blastodisc covers the animal half of the yolk mass, the future body is already well established with notochord, somites and developing neural keel. Apart from these structures, no deep cells can be detected between enveloping layer and yolk surface; not even a germ ring remains behind the advancing edge of the enveloping layer. Epiboly over the greater part of the yolk is achieved only by the enveloping layer and the yolk syncytial layer. As the margin of the enveloping layer begins to reduce its circumference when closing around the vegetal pole, groups of cells in the advancing edge become spindle-shaped, with a single cell in between of each of these groups broadening along the edge. The enveloping layer (called periderm after epiboly) remains intact until after hatching, when, together with the underlying ectoderm, it forms the double-layered skin of the larval fish. Thereafter, cells deriving from the subperipheral ectoderm gradually replace the decaying periderm cells to form the final epidermis. Thus, in the cichlids studied, the enveloping layer alone forms the yolk sac to begin with, and it covers the larval body until some days after hatching.