The cleavage program in the 2d cell lineage of Tubifex embryos

Early development in clitellate annelids is characterized by a highly stereotyped sequence of unequal, spiral cleavages. Cell 2d (i.e., the second micromere of the D quadrant) in the oligochaete Tubifex tubifex also undergoes an evolutionarily conserved sequence of cell division to produce four bilateral pairs of ectodermal teloblasts that act as embryonic stem cells. This study was conducted to characterize each of the 15 rounds of cell division that occur in the 2d cell lineage in this clitellate. After its occurrence, cell 2d undergoes three rounds of highly unequal divisions, giving off the first smaller daughter cell toward the posterior right of the larger daughter cell, the second cell toward the posterior left, and the third cell toward the anterior side of the cell; the larger daughter cell that results from the third division (i.e., the great-granddaughter cell of 2d) then divides equally into a bilateral pair of NOPQ proteloblasts. Cell NOPQ on either side of the embryo undergoes 11 rounds of cell division, during which ectoteloblasts N, Q, and O/P are produced in this order. After its appearance, NOPQ undergoes highly unequal divisions twice cutting off the smaller cells toward the anterior end of the embryo and then divides almost equally into ectoteloblast N and proteloblast OPQ. After its appearance, OPQ undergoes highly unequal divisions twice giving off the first smaller cell toward the anterior and the second smaller cell toward the posterior of the embryo and then divides almost equally into ectoteloblast Q and proteloblast OP. Finally, OP undergoes highly unequal division four times after its birth budding off the smaller cells toward the anterior and then cleaves equally into ectoteloblasts O and P. In the unequally dividing cells of the 2d cell lineage, the mitotic apparatus (MA), which forms at the cell's center, moves eccentrically toward the cortical site where the smaller cell will be given off. The moving MA is oriented perpendicular to the surface it approaches, and its peripheral pole becomes closely associated with the cell cortex. In contrast, the MA involved in the equal divisions remains in the cell center throughout mitosis. The key features of the cleavage program in the 2d cell lineage are discussed in light of the present observations. The mechanical aspects of unequal cleavage in the 2d cell lineage and the modes of specification of MA orientation are discussed. A comparison of the cleavage mode in the 2d cell lineage is also performed among six selected clitellate annelid species.     

Some aspects of spiralian development

Nielsen, C. 2010. Some aspects of spiralian development. —Acta Zoologica (Stockholm) 91 : 20–28 Spiralian development is not only a characteristic early cleavage pattern, with shifting orientations of the cleavage planes, but also highly conserved cell lineages, where the origin of several organs can be traced back to identifiable cells in the lineage. These patterns are well documented in annelids, molluscs, nemertines, and platyhelminths and are considered ancestral of a bilaterian clade including these phyla. Spiral cleavage has not been documented in ecdysozoans, and no trace of the spiral development pattern is seen in phoronids and brachiopods. Origin of the spatial organization in spiralian embryos is puzzling, but much of the information appears to be encoded in the developing oocyte. Fertilization and “pseudofertilization” apparently provides the information defining the secondary, anterior‐posterior body axis in many species. The central nervous system consists of three components: an apical organ, derived from the apical blastomeres 1a¹¹¹‐1d¹¹¹, which degenerates before or at metamorphosis; the cerebral ganglia derived from other blastomeres of the first micromere quartet and retained in the adult as a preoral part of the brain; and the originally circumblastoporal nerve cord, which has become differentiated into a perioral part of the brain, the paired or secondarily fused ventral nerve cords, and a small perianal nerve ring.     

Unequal cleavage in the early Tubifex embryo

Unequal cleavage that produces two blastomeres of different size is a cleavage pattern that many animals in a variety of phyla, particularly in Spiralia, adopt during early development. This cleavage pattern is apparently instrumental for asymmetric segregation of developmental potential, but it is also indispensable for normal embryogenesis in many animals. Mechanically, unequal cleavage is achieved by either simple unequal cytokinesis or by forming a polar lobe at the egg's vegetal pole. In the present paper, the mechanisms for unequal cytokinesis involved in the first three cleavages in the oligochaete annelid Tubifex are reviewed. The three unequal cleavages are all brought about by an asymmetrically organized mitotic apparatus (MA). The MA of the first cleavage is monastral in that an aster is present at one pole of a bipolar spindle but not at the other. This monastral form, which arises as a result of the involvement of a single centrosome in the MA assembly, is both necessary and sufficient for unequal first cleavage. The egg cortex during the first mitosis is devoid of the ability to remodel spindle poles. In contrast to the non-cortical mechanisms for the first cleavage, asymmetry in the MA organization at the second and third cleavages depends solely on specialized properties of the cell cortex, to which one spindle pole is physically connected. A cortical attachment site for the second cleavage spindle is generated de novo at the cleavage membrane resulting from the first cleavage; it is an actin-based, cell contact-dependent structure. The cortical microtubule attachment site for the third cleavage, which functions independently of contact with other cells, is not generated at the cleavage membrane resulting from the second cleavage, but is located at the animal pole; it may originate from the second polar body formation and become functional at the 4-cell stage.     

Cell lineage,axis formation,and the origin of germ layers in the amphipod crustacean Orchestia cavimana

Embryos of the amphipod crustacean Orchestia cavimana are examined during cleavage, gastrulation, and segmentation by using in vivo labelling. Single blastomeres of the 8- and 16-cell stages were labelled with DiI to trace cell lineages. Early cleavage follows a distinct pattern and the a/p and d/v body axes are already determined at the 4- and 8-cell stages, respectively. In these stages, the germinal rudiment and the naupliar mesoderm can be traced back to a single blastomere each. In addition, the ectoderm and the postnaupliar mesoderm are separated into right and left components. At the16-cell stage, naupliar ectoderm is divided from the postnaupliar ectoderm, and extraembryonic lineages are separated from postnaupliar mesoderm and endoderm. From our investigation, it is evident that the cleavage pattern and cell lineage of Orchestia cavimana are not of the spiral type. Furthermore, the results of the labelling show many differences to cleavage patterns and cell lineages in other crustaceans, in particular, other Malacostraca. The cleavage and cell lineage patterns of the amphipod Orchestia are certainly derived within Malacostraca, whose ancestral cleavage mode was most likely of the superficial type. On the other hand, Orchestia exhibits a stereotyped cell division pattern during formation and differentiation of the germ band that is typical for malacostracans. Hence, a derived (apomorphic) early cleavage pattern is the ontogenetic basis for an evolutionarily older cell division pattern of advanced developmental stages. O. cavimana offers the possibility to trace the lineages and the fates of cells from early developmental stages up to the formation of segmental structures, including neurogenesis at a level of resolution that is not matched by any other arthropod system.     

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.     

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.     

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.     

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.     

Cleavage and gastrulation of the euphausiacean<Emphasis Type="Italic"> Meganyctiphanes norvegica</Emphasis> (Crustacea,Malacostraca)

According to the Articulata hypothesis the cleavage of arthropods must be derived from spiral cleavage. However, arthropods show a great variety of cleavage modes with a widespread occurrence of superficial cleavage. In the Malacostraca, holoblastic cleavage occurs in some taxa such as Amphipoda, Euphausiacea and Dendrobranchiata. In particular, the cleavage of euphausiaceans has been proposed to be a modified spiral cleavage. The cell lineage of early stages up to blastoderm formation of the euphausiacean Meganyctiphanes norvegica is reconstructed using recent methods of fluorescent staining. Only the oblique angle of the mitotic spindles during the transition from the 2- to the 4-cell stage resembles the spiral cleavage mode. At the 8-cell stage, four cells each form a pattern of two interlocking bands which is preserved until the 122-cell stage. One blastomere is delayed in division and shows an oblique division from the fourth cleavage on. It is the precursor cell of two enlarged and cleavage-arrested cells at the 32-cell stage. At the 62-cell stage, these two cells are surrounded by eight cells following a specific cell division pattern during the subsequent division cycles. The cleavage pattern of M. norvegica occurs in two mirror images. A comparative approach reveals distinct similarities between the early cleavage patterns of Euphausiacea and Dendrobranchiata which are suggested to be homologous. Furthermore, the relationships to non-malacostracan cleavage patterns are discussed. It is shown that the early cleavage pattern of M. norvegica does not offer an example of a spiral cleavage within arthropods.     

Analysis of the unique geothermal microbial ecosystem of the Blue Lagoon

Cultivation and culture-independent techniques were used to describe the geothermal ecosystem of the Blue Lagoon in Iceland. The lagoon contains both seawater and freshwater of geothermal origin and is extremely high in silica content. Water samples were collected repeatedly in summer and autumn in 2003 and 2005 and in winter 2006 were analyzed for species composition. The study revealed the typical traits of an extreme ecosystem characterized by dominating species and other species represented in low numbers. A total of 35 taxa were identified. The calculated biodiversity index of the samples was 2.1–2.5. The majority (83%) of analyzed taxa were closely related to bacteria of marine and geothermal origin reflecting a marine character of the ecosystem and the origin of the Blue Lagoon hydrothermal fluid. A high ratio (63%) of analyzed taxa represented putative novel bacterial species. The majority (71%) of analyzed clones were Alphaproteobacteria , of which 80% belonged to the Roseobacter lineage within the family of Rhodobacteraceae . Of seven cultivated species, the two most abundant ones belonged to this lineage. Silicibacter lacuscaerulensis was confirmed as a dominating species in the Blue Lagoon. One group of isolates represented a recently identified species within the genus of Nitratireductor within Rhizobiales . This study implies an annually stable and seasonally dynamic ecosystem in the Blue Lagoon.     

Formative and proliferative cell divisions,cell differentiation,and developmental changes in the meristem of Azolla roots

The root of the water fern Azolla is a compact higher-plant organ, advantageous for studies of cell division, cell differentiation, and morphogenesis. The cell complement of A. filiculoides Lam. and A. pinnata R.Br. roots is described, and the lineages of the cell types, all derived ultimately from a tetrahedral apical cell, are characterised in terms of sites and planes of cell division within the formative zone, where the initial cells of the cell files are generated. Subsequent proliferation of the initial cells is highly specific, each cell type having its own programme of divisions prior to terminal differentiation. Both formative and proliferative divisions (but especially the former) occur in regular sequences. Two enantiomorphic forms of root develop, with the dispositions of certain types of cell correlating with the direction, dextrorse or sinistrorse, of the cell-division sequence in the apical cells. Root growth is determinate, the apical cell dividing about 55 times, and its cell-cycle duration decreasing from an initial 10 h to about 4 h during the major phase of root development. Sites of proliferation progress acropetally during aging, but do not penetrate into the zone of formative divisions. The detailed portrait of root development that was obtained is discussed with respect to genetic and epigenetic influences; quantal and non-quantal cell cycles; variation in cell-cycle durations; relationships between cell expansion and cell division: the role of the apical cell; and the limitation of the total number of mitotic cycles during root formation.     

A study on cell lineage,especially the germ cell line,in embryos of the teleost fish,Barbus conchonius

Summary Lucifer Yellow-Dextran labelling of lower layer cells (LLC), sometimes together with upper layer cells (ULC), of the 64-cellBarbus conchonius embryo resulted in labelled primordial germ cells (PGCs) at 12 h after fertilization (a.f.) in about 25% of cases. The presence of labelled PGCs was independent of the location of the injected blastomere with respect to the later orientation of the embryonic axis. After injection of an ULC alone, however, labelled PGCs were never found. Also, the distribution of labelled somatic cells differed between the ULC- and LLC-injected embryos. When we found fluorescent PGCs, only a few of them were labelled, suggesting that either a single predecessor exists earlier than the 64-cell stage or that the formation of germ cells is a polyclonal process. Tracing the fluorescent cells at successive stages of development shows an extensive mixing with unlabelled cells during the epiboly stage, which might well be the cause of partly unpredictable cell lineages. The chance of being committed to a specific fate is different for the ULC and LLC descendants. This might be due to relatively limited cell mixing between these two cell populations.     

Paternal,maternal, and biparental molecular markers provide unique windows onto the evolutionary history of macaque monkeys

Abstract.— We report the results of one of the first intrageneric analyses to simultaneously survey mitochondrial, Y-chromosomal, and autosomal loci from the same individuals representing the same taxa. Phylogenetic trees were constructed for each of these genetic systems from a pool of 63 macaques, representing all 19 recognized species in this genus, and eight outgroup taxa. The mitochondrial locus analyzed here (1.5 Kb) spans the 3' end of 12S rDNA, tRNA-VAL, and the 5' end of 16S rDNA; the Y chromosome dataset (3.1 Kb) consists of the genes SRY and TSPY; the two autosomal datasets include IRBP intron 3 (1.6 Kb) and the 5' half of C4 "long" intron 9 (3.3 Kb). A total of 1.35 million bases were read, revealing 682 variable sites within the genus Macaca. With regard to earlier unresolved issues of macaque evolution, a comparison of topologies reconstructed from each of the three genetic systems suggests: (1) four monophyletic species groups; (2) an initial bifurcation among Asian macaques between the silenus group progenitor and a M. fascicularis -like taxon, with the latter representing the probable common ancestor to all non-silenus group Asian macaques; (3) a possible hybrid origin of M. arctoides from proto- M. assamensis/thibetana and proto- M. fascicularis; and (4) contemporary introgression between M. mulatta and M. fascicularis in Indochina. Inferences 3 and 4 are of particular interest, because episodes of reticulate evolution often go undetected in analyses employing a single genetic system. Finally, divergence calculations suggest that, in female-philopatric taxa, mitochondrial bifurcations may typically predate Y-chromosomal divergences at the same node.     

Embryonic development of the sea butterfly Desmopterus papilio (Gastropoda: Thecosomata)

The embryonic development of the thecosome Desmopterus papilio is described for the first time. The mature individual produced a round-shaped egg mass containing ca. 200 fertilised eggs. First cleavage was observed 15 min after the release of the egg mass. Embryos showed typical molluscan spiral cleavage: macromeres produced the first and second quartets of micromeres in clockwise and counterclockwise directions, respectively. A trochophore larva hatched from the egg capsule 28 h after the release of the egg mass. Thereafter, the larva secreted a primary shell at the posterior part, developing into the veliger stage. These findings may be useful for future work on postembryonic development, especially on the loss of the veliger shell, in the genus Desmopterus which is the only group of thecosome species without a shell in the adult stage.     

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.