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.     

Cleavage and gastrulation of the dendrobranchiate shrimp Penaeus monodon (Crustacea,Malacostraca, Decapoda)

The cleavage pattern of the black tiger shrimp Penaeus monodon was analyzed from the first division until gastrulation. Observations were based on microscopy combined with the use of fluorescent dyes, histological techniques, and computer based three-dimensional reconstructions. Early cleavage is holoblastic and follows a stereotypic pattern, which largely corresponds to what is known from other dendrobranchiate decapods. However, for the first time in this group, we report the presence of an intracellular structure throughout early development. This intracellular body (icb) marks the lineage of one of the two enlarged and division-delayed mesendoderm cells that initiate gastrulation. The identity of the icb as a natural marker and putative determinant of the germ line and its implications on the establishment of the body axes are discussed. The icb as a landmark reveals that the same stereotypic cell division pattern can lead to different fates of individual cells. Hence, the results of this study permit an additional approach to study the relation between cell lineage pattern and the identity of cell lineages.     

Cleavage and gastrulation in Pycnogonum litorale (Arthropoda, Pycnogonida): morphological support for the Ecdysozoa?

The early cleavage and gastrulation of the pycnogonid Pycnogonum litorale is investigated in detail by fluorescence microscopy, confocal laser scanning microscopy, and histology. The cleavage is holoblastic with equally sized blastomeres and an irregular radial pattern. There is no stereotypic cell lineage, and timing and spindle directions of individual mitoses vary to a high degree. Gastrulation begins at the 63-cell stage with the retardation and enlargement of a cell which adopts the form of a bottle and fills the interior of the egg, followed by immigration and epiboly of smaller cells surrounding the large bottle-shaped cell. The gastrulation site marks the dorsal side of the embryo and the stomodaeum forms adjacent to the area of gastrulation. The pattern of the early development of Pycnogonum is compared with that of other Pycnogonida resulting in a putative ground pattern of pycnogonid development. Furthermore, our results are discussed in the wider framework of putative arthropod and cycloneuralian relationships. This comparison implies morphological support for the Ecdysozoa.     

Pre-meiotic bands and novel meiotic spindle ontogeny in quadrilobed sporocytes of leafy liverworts (Jungermannidae, Bryophyta)

Indirect immunofluorescence and confocal microscopy were used to study the nucleation and organization of microtubules during meiosis in two species of leafy liverworts, Cephalozia macrostachya and Telaranea longifolia. This is the first such study of sporogenesis in the largest group of liverworts important as living representatives of some of the first land plant lineages. These studies show that cytoplasmic quadrilobing of pre-meiotic sporocytes into future spore domains is initiated by girdling bands of γ-tubulin and microtubules similar to those recently described in lobed sporocytes of simple thalloid liverworts. However, spindle ontogeny is not like other liverworts studied and is, in fact, probably unique among bryophytes. Following the establishment of quadrilobing, numerous microtubules diverge from the bands and extend into the enlarging lobes. The bands disappear and are replaced by microtubules that arise from γ-tubulin associated with the nuclear envelope. This microtubule system extends into the four lobes and is gradually reorganized into a quadripolar spindle, each half spindle consisting of a pair of poles straddling opposite cleavage furrows. Chromosomes move on this spindle to the polar cleavage furrows. The reniform daughter nuclei, each curved over a cleavage furrow, immediately enter second meiotic division with spindles now terminating in the lobes. Phragmoplasts that develop in the interzones among the haploid tetrad nuclei guide deposition of cell plates that join with the pre-meiotic furrows resulting in cleavage of the tetrad of spores. These observations document a significant variation in the innovative process of sporogenesis evolved in early land plants.     

Morphology,Oviposition, and Embryogenesis in an Australian Population of Acrobeloides nanus

A population of Acrobeloides nanus in Australia is described and illustrated, based on light and scanning electron microscopy. Embryogenesis from egg laying to hatching is followed over a wide range of temperatures. At 15 C, hatching occurs in about 125 hours and at 35 and 37.5 C after about 40 hours. At 40 C, egg development ceases early in cleavage. The capacity of A. nanus to develop over such a range of temperatures, and its anhydrobiotic capabilities, are discussed in relation to its survival and wide distribution in Australia.     

Cleavage and gastrulation in the shrimp Penaeus (Litopenaeus) vannamei (Malacostraca, Decapoda, Dendrobranchiata)

While most malacostracan crustaceans develop through superficial cleavage, in the Amphipoda, Euphausiacea, and Dendrobranchiata (Decapoda) cleavage is complete. Euphausiaceans and dendrobranchiate shrimp share a similar early cleavage pattern, early cleavage arrest and ingression of mesendoderm progenitor cells, a ring of crown cells (prospective naupliar mesoderm) around the blastopore, and hatching as a nauplius larva. Yet recent phylogenies do not support a close relationship between Euphausiacea and Decapoda. In addition, some variation is reported in the timing of mesendoderm cell arrest and number of crown cells for a number of dendrobranchiates. To determine the representative pattern of development in the Dendrobranchiata, embryos of the Pacific white shrimp Penaeus (Litopenaeus) vannamei were stained with Sytox Green to label chromosomes and nuclei and examined with confocal microscopy. The early cleavage pattern, mesendoblast arrest and subsequent ingression at the 32-cell stage, presence of 8 initial crown cells, and fates of the mesendoblasts are the same for P. vannamei (family Peneaeidae) and Sicyonia ingentis (family Sicyoniidae). The lineage of the primordial endoderm cells differs from that reported for P. kerathurus. These characters were discussed in the context of the evolution of development in the Dendrobranchiata and in comparison to the Euphausiacea.     

Formation of pigment cell patterns in Triturus alpestris embryos

The distribution of melanophores and xanthopores in developing tailbud stages of Triturus alpestris was investigated. In stage 27 embryos (curved tailbuds), melanophores are distributed evenly but sparsely over the entire dorsolateral trunk. With progressive development melanophores arrange themselves into compact dorsal and lateral bands present in stage 34 embryos (9 to 10-mm-long larvae). On the inner surface of detached pieces of skin from early tailbuds which were investigated in the scanning electron microscope xanthophores were discovered in addition to and mixed with melanophores. Unlike melanophores they are invisible from outside. Later in development they occupy the zone between the melanophore bands and also the dorsal fin. Thus, formation of pigment cell patterns in Triturus embryos is a process which seems to depend on the differential sorting out of two populations of neural crest-derived chromatophore cell types.     

Electron Micrography of Bud Formation in Metschnikowia krissii

The fine structure of bud formation of Metschnikowia krissii was studied by means of ultramicrotomy and transmission electron microscopy. Bud protrusion and development were observed by scanning electron microscopy. Bud formation in this yeast takes place by an extension of a small localized area of the existing parent wall. The parent cell and its bud are initially separated by the plasmalemma, creating an intercellular site within which the generation of new cell wall (bud and birth scar areas) occurs centripetally. When the dividing wall is complete and new cell wall material is formed, a narrow cleavage plane becomes increasingly defined. This cleavage plane apparently proceeds laterally toward the direction of the existing outer walls which rupture, resulting in the separation of the bud from the parent cell. The bud scar is prominently convex in shape; the birth scar is less conspicuous and initially concave in shape. Comparison of bud formation in M. krissii is made with that observed in Saccharomyces cerevisiae and Rhodotorula glutinis.     

Cleavage in<Emphasis Type="Italic"> Nemertoderma westbladi</Emphasis> (Nemertodermatida) and its phylogenetic significance

Recent phylogenetic analyses of ribosomal and protein coding nuclear genes place the marine worms within the Nemertodermatida as one of the oldest lineages among the bilaterian animals. We studied the early embryonic cleavage in Nemertoderma westbladi to provide the first account of nemertodermatid early development. Live embryos were studied with interference microscopy and fixed embryos were either sectioned or studied with confocal laser scanning microscopy. Initially the divisions in the embryo are radial, but then micromeres are shifted clockwise generating a spiral pattern. The four-cell stage is characterized by duets of macromeres and micromeres and thus resembles the duet cleavage reported from members of the Acoela. However, subsequent stages differ from the acoel duet pattern and also from quartet spiral cleavage. The optimization of the cleavage pattern on current phylogenetic hypotheses with Nemertodermatida and Acoela as early bilaterian branches is discussed.     

Early cleavage in Phoronis muelleri (Phoronida) displays spiral features

The view that early cleavage in Phoronida follows a radial pattern is widely accepted. However, data supporting this characterization are ambiguous. Studies have been repeatedly reporting variation between individual embryos, and the occurrence of embryos exhibiting oblique divisions or nonradial cell arrangements. Such embryos were often considered to represent variation within radial cleavage, or artificial appearances. Cleavage in Phoronis muelleri was previously characterized as “derived radial,” but also oblique spindles and cell elongations, and shifted cell arrangements were observed. We studied the early cleavage in P. muelleri applying 4D microscopy, fluorescent staining, and confocal laser scanning microscopy. To deal with the problem of variation we provide statistical evaluations of our data. These show that oblique divisions do not represent variational abnormalities. In fact, they reveal that most cells divide obliquely from the third cleavage onwards. What is more, in almost all cells the axis of the third cleavage is inclined dextrally. The fourth cleavage is even stronger sinistrally pronounced. Subsequently, the pattern of alternating cleavage orientation is largely restricted to animal and vegetal blastomeres. As a result of the obliqueness of divisions, four cells encircle the poles in most embryos. Cross furrows are occasionally present. We found no indications for radial cleavage in P. muelleri. In contrast, the observed cleavage displays several characters consistent with the pattern of spiral cleavage. A close relation of phoronid and spiralian cleavage is also suggested by molecular phylogenies, allying both groups in the Lophotrochozoa. We suggest our findings to represent morphological support for this lophotrochozoan/spiralian affinity of Phoronida.     

First cleavages,preblastula and blastula in the parthenogenetic mite Archegozetes longisetosus (Acari,Oribatida) indicate holoblastic rather than superficial cleavage

The mode of cleavage in the Acari is generalized as superficial or intralecithal, with a preceding phase of total (holoblastic) cleavage, but the knowledge is fragmentary and conclusions have been inconsistent, even when relating to the same species. Since no data about early embryology is available for the speciose group Oribatida, we studied Archegozetes longisetosus using transmission electron microscopy. We focused on early cleavages and the formation of the blastula, as these are the important and controversial points in early embryology of the Acari. We expected, as postulated for other acarine eggs, the early cleavages to be holoblastic and followed by a superficial preblastoderm stage. The early cleavages of A. longisetosus are holoblastic and blastomeres give rise to yolk-free micromeres and macromeres containing all the yolk. In contrast to expectations, the micromeres do not form a superficial preblastoderm layer. They are scattered along the embryonic surface and form an external, monocellular layer that covers the whole surface of the embryo. Since each of the existing TEM studies of mites shows this same pattern, and since this specialized form of total cleavage seems to be unique in Chelicerata, it may be the general mode of cleavage in Acari. However, the question will require much more investigation, especially since most data relate to the Actinotrichida and very few are currently available for species in the other major group, the Anactinotrichida.     

Development of cell surface activity and cell surface adhesiveness in early embryos of the newt,Cynops pyrrhogaster

The development of cell surface activity and adhesiveness was examined in relation to cleavage number in early embryos of the newt, Cynops pyrrhogaster. Both large hyaline bleb formation and surface adhesiveness to substratum were manifested in presumptive ectodermal cells isolated from embryos after the eleventh cleavage (mid-blastula stage). Scanning electron microscopy of the inner surface of the blastocoelic wall (presumptive ectodermal cell layer) revealed the formation of large blebs after the eleventh cleavage. Treatment with alcian blue and lanthanum nitrate demonstrated the accumulation of an extracellular matrix (ECM) on the surface of large blebs.     

Cleavage and gastrulation in the shrimp Sicyonia ingentis: invagination is accompanied by oriented cell division.

Embryos of the penaeoidean shrimp Sicyonia ingentis were examined at intervals during cleavage and gastrulation using antibodies to beta-tubulin and DNA and laser scanning confocal microscopy. Cleavage occurred in a regular pattern within four domains corresponding to the 4-cell-stage blastomeres and resulted in two interlocking bands of cells, each with similar spindle orientations, around a central blastocoel. Right-left asymmetry was evident at the 32-cell-stage, and mirror-image embryos occurred in a 50:50 ratio. Gastrulation was initiated by invagination into the blastocoel at the 62-cell-stage of two mesendoderm cells, which arrested at the 32-cell-stage. Further invagination and expansion of the archenteron during gastrulation was accompanied by rapid and oriented cell division. The archenteron was composed of presumptive naupliar mesoderm and the blastopore was located at the site of the future anus of the nauplius larva. In order to trace cell lineages and determine axial relationships, single 2- and 4-cell-stage blastomeres were microinjected with rhodamine-dextran. The results showed that the mesendoderm cells which initiated gastrulation were derived from the vegetal 2-cell-stage blastomere, which could be distinguished by its slightly larger size and the location of the polar bodies. The mesendoderm cells descended from a single vegetal blastomere of the 4-cell-stage. This investigation provides the first evidence for oriented cell division during gastrulation in a simple invertebrate system. Oriented cell division has previously been discounted as a potential morphogenetic force, and may be a common mechanism of invagination in embryos that begin gastrulation with a relatively small number of cells.     

Pattern and Time Course of Cleavages in Early Development of the Ovoviviparous Pond Snail, Sinotaia quadratus historica

The pattern and time course of cleavage during early development of the ovoviviparous pond snail, Sinotaia quadratus historica , in in vitro culture were investigated. The fertilized egg, 25–30 μm in diameter, underwent cleavage by repeated constriction and compaction as in blastocyst formation in mammalian embryos. The cleavage was slightly unequal and dextrally spiral, although a slight time lag in cleavages of blastomeres was observed after the 2nd cleavage. A small polar lobe was formed at the 1st cleavage, but not at the 2nd cleavage. Each cleavage proceeded very slowly under the experimental conditions, the 1st, 2nd, 3rd and 4th cleavages taking 22 hr or more, 9 hr and 10 hr, respectively. The cleavage pattern in in vitro cultures observed by light microscopy was confirmed by scanning electron microscopy.     

Effect of environmental pollution on the proteins, allergenic bands, ontogeny and structure of Avicennia marina (Forsk.) Vierh (Avicenniaceae) pollen grains

In Bushehr province of Iran, Avicennia marina trees have grown in Bordekhoon (Mond Protected Area) and Assaluyeh (Marine National Park of Nayband). Contrary to Bordekhoon, Assaluyeh is a petrochemical region with environmental pollution. This study was aimed to studying protein profiles, allergenic bands, ontogeny, structure and elemental composition of tectum of A. marina pollens in Assaluyeh and Bordekhoon. Pollens were collected from two regions and extracted in PBS, and protein profiles of pollens were determined by sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). As an experimental model, 20 female 6–8-week-old Balb/C mice were divided into two groups. The mice of first and second groups were sensitized by Bordekhoon and Assaluyeh pollen extracts, respectively, and mice serum samples were used for immunoblotting. Pollen characteristics were studied using light and scanning electron microscopes. SDS-PAGE showed some differences between pollen protein profiles of two regions. Immunoblotting assay detected that pollens have two allergenic bands and the protein band at 100 KD is the common allergenic protein in two regions. Light microscopy revealed that the development of anther wall was basic type and some abnormalities were observed in microspores and pollens of Assaluyeh. Scanning electron microscopy studies showed that the apertures in considerable numbers of Assaluyeh pollens were closed. The comparison of elemental composition of pollen tectum between two regions showed that pollens of Assaluyeh have accumulated Cu on their tectum. Results obtained indicated that environmental pollution can affect protein profile, allergenic bands, structure and elemental composition of tectum of A. marina pollens.     

Determinative development in the polyclad turbellarian,Hoploplana inquilina

Summary

Blastomere deletion experiments at the two- and four-cell stages were carried out on the embryo of the polyclad turbellarian Hoploplana inquilina to further examine the relationship between spiral cleavage and early embryonic determination in primitive spiralians. Deletion of one cell at the two-cell stage resulted in “half” larvae that were abnormal in body shape, lobe development, and behavior. Deletion of one cell at the four-cell stage produced less abnormal “three-quarter” larvae which were still underdeveloped in one of the quadrants. A 3:1 ratio of one-eyed to two-eyed larvae implies that deletion of any one of three blastomeres results in loss of an eye, with two constituting the eye lineage and the third controlling the development of two eyes. The results demonstrate that the polyclad embryo is determined early in development, though significant cell interactions occur during cleavage, and suggest that determinative development and quartet spiral cleavage are always associated and probably represent a primitive, strongly conserved evolutionary condition.     

Effect of 3'-deoxyadenosine (cordycepin) on the early development of the sand dollar, Dendraster excentricus

The embryos of the sand dollar Dendraster excentricus have been examined with regard to their ability to undergo the early events of larval development in the presence of cordycepin, a reported inhibitor of RNA adenylylation. It has been shown that all the morphogenetic events from hatching to prism are inhibited by cordycepin at a concentration of 25 μg/ml, while the aspects of development prior to hatching (cleavage, blastulation, and the formation of cilia) are not affected by cordycepin. The period of sensitivity of each developmental event to cordycepin has been determined; for the early developmental processes this period substantially precedes the event, while for later processes there is a closer temporal association. In embryos treated with cordycepin, DNA synthesis and cleavage are unaffected prior to hatching, but subsequently are blocked, whereas respiration and total RNA synthesis per cell remain unaffected.